Vitamins and Hormones: Advances in Research and Applications, Volume X

VITAMINS AND HORMONES VOLUME X

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VITAMINS AND HORMONES ADVANCES IN RESEARCH AND APPLICATIONS

Edited by ROBERTS. HARRIS Professor of Biochemistry of Nutrition, Massachusetts lnstitute of Technology, Cambridge, Masaachusetta

G. F. MARRIAN

Professor of Medical Chemistry, University of Edinburgh, Edinburgh, Scotland

KENNETHV. THIMANN Professor of Plant Physiology, Harvard University, Cambridge, Massachusetts

VOLUME X

1952 ACADEMIC PRESS INC. PUBLISHERS NEW YORK

Copyright 1952, by

ACADEMIC PRESS INC. 125 East 23rd Street, New York 10, N. Y. All Rights Reserved NO PART OF THIS BOOK MAY B E REPRODUCED IN ANY FORM,

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OTHER

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Library of Congress Catalog Card Number: (43-10535)

PRINTED IN THE UNITED STATES OF AMERICA

CONTRIBUTORS TO VOLUME X

ERNESTBEERSTECHER, JR., The Department of Biochemistry, The School of Dentistry, The University of Texas, Houston, Texas KENNETH L. BLAXTER, The Hannah Dairy Research Institute, Kirkhill, A yr, Scotland RUDIBORTH,Clinique universitaire de gynkcologie et d’obstJtrique, Geneva, Switzerland ERICK. CRUICKSHANK, University College of the West Indies,-St. Andrew, Jamaica, B. W . I . RALPHI. DORFMAN, Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts BENJAMIN H. ERSHOFF, Emory W . Thurston Laboratories, Los Angeles, and the Department of Biochemistry and Nutrition, University of Southern California, Los Angeles, California ROBERTSON F. OGILVIE,Pathology Department, University of Edinburgh, Scotland LEOT. SAMUELS, University of Utah College of Medicine, Salt Lake City, Utah, and Sloan-Kettering Institute, New York, New York ALBERT EDWARD SOBEL,Department of Biochemistry, The Jewish Hospital of Brooklyn, Brooklyn, New York F. V E R Z ~ The R , Physiological Laboratory, University of Basel, Switzerland HUBERTDE WATTEVILLE, Clinique universitaire de gyn&ologie et d’obstktrique, Geneva, Switzerland CHARLES D. WEST, University of Utah College of Medicine, Salt Lake City, Utah, and Sloan-Kettering Institute, New York, New York

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EDITORS’ PREFACE The Editors of Vitamins and Hormones heartily welcome Dr. G. F. Marrian as a member of the editorial team. They have long felt that these volumes would be strengthened by addition of a third editor with a different scientific and geographic viewpoint. The successive issues of Vitamins and Hormones continue to meet with acceptance and the Editors hope that the almost complete absence of adverse comment indicates that these volumes are filling the need for critical reviews in the vitamin and hormone field adequately. It will be noted that a majority of the articles in the present volume are concerned with hormones. This is a reflection of the greater amount of research being conducted a t present in the hormone than in the vitamin field. The articles come from the fields of biochemistry, endocrinology, experimental zoology, animal husbandry and clinical nutrition. In this respect they typify the breadth of scientific interest in these substances and their functions. THE EDITORS

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CONTENTS CONTRIBUTORS . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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EDITORS’PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . . vii Dietary Neuropathies BY ERICK . CRUICKSHANK, University College of the West Indies. St . Andrew. Jamaica. B . W .I .

I . Introduction . . . . . . . . . . . . . . . . . I1. Beriberi-Peripheral Nerve Lesions Including Neuritis . . . . . . . . . . . . . . . . . . . I11. Painful Feet Syndrome . . . . . . . . . . . . . IV . Wernicke’s Encephalopathy . . . . . . . . . . . V . Niacin Deficiency Encephalopathy . . . . . . . VI . Retrobulbar Neuropathy . . . . . . . . . . . . VII . Cranial Nerve Lesions . . . . . . . . . . . . . VIII . The Cord Syndromes . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . .

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4 18 . . . . . . . . 28 . . . . . . . . . 32 . . . . . . . . 33 . . . . . . . . 38 . . . . . . . . 39 . . . . . . . . 42

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The Problem of the Absorption and Transportation of Fat-Soluble Vitamins BY ALBERT EDWARD SOBEL,Department of Biochemistry, The Jewish Hospital of Brooklyn, Brooklyn, New York

I . Introduction . . . . . . . . . . . . . . . . . . . . I1. Problem of the Absorption of Fat-Soluble Vitamins . . I11. Problem of the Transportation of Fat-Soluble Vitamins IV . Concluding Remarks . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .

. . . . . . . 47 . . . . . . . . 48 . . . . . . . . 56 . . . . . . . 64 . . . . . . . 65

The Nutrition of the Crustacea JR.,The Department of Biochemistry, The School of Dentistry, BY ERNESTBEERSTECHER, The University of Tezas, Houston, Tezas

I . Introduction . . . . . . . . . I1. Culture Methods . . . . . . . I11. Specific Requirements . . . . . 1V.Summary . . . . . . . . . . . References . . . . . . . . . .

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69 71 73 76 76

Nutrition and the Anterior Pituitary with Special Reference to the General Adaptation Syndrome BY BENJAMIN H . ERSHOFF,Emory W . Thurston Laboratories, Los Angeles, and the Department of Biochemistry and Nutrition, University of Southern California, Los Angeles

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 I1. Effects of “ Malnutriture” on the Synthesis and Secretion of Pituitary Hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 ix

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I11. Effects of Miscellaneous Nutrients on Pituitary Structure and Function . 101 IV. Nutrition and the General Adaptation Syndrome . . . . . . . . . . . 103 V. Effects of Nutritive State on Response to Pituitary Hormones . . . . . 126 VI . Effects of Pituitary Hormones on Nutritive State . . . . . . . . . . . 127 VII Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

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Hormone Assays in Obstetrics and Gynecology BY RUDI BORTHAND HUBERTDE WATTEVILLE, Clinique universitaire de gynlcologie et d'obstltrique, Geneva, Switzerland I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 142 I1. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 I11. Menstrual Cycle and Menstrual Disorders . . . . . . . . . . . . . . 152 I V . Sterility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 V. Pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162 VI . Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 V I I . Endocrine Disorders . . . . . . . . . . . . . . . . . . . . . . . . 173 VIII . General Conclusions. . . . . . . . . . . . . . . . . . . . . . . . 174 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 Experimental Glycosuria : Its Production, Prevention, and Alleviation BY ROBERTSON F. OGILVIE,Pathology Department, University of Edinburgh, Scotland I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 184 I1. Insulin Insufficiency . . . . . . . . . . . . . . . . . . . . . . . . 185 I11. Hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196 I V . Diet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206 V . Glycogenolysis . . . . . . . . . . . . . . . . . . . . . . . . . . 207 VI . Kidney . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211 Some Effects of Thyroxine and Iodinated Casein on Dairy Cows, and Their Practical Significance BY KENNETH L. BLAXTER,The Hannah Dairp Research Institute, Kirkhill, Ayr, Scotland I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 I1 The Biological Activity of Preparations . . . . . . . . . . . . . . . 219 111. The Effect of Thyroxine and Iodinated Proteins . . . . . . . . . . . . 224 IV. Homeostatic Effects . . . . . . . . . . . . . . . . . . . . . . . . 229 V The Effect on the Composition of the Milk . . . . . . . . . . . . . . 234 VI. The Effect of Thyroxine and Iodinated Proteins on the Metabolism of the 240 Cow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII . Secondary Effects . . . . . . . . . . . . . . . . . . . . . . . . . 243 VIII . Practical Conclusions . . . . . . . . . . . . . . . . . . . . . . . 245 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247

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The Intermediary Metabolism of the Non-benzenoid Steroid Hormones BY LEOT . SAMUELS AND CHARLES D . WEST, University of Utah College of Medicine, Salt Lake City, Utah, and Sloan-Kettering Institute, New York, New York I Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 11. Intermediary Metabolism of the Androgens. . . . . . . . . . . . . . 252

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I11. Intermediary Metabolism of the Progestins . . . . . . . . . . . . . . 276 IV . Intermediary Metabolism of Steroids of the Adrenal Cortex . . . . . . . 285 V . General Conclusions . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .

289 291

The Influence of Corticdds on Enzymes of Carbohydrate Metabolism

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BY F VERZ~R, The Physiological Laboratory, University of Basel. Switzerland I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 297

I1. General Carbohydrate Metabolism . . . . . . . . . . . . . . . . . . 298 I11. Experiments with Isolated Organs . . . . . . . . . . . . . . . . . . 303 I V. Experiments on Enzymes . . . . . . . . . . . . . . . . . . . . . . 309 V . Histochemical Demonstration of Alkaline Phosphatase . . . . . . . . 316 VI . Phosphoglucomutase . . . . . . . . . . . . . . . . . . . . . . . . 318 VII . Hexokinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319 VIII . Oxidase Systems . . . . . . . . . . . . . . . . . . . . . . . . . . 319 IX . Protein Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . 320 X Reactions between Carbohydrate and Potassium Metabolism in Vitro . . 321 X I . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326

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Steroids and Tissue Oxidation

BY RALPHI. DORFMAN, Worcester Foundation for Experimental Biobgy, Shrewsbury, Massachusetts

I . Introduction . . . . . . . . . . . . . . . . I1. Steroids and Tissue-Enzyme Concentrations . I11. In Vitro Influence on Oxidative Metabolism . I V . Steroids Influencing Specific Enzyme Systems . V . I n Vitro Effects on Metabolism . . . . . . . VI . Conclusions . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . .

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331

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366 368

AUTHORINDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

371

SUBJECTINDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Dietary Neuropathies BY ERIC K. CRUICKSHANK

University College of the West Indies, St. Andrew, Jamaica, B . W . I . CONTENTS

I. Introduction 11. Beriberi-Peri

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g “Alcoholic Peripheral

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1. Pathology . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . .

. . .. . . . . . . . . . . . . . ..... . 3. Summary of Pathology.. . . . . . . . . . . . . . . . . . . . . . 4. Clinical Features Based on Cases in Changi Prisoners-o Singapore. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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b. Motor Features.. . . . . . . . . . . . . . . . , . . , . . . . . . ......................... c. Sensory Features. . . d. Tendon Reflexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . e. Discussion.. . . . . . . . .................. .......................................... f. Summary.. . . . .. ,... ... 111. Painful Feet Syndrome.. . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . ............................. .,,... ... 1. Historical. . . . . . . .........................

4 4 5 10

10 15 15 15 16 16

18 18 18 19

3. Signs..... . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . 20 20 ............................. a. General. . . . . . . . . . . 20 b. Feet and Legs.. . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . 21 c. Nervous System.. . ..................................... d. Cardiovascular System. . . . . . . . . . . . , , . , , . . . . . . . . . . . . . . . . . . . . . . 21 21 .... 4. Treatment ... . . . . . . . . . . . . . . , . . . . . , , . . . . , , . . 22 5. Etiology and Pathology.. . .... .... 22 es . . . . . . . . . . . . . . . . . . . . . . . . . a. Associated Deficiency 23 b. Mechanism of Pain Production. . . . ...................... 24 c. Significance of Exaggerated Reflexes. . . . . . . . . . . . . . . . . . . . . . d. Cause of Hypertension. . . . . . . . . . . . . . . . . . . . . . . . . . 25 . . _ . . _ . . . . 27 6. Summary .... . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 IV. Wernicke’s Encephalop 28 ................. 1. Pathology ....... . . . . . 29 2. Etiology in Relation 29 a. Clinical Evidence b. Experimental Evidence. . . . . . . . . . , . . . . . . , . . . . . . . . . . . . . . . . . . . . . . 30 3. Clinical Features.. . . . . . . . . . . . . . . , , . , . . , , , . . . . . . . . . . . . . . . . . . . . . . . 30 32 4. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . ........................... . . 32 5. Summary .... . . . , , . . , . . . . . . , , . , , . . , , . . , . . . . . . . . . . . . . . . . 1

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V. Niacin Deficiency Encephalopa VI. Retrobulbar Neuropathy ...... 1. Historical ................. 2. Clinical Features.. ......... 3. Summary .......................... . . . . . . . . . . 37 VII. Cranial Nerve Lesions. ........ VIII. The Cord Syndromes.. .......... 1. Pathological and Clinical Fe 2. Relation to Lathyrism.. .... 3. Summary ............................ References........................................................ 42

I. INTRODUCTION Neuropathy in its strictest sense means pathological change in the nervous tissue. Nowadays however, the term is used to describe damage to nervous tissue when the primary lesion is a degeneration of the nerve cell or its processes with little or no evidence of true inflammatory reaction. The cause of the degeneration is disturbance of the normal metaboliim of the cell. This may result from (1) toxic factors, extrinsic or intrinsic, including the “allergies,” or (2) deficiency of substances necessary for normal cell metabolism. Such a deficiency can occur when adequate amounts of one or more of these substances are not available to the cell whether from (a) interference with the blood supply, (b) failure of the body to produce them either by synthesis or from store, or (c) insufficient amounts being present in the gastrointestinal tract or absorbed therefrom. The term “dietary neuropathy” is applied to a variety of clinical syndromes when there is good evidence that lack of a factor or factors essential for normal nerve cell metabolism plays an important part in the cause of the disorder. In the great majority inadequate absorption is responsible but toxic agents, a poor blood supply, deficient synthesis, deficient storage by the body, antienzymes or analogues may be precipitating or aggravating factors. Our knowledge of these syndromes has been greatly extended by observations made on prisoners-of-war and undernourished European populations during World War 11. The majority, however, had been recognized and accurately described before that time. Major advances in neurocellular chemistry and the development of biochemical methods of vitamin assay and of synthesis or extraction have thrown much light on the cause and nature of the lesions. There are, however, still many aspects of the nutritional neuropathies to be explained. Why are only certain persons affected in a large group, when the food intake of each member is often roughly the same? Defective absorption, inadequate

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bacterial syntheses, disturbed liver function, inherent deficiencies of, or excessive metabolic demands on the cells affected, are all attractive theories, but proof as yet is lacking. Why are some of the features so prevalent only in the tropics or subtropics, although the food and vitamin intake may be much less in certain groups in temperate climates? This difference was emphasized by the high incidence of specific deficiency syndromes among prisoners-of-war in the Far East and the very low incidence in grossly undernourished persons in occupied countries and concentration camps in Europe. Abnormal carbohydrate, protein, fat, or vitamin ratios in the diet are a possible explanation, as are high malarial, dysenteric or worm infestation rates, but the evidence is still inconclusive. There are undoubtedly other factors as yet unknown or purely speculative. There is, therefore, still much scope for investigation of these problems, both by accurate observation with controlled experiments in the field and by detailed biochemical or biophysical methods. A clinical classification of the dietary neuropathies is still the most satisfactory, since the cause of some of them is not yet definitely established and they are only believed to be due to a deficiency state. In the patient it is usually easy to show that there is an insufficiency of a number of substances in the diet. It is therefore difficult to attribute the condition to lack of a specific substance. The therapeutic test is valuable when a clear-cut and immediate response is obtained, but when this is not the case, deficiency of the substance used cannot be ruled out as the causative factor. In human nervous system lesions, a prolonged therapeutic test is rarely justifiable without an over-all improvement in the diet so that the results are difficult t o msess. Damage to nervous tissue is seldom quickly reparable. Three grades of damage can be postulated. (a) The structure of the cell and its processes may appear intact but its function is grossly impaired, either from physicochemical disturbances or from pressure of adjacent edema. (b) Demyelinization of the axis cylinder may be present. (c) There may be demyelinization, axon destruction, and even cell death. In the first instance specific therapy may rapidly restore normal function. In the other two the response will depend on whether the nerve processes concerned have a neurilemmal sheathor not. When this is present, as in a peripheral nerve, remyelinization may gradually occur, or there may be a slow re-growth of the axis cylinder; but where a central neuron is involved the damage may be permanent in spite of intensive specitic therapy. The various clinical syndromes can be divided into two main groups. Group I-where the lesion is predominantly in the peripheral nerve. This group comprises (a) beriberi neuropathy, including ‘(alcoholic polyneuritis ’’ and “ polyneuritis gravidarum ” ; (b) the painful feet

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syndrome. Group 11-where the lesion is predominantly in the central nervous tissue. This group includes (a) Wernicke’s encephalopathy ; (b) niacin (nicotinic acid) deficiency encephalopathy, if such a condition exists; (c) retrobulbar neuropathy ; (d) the cord syndromes, namely “spinal ataxia” and spastic paraplegia, or a combination of both. There is a third group of diseases of the nervous system in which deficiency of specific food factors may play a part as precipitating or aggravating elements, but they cannot properly be classified as “ deficiency neuropathies.” Examples in this category are : the relationship of vitamin E t o progressive muscular atrophy, and the relation of the B complex vitamins to Korsakow’s psychosis and depressive states. In the same way subacute combined degeneration of the spinal cord in pernicious anemia cannot be regarded as a “dietary neuropathy ” although the mechanism of its production may be closely related to or identical with that of the cord syndromes. The lesion may result from a defect in the enzyme systems concerned with the metabolism of nerve tissue, and this defect may have a variety of causes as already mentioned. This latter group, however, will not be reviewed here.

11. BERIBERI-PERIPHERAL NERVELESIONSINCLUDING “ALCOHOLIC PERIPHERAL NEURITIS” 1. Pathology

It was only during the latter half of the last century that the study of morbid neuroanatomy became the sine qua non of clinical investigation in this field and the earliest reliable reports of pathological changes observed in the nervous system in beriberi came from Balz (1882). He termed the disease “pan-neuritis endemica’’ and stated that “its nature is that of a true neuritis with degeneration of the fibers quite analogous to that observed in peripheral paralysis or that produced by cutting the nerves.” Scheube (1884) found few changes in the spinal cord, but noted swelling and later degeneration of the medullary sheath into droplets and finally disintegration and complete absorption of medullary sheath and axis cylinder; in the peripheral nerves from cases of paralytic beriberi the changes were most marked in the muscular branches and there was marked atrophy of the associated muscle fibers. Pekelharing and Winkler (1887) gave the first detailed account of the histological findings in a large series of 85 cases. These confirmed Scheube’s findings, but in cases of long duration degenerative changes were found in the posterior root ganglia and these extended centrally into the columns of Goll. The anterior roots showed minimal changes. Some of the anterior and posterior cells of khe spinal cord showed degeneration. These were regarded

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as secondary to damage to the peripheral processes. They also pointed out that these changes were not specific to beriberi, but occurred in all forms of peripheral neuritis. Wright (1901) found changes in the posterior spinal ganglion cells and anterior horn cells, and in the nuclei of the medulla, in cases where the fibers originating from these cells were atrophied. Further detailed examination of the peripheral nerves and spinal root ganglia by Yamagiwa (1899) and Wright (1902) confirmed these earlier observations. Durck (1908) and other workers noted circumscribed recent hemorrhages as almost constant findings in the gray matter. They were regarded by them as inflammatory, but Tsunoda (1909) held that they were purely degenerative in origin and he was thus the first protagonist of what is now the generally accepted view. 2. Etiology and Experimental Pathology

At the turn of the century there was considerable controversy and speculation as to the causes of beriberi. As a result of the pioneer experimental work of Eijkman (1897) the theory that there was a deficiency of some essential substance in the diet was rapidly gaining ground. Eijkman showed that fowls fed on polished rice developed polyneuritis that could be cured or prevented by the administration of a watery or alcoholic extract of rice bran. These experiments were repeated and the results confirmed by subsequent workers, and a detailed description of the histological changes in the nervous system of fowls with polyneuritis induced in this way was published by Vedder and Clark in 1912. I n 1938 Vedder summarized the changes as follows. Every nerve fiber showed some evidence of degeneration though the extent of the lesion varied greatly in different fibers of the same nerve. The myelin was degenerated and there was swelling of the nerve sheath with disintegration of the axis cylinder in 10 to 15% of the cases. The earliest change was slight swelling of the medullary sheath with a tendency towards fragmentation at the circumference. Then large fatty globules that distended the nerve were seen. These became much smaller and finally the neurilemma contained only a few scattered droplets of fat. These changes did not commence in the peripheral nerves but were generalized, affecting both dorsal and ventral nerve roots, all tracts of the spinal cord, medulla, pons, midbrain, and internal capsule. Similar changes in the peripheral nerves have been found in thiaminedeficient animals by other workers since that time (McCarrison, 1919; Findlay, 1921; Voegtlin and Lake, 1919; Woollard, 1927; Zimmerman and Burack, 1932). These findings resemble very closely those already noted in the peripheral nerve in naturally-occurring beriberi in man, and Kimura

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(1920), in a detailed study of the peripheral nerves from beriberi patients and in polyneuritis gallinarum, could find no recognizable difference between the two lesions. Although it is now generally agreed that deficiency of thiamine is an important factor in the production of peripheral neuritis in beriberi, there have been conflicting reports as to whether pure thiamine deficiency under experimental conditions can cause peripheral neuritis in man and animals. That starvation alone can produce changes in peripheral nerves very similar to those resulting from thiamine deficiency was first demonstrated by Chamberlain and his co-workers in 1911 and subsequently by other workers. Vedder and Chinn (1938) found that rats that were starved and given 3 mg. of thiamine daily developed changes in the peripheral nerves identical with those in rats that were starved and given no thiamine, but they were less extensive. The suggestion is made that anorexia -an early symptom of thiamine deficiency-leads to a deficient calorie intake and that the resulting starvation is responsible for the nerve changes. This, however, is unlikely, as signs of peripheral neuritis have been produced experimentally with a diet deficient in thiamine but with an adequate number of calories, and severe polyneuritis often occurs in apparently well-nourished persons. On the other hand, some workers (Engel and Phillips, 1938; Gildea et al., 1930; Prickett, 1934) found no, or few, degenerating nerve fibers in thiamine-deficient rats and expressed the opinion that the symptoms of thiamine deficiency could not be explained on the basis of observable pathological lesions. I n a later paper Prickett et al. (1939) point out that, when rats are rendered acutely thiamine deficient by a thiamine-free diet, no marked changes are found in the peripheral nerves, but when inadequate amounts of thiamine are present in the diet, marked changes in the myelin sheath and axis cylinder appear, particularly when studied with polarized light. Aring and his colleagues (1941) have demonstrated a marked reduction in the number of myelin sheaths per square millimeter, particularly those of larger size, in biopsy specimens of peripheral nerves from pellagrins presenting mild signs of peripheral neuritis. In longitudinal section along with the myelin degeneration there was a considerable amount of beading, fragmentation, and corkscrew formation of the axis cylinders. The signs rapidly disappeared on thiamine administration alone. These histological findings are almost identical with those that Greenfield and Carmichael (1935) described in the peripheral nerves of patients with subacute combined degeneration of the spinal cord. Walshe (1941) points out that, clinically and pathologically, beriberi is a typical polyneuritis and Kinnier Wilson (1940) states “that by whatever diverse and heterogeneous factors nerve substance sustains injury its means of reaction are

DIETARY NEUROPATHIES

7

strictly limited and well defined. A strict division between parenchymatous neuritis and an interstitial neuritis cannot be made as, where parenchymatous degeneration occurs, there is reaction of the interstitial cells and vice versa.” Swank and his associates (1941), however, claim that the changes in the peripheral nerves of experimentally starved rats differ from those seen in the thiamine-deficient animal. I n starvation the changes are confined to the myelin sheath where the myelin droplets diminish in size and eventually disappear. The axis cylinders remain intact and the animals do not exhibit signs of paralysis. I n further experiments Swank and Prados (1942) rendered pigeons acutely and chronically thiamine deficient by careful dietetic measures. I n the acutely deficient birds that received practically no thiamine, opisthotonos rapidly became the outstanding feature. The symptoms were immediately relieved by thiamine. Birds killed when opisthotonic showed few or no degenerative changes in the central nervous system. When the diet was partially deficient in thiamine, opisthotonos did not occur and leg weakness was the characteristic feature. Degenerating nerve fibers were always found in the peripheral nerves, the changes being practically identical with Wallerian degeneration such as occurs in myelinated fibers of the divided sciatic nerve of a rat. The number of degenerated nerve fibers in the sciatic nerve corresponded closely with the degree of paralysis. Degenerative changes were seen earliest in the large and long fibers and appeared first a t the point most distant from the cell body, thereafter proceeding centripetally to within a few millimeters of the dorsal root ganglia in the most severe cases. After about four days chromatolysis appeared in the dorsal root ganglion cells. On the administration of thiamine the total number of myelinated nerve fibers greatly increased and in spite of the apparent severe damage to the peripheral nerve, regeneration of the axis cylinder occurred and few neurons died. The chromatolysis in the cell body disappeared only after complete repair of the axis cylinder i.e., in an average of 55 days. These findings in the peripheral nerves of pigeons are practically the same as those Aring et al. (1941) had already noted in the peripheral nerves of pellagrins, where there was also a rapid response to thiamine treatment alone. Swank also found changes in the spinal cord. These were most apparent in the long and large fibers of the ascending spinocerebellar tract and were most marked a t the peripheral end of the neuron, i.e., from the cervical enlargement upwards. It is of interest that the peripheral processes of the dorsal root ganglion cells degenerated whereas the central processes which enter the cord and form the fasciculus cuneatus and fasciculus gracilis were unaffected, This would indicate that the process of degeneration in these cells was selective,

8

ERIC K . CRUICKSHANK

the order of events being such as to preserve the neuron. Had the central processes degenerated, a permanent loss of function with ultimate death of the neuron would have resulted. The conclusions that Swank draws from these findings are that, when a neuron is subjected to slow depletion of thiamine, its axis cylinder degenerates first a t the point most distant from its trophic cell body because of the apparent difficulty with which this part of the neuron is maintained in its normal state. The myelin sheath also degenerates but this occurs after the axis cylinder is damaged. This observation is in agreement with the much earlier finding of Kimura (1920). This hypothesis of Swank’snamely, that the cell body is the primary site of the disordered metabolism and that it is the changes there which lead to the degeneration a t the distal end of the axon-is more reasonable and more in keeping with physiopathological principles than the conception that local factors produce the damage a t the periphery of the axon. It is supported by the two well established facts, (i) that the longest and the largest axons are the first to degenerate and (ii) that the earliest signs of damage to a peripheral nerve in beriberi appear in the areas most peripheral to the trophic nerve cell, i.e., in the toes and feet and in the tips of the fingers. As there is evidence that the cells of the nervous system utilize only carbohydrate for their energy and that thiamine is an essential agent in this process, it is understandable that, when thiamine is deficient, the nerve cell with its long processes is one of the earliest to show signs of damage. Meiklejohn (1940) in a critical review of the relationship of polyneuritis to vitamin B deficiency concluded that “there is as yet no clear experimental evidence showing that true anatomic polyneuritis is curable by thiamine alone.” Swank’s and Aring’s observations have been published since this statement was made and if their findings are confirmed, the criticism is no longer justified. Thiamine has been administered to all forms of polyneuritis with disappointing results and Walshe (1941), although he is prepared to accept the hypothesis that a defect in carbohydrate metabolism in thiamine deficiency leads to nerve damage, states that the results of treatment with thiamine do not confirm the hypothesis. Improvement in peripheral neuritis, however, with thiamine therapy will depend on two factors, namely (i) that the neuritis is due to deficiency of thiamine and not to any other factor or factors such as a bacterial or chemical toxin and (ii) that the degenerative changes in the nerve are not too far advanced. The significance of a response or otherwise from nervous tissue to a specific therapeutic agent has already been discussed (Section I). The extent and rate of recovery will depend on the number of fibers ’

DIETARY NEUROPATHIES

9

involved and the degree of damage in each. Such a conception may explain the varied results of treatment with thiamine alone in the neuritis of naturally occurring beriberi, in artificially induced thiamine deficiency in human beings and experimental animals, and in ‘(alcoholic” peripheral neuritis, and polyneuritis gravidarum. The relationship between this last condition and the neuritis of beriberi deserves further consideration. Shattuck (1928) first suggested that this latter condition was “caused chiefly by failure to take or assimilate food containing sufficient quantity of vitamin B . . . and might properly be regarded as beriberi.” I n 1933 Strauss and McDonald showed that the neuritis improved markedly on the administration of yeast by mouth and vitamin B concentrates and liver extract parenterally, even if the patients still consumed large quantities of alcohol. Jolliffe et al. (1936) produced evidence to support Strauss’s findings. Brown (1941), however, did not find that thiamine hastened recovery in ((alcoholic”peripheral neuritis. As already pointed out, the response to specific thiamine therapy in polyneuritis due to thiamine deficiency will depend upon the stage which the pathological process in the nerve has reached. Therefore Brown’s results do not exclude thiamine deficiency as the initial cause of the polyneuritis. Chronic alcoholism is capable of producing the full clinical picture of beriberi with edema and cardiac changes that respond rapidly to thiamine therapy alone. Whether all cases of alcoholic peripheral neuritis are due to thiamine deficiency alone is uncertain but there is evidence that it is an important factor in a high proportion of cases. Aring et al. (1939) found that daily doses of 100 mg. of thiamine intravenously produced rapid improvement in function in peripheral neuritis associated with pellagra, beriberi, alcoholism, tuberculosis and pregnancy. Pain disappeared in 24 hours and bedridden patients were able to walk within a few weeks. I n 12 such cases biopsies of a terminal branch of the anterior tibia1 nerve were made both before treatment and several months after treatment with satisfactory improvement, but with residual signs of persisting nerve damage. In all cases there was extensive demyelinization, but in most the axon was intact before treatment and there was little change in the specimens taken months later. These findings suggest that the early rapid improvement in function is due to the ability of thiamine to enhance the effect of acetylcholine in the transmission of the nerve impulse. Minz (1938) had already claimed to have demonstrated such an action of thiamine and it may cause inhibition of cholinisterase. Aring et al. (1939) states that such inhibition was demonstrable in his cases and quotes Antopol and Glick (1939) as having made the same observations with horse and rat serum in vitro. Such being the case, thiamine has the same effect as physostigmine. They do not state

10

ERIC K. CRUICESHANK

whether or not physostigmine produced the same rapid improvement. Moreover their theory demands an intact axon before acetylcholine can be produced at the nerve ending. A functional failure of impulse transmission resulting from a defect in nerve-cell metabolism that can be rapidly corrected by thiamine administration seems a more logical theory. Von Muralt (1948) is of the opinion that acetylcholine is liberated in parts of the nerve cell unit other than the nerve ending and reviews the experimental evidence. He also produces evidence that as well as acetylcholine, a thiamine-like substance, possibly thiamine disulfide is liberated in the nerve, during excitation. He suggests that these two substances may be essential for the transmission of the impulse from one node to the next in medullated nerves by providing the free energy from which the action current is derived. It is therefore an interesting speculation as to whether or not the primary nerve lesion in thiamine deficiency is lack of this thiamine-like substance, which may be necessary for node to node transmission of the impulse. 3. Summary

of Pathology

The pathological findings in the peripheral nerves of patients with beriberi neuropathy range from complete destruction of the myelin sheath and axis cylinder, with the deathof the parent cell, tominor changes in the myelin sheath and axis cylinder. The changes are identical with those seen in the peripheral nerves of animals rendered partly thiaminedeficient under experimental conditions. There is evidence that the administration of thiamine alone can arrest and repair these changes provided the nerve cell is not irreparably damaged. It is suggested that the primary lesion may be in the cell body and that the peripheral changes are secondary. The rate and degree of recovery will depend on the number of neurons affected and the stage which the degenerative changes in the neuron have reached.

4. Clinical Features Based on Cases in Changi Prisoner-of-War Camp, Singapore The neurological features of beriberi are well known from the classical descriptions of the disease (Scheube, 1894; Bdz and Miura, 1905; Vedder, 1913; Shimazono, 1931). There are few detailed analyses of large series of cases to be found in the literature. As the Changi Prisoner-of-War Camp in Singapore provided an excellent opportunity for careful observation of a closed and well-organized community, where the dietary intake of the population could be calculated reasonably accurately, the author feels that some of the information from 400 patients on whom he was able to keep records is worth recording.

DIETARY NEUROPATHIES

11

The incidence of fresh cases of beriberi occurring between March 1942 and August 1945 is shown on Fig. 1. It will be seen that there was an initial period when the total calorie intake was adequate but the thiamine intake was low in proportion to the nonfat calories. During this time numerous cases occurred. There followed a period of 2 years when thiamine and calorie intakes were a t a reasonable level and few new

FIG. 1. Monthly incidence of beriberi compared with estimated total calories and vitamin B1 non-fat-calorie ratio of diet. (From Burgess, R. C., Lancet 2, 41 1, 1946.)

cases appeared. I n the third period both calorie and thiamine intakes were low and fresh cases again became frequent. I n these, protein deficiency and general undernutrition may have been complicating factors but the clinical picture did not differ materially from the early ones. It will be seen that Williams and Spies' (1938) critical level of 0.3 for the thiamine nonfat calorie ratio-empirical though it may be-was of value in predicting the appearance of fresh cases of beriberi. a. Predisposing Factors. The incidence of acute illness preceding the onset of beriberi and therefore probably a precipitating factor was as follows:

12

ERIC K. CRUICKSHANK

Total cases of beriberi.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diarrhea or dysentery as prisoners-of-war.. . . . . . . . . . . . . Malaria as prisoners-of-war. .......................... Other diseases as prisoners-of-war (including pneumonia, dengue and infectious hepatitis). ....................

400 (100%)

154 (38.5%) 68 (17 . O % ) 28 (7 .O%)

Clinical evidence of disease attributed to deficiency of factors other than thiamine was present only in the later cases of the series. Their incidence is shown below: Scrota1 dermatitis.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 (7.75%) Stomatitis (including glossitis, angular stomatitis, and 22 (5.5%) cheilosis) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . “Painful Feet”. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 (0.75%) 1 (0.25%) Retrobulbar neuropathy.. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

The alcohol consumption of the first twenty patients to present with beriberi is of interest. Twelve of these had been consuming half a bottle of whisky or more, or over ten points of beer per day, prior to incarceration. I n the other eight the alcohol consumption was unknown or low. The alcohol consumption of an equivalent age group who had not developed beriberi a t that time was unfortunately not recorded but it would appear that a high alcohol consumption before imprisonment was an important precipitating factor in the early cases. These patients may have been thiamine deficient before imprisonment and the camp diet with possibly associated defective absorption was sufficient to produce frank signs rapidly. As might be expected, the highest incidence of beriberi was in those men doing manual labor. Psychological changes such as irritability and depression, inability to concentrate and uncertain memory (“ Changi Memory ”) were very general mental complaints. Such symptoms have been recorded in experimentally induced thiamine deficiency in man (Williams et al., 1942), but many of the patients with beriberi were mentally alert and optimistic, and there was certainly no state of mind typical of beriberi. Neurological features were present in 229 (57.25%) of the 400 cases diagnosed as suffering from beriberi. The time of incidence of these 400 cases is shown in Fig. 2. They are divided into 3 groups: (1) Those with edema and with or without cardiac abnormalities, and no neurological changes. (2) Those with edema and neuritic signs. (3) Those with neuritic signs only.

The close relationship between the three curves favors a common etiology, viz. thiamine deficiency. A detailed analysis of the neurological

13

DIETARY NEUROPATHIES

OLDLMA O N L Y 30

20

10

0

OEDEMA AND

NEURITIC SIGNS

30

20

I 0

0

NLURlTlC SIGNS

ONLY

30

20

I0

8

1

I

I

'

I

0

1941

I

194a

I

I944

I

FIG.2. Monthly incidence of the reported cases of beriberi presenting with edema only, edema and neuritis, and neuritis only, from February 1942 to February 1944. Note the close relationship between the three curves which favors a common etiology, uiz., vitamin B1deficiency.

14

ERIC K. CRUICKSHANE

features in the first 171 cases (those seen between March 1942 and February 1944) is given in Table I. b. Motor Features. Weakness in the legs in the mildest cases only appeared on walking a short distance (100 yards or more) when the patient TABLE I Analysis of Neurological Findings* Motor symptoms only, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sensory symptoms only.. ........................... Sensory and motor symptoms. . . . . . . . . . . . . . . . . . . . . . .

38 (22.2%) 52 (30.8%) 81 (47.0%)

171 (100.0%) Motor features (with and without sensory changes) Weakness of the legs alone. . . . . . . . . . . . . . . . . . . . . . . . . . 85 (71.4 %) Weakness of the legs and arms. . . . . . . . . . . . . . . . . . . . . . . 10 ( 8 . 4 % ) Winged scapulae.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 (2.6%) Weakness of muscles supplied by ulnar nerve in one hand 2 (1.8 % ) Unilateral foot drop.. .............................. 19 (15.9%) 119 (100.0%) Sensory symptoms (with and without motor symptoms) Paraesthesia alone. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 (43.0%) Numbness alone. .................................. 41 (30 .O%) Numbness and Paraesthesia. . . . . . . . . . . . . . . . . . . . . . . . . 36 (27 .O %)

--

133 (100.0%)

Distribution

Legsalone.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 (60.1%) Trunk, legs and arms.. . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 (28.7%) Arms alone.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 (4.5%) Distribution of lateral femoral cutaneous nerve.. . . . . 5 (3.7%) Ulnar nerve of one hand. . . . . . . . . . . . . . . . . . . . . . . . . . 3 (2.3%) Abdomen round umbilicus. . . . . . . . . . . . . . . . . . . . . . . . 1 (0.7 %) 133 (100.0%) Aching, pain, stiffness, tightness, or cramps in the 46 calves or associated muscles. .................... Associated with motor weakness.. . . . . . . . . . . . . . 37 Associated with sensory changes only.. . . . . . . . . . 9

* Findinga in 400 owes of beriberi.

would tend to drag his feet. The “squatting test” was not a reliable sign of early muscle weakness in the legs, and weakness of dorsiflexion of the feet was much more reliable. Denny-Brown (1947) makes the same comment. I n the others it was of varying severity up to complete inability to stand or to move the ankles or knees. It was usually most marked in the peronei. In five apparently healthy men this symptom appeared suddenly, in three while walking, and in two after running some distance (100 to 400 yards). These five had a marked degree of paresis when they

DIETARY NEUROPATHIES

15

were seen 6-24 hours after the onset and all had absent knee and ankle jerks; three of them had no sensory changes. Thirty-seven cases complained of aching pains, stiffness, tightness, or intermittent cramps in the calves or affected muscles. These usually accompanied, but sometimes preceded, the onset of weakness. Muscle wasting was not a feature of the cases initially, and was dependent on the duration and severity of the disease. Where paralysis was complete, wasting developed rapidly and was well marked within 2 to 3 weeks of the onset; this occurred in only 17 patients. The above series, however, is drawn from mild cases abIe to report as outpatients who received a t an early date such treatment as was available. The most severe cases were admitted directly to hospital or were patients already in hospital with chronic disease such as dysentery or septic war wounds. In such cases wasting was the rule. Trophic changes, coldness, and slight cyanosis were also common findings in these latter patients. Paralysis of the individual muscle groups supplied by one peripheral nerve was seen in three sites, and one purely sensory nerve was affected. (a) Unilateral “drop foot” occurred in 19 cases and in 15 of these it was unassociated with sensory or reflex changes or edema. It is of interest that when the patient was asked to cross his legs, invariably the affected limb was placed uppermost and on questioning this was the patient’s usual habit. It is therefore possible that repeated minor trauma to the peroneal nerve, by pressure between the knee cap of the other leg and the head of the fibula where it lies superficially, was the precipitating cause of paralysis in a nerve rendered less resistant by a minor degree of thiamine deficiency. (b) Three cases of winging of the scapula are included in this series and about 15 cases in all were seen in the camp. Some of these men had been carrying heavy weights on the shoulder of the affected side. (c) I n two patients there was weakness and wasting of the muscles supplied by the ulnar nerve in one hand. (d) The other single nerve to be affected, the lateral femoral cutaneous, is purely sensory and anesthesia limited to its distribution was present in five cases. As with the peroneal nerve, these three nerves are particularly prone t o repeated minor traumata, the nerve to serratus anterior in its course through the scalene muscles in the neck, the ulnar nerve at the elbow, and the lateral femoral cutaneous nerve where it passes through the fascia lata of the thigh about four inches below the anterior superior iliac spine. c. Sensory Features. The sensory symptoms complained of were tingling, pins and needles, and most commonly numbness. Tingling and pins and needles were probably two different terms used by different people to describe the same sensation. Details of the distribution of

16

ERIC K. CRUICKSHANK

sensory loss are given in Table I. Position and vibration sense, although tested in a number of the cases, are not recorded. This is unfortunate in view of the deficiency syndrome described by a number of authors (Scott, 1918; Landor and Pallister, 1935; Spillane and Scott, 1945; Denny-Brown, 1947) where dissociated sensory loss indicative of posterior column damage is present. This condition, discussed later (Section VIII, l),is usually associated with evidence of riboflavin deficiency, retrobulbar neuropathy and a t times spastic paraplegia, which were rare findings (see Section VII) in the above cases. The impression is, from memory, that vibration and position sense were diminished only in proportion to the degree of hypalgesia and hypaesthesia, but this may not have been the case in some of the more ataxic patients. d. Tendon ReJlexes. Ninety-six (56%) of the 171 patients with neuritic features had absent or markedly diminished tendon reflexes. The reflexes involved are shown in the following table: RefEems Absent or Markedly Diminished Knee jerks and ankle jerks.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ankle jerks only.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Knee jerks o n l y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Knee jerks, ankle jerks and arm reflexes.. . . . . . . . . . . . . . . . . . . . . . . . Total . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

59 24 4 9

96

e. Discussion. The neurological features in these cases are in the main the same as those noted in classical descriptions of the disease. Scheube (1894), Balz and Miura (1905), Vedder (1913), and Shimazono (1931), all agree that the earliest symptoms of beriberi neuritis is a feeling of weakness and insecurity in the knees rapidly followed by a tendency to drag the feet on walking. This is, in the majority of cases, accompanied by numbness or paresthesia first felt over the outer or inner surface of the shin and dorsa of the feet and toes, and on examination all forms of sensation appear to be equally involved. They recognize that in some cases motor symptoms and signs alone may be present for some time before the appearance of sensory features or vice versa. They do not give definite figures however, and it is interesting to note that in this series motor features alone were present in as many as 22.2 % of the cases and sensory features alone in 30.8 % of the cases. Little (1914), reporting on 220 early cases in Newfoundland fishing communities, found sensory changes in 217 patients (99%) but only 83 (37 %) complained of weakness in the limbs. Willcox (1916a,b,c), in his:account of beriberi in British soldiers in the Dardenelles, does not give precise’figures but says that the earliest symptom was usually some weakness of the legs with an unsteady gait on marching. On examination he found anesthesia in all cases varying from patches on the dorsa of the feet to involvement of the whole

DIETARY NEUROPATHIES

17

legs. Sprawson (1920), on the other hand, who received a further 54 patients from the Mesopotamian force, states that weakness of the legs was a common early symptom but anesthesia was uncommon. Riddell et al. (1919), in their clinical account of 60 cases occurring among United States troops in Puerto Rico, describe numbness over the shins first gradually spreading over the calves and knees, up the thighs to the abdomen between the umbilicus and symphysis pubis. In some cases the finger tips and hands were also affected. Weakness in the legs was present in 59 (98%) from the onset, and in 31 (53%) the trunk and/or the arms were involved. This spread of anesthesia and muscle weakness from the legs upwards to the trunk and arms is regarded as the usual development of the neuritic manifestations. Schretzenmayr (1941) emphasizes the symmetrical distribution of the hypalgesia, which rarely has a root or specific peripheral nerve distribution but frequently is of a glove and stocking type. Nor is there any clear-cut margin, anesthesia gradually passing from hypalgesia to normal sensation. This spread from the feet up, symmetrical in distribution and indefinite of margin, was characteristic of the majority of cases in this series. The involvement of individual nerves, particularly the peroneal in some cases, has already been discussed. Balz and Miura (1905) noted peroneal paralysis and anesthesia over the distribution of the saphenous nerve in Japanese who sit with their legs crossed underneath them. Pressure was probably also the precipitating factor in these cases. Anesthesia of the legs, arms, and trunk as far as the clavicle, is a feature of severe cases and is usually accompanied by gross paralysis. Hepburn (1920), however, found anesthesia of this extent in 30 % of the 100 early cases he studied in Bangkok and these were ambulant. In the author’s series, which were all mild cases, anesthesia of the trunk was only demonstrated in five patients, and in some of these it was confined to a band round the umbilical region. Seven severe hospital cases, however, who are not included in this series, had anesthesia to the clavicles and had such marked muscle weakness that they were hardly able to move in bed. Two of these cases, both young men, had retention of urine for 5-10 days, which gradually recovered as the muscle power returned with thiamine therapy. This is a rare complication of beriberi and Balz and Miura (1905) state that “only extremely seldom must one recourse to the catheter.” Balz regards the changes in the tendon reflexes as one of the most important signs of beriberi. They may be exaggerated for a brief period and then rapidly disappear. In this series, however, only 53% of the patients with neuritic features had absent or markedly diminished reflexes, and marked exaggeration of the response was only observed to precede depression in six cases. Eighty-seven per cent of the cases described by Riddell et a2. (1919) had diminished or absent knee reflexes but only 30%

18

ERIC K. CRUICKSHANK

had absent ankle jerks, while Hepburn (1920) found absent knee and ankle jerks in 49% of his cases. Other authors (Scheube, 1894; Biila and Miura, 1905; Vedder, 1913) do not give precise figures for the different neurological manifestations but most comment on the variation which may be found in individual cases. f. Summary. The conclusion that can be drawn from the neurological findings in these cases and a survey of the observations of other authors, is that in the early stages of peripheral neuritis in beriberi, whether edema is present or not, most frequently all functions of the nerve are impaired. In a proportion of cases, however, sensory changes only, motor weakness only, and, much less frequently, reflex alterations only, may be present, or any two of these three factors may occur together. It is difficult t o determine what limitations should be placed on the term “beriberi neuritis” or preferably “beriberi neuropathy.” It is reasonable for practical purposes to apply the term to manifestations of peripheral nerve damage occurring in a person or a community that can be shown to have been on a diet low in thiamine with a reasonable calorie intake, as the balance of evidence already discussed (Section 11, 2 ) , is in favor of thiamine deficiency being the major factor responsible. The case is strengthened if edema, which responds dramatically t o thiamine therapy, is concomitantly present in the individual or the community. Deficiency of other food factors in the diet nearly always exists when beriberi neuropathy occurs. It is uncertain how much this may contribute t o the nerve damage, and there is some experimental work in animals t o suggest that pyridoxin or riboflavin deficiencies alone may produce nerve degeneration (Zimmerman, 1943; Wintrobe, 1942). Alcoholic polyneuritis and polyneuritis of pregnancy can justifiably be classified as “beriberi neuropathies.” It is not justified, however, to put under this heading other neurological conditions occurring in malnourished persons, such as retrobulbar neuropathy, spastic paraplegia, posterior column damage, eighth nerve deafness and the painful feet syndrome, as there is little evidence that thiamine deficiency plays much part in their production. The etiology of these disorders is to be discussed later.

111. PAINFUL FEETSYNDROME 1. Historical Burning sensations or shooting pains in the feet have long been recognized as adisorder occurring in tropical countries. Bontius in 1645, in his description of beriberi, refers to radiating pains in the soles of the feet which “attack those who unwarily sleep exposed to the land-winds which

DIETARY NEUROPATHIES

19

issue every morning about sunrise from the neighbouring mountains, suddenly seizing them with a painful sensation in the periosteum of the arms and legs. In some persons the pain abates as the day advances and the air becomes warmer: but in others it continues for a considerable time, attended by weakness of the knees and an uneasy sensation in the calves of the legs and soles of the feet. . . . ,, Denny-Brown (1947) refers to a paper read at Calcutta in 1826 by Grierson on “the burning feet of the natives” observed during the first Burma war. Malcolmson (1835) and Waring (1860) also gave descriptions of the disorder. The former clearly distinguished it from beriberi, and remarked on an accompanying sensation of numbness but without edema or other signs of vascular disorder, and noted intensification of the burning a t night when the sufferer could not bear any covering on his feet. An outbreak of burning sensations in the feet followed by persistent diarrhea, an erythematous rash, and occasionally dark pigmentation of the skin, occurred in Paris in 1828 and 1829 according to Chardon (1830) (quoted by Denny-Brown, 1947), who termed the condition “acrodynia.” Vidal in 1864 reviewed the syndrome and mentioned earlier epidemic outbreaks in Italy and Belgium. The diet was not regarded as responsible and the possibility of ergot poisoning in particular was ruled out. It is doubtful, however, whether this was the same disorder as the one under consideration. Stannus (1911) described a disease in natives of Nyasaland, the main features of which were a pellagrous rash, angular stomatitis, glossitis, and scrota1 dermatitis. A large number of these patients also complained of burning sensations in the soles of the feet. Exaggerated tendon reflexes, dimness of vision, and deafness, were commonly found on examination. Scott (1918) and Sharples (1929) record burning pain in the feet of laborers in the sugar cane plantations of Central America. I n many of their cases these symptoms were accompanied by failure of vision with temporal pallor of the optic discs and an ataxic gait with absent reflexes. The first reports of a syndrome closely resembling the one under review, occurring in Europeans, came as an aftermath of the Spanish Civil war from Peraita (1942) and Grande and Jimenez (1942). More recently other authors have published their observations in prison camps in the Far East (Harrison, 1946; Jackson, 1946; Page, 1946; Smith, 1946). The author was able to keep records on over 500 cases and it is on these that the subsequent information is based (Cruickshank, 1946). 2. Symptoms

The pain experienced is of two types. (1) A dull ache, usuallyfelt below the heads of the metatarsals and the balls of the toes, first makes its

20

ERIC K . CRUICKSHANK

appearance a t night or after a long day of activity. Initially it is noticed just as the patient is dropping off to sleep but as the days pass it is present all day, becoming worse after the patient has gone to bed. A variety of adjectives is used to describe it, such as aching, burning,Lor throbbing. (2) I n over half the cases, sharp or stabbing pains may be superadded. These last a few seconds, travelling parallel to the toes and soles of the feet to the heels and sometimes extending up the shins to the knees. I n a small proportion of cases, similar symptoms develop in the hands, but usually in cases where the pains in the feet are of long duration. As time goes by, the ache or burning sensation becomes persistent, getting worse a t nighttime, when the sharp stabs of pain appear, reaching their maximum particularly after the patients retire, thus preventing sleep. I n severe cases, the patient may get no sleep for weeks on end. Exercise frequently gives relief and it is not uncommon to find the sufferers walking around most of the night. Partial relief may be obtained from massage, or placing the feet in cold water, while others prefer warmth. The constant pain and loss of sleep, however, produce an exhausted, red-eyed, irritable patient; appetite becomes poor with resultant loss of weight, and lassitude is marked. In a high percentage of cases, there is a previous history of stomatitis, glossitis, scrota1 dermatitis or defective vision. 5. Signs

a. General. There may be evidence of recent debilitating disease particularly dysentery, or chronic malaria. In well established cases, the face wears an expression of chronic distress with dark shadows under the eyes. They are often jumpy and are apprehensive a t the prospect of having their feet examined. Epithelial lesions of the scrotum and mouth of a deficiency type are present in nearly half the cases. Diarrhea is not a common associated feature although there is often a history of several attacks of dysentery. b. Feet and Legs. On inspection of the limbs there is no change that can be considered specific to the condition. The feet in many patients, however, show patchy areas of capillary dilatation of the soles which vary in situation. In others, the feet may be pale or even slightly cyanosed. In the cases observed in tropical climates, there is no evidence of vascular spasm or trophic changes, but when sufferers are transferred to cold climates, or if prolonged immersion in cold water occurs, the feet may become pale and cyanosed when dependent. In these cases the posterior tibia1 and dorsalis pedis pulses often disappear and gangrene of toes and loss of a limb can result (Page, 1946). Variations in skin temperature and color are regarded by some writers as indicative of an abnormal capillary circulation. I n the author’s experience, as wide a range of circulatory change is demonstrable in the feet of persons who have no

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21

symptoms of the disorder. Deformities of the feet, such as flat foot and hallux valgus are no commoner than in control groups. In severe cases, usually those showing hyperemia of the soles, there may be excessive sweating of the feet that rapidly reappears after drying. The patients may adopt a characteristic attitude in bed, sitting forward in a half squatting position and firmly gripping their toes. The feet are rapidly withdrawn when lightly touched and hypersensitivity to pinprick is common, but the patient can usually tolerate without distress (although with some apprehension) firm gripping or handling of the feet. c. Nervous System. Sensory changes suggestive of coexisting peripheral neuritis have been described by some authors in as many as 30% of the cases (Smith, 1946; Harrison, 1946). Such findings, however, are probably due to coexisting beriberi, as edema was also common in these patients. In the great majority of cases, there is no demonstrable sensory change when coexisting beriberi neuropathy can be excluded. Ataxia with diminution of vibration and position sense has been described as accompanying the syndrome by some authors (Stannus, 1911 ;Scott, 1918; Landor and Pallister, 1935), but this is probably due to a separate but associated lesion as this feature did not occur in the majority of cases seen in Malaya and Java during the last war. Exaggerated reflexes are found in over one-third of the cases, particularly those of long duration unless there is associated peripheral neuropathy, and the slightest touch from a reflex hammer will produce a very brisk response. Plantar reflexes may be difficult to elicit on account of hypersensitivity, but they are flexor. Frank extensor plantar response and ankle clonus are exceptionally rare and indicate the presence of pyramidal tract damage. These belong to the spastic paraplegia group of the cord syndrome where painful feet may be a precursor. The gait in severe cases is slow and hobbling on account of the pain, but there is no mode of progression that can be regarded as typical of the condition. d. Cardiovascular System. Tachycardia at rest may occur but the heart rate is usually within normal limits. An interesting feature of the syndrome in some reported series (Cruickshank, 1946; Harrison, 1946; and Smitskamp, 1947) is hypertension of a moderate degree appearing during the course of the syndrome. It may be present in from 10 to 20 % of cases and is usually found in severe long-standing sufferers. The blood pressure returns gradually to normal with improvement in the symptoms and there is no initial or residual evidence of any renal damage.

4. Treatment All patients should be rested in bed as far as possible or given work that does not involve long standing or walking. It may be very difficult to enforce bed rest because of the temporary relief gained from exercise.

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Foodstuffs rich in vitamin B complex and protein are added to the diet if possible. This usually leads to gradual improvement and disappearance of symptoms in a few weeks’ time. Rapid and sustained improvement in some cases has been obtained from the intravenous administration of the diethylamide of niacin in doses equivalent to 340 mg. of niacin. This preparation has no vasodilatory effect. Riboflavin and thiamine produce little improvement. Vasodilator drugs produce transient improvement in some patients. Gopalan (1946) claims dramatic and complete cure in all cases in which he administered 20-40 mg. of calcium pantothenate intramuscularly daily. Reports confirming his observations have not yet been forthcoming. 5. Etiology and Pathology a. Associated Deficiency Diseases. The symptom of “burning of the feet” described by writers of the last century was regarded by them as a manifestation of beriberior malaria. Stannus, however, in 1911, noted its relationship with stomatitis, scrotitis, and defective vision, and classified it as a feature of pellagra, and since that time his view has been supported by other authors. This symptom received scant attention in the past and emphasis was laid on the visual, epidermal, auditory, and ataxic features. Grande Covian (1942) classified the various features of the cases he saw in Madrid under five headings : paresthesia alone, paresthesia with burning pains in the feet, retrobulbar neuritis, “funicular myelopathy,” and “cochlear neuritis.” Peraita (1942) gives a graphic description of the pain these patients suffered and quotes such phrases as “terrible pricklings,” “ horrible stabbing pains.” Warmth markedly aggravated the pain and at night in bed the pain became unbearable so that the victims had to walk barefoot on the floor or wrap their feet in cloths wrung out in cold water. Associated features were insomnia, depression, and forgetfulness. The symptoms he describes are identical to those experienced by prisoners-of-war in the Far East. He observed little classical pellagra and no true beriberi. Peripheral motor defects were rare and he does not mention the reflexes or blood pressure. His account is the first to emphasize painful feet as a deficiency syndrome in the absence of other well recognized manifestations of vitamin deficiency. Stomatitis and scrota1 dermatitis precede or accompany a high percentage of cases. Defective vision also frequently occurs. This may be due to one of two lesions. Firstly, a punctate granular change of the cornea. This disorder is now a well recognized feature of B complex deficiency and Metivier (1941) who called the condition corneal epithelial dystrophy” noted its association with burning sensations in the feet of patients in Trinidad. He found rapid improvement on treatment

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23

with riboflavin and therefore regarded the lesion as due to riboflavin deficiency. Secondly, there is retrobulbar neuropathy which has long been recognized as accompanying the painful feet syndrome. Its etiology ie discussed later (p. 33). Smitskamp (1947) found this disorder in 42.8% of the 347 cases of painful feet he studied, but the majority had had painful feet for several weeks or even months before they developed eye symptoms. It is of interest that 75% of the early cases of retrobulbar neuropathy in Changi Prisoner-of-War camp suffered from painful feet, but of the later cases only 2 % had this symptom. b. Mechanism of Pain Production. As pain is entirely a subjective phenomenon, it is difficult to assess its characteristics analytically. The pain of this syndrome has much in common with the pain of causalgia, pink disease, intermittent claudication, erythromelalgia (Brown, 1932; Lewis, 1936), pseudoerythromelalgia (Craig and Horton, 1938), and asthenia crurum dolorosa (Ekbom, 1945). The mechanism of pain production in these conditions is speculative and it is equally difficult to provide an explanation for the pain in this syndrome. It bears a very close resemblance to the early aching and stabbing pains of the irritative stage of peripheral neuritis, but in this condition exercise and pressure usually aggravate the pain and muscular tenderness is present. It is most closely related to the pain of the “immersion feet” syndrome (Ungley et al., 1945), in which severe burning or throbbing pain is felt in the feet and legs during the hyperemic stage. From 7 to 10 days later very acute shooting or stabbing pains appear. These come in bursts like machine-gun fire and radiate from the center of the foot. These are relieved by cold but aggravated by heat and exercise. They are also accompanied by circulatory changes and objective neurological signs which are not present in the painful feet syndrome. Excessive sweating in severe cases is a feature common to both. The close resemblance between the pain of the “painful feet” syndrome, “immersion foot” and “peripheral neuritis,” suggests that they have a common mechanism, but in the case of the painful feet syndrome the process does not progress to a stage where clinical evidence of nerve damage is found. The primary disturbance may lie in the arterioles and capillaries but there is little evidence of local vascular disturbance, although Smitskamp (1947), describes capillary changes when the vessels are observed in vivo by a special technique (see p. 25). It has been suggested that the pain originates from changes in the spinal nerve roots as cases occur where pain is experienced in a phantom limb following amputation. Spinal anesthesia and local nerve block, however, can produce temporary relief from the pain which makes it unlikely that the pain is central in origin. The most attractive theory is that it originates

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from interference with the normal metabolism of the nerve endings at the periphery, either on account of a specific enzyme deficiency or local disturbance of the blood supply due to capillary damage. c. Significance of Exaggerated ReJexes. Exaggeration of tendon reflexes in a high percentage of cases is difficult to explain. It is not

FIQ.3. Calories, carbohydrates, proteins, and fat in daily ration: H, heavy-duty ration; L, light-duty ration; N, no-duty ration. (From Cruickshank, E. K., Lancet 2, 369, 1946.)

usually confined to the affected limbs, but is also found in the arms. I n a great majority of cases, there is no other sign to suggest pyramidal tract lesions, since the abdominal and cremasteric reflexes are active and the plantar responses are flexor. Protracted pain and loss of sleep may be contributing factors. In Changi Prisoner-of-War Camp, however, cases of spastic paraplegia, the result of motor neuron myelinoclasis, occurred when the incidence of the painful feet syndrome was a t its height and the

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25

onset of spasticity in some of these patients was preceded by painful feet. It is possible, therefore, that exaggerated reflexes indicate a minimal but reversible degree of damage to the pyramidal tracts. d. Cause of Hypertension. The mechanism of production of high blood pressure is uncertain. It is undoubtedly closely related to the syndrome and is not incidental. General arteriolar spasm must be present but there is little clinical evidence of this. The feet in the majority of cases appear normal, although some are cold, pale, or even slightly cyanosed, while others are lobster red. Smitskamp (1947) observed microscopically the capillaries in the nail beds of the big toes and fingers in cases of painful feet in Java. He found alterations from the normal and describes three stages. (1) At the onset of the symptoms when the foot was normal, pinkish, or bright red, the arterial limb of the capillaries was normal, but the venous limb was dilated. (2) After the symptoms had persisted for some time and the patient complained mainlyof coldness and shooting pains, the foot was normal or pale and often clammy. The arterial limb was narrow and the venous limb was normal or narrow. (3) In cases with intense aching and radiating pains where the foot felt cold and numb, he found the arterial limb almost invisible and the venous limb narrow. He gives the average blood pressure in his 347 cases as 132/77, which he regards as considerably higher than those usually encountered in the tropics. I n 20 cases there was a definite rise df the systolic as well as the diastolic pressure during the time when the complaints and clinical signs were serious and a lowering when the symptoms disappeared. He considers that these capillary changes are the primary lesion in the disease and are responsible for the pain. Harrison (1946), reporting on the 400 cases of the painful feet syndrome he observed in the military prisoner-ofwar camp in Hong Kong, found considerably raised systolic and diastolic blood pressure in 19% of the patients, and he is also of the opinion that vascular spasm is the primary disorder in this complaint. Page (1946) describes marked circulatory impairment as a late development of the syndrome in men taken to the colder climate of Japan. The feet became pale, cold and cyanotic when dependent. Gangrene of the toes developed in some of the patients. This complication does not occur in tropical countries. There is considerable evidence, therefore, that peripheral vascular changes are an important feature of the syndrome. No definite conclusion can be drawn about the relationship between these changes and the pain of the syndrome, but three possibilities may be considered: (1) Pain may produce a rise in systolic and diastolic pressure. This may be the result of impulses passing directly from the higher pain centers to the

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vasomotor center, but Ranson and Billingsley (1916) demonstrated that pressor impulses pass up the pain fibers in a peripheral nerve in animals when the stimulus is sufficient to elicit pain, with a resultant rise in the systolic and diastolic pressure. Alam and Smirk (1938), on the other hand, found in human experiments that some sensory stimuli cause much pain but little rise in blood pressure, while others cause less pain but much greater increases in blood pressure. It is possible, therefore, that in the painful feet syndrome the mechanism producing the pain impulses is also initiating pressor impulses, which may vary considerably for the individual. No information is available whether long continued pain is associated with a sustained rise in pressure, and hypertension is not a recorded feature of intractable pain from any cause. (2) Vessel spasm may be responsible for pain. Lewis (1942) does not believe that the pain arises from the vessel wall, but that it is secondary to the resultant tissue ischemia. The pain of Raynaud’s disease exemplifies such a state of affairs. I n this condition, there is always clinical evidence of vascular spasm, while in the painful feet syndrome severe pain occurs in the majority of cases without any evident disturbance of the circulation of the feet. It is unlikely, therefore, that vessel spasm, even if present, is the main cause of the pain. (3) A tissue metabolite, such as the hypothetical “ P ” substance of Lewis (1942), which may be responsible for the pain, may also act as a pressor substance and produce vasospasm by direct action on the vessels. These possibilities are only speculative and further carefully controlled investigations under ideal conditions will be necessary before light can be thrown on a syndrome presenting, as its main features, two of the major physiopathological problems of medicine-pain and hypertension. From the data obtained in these cases it is difficult to attribute the symptoms to the deficiency of one specific vitamin. That thiamine given intravenously produces little or no improvement is agreed by the majority of observers. The syndrome can occur when thiamine intake is considered adequate and none of the features of classical beriberi is present. Thiamine deficiency, therefore, cannot be regarded as an important precipitating factor. Landor and Pallister (1935), Peraita (1946) , Grande Covian (1942), and, till recently, Stannus (1946) believed that riboflavin deficiency was responsible for the symptom. Their opinion, however, was based on the fact that it was commonIy associated with scrotitis, stomatitis, and ocular disturbances, and cleared up on the administration of riboflavin-containing remedies. None, however, demonstrated that riboflavin alone could cure the condition and in Changi Prisoner-of-War Camp the syndrome

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had almost disappeared before there was any increase in the calculated riboflavin content of the diet. The intravenous administration of the diethylamide of niacin brought about relief or much improvement in 68.8% of the cases in which it was used in the author’s series (Cruickshank, 1946). The substance has no pharmacological vasodilator effect as has niacin. Other investigators, however, have not obtained such spectaculariresults (Harrison, 1946; Peraita, 1942; Simpson, 1946; Page, 1946; Smith, 1946; Gopalan, 1946). They used niacin in much smaller doses, 50-100 mg. given by mouth, and only employed the drug in a few cases. They attributed the temporary improvement they obtained in some cases to its vasodilatory action. The beneficial effect of a diet or dietary supplements containing adequate amounts of vitamin B complex point to a B complex deficiency. The niacin observations in Singapore suggest that a deficiency of this vitamin is an important factor. But a t a later date of imprisonment when the niacin content of the diet again fell to, and remained at, a level as low as that at which the initial outbreak developed, cases of the syndrome did not again appear in any great number. During this later period, in contrast to the earlier, the riboflavin content of the diet was adequate. A simultaneous deficiency of niacin and riboflavin may therefore be necessary for the production of the syndrome, or an essential substance, the distribution of which in foodstuffs is closely related to that of riboflavin and niacin, may be the missing element. There is suggestive evidence that pantothenic acid is this element. Gopalan (1946) treated 53 natives of southern India with the typical pains in the feet, glossitis, scrotitis, punctate keratitis, and exaggerated reflexes. Twelve cases were given 10 mg. of riboflavin daily for 2-3 weeks. The epidermal lesions rapidly cleared up but there was no change in the pain. Niacin and thiamine were ineffective but rapid improvement and cure in 2 to 3 weeks resulted in all of 10 cases who were given 20-40 mg. of calcium pantothenate intramuscularly daily. The remainder of the cases received 1-l+ oz. of marmite daily and the symptoms disappeared in 4 weeks. He concludes that pantothenic acid is the deficient factor in the painful feet syndrome. This may well be the case, but his findings must be confirmed and completely satisfactory results will have to be obtained in a much larger series of cases. 6. Summary

A syndrome of which the chief features are aching and stabbing pains in the feet is described. It has long been recognized as occurring in persons whose diet is deficient in protein and vitamin B complex. Hyperten-

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sion and exaggerated tendon reflexes are added features which occurred in some prisoners-of-war in the Far East. The factors which may be responsible for the pain and hypertension have been discussed. There is considerable evidence that peripheral vascular changes are an important feature of the syndrome. The relationship between these and the pain is uncertain. Disordered tissue metabolism, the result of vitamin deficiency, may be responsible for them both but the pain may produce the vaso-spasm and vice versa. No satisfactory conclusion is reached. There is some evidence to suggest that the condition may be benefited by large doses of niacinamide intravenously. The syndrome appears when the niacin and riboflavin intake in a community is low. Deficiency of these substances may, therefore, play a major part in the production of the syndrome. There is recent evidence which has yet to be confirmed, that deficiency of pantothenic acid is responsible for the syndrome. IV. WERNICKE’SENCEPHALOPATHY I . Pathology

Within recent years it has been recognized that there is a close relationship between thiamine deficiency and a disorder of the central nervous system first described by Wernicke in 1881 and termed “encephalitis haemorrhagica superior.” In Wernicke’s original description the presenting features were ocular paralysis, a reeling gait, and disturbances of consciousness. At autopsy the dominant lesions were punctate hemorrhages in the tissue lining the third ventricle, in the corpora quadrigemina, and in the retinae. These features were present in three cases, two being chronic alcoholics and the other a case of sulfuric acid poisoning (Jacobiius, 1894). Wernicke considered the lesions to be inflammatory in origin. Nearly 50 years passed before further careful study was made of the condition. I n 1928 Gamper in Germany focussed attention on the syndrome with a detailed account of the pathology, and in succeeding years further descriptions appeared in the German literature (Creutzfeldt, 1928; Ohkuma, 1930; Neuburger, 1931a; Bodechtel and Gagel, 1931; Kant, 1932). The essential changes noted by these authors were focal lesions throughout the gray matter of the brain stem due to hyperemia or small hemorrhages, endothelial and mesenchymal vascular proliferation, and a varying degree of glial proliferation, without inflammatory infiltration and with relatively slight evidence of damage to nerve cells. They had a characteristic localization, being constant and most marked in the corpora mamillaria and less constant in the other hypothalamic nuclei. In the mid-brain they were frequently seen in the

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peri-aqueductal gray matter, involving the oculomotor nuclei. Nothing characteristic was found in the cerebral cortex. 2. Etiology in Relation to Thiamine DeJiciency

a. Clinical Evidence. The German authors referred to considered alcohol to be an important etiological factor and their view was supported by Bender and Schilder (1933), who suggested that the condition should be called “ encephalopathia alcoholica.” Wechsler (1932) ,on the other hand, was apparently the first to propose that vitamin deficiency was the important factor, this being frequently induced by chronic alcoholism. He had no proof to offer for his suggestion. Shattuck in 1928 had suggested that alcoholic peripheral neuritis might properly be regarded as beriberi since it was caused by a failure to take or assimilate food containing a sufficient quantity of vitamin B. Strauss in 1933 showed that alcoholic peripheral neuritis improved rapidly when treated with vitamin B concentrates and liver extracts parenterally, although large quantities of alcohol were still consumed, and his findings were confirmed by Jolliffe, et al. (1936). Polyneuritis of pregnancy was placed in the same category (Strauss, 1933) and the stage was set for the inclusion of Wernicke’s syndrome in this group. It was already recognized that the syndrome could occur in patients taking no alcohol. Berkwitz and Lufkin (1932) had found petechial hemorrhages in the brain and spinal cord of three patients who died from polyneuritis gravidarum. Clinically, they presented the classical picture of Wernicke’s syndrome. Further cases of polyneuritis gravidarum presenting similar clinical and pathological findings were described by Tillman (1934) and later Wagener and Weir (1937). Tanaka (1934) found lesions identical with those of Wernicke’s encephalopathy in an inexplicable cerebral disorder occurring in suckling infants. He attributed it to intoxication from the mother’s milk. It is likely that these were cases of infantile beriberi, as at that time beriberi was common among Japanese nursing mothers. Neubiirger (1937) described three cases of “chronic gastritis ” who had ocular palsies and mental disturbances preceding death. At postmortem examination the typical lesions of Wernicke’s encephalopathy were found. Since there was no history of alcoholism, he suggested endogenous toxins elaborated in an abnormally functioning gut with liver damage preventing adequate detoxication, as the cause. In the discussion he considered nutritional deficiency as a possible cause, but discarded the idea in favor of the toxin theory. By 1939, however, the view that thiamine deficiency was an important factor in the production of Wernicke’s encephalopathy was gaining ground and was supported by Ecker and Woltman (1939), who reported one case where there were associated signs of nutritional defi-

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ciency. They claimed to have cured other patients with large doses of the B complex but did not give details. b. Experimental Evidence. On the experimental side, evidence was also being brought forward in favor of thiamine deficiency as the etiological factor. Pappenheimer and Goettsch (1931) produced foci of edema, necrosis, and petechial hemorrhage, limited to the cerebellum, in chicks fed on a diet deficient in thiamine. The intake of “vitamin B2” was adequate. Prickett (1934), and later Church (1935) made similar observations in rats fed on a thiamine-free diet. Green in 1936 described a disease in foxes known as ‘‘Chastek paralysis,” the features of which were loss of appetite, weakness, ataxia, spastic paralysis, and death. He suggested that the disease was due to thiamine deficiency and was later able to show that the deficiency resulted from the inactivation of thiamine in the diet by a substance present in uncooked fish. The appearance of the disease could be prevented by large doses of thiamine. The histological findings in the brain of these animals were small, symmetrical, punctate hemorrhages in the floor of the fourth ventricle, in the corpora quadrigemina, in certain nuclei ventral and lateral to the aqueduct of Sylvius, in the thalamus, and in certain parts of the cerebral cortex; and, therefore, very similar t o the changes in the rats observed by Prickett (1934) and Church (1935). Alexander and his colleagues (1938,1940), by feeding pigeons a diet deficient in thiamine but otherwise adequate, produced pinpoint hemorrhages within areas of subacute necrosis in the paraventricular and paramedian nuclei of the thalamus and in the nuclei of the eye muscles, with significant regularity. They pointed out that the lesions in these pigeons were identical with those of Wernicke’s encephalopathy in man. 3. Clinical Features The two best known clinical features of Wernicke’s encephalopathy are ophthalmoplegia and mental ‘confusion. They were the outstanding features of the27 cases describedin detail by Joliffeet al. (1941). Twentyfour of these were chronic alcoholics. In 25 of them, peripheral neuritis had developed prior to the appearance of ophthalmoplegia. There was a striking response to thiamine administration in 19 of the cases, the ophthalmoplegia improving markedly or disappearing. No response wa8 obtained with niacin. One of these made a dramatic recovery when given 10 mg. of crystalline thiamine intramuscularly daily for 9 days. There are two early important signs which deserve comment-vomiting and nystagmus. Vomiting, when a prominent feature, has usually been attributed to the primary disease, since the majority of the reported cases of Wernicke’s encephalopathy have occurred in chronic alcoholics or

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in patients with chronic gastrointestinal disorders. In the author% experience, however, vomiting appears early in the syndrome even when there is little evidence of a gastrointestinal disorder. It is usually effortless in nature, suggesting that it is cerebral and not gastrointestinal in origin, and can be rapidly terminated by the administration of parenteral thiamine. Nystagmus, the other important early sign, is not commented on by some authors, but it is possible that it is often obscured by coexisting ophthalmoplegia, e.g., in Jolliffe’s series of 27 patients, 20 had gross ophthalmoplegia. Campbell and Russell (1941) noted it in 5 of their 21 cases but admitted that it might have been overlooked in others. Diplopia is, naturally, an accompanying symptom of ophthalmoplegia and it often precedes the appearance of mental derangement. Psychological disturbances, when present, may appear in a variety of ways, such as dull apathy with mental retardation, confabulation, disorientation in space and time, and occasional visual and auditory hallucinations. One of the distressing aspects of the mental changes is that they may not respond to thiamine therapy, although the other features of the syndrome may disappear or improve markedly. Headaches may be complained of by some patients and occasional fundal hemorrhages are seen. Peripheral neuropathy is a frequent associated feature. Jolliffe and his colleagues (1941) found it in 25 of their 27 cases and it was present in 9 of 21 cases described by Campbell and Russell in 1941. de Wardener and Lennox (1947) described the clinical features of 51 patients seen in Changi Prisoner-of-War camp where the signs of peripheral neuropathy were present in 47. Of 8 cases seen in the same camp and described by the author (Cruickshank, 1950) no peripheral neuropathy or other sign of thiamine deficiency was present in 6. Therefore, the appearance of the triad of effortless vomiting, nystagmus and diplopia, in any patient who may be thiamine deficient, should be regarded as indicating the onset of Wernicke’s encephalopathy, even when there is no other clinical evidence suggestive of thiamine lack. Alexander in 1940, as a result of his experimental work, was of the opinion that smaller quantities of thiamine are required for blood-vessel maintenance than for peripheral nerves. This hypothesis is now generally supported by the clinical observations of other authors (Jolliffe, 1941; De Wardener and Lennox, 1947). Since, however, cases of this syndrome occur in the absence of other signs of thiamine deficiency, it may occasionally be the earliest sign of shortage of this vitamin. Accordingly in thiamine deficient states the first clinical evidence of the deficiency in some is peripheral nerve damage, in others disturbances in the cardiovascular system and finally, there is a small group where Wernicke’s encephalopathy is the presenting abnormality. There is also evidence from the experimental work of Alexander (1940)

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and the observations in Changi Prisoner-of-War Camp (de Wardener and Lennox, 1947; Cruickshank, 1950), that Wernicke’s encephalopathy is more likely to occur when gross thiamine deficiency appears suddenly in a previously healthy individual or community, than when slow thiamine deprivation takes place in association with general undernutrition.

4. Treatment Vomiting and ophthalmoplegia will disappear rapidly with the parenteral administration of small doses (2 to 4 mg.) of thiamine, and this is the strongest evidence that deficiency of thiamine alone is the all-important factor in the production of the syndrome. It should, therefore, be given immediately the diagnosis is suspected. At the same time the electrolyte and fluid balance must be adjusted by appropriate measures and thereafter a properly balanced high calorie, high vitamin diet given when possible. When treatment is initiated early, recovery should be complete, but if delayed, there may be disappearance of the vomiting and ocular manifestations, with persistence of the mental changes. 5. Summary There is good evidence that Wernicke’s encephalopathy results from acute thiamine deficiency. The dominant pathological lesions are focal hemorrhages in the brain stem most marked in the corpora mamillaria and frequently involving the peri-aqueductal gray matter including the oculomotor nuclei. The primary lesion is presumably in the vascular endothelium of that area. Other signs of thiamine deficiency may be present but their presence is not necessary for the diagnosis to be made. Continued vomiting and the appearance of nystagmus in a patient who is not patently thiamine deficient should be regarded as warning signs of Wernicke’s encephalopathy and parenteral thiamine given immediately. Small doses of 2-5 mg. daily are sufficient to produce a dramatic effect if the diagnosis is correct. Other important features are ocular pareses and mental confusion.

ENCEPHALOPATHY V. NIACINDEFICIENCY Mental symptoms are part of the classical picture of pellagra with the triad “diarrhoea, dermatitis and dementia.” The earliest changes are usually mild psychological upsets such as insomnia, irritability, depression, memory defects, headaches, and dizziness, and may be regarded as neurasthenic. Later on there may be marked dulling of intellect and some mental confusion. At this stage the patient looks listless and disinterested, wearing a miserable expression. Some authors believe

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33

that acute niacin deficiency may precipitate a picture of encephalopathy. Jolliffe et al. (1940) describe stupor, cogwheel rigidity of the limbs and uncontrollable sucking and grasping reflexes as typical of such a condition. Some‘of the cases had complete ophthalmoplegia and nystagmus. These latter features were probably due to acute thiamine deficiency. Such patients, however, when treated with hydration and thiamine alone, invariably died; but3when large doses of niacin (up to 1000 mg. per day) were given parenterally, the mortality fell to under one-third with dramatic improvement. Jolliff e suggests that the above picture occurs when there is acute niacin deficiency since some of these patients showed few of the classical signs of pellagra; on the other hand, when there was chronic niacin deficiency, mental dulling, depression and confusion occurred. Cleckley et al. (1939) also report a dramatic response to sodium niacinate in a group of cases of stupor with few physical signs. I n a control group when the cause of the stupor was known, there was no improvement. Sydenstricker (1943) points out that a number of socalled “ toxic” or “exhaustion” psychoses seen in medical or surgical wards may respond strikingly to niacin. Such patients are often middleaged or elderly and have been on a poor diet for some time. The mental changes are attributed to arteriosclerosis, drug intoxication, uremia, and senile dementia. Claims have also been made that niacin produces rapid and marked improvement in delirium tremens and in “toxic” psychoses in chronic alcoholics (Mainzer and Krause, 1939; May, 1939). Other workers have not had nearly such impressive results in the treatment of “toxic” psychoses with niacin. I n view of these claims and the absence of toxic effects of niacin, it is justifiable to give large doses of this vitamin to all patients with unexplained stupor and toxic or exhaustion psychoses, as many such patients have been on a deficient diet for varying periods of time.

VI. RETROBULBAR NEUROPATFIY 1. Historical Deterioration of vision in persons on a deficient diet was first recognized by Japanese workers. Their observations are reviewed by Kagawa (1938) who attributes the first description to Hori (1887). They regarded it as a “beriberi neuritis.” Denny-Brown (1947) in his excellent review of the neurological conditions in prisoners-of-war returning from the Far East gives a detailed survey of the literature. This is the chief source of the author’s knowledge of the historical aspect of the condition, as many of the original articles are not available to him. Denny-Brown notes that retrobulbar neuropathy leading to failing vision is not mentioned in

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I(.

CRUICKSHANK

the earlier descriptions of beriberi in European literature (Malcolmson, 1835; Vedder, 1913; Pekelharing and Winkler, 1887). Kagawa (1938) points out that other Japanese workers did not accept retrobulbar neuropathy as a feature of beriberi but attributed it to other factors mainly toxic in nature. He reviews 200 cases. I n these, signs of, or a previous history of beriberi were present in less than half, and the time of incidence and severity of the condition did not correspond with those of beriberi. Kagawa nevertheless concludes that the retrobulbar neuropathy is part of the beriberi picture. Denny-Brown critically reviews Kagawa’s evidence and observes that it is capable of reverse interpretation, namely, that retrobulbar neuropathy and beriberi are not part of the same syndrome. Signs of optic nerve damage have been observed in “pellagra” by a number of authors over the last hundred years. Pellagra, however, can no longer be regarded as a specific deficiency disease, and although niacin is an important therapeutic agent for some of the classical features, many elements of the syndrome remain unaltered with niacin therapy alone. Sebrell and Butler (1939) point out that the clinical picture may be a mixture of symptoms from three different deficiencies, namely, niacin, riboflavin, and thiamine chloride. To this list may be added other specific vitamins or food factors although, as yet, their exact role has not been determined, and the varied neurological features that may occur i,n “pellrtgra” cannot be attributed specifically to deficiency of one or more of them. Toxic factors alone, toxic factors operative only when tissue metabolism is disturbed on account of deficiency, or anti-vitamins may also play their part. There are numerous accounts of optic atrophy in undernourished subjects. Denny-Brown refers to early observations by Lombroso (1892), Bietti (1901), of optic nerve damage in pellagrins. Stannus (1911) noted that defective vision could be an early occurrence without other gross evidence of pellagra, Scott (1918) described “central neuritis” associated with ataxia and numbness spreading from the legs upwards occurring in Jamaican sugar workers whose diet was practically limited to sugar cane; he considered the condition toxic in origin. I n Malayan convicts receiving a diet adequate in thiamine and vitamin A, Landor and Pallister (1935) reported failing vision going on, in some cases, to optic atrophy as evidenced by temporal pallor of the discs and central vision of 6/60. Other features were sensory ataxia, stomatitis, and scrota1 dermatitis, and they suggested that the picture waa due to deficiency of “vitamin B2.’) Moore (1934) made similar observations in Nigerian school children. There are also records from the West Indies, Ceylon, and Spain in the Civil War (Grande and Jiminez, 1942; Peraita, 1942).

35

DIETARY NEUROPATHIES

2. Clinical Features Prison camps in the recent war, particularly those of the Far East, produced many reports on retrobulbar neuropathy (Wilkinson, 1944 ; Wilkinson and King, 1844; hdolph et al., 1944; Spillane and Scott, 1945; RETROBULBAR NEURITIS KERATITIS ----------- PULAGROID SKIN RASH

!., -

30

SCROTAL DERMATITIS ACHING FEET ANGULAR STOMATITIS & GLOSSITIS

-

--

-

u)

c

0.5

-

Zb 0 4 -

q803-

q

-

0.2

St0.l

-1942

1943 ~ ~ l S 4 4 - - 1 9 4 5

-

Fm. 4. Weekly incidence of various deficiency conditions compared with niacin and riboflavin contents of diet. (From Burgess, R. C., Lancet 2,411, 1946.)

Clarke and Sneddon, 1946; Smith, 1946; Hobbs and Forbes, 1946). Prisoners in Changi Camp, Singapore, first came under observation for failing vision in June 1942. The incidence of fresh cases per month in the population is shown in Fig. 4 along with other deficiency syndromes, as well as the estimated niacin and riboflavin intake. Of 536 cases seen by one ophthalmologist (Major Orr, A.A.M.C.), 293 (54.7%) gave a history of recent acute disease. Details we given in the following table:

36

ERIC K. CRUICKSHANK

Dysentery. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 (23.9%) Malaria.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 (13.5%) Battle casualties. . . . . Dengue.. .. .... Typhus..... .... . . . . 1 (0.2%)

--

Total with recent a . . . . . . . . 293 (54.7%) Total number of cases.. ........................... 536 (100%)

Associated disease was present in 59 (11%)-dysentery in 27 ( 5 % ) , malaria in 18 (3.4%), dengue in 4 (0.7%). The following points can be made about the etiology: (a) the undernourished were more prone to the disease, (b) there was no difference in incidence between smokers and nonsmokers, although the clinical features were indistinguishable from tobacco amblyopia, (c) prolonged physical effort seemed to be a precipitating factor, and when improvement had occurred it could also bring about a relapse, as would an acute febrile illness such as malaria or dysentery, (d) symptoms appeared in some cases as long as 6-9 weeks after a marked improvement in the diet, suggesting a latent period before damage is apparent. The earliest symptom was inability to read for any length of time without the eyes becoming tired. This was shortly followed by blurring of vision both for distance and for reading, usually occurring gradually but sometimes suddenly. There might be shimmering or flickering of images. Occasionally there was slight photophobia, and dull aching or boring pain behind the eyes aggravated by strong light was common. Some patients were aware of a central blind spot. On examination there was lowered visual acuity of one or both eyes with difficulty in reading, especially in picking out words or letters in words. The conjunctivae and corneae were normal except when there was associated punctate keratitis. Central or paracentral scotomata just above or below the point of fixation were constant findings. They varied in size but were usually larger for red than for black or white. A frequent site for the scotoma was the region corresponding with an angle of 3 4 " from the line of fixation towards Marriott's spot. The peripheral field of vision was usually good but might be much contracted, particularly for color. Ophthalmoscopy in the early stages usually revealed a normal fundus, but perivascular infiltration and blurring of the disc margins was seen. It was well marked in a few cases and occasionally engorgement of the veins with small retinal hemorrhages were seen. I n patients with symptoms of long duration, pallor of the temporal side of the disc developed, frequently corresponding with the pupilomacular bundle. In several long standing cases, marked optic atrophy with exposure of the lamina cribosa was seen. Occasionally fine pigmentary

DIETARY NEUROPATHIES

37

disturbances were noted in the region round the macula giving an appearance of fine stippling. This finding was also observed a t times in apparently normal eyes. All subjective tests were carried out with the patients wearing full correction for refractive errors and a mydriatic was used to ensure accurate estimation of such errors and to allow full examination of the fundi and media. When scotomata were mapped, preliminary training on the Bjerrum’s screen was given. The prognosis was variable. I n most early cases, if marmite was given or an over-all betterment in the diet effected, slow improvement occurred, but some, unfortunately, progressed. Severe or longstanding untreated cases made little or no improvement even when full vitamin therapy and a good diet became available. These patients showed evidence of optic atrophy and it can only be assumed that irreparable damage had been done to the optic nerves. Other evidence of dietary deficiency was present in a high proportion of the patients. The commonest associated conditions were the painful feet syndrome, and the orogenital syndrome (glossitis, stomatitis, and scrotal dermatitis). Exaggerated knee and ankle jerks were present in a small proportion of these cases and some of them had equivocal plantar responses. Signs of “wet or dry)’ beriberi were rare.

3. Summary The features of retrobulbar neuropathy described above agree in general with those of other observers, but its etiology still remains uncertain. It is unlikely that thiamine deficiency plays much part in its production. It occurs most frequently in association with conditions attributable to riboflavin and niacin deficiency such as glossitis, stomatitis, and scrotal dermatitis. It rarely occurs where there is gross caloric deficiency, as in the German concentration camps during the last war, and it appears to be very uncommon in temperate climates. There is no evidence that tobacco or any toxic substance in the diet is responsible. Yeast preparation and food stuffs rich in the B complex are the most effective remedies and prophylactics. The effects are never dramatic, possibly because of the limited response of which nervous tissue is capable. It is not possible as yet to attribute the condition to lack of a specific vitamin or other food factor such as an amino acid, or to exclude a dietary anti-vitamin, or a metabolic endotoxin. The mechanism of its production is possibly the same or closely related to that responsible for damage to the spinal pathways which may also occur in deficiency states. These “cord syndromes” are to be discussed. Further studies of the pathological changes in the retinae, the optic nerves, and related cerebral pathways are required.

38

ERIC K. CRUICKSHANK

VII. CRANIALNERVELESIONS The optic nerve appears to be the most susceptible in deficiency states but evidence of damage to other cranial nerves or their cerebral connections may occur. Ocular pareses or paralyses, indicative of damage to the nuclei of the third, sixth, and possibly the fourth nerves, appear in Wernicke’s syndrome, which has already been discussed. Facial paralysis, laryngeal paralysis, and dysphagia are recorded as occasional features of severe beriberi (Denny-Brown, 1947). I n the author’s experience these features were not observed in patients with signs of classical beriberi, but occurred occasionally in association with retrobulbar neuropathy and eighth nerve deafness, or in severe casea of “acute motor neuron myelinoclasis ”-the spastic syndrome (see Section VIII). They cannot, therefore, be properly regarded as resulting from thiamine deficiency and their etiology is more likely t o be closely linked with that of retrobulbar neuropathy and the cord syndromes. The old theory that the cardiac manifestations of beriberi were due to paralysis of the vagal nerves, is no longer acceptable. Deafness of a central type of gradual onset was observed in a number of camps in the Far East. Such a symptom had already been recognized as part of a deficiency syndrome by Stannus (1936) and other medical officers in British Colonies. Grande and Jiminez (1942) and Peraita (1942) termed the condition ‘(cochlear neuritis” when it appeared in patients with retrobulbar neuropathy and signs of cord damage during the Spanish Civil War. In the patients in Changi Camp the deafness was rarely complete and in many appeared to be confined to sounds of certain pitch only, although the range would vary from patient to patient. The defect might be regarded as an “auditory scotoma” analogous to that of retrobulbar neuropathy. Sometimes it was the only sign of deficiency, but more often accompanied defective vision and epithelial lesions. Periodic aural vertigo or “camp dizziness” occurred in some camps. Kuilman (1945) reported 412 cases with attacks occurring a t frequencies of several a day t o once per week. Most were associated with some degree of deafness and, of 43 cases with progressive bilateral deafness, 37 complained of vertigo. De Raadt (1947) claimed that vestibular hyperexcitability to cold water could be demonstrated in 74% of 160 patients complaining of dizziness, deafness and tinnitus: 63% of these also presented the ‘(painful feet syndrome.” It is apparent, therefore, that in dietary deficiency, disturbance of both the cochlear and vestibular functions of the eighth nerve may occur but cannot as yet be attributed to deficiency of a specific food factor. A further condition involving the cranial nerves, termed (‘myasthenic

DIETARY NEUROPATHIES

39

bulbar paralysis” is reviewed by Denny-Brown (1947). It is a rare occurrence and its main features are tiring of the eyes, ptosis of the lids, diplopia, weakness of the muscles of the face and neck, and sometimes dysarthria and dysphagia, all occurring towards the end of the day. Although it appears under conditions of dietary restriction, it is doubtful if it can be regarded as a true dietary deficiency disease.

VIII. THE CORDSYNDROMES 1. Pathological and Clinical Features

Spinal cord damage on both clinical and pathological evidence has long been recognized as occurring in pellagra and again Denny-Brown (1947) gives an exhaustive review of the literature. The authors quoted agree that the dorsal and lateral columns bear the brunt of the damage and the signs usually appear late in the disease. Spastic weakness of the lower limbs and loss of vibration and position sense are found in the chronic cases of long duration in which the epithelial changes have been present for some time. There are few reports of these neurological features appearing in the condition before epithelial damage is well marked. Guillain and his colleagues (1934) quote a case where spastic paraplegia long preceded cutaneous changes and stomatitis. Stannus (1911) considered as pellagrous, neurological signs in African natives although the full picture of pellagra was absent. Ataxia was the outstanding feature associated with defective vision and deafness. Ataxia was also a prominent early sign in the Jamaican sugar workers described by Scott (1918) and these patients later developed amblyopia. Spastic changes in the legs were not present and there were no extensor plantar responses. At postmortem examination the posterior columns and the optic nerves showed diffuse degenerative changes with minimal damage to the lateral columns. Sensory ataxia was a prominent feature in the Malayan prisoners described by Landor and Pallister (1935) and, in these, exaggerated reflexes were common findings. The association of spinal ataxia with retrobulbar neuropathy has been recorded by several other authors (Moore, 1934, 1937a and b, Metivier, 1941)) and in addition to these findings spasticity with extensor plantar responses indicating lateral column damage was noted by Grande and Jiminez (1942) and Peraita (1942) during the Spanish Civil war. Experiences in prisoner-of-war camps produced a number of accounts of simiiar cord damage attributable to nutritional deficiency. Spillane and Scott (1945) described spinal ataxia and retrobulbar neuropathy in 23 German prisoners in the Middle East. Clarke and Sneddon (1946) observed the

40

ERIC K. CRUICKSHANK

same combination in 31 prisoners and internees repatriated from HongKong. Spinal ataxia alone or in combination with retrobulbar neuropathy was not recognized in Changi Camp, Singapore, but some of the patients regarded as having signs of peripheral nerve damage may have in fact had spinal cord damage. Retrobulbar neuropathy and “neuritic beriberi ” were a rare combination. From June 1942 to February 1943, when there was a high incidence of the painful feet syndrome, a condition appeared with signs of acute upper motor neuron damage. Details of these cases have been recorded by Graves (1947). Over 60 cases occurred, varying from minor signs such as exaggerated reflexes, ankle clonus and extensor or equivocal plantar responses, to a rapidly developing acute confusional or comatose state with signs of extensive pyramidal tract involvement and death. The earliest symptoms were difficulty in walking and pains in the legs and spine. These were followed by mental dulling, dimness of vision, fleeting diploplia in some, and spasticity. The six fatal cases rapidly or suddenly became confused and passed into a coma with marked spasticity of limbs. Epileptic seizures occurred in two. Four died within 18 days of onset, and the other two more than a year later. The blood picture and cerebrospinal fluid were normal, and free acid was present in the gastric juice of all those tested. Sensory changes were not demonstrable in any of the patients. No strabismus or nystagmus was observed. In one there was a transient facial paralysis lasting a few hours and in another aphonia with palatal paralysis developed. Fever was a common accompaniment of the delirious phase and one patient died in hyperpyrexia. Thiamine (2 mg.) and niacin (340 mg. given as the diethyl amide) intravenously, liver extract intramuscularly, and marmite orally failed to produce any obvious improvement. Autopsy in four of the fatal cases showed small, gray, translucent, well-defined areas neither raised above nor depressed below the cut surface of the brain, and most marked in the corona radiata and occipital poles where as many as ten to the square centimeter occurred. No histology was possible but the findings were interpreted as areas of acute demyelinization or “myelinoclasis.” Three of the four cases had acute dysenteric lesions of the bowel but none were severe. Six other severe cases who were mentally confused recovered their normal senses and slow improvement occurred, but evidence of residual upper motor neuron damage persisted and in several cases was sufficiently severe to be permanently crippling. This condition may be related to niacin encephalopathy described by Jolliffe et al. (1940) and Sydenstricker (1943) (see Section V) but did not show any improvement with intravenous niacin therapy. Larger doses, however, might have produced some improvement.

DIETARY NEUROPATHIES

41

The classical picture and pathological changes of this syndrome bore no relation to Wernicke’s encephalopathy and the maximum incidence occurred a t a time when few fresh cases of beriberi were appearing. The deficiency states associated with the condition were as follows: (a) amblyopia, probably from retrobulbar neuropathy46 % (b) “painful feet”-38% (c) stomatitis and scrota1 dermatitis-18 %. 2. Relation to Lathyrism

As this syndrome did not occur in any of the other prisoner-of-war camps in the Far East, it is possible that it was not due to dietary deficiency but to an ingested toxic factor. Spastic paraplegia with a similar onset occurs in lathyrism. I n this condition there is evidence that a toxic substance in one of the pea family, Lathyrus sativus, is responsible for the cord damage as normally it is a component in the diet of a community where lathyrism occurs. Only a proportion of those who consume the pea develop signs of the disease and it may be that dietary deficiency or individual idiosyncrasy are necessary precursors. Although horses and rabbits fed on the pea have developed paraplegia (Proust, 1883; Fumarola and Zanelli, 1914), research work has failed to isolate from the pea any substance which could be consistently demonstrated to have any toxic action, or to produce damage in the nervous tissue of animals. Lathyrus sativus has been shown to be contaminated by a vetch, Vicia sativa, and claims have been made that this contains the noxious agent (Anderson et al., 1925). Shah (1939) describes an outbreak of lathyrism in the Punjab in which contamination of the diet with Lathyrus sativus could be excluded, and later Shourie (1945) records a group of cases in which Vicia sativa could not have been ingested. I t appears, therefore, that lathyrism or a clinical picture identical to it can occur when neither of the substances allegedly responsible is present in the diet. Whether there is any relationship between lathyrism and the spastic syndrome observed in Changi Camp remains speculative. Certainly neither Lathyrus sativus nor Vicia sativa was included in the diet in Singapore. 3. Summary

It can be said that under conditions of dietary deficiency, particularly in the tropics, signs of spinal cord damage may develop. Either the posterior or lateral columns alone may be affected, or varying degrees of damage to both may be present. Evidence of optic nerve degeneration is a frequent accompaniment and the maximum incidence of these disorders in a community is associated with a high incidence of epithelial lesions. There is evidence that the latter are attributable to riboflavin and niacin

42

ERIC K. CRUICKSHANK

deficiency, but the importance of deficiency of these vitamins in the production of the cord syndrome and retrobulbar neuropathy is uncertain. There is no satisfactory evidence a t present, that deficiency of other food factors, antienzymes, vitamin analogues, or toxic factors play a part.

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Strauss, M. B., and McDonald, W. J. 1933. J . Am. Med. Assoc. 100, 1320-1323. Strauss, M. B. See Minot, G. R., and Cobb, S. 1933. New Engl. J . Med. 208, 1244-1249.

Swank, R. L. 1940. Arch. Path. SO, 689-700. Swank, R. L., Porter, R. R., and Yeomans, A. 1941. Am. Heart J . 22, 154-168. Swank, R. L., and Prados, M. 1942. Arch. Neurol. Psychiat. 47, 97-131. Sydenstricker, V. P. 1943. Proc. Roy. SOC.Med. 36, 169-171. Sydenstricker, V. P., and Cleckley, H. M. 1941. Am. J . Psychiat. 98, 83-92. Tanaka, T. 1934. Am. J. Diseases Children 47, 1286-1298. Tillman, A. J. B. 1934. Am. J . Obstet. and Gynecol. 27, 240-247. Tsunoda, T. 1909. Centr. allgem. Path. u. path. Anat. 20, 337-345. Ungley, C. C., Channell, G . D., and Richards, R. L. 1945. Brit. J . Surg. 33, 17-31. Vedder, E. B., and Clark, E. 1912. Phillipine J. Sci. 7, 415 (quoted by Vedder, 1913).

Vedder, E. B. 1913. Beriberi. John Bale, Sons & Danielsson, London. Vedder, E. B., and Chinn, A. B. 1938. Am. J . Trop. Med. 18, 469-475 (quoted by Williams and Spies, 1938). Vedder, E. B. 1938. J. Am. Med. Assoc. 110, 893-896. Vidal, E. 1864. Acrodynie. Dictionnaire Encyclopedique des Sciences Medicales. Vol. I, p. 654 (quoted by Denny-Brown, 1947). Voegtlin, C., andLake, G. C. 1919. Am. J . Physiol. 47, 558. Wagener, H. P., and Weir, J. F. 1937. Am. J . Ophthalmology 20, 253-259. Walshe, F. M. R. 1941. Lancet 1, 33-35. de Wardener, H. E., and Lennox, B. 1947. Lancet 1, 11-17. Waring, E. J. 1860. Madras Quart. J. Med. Sci. 1, 300-306 (quoted by DennyBrown, 1947). Wechsler, I. 1932. Communication before the Neurological Section of New York Academy of Medicine (quoted by Bender and Schilder, 1933). Wernicke, C. 1881. Lehrbuch der Gehirnkrankheiten fiir Aerete und Studierende. Theo. Fischer, Kassel, pp. 229-42. Whaley, E. M. 1909. Eye Symptoms in Pellagra (quoted by Denny-Brown, 1947). Wilkinson, P. B. 1944. Lancet 2, 655-658. Wilkinson, P. B., and King, A. 1944. Lancet 1,52&531. Willcox, W. H. 1916a. Lancet 1, 553. Willcox, W. H. 1916b. J . Roy. Army Med. Corps 27, 191. Willcox, W. H. 1916c. J. Trop. Med. Hyg. 19,84. Williams, R. D., Mason, H. L., Smith, B. F., and Wilder, R. M. 1942. Arch. Znternal Med. 69, 721-738. Williams, R. R., and Spies, T. 1938. Vitamin Bl (Thiamin) and its use in Medicine. Macmillan, New York, p. 411. Wilson, S. A. K. 1940. Neurology. Vol. I, Arnold, London, pp. 751 (753-1838). Wintrobe, M. M. 1942. J . Nutrition 24, 345-366. Woollard, H. H. 1927. J . Anat. 61,283-297. Wright, H. 1901. Brit. Med. J . 1, 1610. Wright, H. 1902-3. Studies Inst. Med. Research Federated Malay States 2, NO. 1, 1; No. 2, 1. Yamagiwa, K. 1899. Arch. path. Anat. (Virchow’s) 166, 451-506. Zimmerman, H. M., and Burack, E. 1932. Arch. Path. 18, 207-232. Zimmerman, H. M. 1943. Proc. Assoc. Research Nervous Mental Diseases (1941) 22, 51-79.

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The Problem of the Absorption and Transportation of Fat-Soluble Vitamins BY ALBERT EDWARD SOBEL Department of Biochemistry, The Jewish Hospital of Brooklyn, Brooklyn, New York CONTENTS

Page I. Introduction ....................................................... 47 11. Problem of the Absorption of Fat-Soluble Vitamins.. . . . . . . . . . . . . . . . . . . . 48 1. Evidence of Impaired Absorption on Adequate Intake. . . . . . . . . . . . . . . . 50 2. Improved Absorption with Aqueous Dispersions. . . . . . . . . . . . . . . . . . . 3. Path of Absorption of Aqueous Dispersions.. . . . . . . . . . . . . . . . . . . . . . 111. Problem of the Transportation of Fat-Soluble Vitamins.. . . . . . . . . . . . . . . . . 56 1. Examples Indicating the Possibility of Deficiencies Due to Impaired Transportation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 2. Transportation Problems in Skin Diseases.. . .................... 57 3. Enrichment of Milk Vitamin A and Blood Se Levels . . . . . . . . . . . . . . . 59 4. Transfer from Mother t o Growing Embryo.. . . . . . . . . . . . . . . . . . . . . . . . . 61 5. Transfer of Fat-Soluble Vitamins to the Nursing Young.. . . . . . 6. Possible Approach to Some Clinical Problems. . . . . . . . . . . . . . . . IV. Concluding Remarks.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 References ......................................................... 65

I. INTRODUCTION The absorption and transportation of the fat-soluble vitamins is related t o absorption and transportation of the fats in that both are insoluble in water, which is the basic solvent of the body. Thus, for both absorption and transportation, the fat-soluble vitamins are converted to water-soluble forms by means of derivatives or dispersing agents. This problem is not encountered for water-soluble vitamins. It is not surprising, therefore, that deficiencies of the fat-soluble vitamins on adequate intake manifest themselves frequently, due to poor absorption or transportation in the body. Owing to the earlier availability of satisfactory chemical methods, the absorption and transportation of vitamin A was more thoroughly investigated than that of the other fat-soluble vitamins, namely, D, E, and K, and thus experimental data to be cited will center around “A.” Most of the scattered bits of evidence available indicate that vitamin A serves as a good model for the absorption and transportation of the other 47

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ALBERT EDWARD SOBEL

fat-solubIe vitamins. However, as would be expected, their behavior is not identical with “A.” There are known differences in a t least one aspect of the transportation problem, namely, the organs utilized for storage in the body. The main store of vitamin A is in the liver (Moore, 1950), that of vitamin E seems to be centered in the fat depots (Mason, 1951 ; Quaife and Dju, 1949), while there is no well-established reservoir for vitamin D, although we know that some of it can be stored (Morgan and Shimotori, 1943; Polskin et al., 1945; Remp, 1941; Vollmer, 1939). Similarly, a storehouse of vitamin K is not well established (Dam, 1948).

11. PROBLEM OF THE ABSORPTION OF FAT-SOLUBLE VITAMINS The problem of absorption and transportation is of special interest to the clinician, since deficiency of fat-soluble vitamins on adequate intake

-CHILDREN FROM 14 MOS TO 12 YEARS

I

I

I

I

0 3 6 9 HRS. AFTER TEST DOSE OF 6000 USP u/LB BOOY W.

FIG. 1. Vitamin “ A ” tolerances with oleum percomorph in newborns and older children. (Sobel el al., 1949b.) (Sobel et al., 1948b.)

occurs frequently. Whenever there is a breakdown in any of the mechanisms required for absorption, such as insufficient bile acids, lack of pancreatic enzymes, and other modalities as yet not recognized, such fat-soluble vitamins cannot be absorbed and transferred to the site of action. The problem was noticed in the premature, in newborn infants (Fig. I), patients with celiac syndrome (Fig. 2), biliary block, liver disease, sprue, gastrectomy, diarrhea, cretinism, and chylous ascites (Adlersberg,

49

FAT-SOLUBLE VITAMINS

1944; Adlersberg et al., 1948; Barnes et al., 1950; Bercovitz and Paye, 1944; Chesney and McCoord, 1934; Filer et al., 1951; Gribetz and Kanof, 1951; Jones et al., 1948; Kramer el al., 1947,1951; May et al., 1940; May and Lowe, 1948; Popper et aZ., 1947, 1948a,b, 1949; Popper and Volk, 1948; Sobel et al., 1949b,c, 1950). Clausen has shown the pathway of vitamin A when administered in oil. This consists of hydrolysis of vitamin A esters and dispersion with bile acids prior to absorption and reesterification in the wall of the small AQUEOUS DISPERSION OLEUM PERCOMORPH

I

1

I

0 3 6 9 HRS AFTER TEST OOSE O f 6000 USP WLB. BOW WC

FIQ.2. Vitamin “A” tolerances with oleum percomorph and aqueous dispersion in celiac syndrome. (Kramer et al., 1947.)

intestine. Most of the esters pass by way of the lymphatics through the thoracic duct, into the blood stream. His work was since then amply confirmed (Clausen, 1943; Clausen et al., 1946; Eden and Sellers, 1950; May and Lowe, 1948; Popper et al., 1948b). A clinical proof of this is in pancreatic dystrophy, where vitamin A as the alcohol is well absorbed, whereas the ester is not absorbed. In contrast to this, in celiac disease, where the fault lies in the intestine, there is impaired absorption of both the alcohol and the ester (Clausen et al., 1946; May and Lowe, 1948). That bile acids are also needed for absorption is indicated by the almost complete lack of absorption of the fat-soluble vitamins in cases of biliary block (Altschule, 1935; Filer et al., 1951; Lewis el al., 1947; Melnick and Oser, 1947; Stone, 1941).

50

ALBERT EDWARD SOBEL

I . Evidence for Impaired Absorption on Adequate Intake Blackfan and Wolbach (1933) have reported that histological evidences of A avitaminosis may occur in infants who are receiving an amount of vitamin A in their diets usually accepted as meeting the minimal needs of the body. Histological changes of the same type have been observed in our hospital in children with chronic diarrhea and cystic fibrosis of the pancreas, who received amounts of vitamin A much larger than the dose considered adequate (Kramer et al., 1947). Similarly, there have been many reports of incurable rickets in children, that had no response until millions of units of vitamin D were given (Kramer, 1941). Vitamin K deficiency as measured by prolonged prothrombin time was characteristically observed in patients with liver disease, especially biliary block (Quick, 1942). It has been shown by May and his colleagues (1940), and Chesney and McCoord (1934), that impaired intestinal absorption of vitamin A exists in children with these diseases. I n an effort to circumvent the barrier to the absorption of vitamin A, the latter was administered by intramuscular injection in sesame oil (Cienfugos, 1946; Kramer et al., 1947). Although McCoord and Breeze (1943) obtained an elevation of the vitamin A level in the serum of dogs following intramuscular injection of vitamin A, no such result was obtained in children. Lemley et al. (1947) have shown that liver stores of rats given intramuscular injections of vitamin A in oil are 1.7% of those given equal amounts of the same vitamin A orally. 2. Improved Absorption with Aqueous Dispersions

A solution to the problem of poor absorption of fat-soluble vitamins was indicated in the curative response to vitamin D-containing milk. As little as 40 Steenbock units (approximately 108 I.U.) contained in the milk formula cured rickets (Kramer and Gittleman, 1933). This amount of vitamin D in cod liver oil will not assure a cure of the rachitic condition, Shelling and Hopper (1936) pointed out the importance of larger amounts of vitamin D for the prevention of rickets in the premature, and Shelling (1937) indicated that the addition of viosterol to milk in the form of an emulsion enhances the efficacy in curing moderate and severe rickets tenfold. He further suggested that this was because the vitamin D was carried in lipoid particles of small size, and hence more easily absorbed from the gastrointestinal tract. It is very likely that if a satisfactory chemical method had been available for determining vitamin D, the discovery of overcoming impaired absorption by means of aqueous dispersions would have been made much earlier. This author noted in 1933 that colloidal dispersions of irradiated

FAT-SOLUBLE VITAMINS

51

ergosterol appeared t o be more effective than vitamin D in oily solution. This impression was not sufficiently decisive to be made the subject of a report, but is referred to in one of the papers published (Natelson et al., 1934). The groundwork was thus prepared for proposing the logical solution t o the problem of impaired absorption of fat-soluble vitamins, namely, the preparation of an aqueous dispersion with fine particle size by means of harmless dispersing agents, which at the same time prot,ects the vitamins from rapid deterioration.

HRS AFTER E S T DOSE OF 6000 USp u/LB. BODY WT

FIQ.3. Vitamin “ A ” tolerances with oleum percomorph and aqueous dispersion in the newborn. (Sobel et ul., 1948b.) (Sobel et ul., 194913.)

When such a preparation was given t o a series of children with the celiac syndrome, there was a marked elevation of the blood vitamin A, whereas there was hardly any change in the blood level when the same amount of vitamin A was administered in oil (Kramer et al., 1947; Sobel et al., 1946) (Fig. 2). Other studies indicate that these high blood levels indeed represent high absorption (Figs. 3, 4, and 5).

(A) The liver storage of vitamin A is three times as great in normal rats fed aqueous dispersions of “ A” as in similar rats fed “ A ” in oil (Lewis et al., 1947; Sobel et al., 1948a) (Fig. 6). (B) Vitamin A fluorescence studies indicate that three times as much vitamin A passes through the intestine of rats given aqueous dispersions (Popper and Volk, 1948). Most of this absorption takes place

52

ALBERT EDWARD SOBEL

0 3 6 9 HRS AFTER TEST DOSE OF 6000 USP u/LB BODY

wr:

FIG.4. Vitamin “ A ” tolerances with oleum percomorph and aqueous dispersion in the normal older child. (Kramer et al., 1947.)

HRS. AFTER TEST DOSE OF 1000 USP WLB BODY WT.

FIQ.5. Vitamin “A” tolerances with oleum percomorph and aqueous dispersion in normal adults. (Sobel et al., 194813.) (Sobel et at., 1949b.)

FAT-SOLUBLE VITAMINS

53

in the upper jejunum with aqueous media and in the lower jejunum with oily media. (C) To produce a corresponding rise in serum vitamin A in normal lactating women, three times as much oily vitamin A as aqueous dispersions is required (Sobel et al., 1950) (Fig. 7). (D) Vitamin A balance studies showed that far more vitamin A is excreted in the feces when this vitamin is administered in oil as compared to aqueous dispersions (Lewis et al., 1947).

I ~ A Q U E O U SDISPERSION -0LEUM

PERCOMORPH

BT WOROQRAYQ

29.4%

I84 YIOROORAYI

WHOLE LIVER

Fro. 6. Liver storage of vitamin “ A ” in normal rats given oleum percomorph (1250 units) and aqueous dispersion (1200 units). (Sobel et al., 1948a.)

This improved absorption with aqueous dispersions is not inevitable merely because there is vitamin A dispersed in water. Apparently the percentage of dispersing agent, which was sorbitan polyoxyethylene laurate, is an important factor (Kramer et al., 1947; Sobel et al., 1948b, 194913). This was interpreted to mean that the production of a smaller particle size, proposed by Frazer as an important aspect of fat absorption, represents one method of causing improved absorption (Frazer, 1944; Frazer et al., 1947). Moreover, it was proposed that the interfacial tension between the fine particle and the intestinal wall is also important in deciding improved absorption (Sobel et al., 1948a). When the interfacial tension between a liquid and a solid surface is reduced to zero, complete penetration takes place, and one cannot any longer distinguish two phases. In contrast to this, when the interfacial tension is high, as

54

ALBERT EDWARD SOBEL

would be the case with mercury droplets on most surfaces, even a fine particle will not penetrate (Fig. 8). Improved absorption of these aqueous dispersions does not depend on whether the vitamin A is the alcohol or the ester (Sobel et al., 1948a). Finely dispersed esters are about as well absorbed as finely dispersed alcohols, and in both cases aqueous dispersions are better absorbed than the corresponding “ A ” in oily solution. Moreover, in pancreatic dystrophy, where the alcohol administered in oil is well absorbed, but the

-AQUEOUS DISPERSION,1000 u/LB. OLEUM PERCOMORPH,3000 u/LB.

I 0

I 6

I

I I2

I

I

I8 HRS. AFTER TEST DOSE

I

I 24

FIG.7. Vitamin “ A ” tolerances with 3 times as much oily as aqueous vitamin “ A ” in normal lactating women. (Sobel et al., 1949c.)

ester is not, esters dispersed in aqueous media are absorbed about as well as alcohol dispersed in aqueous media (May and Lowe, 1948). In considering the absorption of vitamin A alcohol in pancreatic dystrophy, one must remember that the unsaponified fraction of fish oil was used, which does not contain any neutral fat at all. When such vitamin A is dissolved in an oil which is an ester, like maize oil, the vitamin A alcohol dissolved in it is not preferentially absorbed. Before it was recognized that finely dispersed aqueous dispersions are better absorbed than oily solutions, a wide variety of studies were undertaken on the problem of vitamin A absorption in various clinical conditions (May, 1940; McCoord and Breeze, 1938). So far most of the reports indicate that when there is an impairment of absorption, aqueous dispersions provide a means for improved absorption (Davidson and

FAT-SOLUBLE VITAMINS

55

Sobel, 1949; Halpern el al., 1947; Halpern and Biely, 1948; Kagan et al., 1950; Kern and Antoshkiw, 1950; Lewis et al., 1947; Sobel et al., 1948a,b, 1949b; Sobel and Rosenberg, 1950; Stanletz and Scharf, 1945; Weick and Tsao, 1947). This improvement depends at least in part on the fineness of the particle (Kramer et al., 1947; Lewis et al., 1950; Morales el al., 1950; Sobel et al., 1949b). While the evidence for the better absorption of vitamin A in aqueous dispersion is decisive, that for the other fat-soluble vitamins is not so clean

............... ...............

. . . . . . . . .. .. .. .. .. .. .. .. .. .

FIG.8. Schematic diagram of fine particle size and intestinal absorption. 1. Kramer el al., (1947). 2. Sobel et al., (1948a). 3. Frazer et al., (1944). 4. Frazer (1947). 5. Frazer (1946).

cut. However, evidence bearing on the prophylactic and curative effect and serum calcium levels indicate that aqueous dispersions of “ D ” are better absorbed (Kramer et al., 1951). Changes in blood vitamin E indicate that “ E ” is also better absorbed (Kramer el al., 1951). Vitamin K administration in the presence of bile acids produces decreased prothrombin time (Quick, 1942). This observation may be regarded as implying improved absorption of vitamin K when given in an aqueous dispersion. At present the dispersing agents employed are sorethytan laurate, sorethytan oleate, lecithin, bile acids, “ methocel,” and “ carbowax.” In addition, alcohols like propylene glycol have also been employed. In the

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ALBERT EDWARD SOBEL

case of vitamin K, water-soluble derivatives proved efficacious (Quick, 1942), but in the case of vitamins A, D, and E, such derivatives have not been employed.

3. Path of Absorption of Aqueous Dispersions One of the questions that arises is whether finely dispersed fat-soluble vitamin particles are absorbed through a different path than an oily solution of the same metabolite. This cannot be decisively answered a t present. Most of the rise in the blood is due to the ester, whether given as the alcohol or the ester, just as was the case for vitamin A given in oil (Popper et al., 194813). Thus, a t least in the blood, the forms appear to be the same. That aqueous dispersions of vitamin A may be able to penetrate through a different route than the lymph system was indicated in a case of chylous ascites, where there is a block in the transportation through the lymph system, and no change of blood “ A ” following the administration of “A” in oil took place a t the height of the disease; but there was a considerable rise following administration of vitamin A in aqueous dispersion (Gribetz and Kanof, 1951). That this route may be the portal circulation is indicated in the studies of Kowalewski el al. (1951) in six dogs. When carotene was given in oily solution, substantial proportions of the carotene were absorbed by the lymphatic ducts. However, carotene given in an aqueous emulsion appeared to be absorbed rapidly into the portal vein, The percentage increase in both the carotene and vitamin A in the blood was greater with emulsified carotene. 111.

PROBLEM O F THE TRANSPORTATION O F

FAT-SOLUBLE VITAMINS

I. Examples Indicating the Possibility of Dejiciencies Due to Impaired Transportation While it is recognized that poor intestinal absorption can cause a frank avitaminosis, it is not generally recognized that similar vitamin deficiencies can be the result of the breakdown of the transportation system to the site of action. Theoretically, it is evident that if the membranes of the blood capillaries or the lymph were completely impervious to the passing of fat-soluble vitamins, there should be deficiencies in the tissues requiring the vitamin. That such a possibility actually exists is hinted a t by a wide variety of skin diseases, in which keratosis exists resembling vitamin A deficiency, and which respond only to huge amounts of this vitamin (Davidson and Sobel, 1949; Harris, 1949; Sobel el al., 1950; Tolentino, 1950). Park (1951) recently reported a series of children suffering from infectious diseases who developed rickets that does not respond to vitamin

FAT-SOLUBLE VITAMINS

57

D therapy until the infection is over. Avitaminosis A reported by Blackfan and Wolbach (1933) on adequate intake might fall into the same category. That this is a real possibility can be surmised from two observations. In a wide variety of febrile conditions the serum vitamin A level falls, in many cases reaching zero levels (Leitner, 1951; Moore and Sharman, 1951). I n the study of the transfer of vitamin A to milk it was indicated that the critical factor is the serum level. The higher the serum level, the greater the degree of transfer (Sobel et aE., 1950). Extrapolating this to lower levels, one would reach the conclusion that there is a serum level a t which transfer is almost nonexistent or exists to a low degree. If making the most favorable postulate, that vitamin A transfer occurs across the capillaries and tissue cell walls by a chemical mechanism independent of serum levels, it certainly could be visualized that this mechanism can break down in a manner analogous to the mechanism of gastrointestinal absorption. One can readily admit that blood levels will not be the only factor in transfer. For example, whether the vitamin is ultrafiltrable or not will be a factor. Evidence on ultrafiltrability of the fat-soluble vitamins is practically nonexistent, except for the studies of Klopp et al. (personal communication), who have shown that when the serum level of vitamin A reaches approximately 400 I.U./lOO ml., the vitamin becomes ultrafiltrable. The conditions that result in ultrafiltrability merit study for the further understanding of vitamin transportation. 2. Transportation Problems in Skin Diseases

It has been observed that a wide variety of skin diseases that can be defined by a common denominator of hyperkeratosjs improve on prolonged treatment with high doses of vitamin A. Dermatological abnormalities like acne vulgaris, atypical lichen planus, pityriasis, leukoplakia, xeroderma, lupus vulgaris, and acanthosis nigricans have in common symptoms of vitamin A deficiency. Administration of vitamin A was necessary in large doses, 200,000 to 400,000 U.S.P. units daily over considerable periods, before improvement was noted. “The consensus of opinion seemed to be that vitamin A deficiency was the cause of the skin abnormality even in those cases where the diets were analyzed and a perfectly adequate intake of vitamin A and carotene was found” (Harris, 1949). It was reasoned that if vitamin A deficiency is a factor, aqueous dispersions of vitamin A should show earlier therapeutic results since it is better absorbed than the oily preparation. The particular skin condition that was taken as the test case was acne vulgaris, not of the adolescent type, but in which the subjects had this condition anywhere from one to nine years. The subjects were

58

ALBERT EDWARD SOBEL

given about 50,000 units of vitamin A in 4-5 ml. of solution containing 1 ml. of sorethytan laurate. This treatment continued up to 6 months with the patients. In all but one case, definite and distinct signs of improvement were shown from 2 to 6 weeks after the beginning of the management. In most cases when the administration of the vitamin A dispersion was discontinued, the condition recurred 2 to 3 weeks after discontinuing the supplement. On a second period of supplementation there was again improvement. On examining the question of whether there is impaired absorption in such cases, and whether there is a vitamin

I

0

-AQUEOUS

3

8

DISPERSION

9

HRS. AFTER TEST DOSE OF 3!500 USP urLaBOOY WT

FIG.9. Vitamin “ A ” tolerances with oleum percomorph and aqueous dispersion in acne vulgaris. (Davidson and Sobel, 1949.)

deficiency due to poor absorption, it was found that as a group these patients were poorer absorbers than a control group, but certainly did not have a complete deficiency of vitamin A in their systems, and thus a second postulate was made to explain why high doses of vitamin A were required in the management of the skin condition. The clue was given by a patient who received the same total dose, but had taken it in divided portions during the day. Such patients did not respond. Thus it was postulated that the problem here must be transfer to the organ requiring vitamin A, in this case the skin. With one single dose of the vitamin A dispersion, very high blood levels are obtained, a t least temporarily (Fig. 9). In giving the same total vitamin A, but in a number of smaller doses throughout the day, no such high blood levels are obtained. Since

59

FAT-SOLUBLE VITAMINS

the transfer across a membrane is proportional to the concentration of the diffusing substance, it was reasoned that the high blood levels provide a high diffusion potential which permits the penetration of tissues that normally would block penetration by lower concentrations (Davidson and Sobel, 1949). Thus the postulate was made that the possibility of a vitamin A deficiency may exist in one or more tissues due to impaired membranes of cells and capillary walls with which they are in contact. 3. Enrichment of Milk Vitamin A and Blood Serum Levels

To find physiological proof for the postulate, made as a result of these studies in acne vulgaris, the enrichment of milk was critically investigated.

-1

AQUEOUS DISPERSION

---- OLEUM PERCOMORPH

cn

fa

p

100-

P I

I

I

I

I

HRS. AFTER TEST DOSE OF 1000 USP u/LB. BODY WT

FIG.10. Milk vitamin “ A ” levels with oleum percomorph and aqueous dispersion in normal lactating women. (Sobel et al., 1949c.) (Sobel et al., 1950.)

It is well known that the transfer of the fat-soluble vitamins to milk is very poor from the usual source of vitamin A and D, which is in oil (DeHaas and Meulemans, 1938; Hrubetz et al., 1945; Polskin et al., 1945; Thalberg, 1883; Thatcher and Sure, 1932). When human mothers were given a single large dose of a vitamin A dispersion there was a marked increase in the milk vitamin A. The same vitamin A dissolved in oil caused practically no change in the milk levels (Fig. 10). When, however, the amount of oily vitamin A was increased to the point where blood levels were the

60

ALBERT EDWARD SOBEL

same (Fig. 7) as with a smaller dose of aqueous ((A,” the enrichment of milk vitamin A was about the same (Fig. 11). The foregoing indicated that it is not the total vitamin A in the body that decides transfer to milk, but the concentration of serum vitamin A. While it is admitted that this is not the only factor, it seems to be one of the key factors in determining the enrichment of milk with vitamin A. To prove the point further, the body storage of suckling rats was determined, where the nursing dams were given one dose of vitamin A dispersed in water or the same vitamin

i

250

-

-

AQUEOUS DISPERSION, D O 0 UILB.

---- OLEUM PERCOMORPH, 3000 u/LB

HRS. AFTER TEST DOSE

FIG.11. Rises in milk vitamin “A” levels with 3 times as much oily as aqueous vitamin “ A ” in normal lactating women. (Sobel et al., 1949c.) (Sobelet al., 1950.)

A in oil. There was four times as much vitamin A in the sucklings where the dams received the aqueous preparation (Fig. 12). These experiments certainly suggested that blood concentrations are an important factor in transfer to milk. If such considerations are true it follows that one should produce maximum transfer of vitamin A to the milk by injecting intravenously a dose of vitamin A. Since maximum levels are obtained with intravenous injections, the milk levels should be higher and should reach their maximum levels earlier. This was indeed the case when milch cows were given: (1) Intravenous injection of aqueous dispersion of vitamin A; (2) Oral doses of the same aqueous dispersion; (3) Oral doses of the same vitamin A in oily solution.

FAT-SOLUBLE VITAMINS

61

The total increase in milk vitamin A in the 48-hour collection period following the test dose was ten times as great with the intravenous injection and four times as great with the aqueous dispersion given orally as with the orally administered oily solution. (The aqueous dispersions given orally and injected intravenously were the same. They contained sorethytan oleate in addition to vitamin A. The oily “ A ” was the same vitamin A concentrate dissolved in maize oil.) The maximum rise, just as was expected from theoretical considerations, took place much

-

0AQUEOUS DISPERSION OLEUM PERCOMORPH

-

1

6.4

40.4

pJ PER SUCKLING

PER LITTER

FIG.12. Vitamin “ A ” storage of suckling rats of post-partum mothers fed oleum percomorph or aqueous dispersion for 4 days. (Sobel and Rosenberg, 1950.)

earlier with intravenous injections than with oral administration. These findings are in harmony with the concept that the laws of diffusion are important in defining the amount and rate of vitamin A transfer from blood t o another organ, in this case to milk (Sobel et al., 1949a,c, 1950; Sobel and Rosenberg, 1952).

4. Transfer from Mother to Growing Embryo An interesting problem in clinical deficiency of fat-soluble vitamins consists of the transfer of these vitamins from the mother to the growing embryo. Presumably the growing embryo requires these vitamins. I n fact, young pigs and rats whose mothers were given diets deficient in vitamin A during pregnancy are born with congenital anomalies described by Hale (1938) and Warkany (1944). That this transfer can vary is

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indicated by studies where feeding a diet high in cholesterol to rats led to a lesser amount of vitamin A in the stores of the newborn young than in the controls (Phillips, 1951; Popjak, 1946; Williamson, 1948). Phillips (1951) and Popjak (1946) presented evidence to show that high lipids in the placenta retard maternal nutrients to the fetus. One can readily perceive that in some diseased conditions such transfer is impaired and may lead to abnormally low stores of the young. I n young cows there is a disease called scours in which vitamin A deficiency is a factor. These young are born with insufficient stores, and do not benefit by direct administration of vitamin A. Not unless the milk is fortified with “A,” which requires millions of units given t o the nursing cow, is this rectified (Oser, 1948). The vitamin A stores of the newborn young can be influenced to a mild degree by feeding the parent, but the amounts are actually very low (Henry et al., 1949; Phillips, 1951). Whether the administration (especially by the intravenous route) of aqueous dispersions would result in a greater transfer remains to be further investigated. Massive dietary doses of oily “ A ” in cattle resulted in increased stores in the liver. of the young. Massive doses would produce high blood levels, just as would smaller doses of aqueous “A.” Esh et al. (1947) fed lecithin with vitamin A and found greater liver stores in the young. With lecithin one produces high blood levels, as it forms a n aqueous dispersion (Adlersberg, 1944; Adlersberg et al., 1948). Moreover, a paper by Lewis and his colleagues (1947) suggests that liver stores of the young are higher when mothers are fed aqueous dispersions. The above suggests that aqueous dispersions administered t o the pregnant female, especially when given by the intravenous route, would result in higher stores of “ A ” in the newborn young. 6. Transfer of Fat-Soluble Vitamins to the Nursing Young

I n view of these low stores in the newborn, which usually are associated with typically low blood levels of vitamins A and E, supplements of these fat-soluble vitamins are probably quite important (Mason, 1951 ; Phillips, 1951). I n the nursing young the only source of these vitamins is that present in the milk. That milk content affects the vitamin A stores was indicated in several studies (Henry et al., 1949; Sobel and Rosenberg, 1950). When mothers are given aqueous dispersions instead of oily solutions, not only is the transfer t o the milk greater, but the storage in the nursing young is also greater. I n the case of vitamin E, human mothers’ milk is about five times richer than cow’s milk, and about ten times as rich as cow’s milk diluted into a formula (Wright et al., 1951; Mason, 1951). I n following the vitamin E levels of the young, it has been shown that vitamin E levels are

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distinctly higher in the children receiving human mother’s milk. The placental transfer from the mother to the young indicates the desirability of women m’aintaining fatty depots through pregnancy, as the storage of vitamin E seems to be limited to the fat depots (Mason, 1951; Quaife and Dju, 1949). It is possible that the practice of some obstetricians of administering vitamin E helps to counteract a lack of depots in women following the present fashion of minimum weight. 6. Possible Approach to Some Clinical Problems

It is possible that deficiencies of the fat-soluble vitamins due not only t o defective absorption, but to poor transportation, exist in some diseases not discussed above. The examples cited below only suggest that more intensive investigation may lead to a solution of some of the problems of the clinician. There have been many attempts to interrelate the appearance of kidney stones in vitamin A deficient animals and the appearance of kidney stones in humans. It is an interesting fact that a vitamin A depleted animal, when given small amounts of vitamin A, stores the “ A ” first in the kidney (Johnson and Bauman, 1947a, 1947b; Moore, 1950; Moore and Sharman, 1951). The mitochondria from rabbit kidney contain vitamin A. This complex has been suggested as essential to hydrogen transport by Ernster et al. (1950). This observation, besides suggesting the manner in which vitamin A works in the kidney, further indicates its essential role. It has been observed and confirmed that the majority of patients suffering from kidney stones have a lower citrate content than the urine in control groups (Kissin andLocks, 1941; Scott et al., 1943). The hydrogen transport system does enter in the citric acid cycle. In light of the discussion of poor transportation due to impaired membranes, one might postulate that some kidney stones may be due to a vitamin A deficiency due to poor transportation. T o test this hypothesis, two patients who had repeated histories of kidney stone formation after removal by surgery were given about 40,000 units of vitamin A daily for 2 years. No new stones have formed, and the old stones present in one of the patients a t the beginning of the experiment have shrunk to about one half the original size and have disintegrated (Segal and Sobel, 1949-1951). Parallel with this observation i t was noted that the citrate content of the urine was increased to a normal level. We do not consider these observations as conclusive, but only as suggestive. The observations of Rafsky and Newman (1948) and Yiengst and Shock (1949) indicate that in the aged, especially above seventy years of age, the rate of vitamin A absorption is slower. This may indicate that part of the geriatric problem may be malnutrition due to poor

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absorption. This may be extrapoIated to mean that poor transportation is also involved, especially since vitamin A levels tend to be low. It had been noted by Moore and Sharman (1951)) Leitnel: (1951), and Bodansky and Markardt (1951) that serum vitamin A levels are sensitive to hormone injections. This may indicate an influence of hormones on the transportation system of vitamin A. It wa's observed that normal women have a cyclic change in serum vitamin .A which reaches a minimum around the period of menstruation and a maximum in between menstrual periods (Lipschitz and Sobel, 1952). Women with abnormal cycles showed no such regularity. Since high serum levels are correlated with greater transfer, it is possible a t least tentatively to postulate that these changes in vitamin A are induced by the hormone cycle and may play a role in building up the endometrium and the corpus luteum, which require vitamin A (Ragins and Popper, 1942). The report of successful treatment of premenstrual tension with 200,000 units of vitamin A daily (Argonz and Abinzano, 1950) may be related to the lack of the cyclic elevation of vitamin A characteristic of normal women and suggests the possibility of a transportation problem, since these patients did not suffer from hypovitaminosis otherwise. Leukoplakia vulvae is reported to have been successfully treated with large doses of vitamin A (Hyams and Gallaher, 1950). Vaginal cornification in the rat was eliminated by topical application of vitamin A but not by high oral doses (Kahn and Bern, 1950). In all these cases we are probably dealing with transportation problems across membranes. One must be cautious in interpreting the above phenomena as they might depend on other factors. It is probably true, however, that careful study of the fat-soluble vitamins will reveal in the future deficiencies in some organs a t times when the body stores contain these vitamins.

REMARKS IV. CONCLUDING The evidence presented indicates that, as one would expect from the lack of solubility of the fat-soluble vitamins in water, a breakdown of the system responsible for absorption and transportation can often occur, and frank or subacute symptoms of vitamin deficiencies manifest themselves. It is the author's opinion that investigating disease with this concept in mind will continue to be a fruitful subject of research. Moreover, it should provide the solution to some of the nutritional problems encountered in animal husbandry. ACKNOWLEDQMENTS

The author is indebted to Miss Penni A. Lipschitz for her invaluable assistance in the preparationof this review, and to Dr. Samuel M. Gordon for his helpful suggestions.

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REFERENCES

Adlersberg, D. 1944. N . Y.State J . Med. 44, 606-610. Adlersberg, D.,Kann, S., Maurer, A. P., Newerly, K., Winternitz, W., and Sobotka, H. 1948. Gastroenterology 10, 822-830. Altschule, M.D. 1935. Arch. Path. 20, 845-856. Argonz, J., and Abinzano, C. 1950. J . Clin. Endocrinol. 10, 1579-1590. Barnes, B., Wollaeger, E. E., and Mason, H. L. 1950. J . Clin. Invest. 29,982-987. Bercovitm, Z., and Paye, R. S. 1944. Ann. Znternal Med. 20, 239-253. Blackfan, K.D., and Wolbach, S. B. 1933. J . Pediat. 5, 679-706. Bodansky, O.,and Markardt, B. 1951. Federation PTOC.10, 164. Chesney, J., and McCoord, A. 1934. PTOC. SOC.Exptl. Biol. Med. 51,887-888. Cienfugos, S. 1946. J . Pediat. 28, 191-192. Clausen, S. W. 1943. Harvey Lectures S8, 199-226. Clausen, S. W., McCoord, A. B., and Goff, B. L. 1946. Federation PTOC. 6, 129. Dam, H. 1948. Vitamins and Hormones 6,27-53. Davidson, D. M., and Sobel, A. E. 1949. J . Invest. Dermatoi. 12,221-228. DeHaaa, J. H., and Meulemans, 0. 1938. Lancet 1, 1110-1111. Eden, E.,and Sellers, K. C. 1950. Biochem. J . 46,261-266. Ernster, L., Zetterstrom, R., and Lindberg, 0. 1950. Exptl. Cell Research 1, 494496. Esh, G. C., Sutton, T. S., Hibbs, J. W., and Krauss, W. E. 1947. J . Animal Sci. 6, 485. Filer, L. J., Wright, S. W., Manning, M. P., and Mason, K. E. 1951. Pediatrics 8, 328-339. Frazer, A. C. 1947. Chemistry & Industry 27, 379-382. Frazer, A. C., Schulman, J. H., and Stewart, H. C. 1944. J . Physiol. 103,306-316. Gribetz, D., and Kanoff, A. 1951. Pediatrics 7,632-641. Hale, F. 1937-1938. Tez. State J . Med. SS, 228-232. Halpern, G. R.,Biely, J., and Hardy, F. 1947. Science 106,40-41. Halpern, G. R., and Biely, J. 1948. J . Biol. Chem. 174,817-826. Harris, P. L. 1949. Ann. Rev. Biochem. 18, 391-434 (see pp. 391 and 396). Henry, K. M., Kon, S. K., Mawson, E. H., Stanier, J. E., and Thomson, S. Y. 1949. Brit. J . Nutrition 5, 301-319. Hrubetz, M. C., Deuel, H. J., Jr., Hanley, B. J., and Fairclough, M. 1945. J . Nutrition as, 245-254. Hyams, M. N., and Gallaher, P. D. 1950. Am. J . Obstet. Gynecol. 69,1346-1350. Johnson, R. M., and Bauman, C. A. 1947s. Arch. Biochem. 14,361-367. 6,265. Johnson, R. M., and Bauman, C. A. 1947b. Federation PTOC. Jones, C. M., Culver, P. J., Drummer, G. D., and Ryan, A. E. 1948. Ann. Znternal Med. 29, 1-10. Kagan, B. M., Thomas, E. M., Jordan, D. A,, and Abt, A. F. 1950. J . Clin. Invest. 29, 141-145. Kahn, R. H., and Bern, H.A. 1950. Science 111,516-517. Kern, C. J., and Antoshkiw, T. 1950. Ind. Eng. Chem. 42, 709-713. Kissin, B., and Locks, M. S. 1941. PTOC. SOC.Exptl. Biol. Med. 46,216-218. Klopp, C. T., Danish, A,, and Tabor, C. W. 1951. Personal communication. Kowalewski, K., Henroitin, E., and van Geertruyden, J. 1951. Acta Gastroenterol. belg. 14,7-15. Kramer, B. 1941. J. Mt. Sinai Hosp. 8, 188-209.

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Kramer, B., Sobel, A. E., and Gottfried, S. P. 1947. Am. J . Diseases Children 73, 543-553. Kramer, B., Gordon, S. M., Berger, H. M., and Sobel, A. E. 1951. Am. J. Diseuses Children 82, 17-27. Kramer, B., and Gittleman, I. F. 1933. New Engl. J. filed. 209, 906-916. Leitner, Z. A. 1951. Brit. J. Nutrition 6, 130-142. Lemley, J. M., Brown, R. A., Bird, 0. D., and Emmet, A. D. 1947. J. Nutrition 33,53-64. Lewis, J. M., Bodansky, O., Lilienfeld, M. C. C., and Schneider, H. A. 1947. J . Pediat. 31, 496-508. Lewis, J. M., Cohlan, S. Q., and Messina, A. 1950. Pediatrics 6, 425-436. Lipschitz, P. A., and Sobel, A. E. 1952. Vitamin A Levels during the Human Menstrual Cycle, Abstr., 121st Meeting, A.C.S., Div. Biol. Chem., p. 29C. Mason, K. E. 1951. Vitamin E Early in Life. Presented at the 4th Annual Symposium Sponsored by the Robert Gould Foundation of Cincinnati, Ohio, Sept. 10-11, Baltimore, Maryland. May, C. D., Blackfan, K. D., McCreary, J. F., and Allen, F. H., Jr. 1940. Am. J . Diseases Children 69, 1167-1184. May, C. D., and Lowe, L. 1948. J . Clin. Invest. 27, 226-230. McCoord, A., and Breeze, B., cited by Clausen, S. W. 1943. Harvey Lectures, 1942-1943, 38, 199 and 216. Melnick, D., and Oser, B. L. 1947. Vitamins and Hormones 6,57-92. Moore, T. 1950. Ann. Rev. Biochem. 19, 319-338. Moore, T., and Sharman, I. M. 1951. Brit. J. Nutrition 6 , 119-129. Morales, S., Chung, A. W., Lewis, J. M., Messina, A., and Holt, L. E., Jr. 1950. Pediatrics 6, 644-649. Morgan, A. F., and Shimotori, N. 1943. J . Biol. Chem. 147, 189-200. Natelson, S., Sobel, A. E., and Kramer, 3. 1934. J. Biol. Chem. 106, 761-765. Oser, B. 1948. Ann. Rev. Biochem. 17, 381-448 (see p. 385). Park, E. A. 1951. Rickets in Baltimore Since the Advent of Vitamin D Treatment. Presented a t the 4th Annual Symposium Sponsored by the Robert Could Foundation of Cincinnati, Ohio, Sept. 10-1 1, Baltimore, Maryland. Phillips, P. A. 1951. Vitamin A in the Newborn. Presented a t the 4th Annual Symposium Sponsored by the Robert Could Foundation of Cincinnati, Ohio, Sept. 10-11, Baltimore, Maryland. Polskin, L. J., Kramer, B., and Sobel, A. E. 1945. J . Nutrition 30, 451-466. Popjak, G. 1946. 3. Physiol. 106,236-254. Popper, H., Steigmann, F., and Dyniewicz, H. A. 1947. J . Lab. Clin. Med. 32,1403. Popper, H., Steigmann, F., and Dyniewicz, H. A. 1048a. Gastroenterology 10, 987- 1000. Popper, H., Steigmann, F., Dubin, A., Dyniewicz, H. A., and Hesser, F. P. 1948b. Proc. SOC.Exptl. Biol. Aled. 68, 676-680. Popper, H., Dubin, A., Steigmann, F., and Hesser, F. P. 1949. J. Lab. Clin. Med. 32, 648-652. Popper, H., and Volk, B. W. 1948. Proc. Soc. Exptl. Biol. Med. 68, 562-564. Quaife, M. L., and Dju, M. J. 1949. J . Biol. Chem. 180, 263-272. Quick, A. J. 1942. The Hemorrhagic Diseases. Charles C Thomas, Springfield, Illinois. Rafsky, H. A., and Newman, B. 1948. Gastroenterology 10, 1001-1006. Ragins, A. 3.,and Popper, H. 1942. Arch. Path. 34, 647.

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Remp, D. G. 1941. J . Biol. Chem. 140,Sci. Proc., SOC.Biol. Chemists, pp. cv-cvi. Scott, W. W., Huggins, C., and Selman, B. C. 1943. J. Urol. 60, 202-209. Segal, A. D., and Sobel, A. E. 1949-1951. Unpublished data. Shelling, D. H. 1937. J. Pediat. 10, 748-761. Shelling, D. H., and Hopper, B. K. 1936. Bull. Johns Hopkins Hosp. 68, 137-211. Sobel, A. E., Gottfried, S. P., and Kramer, B. 1946. A Comparison of Vitamin A Serum Levels in Children Following the Oral and Intravenous Administration of Vitamin A in Oily and Aqueous Media., Abstr., 110th Meeting, A.C.S., Div. Biol. Chem., Sept. 11. Sobel, A. E., Sherman, M., Lichtblau, J., Snow, S., and Kramer, B. 1948a. J. Nutrition 36, 225-238. Sobel, A. E., Besman, L., and Kramer, B. 1948b. Federation Proc. 7, 373. Sobel, A. E., Rosenberg, A., and West, M. M. 1049a. Intravenous Injections of Aqueous Dispersions of Fat Soluble Vitamins, Abstr., 116th Meeting, A.C.S., Div. Biol. Chem., Sept. 18. Sobel, A. E., Besman, L., and Kramer, B. 1949b. Am. J. Diseases Children 77, 576-59 1. Sobel, A. E., Rosenberg, A,, Geduldig, R., Engel, E., West, M., and Kramer, B. 1 9 4 9 ~ . Federation Proc. 8, 253. Sobel, A. E., Rosenberg, A., and Kramer, B. 1950. Am. J. Diseases Children 80, 932-043. Sobel, A. E., and Rosenberg, A. 1950. J. Nutrition 42, 557-564. Sobel, A. E., and Rosenberg, A. 1952. J. Nutrition, in press. Stanletz, C. A., and Scharf, A. J. 1945. J. Nutrition 30, 239-243. Steinberg, C. L. 1946. Med. Clinics N . Amer. 30,221-231. Stone, S. 1941. J. Pediat. 18,310-316. Thalberg, J. 1883. Arch. Augenheilk. 12, 315-332. Thatcher, H.S., and Sure, B. 1932. Arch. Path. 13,756-765. Tolentino, J. C. 1950. J . Philippine Med. Assoc. 26, 564-566. Vollmer, H. 1939. Am. J . Diseases Children 67, 343-348. Warkany, J. 1944. J. Pediat. 26, 476-480. Weick, G., and Tsao, M. 1947. Univ. Hosp. Bull., Ann Arbor 13, 114-116. Williamson, M. B. 1948. J. B i d . Chem. 174,631-636. Wright, S. W., Filer, L. J., and Mason, K. E. 1951. Pediatrics 7, 386-393. Yiengst, M. J., and Shock, N. W. 1949. J. Gerontol. 4, 205-211.

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The Nutrition of the Crustacea BY ERNEST BEERSTECHER, JR. The Department of Biochemistry, The School of Dentistry, The University of Texas, Houston, Texas CONTENTS

Page I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 11. Culture Methods.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 111. Specific Requirements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 IV. Summary .......................................... . . _ . 76 References .................................................. 76

I. INTRODUCTION Studies on the variations in any biological function over a range of species or phyla have long been recognized as an important approach to the elucidation of vital phenomena (Florkin, 1949). In the field of nutrition, the study of the specific dietary requirements of various invertebrates and lower vertebrates has produced much of our present knowledge, particularly with respect to the B vitamins (Williams et al., 1950). Major emphasis in nutritional studies has come to rest upon certain conveniently manipulable groups of organisms, i.e., the bacteria, protozoa, insects and vertebrates. While considerable progress has been made with these organisms, almost nothing has been done with the several other important groups of animals, and as a result there exist severe limitations upon the possibilities of evolving and applying principles of comparative physiology and biochemistry. This is true despite the fact that many of the gaps which exist in our knowledge of invertebrate nutrition present major economic and public health problems quite aside from their more academic aspects. The most acute defect in this regard probably concerns the large and highly evolved subphylum of crustaceans. Within this group may be found a tremendous variation in morphology such as should suit the technical requirements of most experimental disciplines. A t the same time a great many of the major physiological systems to be found in the vertebrates are present in a reasonably advanced stage of evolutionary development. Ranging in size and form from the minute water fleas to the barnacles, crabs, and lobsters, and existing in both aquatic and ter69

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restrial forms, the Crustacea present highly interesting possibilities to the experimental biologist. Practical interest in the group is broad. The barnacle presents major problems in marine engineering which are yet to be solved and the solution of which may well rest upon a greater understanding of barnacle physiology. The numerous edible crustaceans, because of their tremendous economic importance, present continual problems in population nutrition because of the recurrent threats which occur to their own populations. Many larger food fishes depend upon crustaceans to a considerable degree for their nutrition, and the importance of the smaller crustaceans in general ecological problems has probably never been adequately assessed. Despite these well recognized facts, there is at present only a very limited effort being put forth on crustacean physiology. For this reason, it is the purpose of this review not only to summarize the existing status of knowledge regarding the nutrition of the Crustacea, but also (at the risk of seeming speculative) to point out some of the attractive possibilities which exist for further estensive study in this area. The history of crustacean nutrition is scant. While studies of the general physiology of the Cladocera, and particularly Daphnia, have occurred in the literature over the past half century with some frequency, it was not until the last thirty years that any serious attention was paid to the nutritional content of the medium in which they live. The study of Viehoever and Cohen in 1938 on the vitamin E requirement of Daphnia is apparently the only published study to date on the requirement by any crustacean for any single nutritional substance. At present all other information concerning crustacean nutrition is of a presumptive nature and lacking experimental verification. Several reviews exist on the general subject of invertebrate nutrition (Galtsoff , 1939; Trager, 1941), and there is of course considerable general knowledge concerning the kinds of food utilized by many species, but this knowledge is in no case sufficiently detailed to indicate, by deficiency data or other means, anything of the essential components of the diet of crustaceans. A number of studies also exist on the vitamin and amino acid content of a variety of edible crustaceans (Santa and Bacesco, 1942; Lubitx et al., 1943; Fontaine et al., 1943; Pottinger and Baldwin, 1946; Icon and Thompson, 1949, Camien, 1951). There are two principal reasons for the lack of information in this field. Experimental nutritionists have generally been able to find for study an adequate variety of lower animals which have required culture techniques that were well enough developed to obviate the long periods of preliminary work involved with relatively unstudied groups of animals. Thus tlhe techniques for controlled laboratory cultivation of rats, insects, and

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protozoa were well known by the time nutritional studies were commenced on these animals. Biological studies with crustaceans, on the other hand, have generally centered around foci of interest wherein nutritional control was not ostensibly a critical variable. The attention of both groups of workers is gradually turning to crustacean nutrition, however, and the considerations above suggest that the importance of this field is truly great. For that reason a summary of the scattered background material for crustacean nutritional studies now seems expedient. The tremendous variation in morphology among the members of this subphylum dictates a necessity for a number of types of technical approaches. Thus, whereas the Cladocera (water fleas) may be handled in many ways much like protozoa, the Malacostraca (higher forms) must be studied by techniques similar to those used with the larger laboratory animals. In each case the major problem involves preparation of the diet in adequate physical form to meet the feeding habits of the particular species. This is particularly true for the Cladocera where minute particles of food material are swept in a current to the oral opening. Experiments involving the latter group must also be conducted under conditions where there is an adequate oxygen tension, in media of suitable tonicity (Macovski, 1946), and in complex media attractive to bacteria and yet remaining sterile. These limitations can be met,, however, and under these circumstances the Cladocera provide convenient experimental animals that may h e handled in large numbers with a minimum of space and difficulty. Proper growth, ecdysis, and reproduction may all be used as criteria of nutritional sufficiency in crustaceans, and in the Cladocera the rapid and readily observable reproductive cycle makes an attractive means of measuring sustained reproductive ability. The ubiquitous occurrence of many crustaceans further provides an advantage to the study of this group. 11. CULTURE METHODS The numerous practical difficulties which have been encountered in the culture of the larger aquatic crustaceans under controlled conditions are so great as largely to preclude the use of these animals as objects of detailed nutritional experiments (Scattergood, 1949). The use of terrestrial forms (e.g. crabs) is much simpler, since individual animals may be placed in cages or aquaria, supplied with water and the desired food, and weighed at suitable intervals. The procurement of young from a single clutch when possible should provide an excellent opportunity for adequate controls, and the relative lack of dietary discrimination among most crabs should insure the success of most synthetic diets provided these are made sufficiently plastic to allow manipulation and

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insertion into the oral cavity. Despite these facts, published reports employing this technique are apparently nonexistent. I n the case of the Cladocera many experimental details have been quite thoroughly studied. It has been shown that these animals may be made bacteria-free by washing techniques, an advantage not so readily attained in many other animals. Fifteen serial washings in sterile medium have been shown to render SO-SO% of Moina rnacrocopa bacteriologically sterile (Stuart et al., 1931a,b) and similar procedures are effective with most other Cladocera (Gellis and Clarke, 1935). Sterile Cladoeera may be obtained by the release of young from the brood pouch of a sterile female. Concentrations of penicillin G of from 100-3000 units % and concentrations of streptomycin of from 1-30 mg. % cause appreciable decreases in the bacterial count of Cladocera media, but also exert a distinctly inhibitory effect on crustaceans. The survival time of Daphnia in such media varies with the logarithm of the antibiotic concentration (Beerstecher and Harper, 1951). The control of bacterial growth by the use of antibiotics has apparently not been reported in the literature, but the use of sulfa type drugs for this purpose is difficult due to their effect on the tonicity of the medium. Early experiments suggesting that Daphnia could utilize dissolved organic matter (Knorrich, 1901; Wolff, 1909; Krogh, 1930; Putter, A., 1907, 1909) have generally been discredited (Kerb, 1910; Gellis and Clarke, 1935) due to the presence of filterable particles such as bacteria in the earlier media. Gellis and Clarke’s study showed that when “colloids ” were removed from the autoclaved media by ultrafiltration through collodion membranes the medium ceased to support the growth of Daphnia. These same workers presented data to show that Daphnia were incapable of any appreciable uptake of glucose from solution as evidence for the inability of Cladocera to utilize dissolved organic matter. The conclusion from all of these studies seemed to be that if dissolved material was assimilated, the amount was far less than was necessary to support growth. This contention seems compatible with the almost certain fact that real, though very small, amounts of dissolved material are utilized. This consideration is indeed a critical one in view of the minute amount of many growth factors required. Thus, whereas the relatively large protein and carbohydrate requirements may only be met by a particulate nutritional source, and whereas media containing only moderate levels of dissolved vitamins might not suffice, adequate amounts of vitamins might well be absorbed from synthetic media highly fortified with vitamins. The necessity for using nutritionally inactive adsorbents (Kaolin, Fuller’s earth, charcoal) to provide particulate form to synthetic diets may thus perhaps be obviated by using relatively high levels of the

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individual vitamins and vitamin-free protein and starch. The total dissolved material in Daphnia medium should apparently not exceed 100 mg./liter. A considerable variety of media has been employed in the laboratory culture of Cladocera and more particularly of Daphnia, but none could be even remotely described as “defined.” Most commonly Daphnia have been cultured in manure infusions (Viehoever and Cohen, 1938a,b). Other media include pond water, aquarium water (Wolff, 1909), Soybean flour-urea infusion (Viehoever and Cohen, 1938a,b), hay infusions (Knorrich, 1901), lettuce infusion (Gellis and Clarke, 1935), bacterial cultures (Brown and Banta, 1935; Stuart et a!., 1931b) and Fleischman’s yeast (Banta, 1921). An excess of organic matter is inimical to growth (Stuart and Banta, 1931), as are extremes of pH, temperature and crowding. Assay containers should be selected so as to provide a large enough surface area for the volume of medium and number of animals employed since Cladocera have a high rate of aerobic metabolism (MacArthur and Baillie, 1929). I n the culture media mentioned, the size of the clutch, the gestation period, and the sex of the young vary with the concentration of the medium and number of animals in a given volume of medium, and these variables must therefore be considered in nutritional studies employing reproductive activities as criteria of dietary adequacy.

111. SPECIFICREQUIREMENTS It is generally recognized that crustaceans have the same general mineral requirements as most other animals, although their exact mineral requirements are unstudied. In most synthetic media made with distilled water, 5 t o 10 mg. of U.S.P. Salt Mixture No. 2 per liter seems to satisfy the mineral requirements of Daphnia. Requirements for organic metabolites are completely unknown with the possible exception of Viehoever and Cohen’s study (1938a) on the vitamin E requirement of Daphnia magna. Working with animals raised in sheep manure infusion a t 21”C., these authors showed that in petroleum ether-extracted media the growth, rhythmic ovarian function, and reproductive activity were inhibited. There was a lack of vigor and a high mortality rate. When cold-pressed wheat germ oil was added to the medium, growth was accelerated within 48 hours, the ovarian rhythm was restored, the number of young per clutch was increased, their vigor was improved, and the mortality rate dropped. Daphnia in stagnated cultures were likewise seen to have a reduced reproductive rate and in some cases developing young were seen to regress and resorb, as they also did in the ether-extracted medium. Addition of the wheat germ oil to such stagnated cultures reverses these symptoms. These authors also claim that the vitamin E oil has a

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beneficial effect on the fertility of young Daphnia in nondeficient media. A vitamin E deficient rat diet medium was also used and “yielded valuable results,” although these were not reported. Based on these results this organism was suggested as an assay animal for vitamin E activity, and similar studies were reported in progress on vitamin A, but none have apparently been published. All of these results certainly merit repetition with the purified tocopherols now available and using aseptic animals before the claim for a vitamin E requirement in Daphnia can be fully accepted, but the authors point out that their results suggest “ a possible analogy with the resorption of young in utero in rats maintained under vitamin E deficiency.” It is worthy of note that these workers also employed Daphnia for the study of the activity of a number of other interesting groups of biological compounds (Viehoever and Cohen, 1938b), and have given detailed instructions for the culture .of this species in a variety of media and for their use in biological assays. The fact that there are few qualitative differences between the B vitamin requirements of mammals, insects, and the higher protozoa would suggest that crustaceans probably have similar requirements for this group (Beerstecher, 1950). When Daphnia are cultured in a synthetic medium containing vitamin-free casein, sucrose, vegetable oil, and a suitable salt mixture, their survival is greatly prolonged by the addition of small amounts of yeast extract (Beerstecher and Harper, 1951). In the range from 1 to 10 mg./liter their life span is approximately proportional to the logarithm of the yeast extract concentration, but amounts in excess of this appear to be toxic. A mixture of all of the known B vitamins in concentrations equivalent to that present in the extract has little or no value in supporting growth in the basal medium, however, although it does promote prolific bacterial growth. The growth factor in the yeast extract is not one of the known fat-soluble vitamins or ascorbic acid. Busnel (1943) states that the amount of riboflavin in the hypodermis of Brachyura varies during the various life stages with the maximum occurring during the period of maximum food intake, suggesting that the tissue levels are dependent upon the diet and that the riboflavin source therefore is not endogenous. Higher levels are reported in euryhaline crabs than stenohaline crabs, and in species living part of the time out of the water than in obligate marine forms (Fontaine et al., 1943). The high xanthine oxidase activity of crayfish and lobster “liver” (Florkin and Duchateau-Bosson, 1943) suggests a considerable requirement for riboflavin for this purpose alone in these animals. The reported coexistence of riboflavin and melanin in the melanocytes of Brachyura (Cancer, Carcinides, Eriocheir) (Busnel, 1943; Verne and Busnel, 1943)

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suggests a possible photosensitive function for riboflavin in these animals similar to that believed to exist in plants in the photo-oxidation of indole derivatives and in the avascular mammalian cornea. The role of thiamine in crustacean nutrition merits particular investigation because of the presence of a thiaminase in many shrimps (Carassius, Penaeus, Tellinae) (Jacobsohn and Azevedo, 1947) and the consequent low levels of thiamine to be found in their tissues after death (Lubitz et al., 1943). The enzyme is apparently not present in crabs. 3-Acetylpyridine, which has been employed as an inhibitory analogue of niacin in chick embryos, mice, and dogs, is toxic to Daphnia at concentrations above 0.3 mg./ml., which correspond well with values in other species (Beerstecher and Harper, 1951). Its toxicity, however, is not reversed by nicotinamide. The fact that the y-isomer of hexachlorocyclohexane is more toxic to Daphnia than the a- or @-isomermay be taken with much reservation as evidence for an exogenous requirement for inositol (Poussel, 1949). The presence of acetylcholine and its esterase in crustaceans (Marnay and Nachmansohn, 1937) implies an important role for choline and pantothenic acid in these animals. The relatively large amounts of ascorbic acid which have been reported in several crustaceans (Paramysis and Parenosa) (Santa and Bacesco, 1942) seem to vary with the species, age, and sex. The levels decrease rapidly when there is nutritional deprivation, again suggesting an exogenous origin, but it is unknown whether a physiological function exists for vitamin C in crustaceans. Certainly many Cladocera have been reared in its absence. The presence of vitamin A in a number of marine crustaceans has been extensively investigated (Kon and Thompson, 1949; Henry et al., 1951). I n some species (Meganyctiphanes norwegica) the vitamin A has been shown to be concentrated almost exclusively (98%) in the eyes (Fisher et al., 1951), while in others (Palaemonetes) there is a large concentration in the exoskeleton but little in the cephalothorax or telson. Cladocera have been cultured with varying success on a number of rather specialized media that deserve passing mention as indicating something about the fastidiousness of crustacean nutritional requirements. Stuart et al. (1931b) found that bacteriologically sterile Moina macrocopa could survive and multiply on dead cells of Escherichia coli for only limited periods of time, requiring viable cells for indefinite survival. Sterile Moina were also raised in pure cultures of living E. coli, Aerobacter aerogenes, Staphylococcus albus, Alkaligenes faecalis, Bacillus subtilis, Sarcina lutea, Spirillum rubrum and Chromobmter violaceum. Growth was supported by all except B. subtilis and the pigmented strains of Chromobwterium and Sarcina. The pigment of Chroniobacler (vio-

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lacein, a quinone imine) is apparently toxic to the Moina since nonpigmented strains could be used as food. A similar consideration apparently applies to the carotenoid pigments of Sarcina. Gellis and Clarke (1935) found that sterile Daphnia could not molt in solutions of glucose, double proteose peptone broth, bactopeptone, or yeast autolysate.

IV. SUMMARY While very little is known at present with regard to the nutritional requirements of crustaceans, there is a small body of evidence suggesting that their requirements are similar in general to those of higher animals. The only specific requirement that has a t present been demonstrated with any degree of certainty is that of Daphnia for vitamin E . The Cladocera represent the highest form of animal life that can be conveniently cultured in aqueous media under sterile conditions, and theref ore present an interesting challenge to nutritional research. Many of the experimental details for culturing these animals have already been developed, while the general techniques for studying the higher terrestrial crustaceans may be borrowed from the experimental procedures long established with higher animals. REFERENCES

Banta, A. M. 1921. Science 63, 557. Beerstecher, E.,Jr. 1950. Science 111, 300-302. Beerstecher, E.,Jr., and Harper, R. M. 1951. Unpublished data. Borradaile, L. A., and Potts, F. A. 1935. The Invertebrata. University Press, Cambridge. Brown, L. A., and Banta, A. M. 1935. Physiol. 2001.8, 138-155. Busnel, R. G. 1943. Compt. rend. 216, 85-86, 162-164. Camien, M. N., Sarlet, H., Duchateau, G., and Florkin, M. 1951. J . Biol. Chem. 193, 881-885. Fisher, L. R., Kon, S. K., and Thompson, S. Y. 1951. Biochem. J . 49, xv. Florkin, M. 1949. Biochemical Evolution. Academic Press, New York. Florkin, M., and Duchateau-Bosson, G. 1943. Enzymologia 11, 24-25. Fontaine, M.,Raffy, A,, and Collonge, S. 1943. Compt. rend. SOC.biol. 137, 27-29. Galtsoff, P. 1939. Culture Methods for Invertebrate Animals. Comstock Publishing Co., Ithaca. Gellis, S. S., and Clarke, G. L. 1935. Physiol. 2001.8, 127-137. Henry, K. M., Kon, S. K., and Thompson, S. Y. 1951. Biochem. J . 48, x. Jacobsohn, K. P., and Azevedo, M. D. 1947. Arch. Biochem. 14, 83-86. Kerb, H. 1910. Intern. Rev. ges. Hydrobiol. Hydrog. 3, 496. Knorrich, F. W. 1901. Ploner Ber. 8, 50. Kon, S. K., and Thompson, S. Y. 1949. Arch. Biochem. 24, 233-234. Krogh, A. 1930. Z . uergleich. Physiol. 12, 668-681. Lubitz, J. A., Fellers, C. R., and Parkhurst, R. T. 1943. Poultry Sci. 22, 307-313. MacArthur, J. W., and Baillie, J. 1929. J . Ezptl. 2001.63, 243-268. Macovski, E. 1946. Bull. sect. sci. acad. roumaine a@,86-90.

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Marnay, A., and Nachmansohn, D. 1937. Con@. rend. SOC. biol. 126, 1005-1007. Pottinger, S. R., and Baldwin, W. H. 1946. Com. Fisheries Rev. 8, &9. Poussel, H. 1949. Compt. rend. 228, 1533-1535. Pratt, H. S. 1935. A Manual of the Common Invertebrate Animals. Blakiston, Philadelphia. Putter, A. 1907. Z . allgern. Physiol. 7 , 16-61. Putter, A. 1909. Die Ernahrung der Wassertiere und der Stoffhaushalt der Gewasser. Fischer, Jena. Santa, N., and Bacesco, M. 1942. Chem. Centr. 11, 2712. Scattergood, L. W. 1949. U.S. Fish Wildlije Service, Special Scientific Rep1 Fisheries No. 6 . Stuart, C. A., and Banta, A. M. 1931. Physiol. 2001.4, 72-86. Stuart, C. A., Cooper, H. J., and Tallman, J. 1931a. Physiol. Zool. 4, 594-603. Stuart, C. A., McPherson, M., and Cooper, H. J. 1931b. Physiol. Zool. 4, 87-100. Stuart, C. A., Tallman, J., and Cooper, H. J. 1931c. Physiol. Zool. 4, 581-593. Trager, W. 1941. Physiol. Rev. 21, 1-35. Verne, J., and Busnel, R. G. 1943. Compt. rend. SOC. biol. 137, 6-7. Viehoever, A., and Cohen, I. 1938a. Am. J . Pharm. 110, 296-315. Viehoever, A., and Cohen, I. 193810. Am. J. Pharm. 110, 526-532. Williams, R. J., Eakin, R. E., Beerstecher, E., Jr., and Shive, W. 1950. Biochemistry of the B Vitamins. Reinhold, New York. Wolff, M. 1909. Intern. Rev. ges. Hydrobiol. Hydrog. 2, 715.

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Nutrition and the Anterior Pituitary with Special Reference to the General Adaptation Syndrome* BY BENJAMIN H. ERSHOFF Emory W . l’hurston Laboratories, Los Angeles, and the Department of Biochemistry and Nutrition, University of Southern California, Los Angeles CONTENTS

Pa y e I . Introduction.. . . . . . . . . . . . . . . . . .......................... 11. Effects of “Malnutriture” on t ynthesis and Secretion of Pi Hormones. . . . . ............................................. 81 1. Effects of Caloric Restriction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 2. Effects of Vitamin Deficiencies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 3. Effects of Protein and Amino Acid Deficiencies.. . . . . . . . . . . . . . . . . . . . . 95 4. Effects of Mineral Deficiencies.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 111. Effects of Miscellaneous Nutrients on Pituitary Structure and Function.. . 101 IV. Nutrition and the General Adaptation Syndrome.. . . . . . . . . . . . . . . . . . . . . . 103 1. Proteins and Amino Acids. . . . 2. Vitamins.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 a. Ascorbic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 b. Pantothenic Acid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 1 c. Thiamine.. . . . . . . .................. . . . . . . . . . . . . . . . . . . 115 d. Choline.. . . . . . . . .......................................... 116 e. Riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 f. Pyridoxine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 g. VitaminA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 3. Minerals. . ........................................ 122 a. Sodium.. . . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . 122 b. Potassium .................... . . . . . . . . . . . . . . . . . . . 122 4. Unidentified Nutrients. . . . . . . . . . . . ....................... 123 5. Miscellaneous Factors.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 V. Effects of Nutritive State on Response to Pituitary Hormones.. . . . . . . . . . 126 . . , . 127 VI. Effects of Pituitary Hormones on Nutritive State. . . . . . VII. Summary.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 References. ....... ............ . . . . . . . . . . . . . . . . . . . . . . . . . . 129

I. INTRODUCTION Considerable data are available indicating that the functions of the anterior pituitary and the target organs of its secretions are largely

* Communication No, 298 from the Department of Biochemistry and Nutrition, University of Southern California. 79

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dependent on the nutritional state and the composition of the diet fed. Conversely the various hormones of the anterior pituitary and its target organs profoundly affect the absorption, utilization and excretion of nutrients and body requirements for specific dietary factors. The object of the present review is to summarize available data on the nutritionendocrine interrelationships of the mammalian organism, particularly as they pertain to the anterior pituitary and with special reference to their role in the general adaptation syndrome. It is becoming increasingly apparent that the nutritional state or “nutriture” of an organism is dependent on considerably more than its food intake. As pointed out by Kruse (1942) nutritional disease, or (‘malnutriture” as Sinclair terms it (1948a, 1948b), denotes a deficiency of essential nutrients in the tissue cell. Any factor, therefore, that interferes with the digestion, absorption or utilization of nutrients or increases their destruction or excretion may result in malnutriture despite the apparent adequacy of the diet fed. Body requirements for essential nutrients may furthermore be significantly increased for purposes of detoxification or by factors such as physical exertion, fever, drugs, toxins, abnormal environmental conditions, pregnancy, lactation and related conditions which result in an increased metabolic requirement on the part of the tissue cell. The net result of such factors is an increased body requirement beyond the usual or average range and the precipitation of nutritional deficiencies on diets that would otherwise be adequate (Jolliffe, 1942, 1943; Starr, 1942; Hickman and Harris, 1946; Ershoff, 1948a, 1951a). Such factors as an inadequate supply of water and oxygen within the cell and impaired removal of waste products may also contribute to the development of a malnutritive state. It is apparent that a discussion of nutrition-endocrine interrelationships cannot properly be divorced from a consideration of the factors indicated above, for any condition which affects the nutriture of the endocrine cell, the target organ or the body as a whole affects the formation, release, metabolism and excretion of hormones and the response of target organs and other tissues thereto. A number of reviews dealing with various phases of nutrition-endocrine interrelationships have been published. The following are particularly meritorious in their field: nutrition and the anterior pituitary (Samuels, 1947, 1950), vitamin-thyroid interrelationships (Drill, 1943), nutritional therapy of endocrine disturbances (Biskind, 1946), B vitamins and reproduction (Hertz, 1946, 1948), vitamins and the sex glands (Mason, 1939), nutrition and reproduction (Mason, 1949).*

* The text of the present manuscript was completed in July, 1951. The following reviews which appeared while this manuscript was in press are recommended for their excellent presentation of additional data pertinent to the subject matter of the present

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11. EFFECTS OF “MALNUTRITURE” ON THE SYNTHESIS AND SECRETION OF PITUITARY HORMONES 1. Effects of Caloric Restriction Prolonged restriction of caloric intake depresses the formation and release of a number of anterior pituitary hormones. The effects of caloric restriction are particularly marked in respect to gonadotropic secretion. Ovarian atrophy, uterine atrophy and anestrus vaginal smears have been observed by numerous authors during inanition (Jackson, 1917,1925; Papanicolaou and Stockard, 1920; Loeb, 1921; Evans and Bishop, 1922, 1923; Marrian and Parkes, 1929; Guilbert and GOSS,1932; Selye and Collip, 1936; Werner, 1939; Mulinos et al., 1939; Mulinos and Pomerantz, 1940; Stephens and Allen, 1941; Drill and Burrill, 1944; Maddock and Heller, 1947; Boutwell et al., 1948; Rinaldini, 1949; and others). Ovarian hypofunction (with resultant uterine atrophy and anestrus vaginal smears) appears to be due to the absence or decrease of circulating gonadotropins and not to a refractory state of the ovaries. This is indicated by the re-establishment of normal ovarian (and uterine) weight in starved animals by the injection of hypophyseal or chorionic gonadotropins (Marrian and Parkes, 1929; Mulinos et al., 1939; Werner, 1939; Stephens and Allen, 1941; Drill and Burrill, 1944; Rinaldini, 1949). Similar findings occur in the male. Atrophy and hypofunction of the testes have been repeatedly observed during inanition (Stewart, 1918; Siperstein, 1921 ; Jackson, 1925; Mason, 1933 ; Mulinos and Pomerantz, 1941a, 1941b, Quimby, 1948; and others). The accessory sex organs (prostate and seminal vesicles) are particularly sensitive to restriction of caloric intake with shrinkage of these structures (and accompanying castrate-type changes) occurring before the testes are appreciably reduced in size (Moore and Samuels, 1931). As in the case of the females the weight of the gonads and accessory sex organs can be restored to normal in starved animals by the injection of hypophyseal (Moore and Samuels, 1931) or chorionic (Mulinos and Pomerantz, 1941a) gonadotropins. It would appear from the above findings that under conditions of caloric restriction (associated with a loss in body weight of 25 to 40%) hypophyseal gonadotropin is secreted in insufficient amounts to maintain the review: Nutritional factors and hormones in stress reactions” (Samuels, L. T . 1951! Nutrition Fronts in Public Health, Nutrition Symposium Series No. 3. The National Vitamin Foundation, Inc., New York, 142-166) and “The effect of vitamin deficiencies on adrenocortical function” (Morgan, A. F. 1951. Vitamins and Hormones 9, 161-2 12).

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endocrine function of the gonads. As a result these organs atrophy and the accessory sex organs become castrate in type. The gonadotropic content of the hypophysis has been assayed by a number of investigators during inanition. Mason and Wolfe (1930) implanted the pituitaries of chronically starved male rats into immature female rats and found that the resulting increase in ovarian and uterine weight was significantly less than that resulting from hypophyseal implants obtained from littermate brothers fed the same diet ad libitum or from young male rats whose weight was comparable to that of the chronically starved animals. Similar results were obtained by Werner (1939) with the pituitaries of chronically starved female rats employing the immature Swiss mouse as the assay animal. Marrian and Parkes (1929), however, found no reduction in the gonadotropic content of the pituitary of chronically starved rats. Maddock and Heller (1947) also observed no reduction in the pituitary content of gonadotropin after 12 days of starvation in the rat. In fact, judged as potency per milligram of gland tissue, the starved rats’ pituitary glands (anterior pituitary weight had fallen about 40% during starvation) were more potent than normal. Similar findings have been reported by Meites and Reed (1949). A marked increase in the gonadotropic potency of the pituitary after chronic under-feeding was obtained by Rinaldini (1949) when hypophysectomized young female rats were employed as the assay animal. The ovaries of animals injected with the pituitaries of underfed rats were four times as large as those obtained from animals injected with the pituitaries of normal controls (108 mg. vs. 26 mg.). In summarizing the above data it would appear that inanition (either chronic or acute) is not necessarily associated with a decreased hypophyseal content of gonadotropins. On the contrary the pituitary content of gonadotropins is more likely to be increased, a t least as judged by potency per milligram of gland tissue. Data such as the above, however, are difficult to interpret, for in the final analysis they provide no information as t o the functional state of a gland. An increase in the hormonal potency of a gland might result either from increased production of a hormone or decreased secretion; similarly, a reduction in hormonal potency may indicate either decreased formation or increased elaboration. A better indicator of the functional state of a gland would be the blood level of the hormone i t produces. However, an increase in the blood level of a hormone (for example, estrogen) may result not only from increased secretion but from impaired inactivation as well (Biskind, 1946; Hertz, 1946, 1948) ;similarly a reduction in blood level (as in the case of thyroglobulin) may result not only from reduced secretion but from augmented utilization or elimination (Ershoff and Golub, 1951).

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The level of circulating gonadotropin during inanition is reduced. This is indicated by the fact that both the gonads and accessory sex organs atrophy during this condition although the ovaries retain apparently normal sensitivity t o gonadotropic stimulation (Werner, 1939; Maddock and Heller, 1947; Rinaldini, 1949). A clear-cut dichotomy thus exists between the pituitary content of gonadotropin (normal or increased amounts) and the amount of circulating gonadotropins (low levels). It would appear, therefore, that during inanition the release of hypophyseal gonadotropin is impaired and that failure of the secretory mechanism and not reduction in hormone production is responsible, at least initially, for the gonadal atrophy observed during periods of caloric restriction (Maddock and Heller, 1947 ; Meites and Reed, 1949; Rinaldini, 1949). I n the final analysis, however, the production of gonadotropin must also be reduced, since despite failure of the release mechanism the hypophyseal content of gonadotropin during inanition is not materially increased. I n general, observations on human populations during periods of food restriction are in accord with the above findings. Testicular atrophy and loss of gonadal function were found in many of the male prisoners of war (Jacobs, 1948). I n women amenorrhea and reduced fertility are general concomitants of chronic inanition (Jackson, 1925; Zimmer et al., 1944; Sydenham, 1946; Keys el al., 1950). Such observations, however, are difficult to evaluate, since it is impossible to determine to what extent findings were due to caloric restriction per se or to what extent they may reflect a deficiency of protein or other dietary constituents. Depressed fertility and gonadal pathology may, furthermore, result from factors other than inadequate nutriture. Available data indicate, however, that a limitation in food intake (either calories, nutrients or both) will, if sufficiently prolonged, reduce fertility in man, and that reduced fertility is associated, at least in some cases, with a decreased secretion of hypophyseal gonadotropin (Klinefelter el al., 1943). There is evidence, furthermore, that impaired function of the pituitary may continue long after the deficiency state has apparently been corrected. The clinical significance of these findings has been pointed out by a number of investigators (Sutton and Ashworth, 1940; Vollmer, 1943; Samuels, 1948) ;their importance, however, is not generally appreciated. Gonadotropin is not the only pituitary hormone secreted in reduced amounts during inanition, although it is probably the first to be affected. The secretion of thyrotropin, somatotropin (growth hormone), and luteotropin (lactogenic hormone) may also be impaired. Morphological alterations in the thyroid gland, characterized by atrophy, involution and degeneration have been described by a number of investigators during

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inanition, both in the experimental animal (Jackson, 1916; Morgulis, 1923; Rabinovitch, 1929; Mulinos and Pomerantz, 1940; Stephens, 1940), and in man (Meyers, 1917; Stefko, 1931). As in the case of the gonads, it would appear that thyroidal atrophy during inanition is due to impaired function of the anterior pituitary. This is indicated by the restoration of normal thyroid structure in undernourished guinea pigs by the administration of an anterior pituitary extract containing the thyrotropic principle (Stephens, 1940). In addition, the thyroids of undernourished guinea pigs were markedly sensitive to thyrotropic stimulation (structural changes were demonstrated in the glands of such animals with as little as one-tenth the minimal effective dose for normal animals) (Stephens, 1940). A similar increase in the sensitivity to thyrotropic principle (Anderson and Collip, 1934) and morphological alterations in the thyroid gland comparable to those reported above (Smith, 1930; Collip and Anderson, 1935; Hertz and Oastler, 1936) have been observed after hypophysectomy. It would appear, therefore, that a reduced secretion of thyrotropin was responsible, at least in part, for the regressive changes in the thyroid gland during inanition and, perhaps, indirectly for the attendant reduction in basal metabolism as well. The decreased morphogenetic response of the thyroid gland to goitrogenic agents in partially starved rats (Gomez Mont et al., 1947; Meites and Agrawala, 1949), the reduced uptake of radioactive by the thyroids of underfed rats and mice (Meites, 1949; Meites and Wolterink, 1950), and the demonstration of a reduced thyrotropic hormone level in the blood of acutely starved male rats and mice (D’Angelo, 1951) all provide additional evidence that thyrotropin secretion is reduced during inanition. If an immature animal is placed on an insufficient food intake, growth, as represented by skeletal elongation and gain in body weight, is reduced. Administration of somatotropin to such an animal results in more rapid growth associated with reduced nitrogen excretion (Lee, 1938; Li et al., 1949). These findings suggest that the growth retardation (or loss in body weight) during conditions of reduced caloric intake may be due in part to a decreased secretion of somatotropin. Recent data indicate that the synthesis of luteotropin may also be impaired during inanition. Meites and Reed (1949) determined the lactogenic potency of the pituitaries of adult female rats which had been maintained under conditions of ad libitum and reduced caloric intake (75%, 50%, 25% and 0% of the ad libitum controls). Lactogenic potency was assayed by injecting the equivalent of two pituitaries from each group intradermally over the crop glands of five mature White Carneau pigeons during a 4-day period, and rating the crop responses by the Reece-Turner (1937) method on the fifth day. The pituitaries of rats maintained on a reduced caloric intake

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showed a decrease in lactogenic potency, which was proportional in general to the decrease in food consumption of the animal, and was interpreted as indicating a reduced formation of hypophyseal luteotropin. Mulinos and Pomerantz (1940, 1941a, 1941b) were among the first to emphasize the depression in pituitary function occurring during inanition in the rat. These workers demonstrated that prolonged caloric restriction resulted in a number of changes similar to, but less marked, than those occurring after hypophysectomy ; and they suggested the term “pseudohypophysectomy ” to describe the impairment in anterior pituitary function occurring during inanition in the rat. Pseudo-hypophysectomized animals, however, differ from those that have had their pituitary removed surgically in a number of respects. Chorionic gonadotropin, for example, is capable of stimulating the atrophic germinal epithelium of the testes of the chronically underfed rat but not that of the hypophysectomized rat (Mulinos and Pomerantz, 1941a). Comparable findings have been described in the female rat (Werner, 1939). Furthermore, although secretion of gonadotropin is impaired during chronic inanition, some release of this hormone does take place. This is indicated by the fact that the testes of immature rats continue to gain in weight, although a t a reduced rate, during periods of prolonged caloric restriction, and further, by the fact that in over 200 young male rats, chronically starved from 30 days of age, there were only two instances of undescended testes (Quimby, 1948). I n immature hypophysectomized rats, on the other hand, the testes do not descend nor do they gain in weight. Pseudo-hypophysectomized animals still have a pituitary gland; and although it is true that in inanition pituitary function is depressed, some activity is still present. This is particularly true in respect to adrenocorticotropin, which appears to be elaborated in increased amounts, a t least initially, during periods of caloric restriction. An increase in the ratio of adrenal to body weight during chronic inanition has been reported by Quimby (1948) in the rat, D’Angelo et al. (1948a) in the guinea pig and by Boutwell et al. (1948) in the mouse. The latter workers also observed (1) an involution of the thymus gland (2) a decrease in the lymphocyte count and (3) increased gluconeogenesis. These latter findings (Ingle, 1938; Dougherty and White, 1947;Long et al., 1940) as well as the increase in adrenal weight (Selye, 1936; Selye et al., 1940) are all manifestations of intensified adrenal cortical activity. Further data indicative of an activated pituitary-adrenocortical mechanism during inanition has been observed by D’Angelo et al. (1948a) in the guinea pig. I n this animal the characteristic response of the adrenal gland to starvation is hypertrophy, and this occurs whether the inanition is chronic or acute. The increase in adrenal size is both relative and absolute and is

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roughly proportional to the degree of body weight loss and the duration of the inanition. The adrenal hypertrophy involves the inner cortical zones, particularly the fasciculata and to a lesser extent the reticularis zone, whereas the zona glomerulosa is characteristically atrophic. These findings are similar to those observed in the hypophysectomized animal following the administration of adrenocorticotropin. Cortical hypertrophy, however, fails to occur in the starved hypophysectomized guinea pig. Moreover, the cytological features of the starved pituitary are such as t o indicate that some of the basophilic cells are in high secretory activity as shown by their enlargement, degranulation, mitochondria1 content, and hypertrophied Golgi apparatuses (D’Angelo et al., 1948a). This heightened secretory activity is occurring at a time when the gonadal system is atrophic, and the thyroid gland resting or inactive. These considerations seem to indicate that during inanition a shift occurs in the production of anterior pituitary hormones (general adaptation syndrome (Selye, 1946)), resulting in an increased secretion of the essential adrenocorticotropin at the expense of less critical factors. In contrast to the data reported above, Mulinos and Pomerantz (1940) described atrophic changes in the adrenals of chronically underfed rats with a marked diminution in the cytoplasm of cortical cells (especially in the zona reticularis) and an increased nucleus-plasma ratio. Since pituitary implants restored the atrophic adrenals to normal despite continued underfeeding (Mulinos and Pomerantz, 1941c), these workers interpreted the adrenal atrophy as indicative of a reduced adrenocorticotropin secretion. The apparent discrepancy between the findings of Mulinos and Pomerantz and those reported by other workers may be due, a t least in part, to differences in the diets fed. It is obvious that restricting the caloric intake may lead not only to simple caloric restriction but to deprivation of specific dietary factors. It is possible, therefore, that the adrenal atrophy noted by Mulinos and Pomerantz under conditions of chronic undernutrition may reflect not caloric restriction per se but associated dietary deficiencies (no data are given by Mulinos and Pomerantz on the vitamin content of their diet). I n line with the above, Selye (1945) observed that the adrenal glands atrophied under conditions of reduced caloric intake when a high carbohydrate-low protein diet was fed but not when animals were fed a similar caloric intake of a high protein diet. Astrain difference in the adrenal response to acute starvation has been reported. I n the rat, adrenal enlargement occurs only in the advanced stages of starvation (if at all) and is due primarily to increased water retention, with the solid content unchanged or reduced (Mulinos and Pomerantz, 1940; Cameron and Carmichael, 1946; D’Angelo e‘ al., 1918b). I n the guinea pig, however, the adrenals enlarge progressively during

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starvation and increase not only in size but in solid content as well (D’Angelo et al., 1948a, 1948b). Histological differences in the adrenals of the two species during acute starvation have also been reported. In the guinea pig adrenal enlargement is associated with an actual increase in cortical mass, confined largely to the zona fasciculata but involving the zona reticularis as well. I n the rat, adrenal enlargement is associated with regressive changes in all cortical layers. The cause of this differential response is not readily apparent. Since no data are available on the level of circulating adrenocorticotropin during inanition in either species, it is impossible t o state to what extent differences in (1) the rate of formation or release of adrenocorticotropin (2) the rate of destruction or elimination of this hormone or (3) the sensitivity of the adrenals to corticotropic stimulation, were responsible for the observed results. d . Efects of Vitamin Dejiciencies

Considerable data are available on the effects of diets deficient in various nutrients on the morphology and function of the pituitary gland. Since many of these experiments were conducted prior to the isolation and identification of nutrients that we now recognize to be essential for optimal health, there is some question as to the significance of the findings obtained, I n many cases animals were deficient in more than one dietary factor, and the effects described were due to a multiple dietary deficiency. Such shortcomings, of course, were characteristic of all nutritional experiments conducted during the early days of vitamin research; and since unidentified nutritional factors still exist, there is still some question as to what constitutes an adequate basal diet. Evans and Bishop (1922a, 192213) were among the first to show that diets deficient in one or more vitamins may impair gonadal function in the rat. These workers demonstrated that rats deficient in “vitamin B ” ovulated infrequently if at all. Similar results were obtained by Parkes (1928), Shin (1933) and others. The above experiments were conducted with rats fed diets deficient in the various B vitamins; and it is not clear from these studies whether the resulting anestrus was due to a deficiency of B complex factors or of thiamine itself. In subsequent work, however, similar results have been reported for rats fed a diet adequate in all respects except for the absence of thiamine (Coward and Morgan, 1941; Drill and Burrill, 1944). Gonadal dysfunction in the above experiments was apparently due to an impaired secretion of pituitary gonadotropin. This is indicated by the fact that (1) implants of normal hypophyseal substance provoked follicular ripening, oestrus and ovulation in rats deficient in “antineuritic vitamin B ’’ (Evans and Bishop, 1922a, 1922b, 1923; and Marrian and Parkes, 1929), and (2) by the demonstration that

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the gonadotropic potency of the pituitary of male rats fed a diet deficient in “antineuritic vitamin B ” was significantly less than that of adequately fed litter mates or of younger animals fed an adequate diet but whose body weight was comparable to that of the deficient rats (Evans and Simpson, 1930). Similar results have been reported by Teresa (1937) for the “vitamin B ” deficient mouse and by Hundhausen (1939b) for the “vitamin B1-deficient” rat. The latter investigator found that the pituitaries of rats kept on a diet deficient in vitamin BIhad between onefourth and one-eighth of the gonadotropic potency of the pituitaries of control rats. This reduction was apparently not due to inanition, for animals kept on a complete diet restricted in amount to that eaten daily by the deficient rats had the same gonadotropic potency in their pituitaries as normal controls. Other workers, however, have indicated that the impaired secretion of hypophyseal gonadotropin and the resulting gonadal dysfunction observed in thiamine-deficient rats were caused not by a vitamin deficiency per se but by the concomitant inanition. Thus Moore and Samuels (1931) demonstrated that although the secondary sex glands of male rats atrophied when animals were fed a thiamine-deficient diet, atrophy also occurred in male rats fed a complete diet but restricted in caloric intake to that ingested daily by the thiamine-deficient animals. I n both the thiamine-deficient and chronically underfed rats, normal testes and secondary sex organs could be maintained by the administration of gonadotropic extracts. Similar results have been reported in the female rat. Drill and Burrill (1944) observed that restricting the caloric intake of a complete ration to that consumed daily by animals fed a thiamine deficient diet produced the same reduction in ovarian activity and cessation of estrous cycles as occurred in thiamine-deficient rats. As in the case of male rats, gonadal function was restored by the administration of gonadotropic extracts both in thiamine-deficient and underfed rats. Diets deficient in pantothenic acid also impair the secretion of gonadotropic hormone in the rat. Figge and Allen (1942) pointed out that the genital organs remain infantile in female rats maintained from weaning on diets deficient in pantothenic acid. I n 35 out of 36 rats on a pantothenic-acid-deficient diet, the vagina had not opened a t the age of 88 days. In 24 controls maintained on the same diet plus pantothenic acid, the vagina opened between 40 and 45 days of age and cornified smears indicated regular estrous cycles a t intervals of 4 to 6 days. The genital organs of the pantothenic acid deficient animals were extremely atrophic. The vaginal epithelium was only 2 layers thick and the uterus was tiny and anemic. The ovaries were poorly developed and were similar in appearance to those of hypophysectomized rats. It would

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appear that these effects were due, at least in part, to a reduced secretion of pituitary gonadotropin since injections of gonadotropic hormone caused typical responses in pantothenic acid-deficient rats. Testicular degeneration has also been observed in rats fed a diet deficient in pantothenic acid (Daft and Sebrell, 1939). Since the food intake was reduced in the above animals, it is not clear, however, to what extent results were due to inanition as distinct from a deficiency of pantothenic acid per se. Delayed sexual maturity, anestrus, atrophy of the ovaries and uteri or testes and secondary sex organs have also been observed in animals deficient in other B vitamins including riboflavin (Warkany and Schraffenberger, 1944, Coward et al., 1941, Schuyl and Groen, 1938, and Colonge and Raffy, 1947), pyridoxine (Emerson and Evans, 1940), biotin (Okey et al., 1950; Manning, 1950) and vitamin B I ~(Hartman el al., 1949). Gonadal and reproductive abnormalities have similarly been observed in rats deficient in the essential fatty acids (Burr and Burr, 1929, 1930; Evans et al., 1934a, 1934b, 1934c; Maeder, 1937), manganese (Shils and McCollum, 1942, 1943; Boyer et al., 1942), and calcium (Boelter and Greenberg, 1943). Further work is needed to determine to what extent an impaired secretion of gonadotropin was responsible for these effects and whether such factors as inanition and an impaired efficiency of food utilization contributed to the results. It is well established that pyridoxine deficiency impairs reproduction in the rat. Nelson and Evans (1948) found that the addition of a pyridoxine antagonist (desoxypyridoxine) to a pyridoxine-deficient diet resulted in a high incidence of resorptions when normal adult rats were placed on such a diet 10 to 20 days prior to breeding. Supplementation with pyridoxine on the day of breeding counteracted the adverse effects of the antagonist. This was true even in pair-fed control animals whose food consumption averaged 56 % of that of control rats fed the same diet ad libitum (Nelson and Evans, 1951). In a subsequent report Nelson et al. (1951) observed that daily injections of 1 Mg. estrone plus 4 mg. progesterone were also effective in maintaining pregnancy in pyridoxine-deficient rats fed a diet similar to the above but that such treatment was ineffective in maintaining pregnancy in rats deficient in pantothenic acid or pteroylglutamic acid. These findings clearly indicate an inadequacy in the secretion of ovarian hormones in the pregnant pyridoxine-deficient rat. Nelson et al. (1951) suggest that an impaired formation or secretion of pituitary gonadotropins may have been responsible, a t least in part, for the observed results. Considerable data are available on the morphology and the hormonal potency of the pituitary of animals deficient in vitamins A and E. Such studies were stimulated by the premise that dysfunction of the anterior

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pituitary may have been responsible for the testicular degeneration and impaired reproductive performance of rats deficient in these factors. Although a number of changes have been observed both in the morphology and hormonal potency of the pituitary of rats deficient in these nutrients, it is now generally believed that such changes might more justifiably be considered the result, rather than the cause, of the reproductive disturbances characteristic of a deficiency of vitamins A and E. Sutton and Brief (1938) have described the morphological alterations in the pituitary of rats deficient in vitamin A. A significant increase in the percentage of beta (basophilic) cells, together with an increase in the number of beta cells containing a macula, was observed in the anterior pituitary of both male and female rats deficient in vitamin A. The increase was particularly marked in the males. The changes noted approached those of the castrate animal. Changes have also been observed in the gonadotropic potency of the pituitary of vitamin A-deficient rats. Mason and Wolfe (1930) placed a number of 25-day-old male rats on a vitamin A-deficient diet, castrating half the number. When xerophthalmia was well advanced, their pituitaries and the pituitaries of normal control rats of the same weight and sex were transplanted into immature littermate females. Using the weights of the ovaries of these recipients to indicate relative degrees of gonadotropic potency in the grafted pituitaries, they found that the pituitaries of noncastrated vitamin A-deficient rats were 43% more potent, and those of the castrated vitamin A-deficient rats 100% more potent, than those of normal controls. An increase in the gonadotropic potency of the pituitary has also been reported by Sutton and Brief (1939) for rats of either sex deficient in vitamin A; this was particularly marked in the males. An increase in the gonadotropic potency of the pituitary was also reported for young bulls fed a vitamin A deficient diet for a period of 1 year (Sutton et al., 1940). Since the hypophyses of castrated rats also exhibit an increased gonadotropic potency (Engle 1929; Evans and Simpson 1929), and since such animals also show an increased number of basophiles in their pituitary (Severinghaus, 1939), Sutton and coworkers suggest that both the morphological and physiological changes in the anterior pituitary of A-deficient animals are compensatory changes similar to those which occur following castration and resulting indirectly from damage to the gonads. The validity of this hypothesis, however, may be questioned for several reasons. I n the first place although atrophy of the testes and degenerative changes in the seminiferous tubules as a result of vitamin A-deficiency have been reported in the rat (Mason, 1930,1933), mouse (Wolfe and Salter, 1931), guinea pig (Wolbach and Howe, 1928) and in cattle (Guilbert and Hart, 1935), the ovaries in general show no pathological changes either in the rat

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(Evans, 1928; Mason, 1935), mouse (Wolfe and Salter, 1931) or sow (Hughes et aE., 1928). Furthermore, since the successful completion of ovulation, implantation, gestation and parturition is the rule, rather than the exception, in A-deficient animals (Mason, 1939a), it would appear that little if any impairment of ovarian function occurred. The suggestion that gonadal damage was responsible for the morphological and physiological changes in the A-deficient rat’s pituitary does not, therefore, appear to be valid, a t least for the female rat. Another objection that may be raised is the apparent difference in the amount of gonadotropin secreted by the pituitary of castrate as distinct from vitamin A-deficient rats. Although the gonadotropic potency of the pituitary is increased in both these groups the amount of circulating gonadotropin appears to be increased in the castrate and reduced in the A-deficient animal. An increased secretion of gonadotropin by the castrate animal’s pituitary has been demonstrated both in the rat (Kallas, 1930; Martins et al., 1931; Hill, 1932, 1933) and mouse (Martins, 1929) by means of the parabiosis technique. An increase in the gonadotropin content of blood (Fluhmann, 1929) and urine (Zondek, 1935; Hamburger, 1933; Leonard and Smith, 1934) has also been reported in castrated men and women. The secretion of gonadotropin, however, is apparently reduced in the vitamin A-deficient animal, particularly in advanced stages of depletion. This is indicated by the presence of atrophic changes in the prostate and seminal vesicles of the A-deficient rat (Wolbach and Howe, 1925; Manville, 1925, Green and Mellanby, 1928; Arons et al., 1932, Moore and Mark, 1936), mouse (Wolfe and Salter, 1931), and guinea pig (Wolbach and Howe, 1928) ; and the restoration of normal weight and structure in these organs by the administration of physiological levels of gonadotropin (Van Os, 1936; Mason, 1939a; Mayer and Goddard, 1951). Further data would be desirable on the actual content of circulating gonadotropin in the blood of castrate and vitamin A-deficient rats. Atrophic changes in the prostate and seminal vesicles (Moore and Samuels, 1931) and an increased gonadotropic potency of the pituitary (Rinaldini, 1949), however, have also been observed on a complete ration under conditions of reduced caloric intake. Since food intake is reduced in the vitamin A-deficient animal, particularly in advanced stages of depletion, it is questionable, therefore, to what extent changes in the pituitary, prostate and seminal vesicles of A-deficient animals were caused by vitamin A-deficiency per se or to what extent they may reff ect concomitant caloric restriction. The observation that restricting the caloric intake of vitamin A-deficient rats retarded the curative effects of vitamin A in restoring the accessory sex glands of the A-deficient rats is pertinent in this regard (Mason, 1939a). Changes in the morphology and hormonal potency of the pituitary of

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vitamin E-deficient rats have been reported by a number of investigators. The anterior pituitary of male rats maintained for prolonged periods on an E-deficient ration exhibits an increase in the number and size of basophiles with cytological changes similar to that produced by cryptorchidism and castration (Van Wagenen, 1925; Nelson, 1932, 1933; Geller, 1934; Gierhake, 1933,1935; Koneff, 1939; Biddulph and Meyer, 1941). Anincrease in the number of chromophobes and a reduction in the percentage of acidophiles have also been noted (Koneff, 1939). In the female rat, results are conflicting. Joel (1943) reported that the pituitary of female rats deficient in vitamin E exhibited changes characteristic of the castrate animal; other investigators, however, found no morphological changes in the pituitary of female rats (either pregnant or nonpregnant) deficient in this vitamin (Nelson, 1933; Stein, 1935; Muller and Muller, 1937). Barrie (1937) and Underhill (1939) on the other hand reported degranulation and degeneration of the acidophiles. A sex difference in pituitary weight has been noted in rats deficient in vitamin E, male rats showing a significant increase (Biddulph and Meyer, 1941) and females showing no change (Nelson, 1933; Stein, 1935; Biddulph and Meyer, 1941). In view of the fact that wheat germ oil and a-tocopherol were only partially effective in counteracting the microscopic changes in the pituitary of E-deficient male rats (Koneff, 1939), and since wheat germ oil failed to prevent the increase in pituitary weight of such rats (Biddulph and Meyer, 1941), it is questionable to what extent the morphologic alterations in the pituitary of vitamin E-deficient rats were caused by E-deficiency per se and to what extent they resulted from lack of some other dietary factor. Studies concerning the effects of vitamin E-deficiency on the hormonal potency of the rat pituitary are conflicting. An increased gonadotropic potency has been reported in the pituitary of both male (Nelson, 1933; Mason and Wolfe, 1930; Drummond et al., 1939; P’An et al., 1949) and female (McQueen-Williams, 1934; P’An et al., 1949) Edeficient rats. A sex difference was noted by Nelson (1933) and Drummond et al. (1939), who observed increased potency in the male and either normal or reduced potency in the female. Biddulph and Meyer (1941), however, found no significant increase over the normal in the gonadotropic potency of either male or female E-deficient rats. Rowlands and Singer (1936), who used the estrous rabbit instead of the immature rat (employed by the other investigators indicated above) as a test animal reported that gonadotropic potency was decreased in the pituitary of E-deficient female rats. The latter results were interpreted as indicating a reduced content of luteiniaing hormone in these glands. Drummond et al. (1939) also reported a decreased amount of luteinizing hormone in the pituitary of E-deficient female rats. In the male, however, the luteinizing potency

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of the pituitary was increased (Biddulph and Meyer, 1941). The cause for these discrepancies is not readily apparent. Differences in the composition of the diets employed and differences in the length of time the various rations were fed may have been responsible, at least in part, for the diverse results; differences in the sex and strain of the assay animal or differences in the technique of gonadotropic assay may also have contributed. Inanition does not appear to have been a factor in the results cited above as judged by the occurrence of apparently normal estrus, ovulation and implantation, and the maintenance of normal structure and function in the prostrate and seminal vesicles of E-deficient rats (Mason, 193913). In spite of the fact that changes have been observed both in the structure and hormonal potency of the anterior pituitary of E-deficient rats, it is the current consensus that dysfunction of the pituitary is not the cause of the testis injury or resorption-gestation of E-deficient rats. The degeneration of the seminiferous tubules with the maintenance of apparently normal interstitial tissue in E-deficient rats is quite unlike the testicular degeneration resulting from absence or dysfunction of the pituitary (Mason, 1933). Furthermore, gonadotropic hormones were ineffective in repairing the testis injury of E-deficient rats (Mason, 1933; Geller, 1934; Drummond et al., 1939). Also Drummond el al. (1939) observed that administration of gonadotropic hormones to male rats that were hypophysectomized after 12 months on an E-deficient diet maintained the weight of the prostates and seminal vesicles but failed to repair the injury to the spermatogenic tubules. It appears, therefore, that the interstitial cells of the E-deficient testis are able to respond to gonadotropic stimulation by the liberation of androgens, whereas the spermatogenic tissues atrophy and become functionless. Endocrine therapy has also proved ineffective in preventing the reproductive pathology of vitamin E-deficient female rats. Neither implantation of fresh pituitary tissue (Evans, 1932; Drummond et al., 1939), injections of anterior pituitary or pregnancy urine extracts (Diakov and Krizenecky, 1933, 1935; Geller, 1934; Drummond el al., 1939), nor administration of chorionic gonadotropin (Drummond et al., 1939) enabled pregnant vitamin E-deficient rats to produce living young. Treatments with follicular hormone (Csik, 1932) corpus luteum extracts (Nelson, 1931), progesterone (Drummond et al., 1939; Ershoff, 1943), testosterone propionate (Drummond et d.,1939), estrone (Ershoff, 1943) and lactogenic (luteotropin) hormone (Ershoff, 1943) were similarly ineffective. Claims that E-deficiency sterility can be cured in the female rat by injection of follicular hormone (Bisceglie, 1939), pituitary and pregnancy urine extracts (Agnoli, 1930; Marchesi, 1935a), placental extracts (Marchesi, 1935b) or by grafting of normal ovaries (Marchesi, 1935c) have not been

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confirmed. It has been demonstrated that administration of vitamin E as late as the 10th or 11th day of pregnancy will enable vitamin E-deficient rats to produce living young (Mason, 1944). This finding suggests that the processes of ovulation, fertilization, and implantation are normal in the E-deficient female rat and that the irreversible changes responsible for the death of the embryo occur after placentation is well established, i.e., a t a time when maintenance of luteal function has been taken over a t least in the rat by the fetal placenta (Astwood and Greep, 1938). As pointed out by Mason (1939b), these facts constitute a serious objection to the hypothesis that anterior pituitary dysfunction is the primary cause of the resorption-gestation of E-deficient rats. Hypoplasia of the thyroid gland has been reported by a number of investigators in the E-deficient female rat (Singer, 1936; Barrie, 1937; Underhill, 1939). The suggestion has been made that these effects were due to a decreased secretion of thyrotropin (Barrie, 1937; Underhill, 1939). Other workers, however, failed to confirm these findings and report that the thyroid glands of E-deficient female rats were normal in weight, function, and appearance (Telford et al., 1938; Biddulph and Meyer, 1941, 1942; and others). I n contrast, a marked increase in thyroid weight with histological evidence of increased activity was noted by Biddulph and Meyer (1941, 1942) in male rats depleted of vitamin E. Correlated with these thyroid changes there was a statistically significant increase in the basal metabolic rate of male rats maintained on an E-deficient diet for 6 months or more. Analysis of the food and water intake of E-deficient and normal rats showed, however, that the E-deficient rats received only about one-fourth as much iodine as the normal rats. The addition of sufficient iodine to the E-deficient diet to provide the same amount as was present in the normal diet maintained normal thJ roid weight and histology and the metabolic rate at a normal level. 14 heat germ oil was also effective although less so than K I in counteracting the thyroid changes in weight, function and appearance indicated above. Biddulph and Meyer (1942) suggested that the stress of E-deficiency increased iodine requirements in the male rat with the result that a relative iodine deficiency developed on the E-free low iodine diet. As a result of the relative iodine deficiency, the pituitary gland secreted an increased amount of thyrotropin, which in turn caused hypertrophy and hyperplasia of the thyroid gland. When the diet of the rats was supplemented with iodine, the increased secretion of thyrotropin was prevented and the thyroids remained normal although typical E-deficiency symptoms persisted. Supplementing the diet with wheat germ oil prevented the development of E-deficiency and the attendant stress on the thyroid, Under these conditions, according to Biddulph and Meyer

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(1942), a relative iodine deficiency did not appear, and consequently the thyroids remained essentially normal. It is possible, however, that the protective effect of wheat germ oil was due not to its content of vitamin E (as suggested by Biddulph and Meyer) but rather to traces of iodine which it may have contained. In regard to this point further data would be desirable on the effects of a-tocopherol on rats maintained under conditions similar to those employed above. It is not clear why female rats did not exhibit thyroid changes comparable to those in the male when fed an identical E-free low iodine diet. Changes in the thyrotropic potency of the pituitary have also been observed in rats deficient in other vitamins. Hypoplasia of the thyroid gland associated with reduced pituitary weight (particularly in females) and a decreased thyrotropic potency of the pituitary has been reported in thiamine deficient rats (Hundhausen and Schulze, 1939). The reduction in thyrotropic potency was apparently not due to inanition, for rats kept on a complete diet restricted in amount to that eaten by the deficient animals had the same thyrotropic potency in their pituitaries as normal controls and their thyroids showed no visible changes from normal (Hundhausen, 1939). A reduction in the thyrotropic potency of the pituitary was also observed in rats deficient in vitamin D (Schulze and Flach, 1939). In contrast to the above the thyrotropic potency of the pituitary was considerably increased in rats deficient in vitamin A (Schulze and Hundhausen, 1939a) and unchanged from the normal in rats deficient in riboflavin (Schulze and Hundhausen, 1939). 3. Egects of Protein and Amino Acid Deficiencies

Considerable data are available indicating that diets deficient in protein impair gonadotropin secretion, a t least in the rat. In this animal restriction of dietary protein, quantitatively or qualitatively, leads to a cessation of estrous cycles (Evans and Bishop, 1922a, 192213; Courrier and Raynaud, 1932; Guilbert and GOSS,1932; Pearson, 1937; hrvy et al., 1946) and atrophy of the seminal vesicles and prostate (Samuels, 1950). These effects were apparently due to impaired secretion of gonadotropin since pituitary implants or gonadotropic extracts stimulated the ovaries (Courrier and Raynaud, 1932) and counteracted the reduction in seminal vesicle and prostate weight (Samuels, 1950) of rats fed protein-restricted diets. Decreased food intake was apparently not the cause of the lowered gonadotropin secretion since gonadal function was impaired in rats fed low protein diets even when total caloric intake was not reduced (Guilbert and Goss, 1932; Samuels, 1950). The minimal protein dietary level capable of maintaining estrus in the protein-restricted rat was approximately 7 % (Guilbert and GOSS, 1932). In the rabbit, however, it

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appears that the reproductive system is much more resistant to protein deprivation. Even after prolonged periods of protein restriction (associated with a reduction in food intake exceeding 50%), the incidence of anestrous in protein-depleted rabbits was less than 25 % (Friedman and Friedman, 1940) in contrast to values approaching 100% in rats maintained under similar conditions. Pituitary assays, furthermore, revealed that rabbits on a completely protein-free diet were able t o synthesize apparently normal amounts of gonadotropin when assayed 8 days after mating with a vasectomized male (Friedman and Friedman, 1940). It has been demonstrated that the gonadotropic hormone of the rabbit pituitary is almost completely discharged by the act of coitus (Hill, 1931; Friedman and Friedman, 1939) and that for the first 4 to 6 days of pseudopregnancy the hormone content of the gland remains a t the depleted level; i.e., the amount of hormone farmed is not much in excess of that released. Between the 6th and 10th days of pseudopregnancy, the hormone content rises sharply toward the initial estrous level, and this increase in hormone content must represent the excess of the amount formed over the amount released from the gland. Since the process of gonadotropic hormone restitution after coitus was apparently unimpaired in the protein-restricted rabbit, it would appear that the protein requirements for the formation of gonadotropic hormone in this animal can be fully met by the nitrogenous products derived from the rabbit’s own tissues, even during the stress of serious nitrogen deficit. Similarly, the pituitaries of male rats fed a diet containing 30% protein did not differ significantly in gonadotropic potency from that of animals similarly treated but fed a 15% protein ration (Weatherby and Reece, 1941). Impaired gonadal function has also been observed on rations deficient in specific amino acids. Testicular damage has been reported in rats fed diets deficient in phenylalanine (Maun et al., 1945a), leucine (Maun et al., 1945b), histidine (Maun et al., 1946), and tryptophan (Albanese and Buschke, 1942; Adamstone and Spector, 1950). The effects observed appear to be due, a t least in part, to factors other than inanition since (1) the testicular atrophy of phenylalanine, leucine or histidine-deficient rats was more severe than that of paired-fed controls (Maun el al., 1945a, 1945b, 1946) and (2) tryptophan deficiency led to degeneration of the seminiferous tubules and primary spermatocytes in animals that were force fed to eliminate the possibility of inanition playing a part in the lesions found (Adamstone and Spector, 1950). It is possible, however, that efficiency of food utilization was impaired in the deficient animals, and hence that the total caloric utilization of these rats was, therefore, less than that of controls fed a similar caloric intake of a complete ration. Gonadal atrophy was also observed by Samuels (1950) in rats force fed a

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tryptophan-deficient diet in amounts sufficient to provide the total amino acid and caloric intake of controls maintained on a complete ration. Albanese et al. (1943) found that diets deficient in tryptophan caused resorption-gestations in the female rat. Inanition was quite marked in the latter experiment, however; and it is questionable to what extent tryptophan deficiency per se was responsible for the observed results. I n view of the finding that arginine is present in considerable concentration in the nucleoproteins of the sperm head, considerable interest has been shown in the possible role of this amino acid in testes physiology. Men on an arginine-deficient diet rapidly developed hypospermia and azospermina, which abnormalities were promptly corrected by the administration of arginine (Shettles, 19-12) or after a period of several weeks by the resumption of an adequate diet (Holt et al., 1912). Arginine-deficient diets have also been reported to cause testis injury in the rat (Shettles, 1942; Holt, 1944; Holt and Albanese, 1944), but Williams and Watson (1944, 1945) found that arginine-free diets did not cause testis injury in this species and that exogenous sources of this amino acid were dispensable for reproduction in either sex. The above findings indicate that testis injury and impaired reproduction may occur on diets deficient in specific amino acids. They provide no information, however, as to the mechanisms involved. Since the hormones of the anterior pituitary are believed to be protein or a t least polypeptide in nature, it has been suggested that a diet deficient in one or more of the essential amino acids might not provide the building material necessary for normal hormone production. Insufficient data are available, however, to show whether or not the formation or secretion of gonadotropin (and other anterior pituitary hormones) is impaired in animals deficient in specific amino acids. It is possible that the testis injury (and other pathological effects) observed on diets deficient in essential amino acids may be due to (1) direct tissue injury or (2) decreased sensitivity of target organs to pituitary stimulation. Further work is needed to determine whether the various amino acids are equally essential for gonadal function and reproduction. It has been demonstrated that protein synthesis occurs only if all the “essential” amino acids are present and only when they are simultaneously available (Geiger, 1947, 1950). Hence, a deficiency of any one of the essential amino acids may cause an animal to go into negative nitrogen balance despite the fact that its total amino acid and caloric intake may be increased (for example, by force feeding). On theoretical grounds one might expect that a deficiency of any one of the essential amino acids, if sufficiently prolonged, would result in many if not all of the effects of a quantitative protein deficiency. It remains for future investigations to determine whether the

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testicular injury (and other pathological effects) observed on diets lacking in a single essential amino acid were specific lesions characteristic of the deficient amino acid or general manifestations of an impaired protein metab olism. 4. Efects of Mineral Deficiencies

It is well established that iodine deficiency results in morphological and functional changes in the anterior pituitary gland. This deficiency may be either absolute or relative and may be caused by a number of factors other than the ingestion of an iodine-poor diet. An impaired absorption or utilization of iodine or an increased excretion or requirement for this nutrient may result in a relative iodine deficiency even if this element is ingested in normally sufficient amounts. Insufficiency of thyroid hormone resulting from impaired formation or increased requirements may also lead to pituitary changes similar to those indicated above. It would appear, therefore, that the pituitary alterations observed on iodine-deficient rations were caused in the final analysis not by iodine-deficiency per se but rather by the accompanying reduction in circulating thyroid hormone. A relative iodine deficiency may be precipitated by any of the following “conditioning factors” : (1) High intake of calcium salts. The literature on this point is contradictory but it has been demonstrated that goiter often occurs in limestone regions (Orr, 1931; Stott, 1932). Thompson (1936) found that the thyroid size varied directly with the calcium concentration of the diet. Hibbard (1933) and Mahorner (1937) obtained a goitrogenic effect from calcium chloride; but Hibbard concluded from studies with sodium chloride that chloride and not calcium was the cause of the enlarged thyroids. Other investigators, however, reported that the calcium content of the diet had no effect on the iodine requirement and that neither calcium chloride nor other calcium salts had any goitrogenic effects whatsoever (Levine et al., 1932; Sharpless et al., 1943). These discrepancies may be due to differences in the diets employed. Sharpless el al. (1943), for example, found that although calcium chloride itself had no goitrogenic effect, when fed together with vitamin D it acted as a goitrogenic agent. However, when the carbonate rather than the chloride salt of calcium was employed together with vitamin D, no goitrogenic effects were observed. (2) Ingestion of goitrogenic foods. The existence of goitrogens in soybeans was shown by McCarrison (1933) who observed thyroid enlargement in young rats fed a soybean diet. These results were confirmed by Sharpless (1938) who found glands as large as five times normal in rats given a diet containing unprocessed soybean flour. Other foods have also

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been shown to exert goitrogenic effects, including cabbage leaves, other members of the Brassica group such as Brussels sprouts and cauliflower, seeds of swede, soft and hard turnip, chou moellier, rape and others (for references and review of literature in this field refer to Charipper and Gordon (1947) and Greer et al. (1949)). Considerable differences exist among the above goitrogens, however, insofar as their effect on iodine metabolism is concerned. The goitrogenic effects of soybeans and Brassica leaves may be completely counteracted by increasing the iodine content of the diet (Webster and Chesney, 1928; Webster, 1929; Sharpless et al., 1939; Halverson et al., 1949). These substances, therefore, induce a relative iodine deficiency by increasing requirements for this element. Iodine, however, was without significant effect on the hyperplastic thyroids of animals fed goitrogens derived from various Brassica seeds, including rape, although some inhibition of the gross thyroid enlargement was obtained (Kennedy and Purves, 1941). The thyroid hyperplasia caused by rape seed could be inhibited, however, by very small doses of thyroxine (Purvis, 1943). Brassica seed goitrogens therefore do not actually induce a relative iodine deficiency since their effect cannot be counteracted by the administration of increased amounts of this element. Their effect, however, is similar in so far as impairment of thyroid hormone synthesis is concerned. Crotti (1938) has observed that a fungus present in goitrogenic cabbage, water supplies in endemic goiter areas and in fresh goiter material also produced goiters in rabbits, guinea pigs and dogs which were not prevented by administration of iodine. As in the case of Brassica seed goitrogens, therefore, the fungus did not actually cause a relative iodine deficiency but its effects were similar. (3) A deficient mechanism of acceptance. The acceptance or concentration of iodine by the thyroid gland may be impaired as a result of “genetotrophic factors” (Williams, 1950; Williams et al., 1950), Addison’s disease (Hertz, 1950), pathologic processes in the thyroid and other factors. (4) Increased iodine excretion. An increased excretion of iodine may occur under conditions of impaired renal function as in patients with lipoid nephrosis (Hertz, 1950) or following the administration of certain drugs such as sulfanilamide (Hertz, 1950). (5) Factors that increase thyroid hormone requirements. Requirements for thyroid hormone and indirectly, therefore, for iodine which is a constituent of this hormone are increased under conditions of puberty, pregnancy and the menopause, by certain infections and intoxications, exposure t o cold, inadequate oxygen supply t o the tissues (e.g., anemias, life a t high altitudes) and various other factors.

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So far as is known, iodine deficiency, whether absolute or relative, results in similar changes in the anterior pituitary; and since these changes in the final analysis were caused by a reduction in the circulating thyroid hormone level and not of iodine per se, similar changes occur when thyroid hormone synthesis is impaired by other factors such as Brassica seed goitrogens, antithyroid drugs, “primary atrophy” or neoplastic invasion of the thyroid, and surgical or X-ray destruction of the gland. Functionally the anterior pituitary responds to a reduction in the level of circulating thyroid hormone by an increased elaboration of thyrotropic hormone. Histologically, there is a decrease in the number of acidophiles and a degranulation of these elements accompanied by a hypertrophy and increase in the number of basophiles. The extent of these changes will vary depending on the degree and extent of thyroid hormone depletion. In all cases, however, they appear to be completely counteracted by the administration of adequate amounts of thyroid substance. Sharpless and Hopson (1940) and Griesbach (1941) were among the first to report pituitary changes in rats fed a soybean or Brassica seed-containing diet. The effects of antithyroid drugs on pituitary structure have been reviewed by Charipper and Gordon (1947). The foregoing discussion has dwelt a t some length on the role of “conditioning factors” in the etiology of iodine deficiency. I n view of the close interrelationship between this element and the thyroid gland, a relative deficiency of this nutrient may readily be observed in terms of impaired thyroid function. “ Conditioning factors” may also precipitate other deficiencies as well, despite the ingestion of normally adequate amounts of the various nutrients (Jolliffe, 1942, 1943; Starr, 1942; Hickman and Harris, 1946; Ershoff, 1948a, 1951a); the specific effects of these deficiencies on endocrine function, however, are not so readily apparent. Virtually no data are available on the effects of other mineral deficiencies on anterior pituitary structure and function. Testicular atrophy and loss of sex interest has been observed in male rats fed manganese-low diets (Orent and McCollum, 1931;Boyer et al., 1942;Shils and McCollum, 1943). The reports on manganese deficiency in the female, however, are conflicting. One group of investigators found that neither the estrous cycles, sex interest, nestling behavior nor lactation capacity were disturbed in either rats or mice on diets deficient in this element (Orent and McCollum, 1932; Daniels and Everson, 1935; Shils and McCollum, 1943), although when such animals were mated to normal males many of the young were born dead or died within a few hours after birth (Daniels and Everson, 1935; Shils and McCollum, 1943). Other workers, however, reported irregularity or complete absence of estrous cycles in both rats

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(Waddell et al., 1931; Skinner et al., 1932; Boyer et aE., 1943) and mice (Kemmerer et al., 1931) on manganese-low diets, a marked delay in the opening of the vaginal orifice, loss of sex interest, and failure to conceive and bear young (Boyer et al., 1942). Differences in the composition of the basal diets and the degree of manganese deficiency produced may have been responsible, at least in part, for these diverse results. Absence of estrous cycles and failure to mate were also noted in female rats raised to maturity on a calcium deficient diet; whereas decreased fertility was observed in adult rats transferred to a similar ration after bearing a previous litter (Boelter and Greenberg, 1943). Data such as those indicated above, however, are difficult to evaluate. Paired-fed controls were not employed; and since food intake was reduced in the deficient animals, it is difficult to evaluate to what extent inanition (and possibly other dietary deficiencies) contributed to the above results. The changes observed suggest the likelihood of an impaired gonadotropin secretion. It is not readily apparent, however, to what extent alterations in anterior pituitary function were responsible for the observed effects. In addition to manganese, certain of the elements (cobalt, copper, iron, and zinc) are indispensable constituents of enzyme systems. One might expect that a deficiency of these minerals would affect the pituitary in addition to other tissues but there is no evidence for this.

111. EFFECTS O F MISCELLANEOUS NUTRIENT8 AND

ON PITUITARY STRUCTURE

FUNCTION

A number of workers have reported changes in the structure of the anterior pituitary following the administration of various nutritional factors. An increase in the number of acidophiles and to a lesser extent in the number of basophiles was observed in rabbits receiving repeated subcutaneous injections of a solution containing vitamin A (Giedosz, 1935). Intravenous injections of a solution of vitamin C were followed by similar changes. An increase in the number of acidophiles and a reduction in the number of basophiles were observed by Julesz (1945) in rabbits administered a vitamin B1-containing preparation. A considerable increase was also noted in the amount of acidophilic colloid substance. Similar findings have also been reported in the rat (Julesz and WinklerJulesz, 1946). The administration of niacin by injection or orally to virgin guinea pigs produced a 20 % decrease in pituitary weight (Hawker, 1916). A decrease in the number of acidophiles and an increase in the number of basophiles were observed after the administration of large doses of vitamin D (Itoh, 1938). Similar findings have been reported in female rats kept for 4 months on a high protein (90% casein) diet (Tuchmann-Duplessis and Aschkenasy-Lelu, 1948). The significance of

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such data is not readily apparent. It would appear that many of the changes observed are not specific and many reflect temporary alterations of pituitary function in response to stress (particularly in those animals that received parenteral injections of the crude vitamin concentrate). There is considerable evidence that massive doses of vitamin A inhibit thyrotropic hormone secretion (Elmer et al., 1935; Fellinger and Hochstadt, 1936; Schulze and Hundhausen, 1939; Balassa and Szanto, 1939; Truscott and Sadhu, 1948; Sadhu and Truscott, 1948). Large doses of vitamin A reduce thyroid weight (Sadhu and Brody, 1947), produce thyroid hypoplasia and increase the amount of thyrotropic hormone in the anterior pituitary (Schulze and Hundhausen, 1939; Balassa and Szanto, 1939). Associated with these changes a decrease occurs in the proteinbound iodine of liver and thyroid and an increase in the protein-bound iodine of blood (Sadhu and Truscott, 1948). These findings indicate that massive doses of vitamin A impair the hepatic destruction of thyroxine, (at least in the rat) resulting in consequent hyperthyroxinemia; the latter depresses thyrotropic hormone secretion, producing the changes in thyroid weight, morphology and protein-bound iodine content indicated above (Sadhu and Truscott, 1948). Large doses of vitamin C have been reported to prevent or reduce the histological activation and increase in thyroid weight evoked by thyrotropic hormone (Marine et al., 1934; Elmer el al., 1935; Loeser, 1936; Terbruggen, 1937; Sturm et al., 1938). Other investigators, however, failed to confirm these findings (Spence and Scowen, 1935; Schafer, 1936; Schober, 1937; Monetti, 1941; Bore11 and Holmgren, 1946). Iodide in the form of the potassium or sodium salt has also been reported to lower thyroid size, depress thyrotropic hormone secretion and inactivate or neutralize the effects of thyrotropin administration (Sadhu, 1948; Crepax, 1948a, 194813). Regressive changes in the thyroid (apparently due to decreased thyrotropin secretion) have also been observed in rats (Itoh, 1938) and dogs (Goormaghtigh and Handovsky, 1935) fed massive doses of vitamin D. Vitamin ( ( P ”as catechin may also exert an antithyrotropic effect. The hyperplasia of the thyroid gland induced in guinea pigs and rats by thiourea was significantly reduced in these animals by the concurrent administration of catechin (Gabe and Parrot, 1950). Inasmuch as thyroid hyperplasia is believed to occur only in response to thyrotropin stimulation, it would appear that catechin was either inhibiting the release of thyrotropic hormone by the pituitary, reducing the sensitivity of the thyroid to histological activation, or facilitating the neutralization, inactivation or excretion of the thyrotropic hormone. Vitamin (‘P,’’ however, appeared to exert this antithyrotropic action only in the presence of ascorbic acid since when given to scorbutic guinea pigs it had no such effect (Gabe and Parrot, 1950).

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Atrophy and reduction in the fat content of the adrenal cortex (apparently due to impaired ACTH secretion) has been reported for rats with hypervitaminosis D (Itoh, 1938). An increase in adrenal weight was noted for rats fed large doses of vitamin A (Sadhu, 1948). Potassium chloride (in large doses) was observed to counteract the increase in urinary nitrogen excretion that normally occurs after ACTH administration in the rat (Whitney and Bennett, 1950). A puzzling report (and one that needs confirmation) is the diverse effects of carotene and vitamin A (when administered in massive doses) on the estrous cycle of the rat. Sherwood et al. (1936) observed that large doses of carotene (orally administered) suppressed estrous cycles in the rat and that cycles were not resumed during the period of carotene administration (15 days) nor for as long as 20 days aftei. cessation of treatment. Burrill and Greene (1941), however, found that massive doses of vitamin A (as distinct from carotene) did not interfere with the estrous cycle of the rat. It has been demonstrated that large but subtoxic doses of vitamin D increase the metabolic rate of normal dogs (Reed et al., 1933; Goormaghtigh and Handovsky, 1935) and rats (Reed, 1934), and are also capable of increasing the oxygen consumption of isolated peripheral tissues in a manner comparable to that observed after thyroxine administration (Gelfan, 1935; Presnall, 1937). No increment in metabolic rate was observed, however, after the administration of vitamin D to thyroidectomized (Deutsch et al., 1936) or hypophysectomized (Bartoli et al., 1939) degs in doses sufficiently large to produce a marked response in normal dogs. It would appear, therefore, that the calorigenic effects of vitamin D were mediated through the pituitary (presumably by an increased secretion of thyrotropin) and resulted from the increased elaboration of thyroid hormone. No change from normal was observed in the thyrotropic potency of the pituitary of male rats fed massive doses of vitamin D (Schulze, 1940).

GENERALADAPTATION SYNDROME The requirements for a number of nutrients are markedly increased under conditions of stress (Ershoff, 1948a, 1951a). Since activation of the pituitary-adrenal system also occurs under these conditions (Selye, 1946, 1950), the question arises as to what relationship if any exists between the two phenomena and what effects the “nutriture” of an animal might have on the course of the general adaptation syndrome. Diets which may be adequate under ordinary circumstances may contain insufficient amounts of certain nutrients to meet the increased requirement for these factors under conditions of stress. A relative deficiency of these nutrients may therefore occur which seriously interferes with the IV. NUTRITION AND

THE

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functioning of the pituitary-adrenal system. The following nutrients appear to be of particular importance in this regard. 1 . Proteins and Amino Acids

Data concerning the effects of protein restriction on ACTH synthesis and secretion during stress are conflicting. Sayers and coworkers (1949, 1950) have reported that neither the formation nor liberation of adrenocorticotropin is significantly altered from normal in the proteindeficient rat. Determinations of the ACTH content of the pituitary 24, 48, and 72 hours after subjecting rats to a standard scald revealed no significant differences between the pituitaries of rats on a protein deficient and control ration. In both groups the content of ACTH in the pituitary was reduced by approximately 50% after scalding; and was restored to normal a t 48-72 hours, despite continued ACTH discharge. Furthermore, no significant difference was observed between the two groups in the rate of ACTH secretion as judged by increase in adrenal weight and reduction in adrenal cholesterol and ascorbic acid after scalding. Similar findings were obtained in rats under conditions of acute fast (complete withdrawal of food but given water), chronic inanition and tryptophan deficiency (Sayers et al., 1950). It was concluded by these workers that in rats malnutrition affects the functional capacity of the pituitary-adrenal system only slightly, in contrast to its marked effect on the pituitary-gonad system. These results, however, are a t variance with those reported by other investigators. Moya et ad. (1948) observed that the reduction in adrenal ascorbic acid concentration (which Sayers and Sayers (1947) had demonstrated to be a useful indicator of endogenous corticotropin activity) was significantly greater 1 hour after exposure to cold in rats fed a high (30%) protein diet than in those receiving a low (15%) protein diet. Furthermore, the degree of compensatory adrenal hypertrophy after unilateral adrenalectomy was significantly greater on the high protein ration than on the low (Moya et al., 1948). Injections of ACTH, however, into hypophysectomized rats which had been pretreated for 3 weeks with the above diets revealed no significant difference in the adrenal ascorbic acid concentration of the two groups. These findings indicate that differences in the adrenal response of rats on high or low protein diets under conditions of stress (exposure to cold or unilateral adrenalectomy) were due not, to differences in the adrenal response to endogenous corticotropin but rather to (1) increased ACTH secretion or (2) the elaboration of increased amounts of some corticotropin-synergizing hypophyseal principle by rats on the high protein diet. It is not possible to conclude with certainty whether the formation or merely the discharge of preformed pituitary hormone was

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altered by the diet. In the cold stress experiment animals were autopsied 1 hour after exposing them to a low environmental temperature. The differences observed between the two dietary groups might have been due, therefore, to differences in the rate of hormone liberation. It would be difficult, however, to explain the more rapid regeneration of adrenal tissue on the high protein diet (which occurred over a period of 10 to 15 days) in terms of increased discharge only; hence increased synthesis probably also occurred. * Similar results have been obtained in animals subjected to other forms of stress. For example, the administration of lyophilized anterior pituitary to intact rats results in marked adrenal enlargement and hypertension in animals fed a high protein ration while slight stimulation of the adrenals and a normal blood pressure were recorded in similarly treated rats on a low protein diet (Dontigny et al., 1948; Hay et al., 1948). Further experiments with protein hydrolyzates (DeGrandpre et al., 1948) and amino acids (Henriques et al., 1948) showed that the latter were responsible for the increased corticotropic action of the hypophyseal material (hypertension was believed to result from excessive cortical hormone production following LAP administration). The ACTH activity of purified anterior pituitary preparations, however, was independent of the dietary protein level (Ingle et al., 1947; Moya et al., 1948). Thus some factor in lyophilized anterior pituitary other than ACTH was apparently responsible for the increased corticotropic activity of this material in intact rats fed a high protein diet. Recent findings by Henriques et al. (1949) are pertinent in this regard. These workers found that * It is well established that the various indices of pituitary-adrenal function may yield confiicting results to any given stressor agent. There is evidence that a number of corticoids are present in the adrenal gland and these may vary both in amount and kind in the cortical secretions produced in response to different stressor agents. Evidence of adrenal activation obtained with one index, therefore, does not rule out the possibility of an impaired production of corticoids which may be involved in other indices. There is also evidence that more than one adrenocorticotropic hormone may be produced. Thus as far back as 1942 Golla and Reiss reported the presence of two corticotropic factors in the pituitary-one affecting adrenal weight, the other the distribution of lipoid in the adrenal cortex. More recently it was noted that certain pituitary extracts are especially active in reducing the ascorbic acid content of the adrenals of hypophysectomized rats, while others mainly cause an increase in adrenal weight under similar conditions (Young, 1951). Talbot el al. (1951) have recently postulated the presence of two ACTH’s in man, one concerned with ll-17-oxycorticosteroid, the other with 17-ketosteroid formation. It is possible, therefore, that nutritional deficiencies may condition not only the response of the adrenal cortex and the target organs of its secretions but also the production and secretion of corticotropic hormones (or other substances-possibly somatotropin-released simultaneously from the pituitary with ACTH as a result of a given stressing situation that may modify the action of ACTH).

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in the hypophysectomized rat, as distinct from the intact animal, the corticotropic action of lyophilized anterior pituitary was not influenced by the dietary protein level. I n other words the ability of high-protein diets to increase the corticotropic effect of lyophilized anterior pituitary was dependent on the integrity of the hypophysis. Itj would appear, therefore, that the increased corticotropic activity of lyophilized anterior pituitary in rats fed a high protein diet was due to the presence of “toxic” components in this material which acted as a non-specific stress. The pituitary responded to this form of stress just as it did to exposure to cold or unilateral castration, i.e., by increasing either (1) ACTH secretion or (2) the elaboration of some corticotropin-synergizing hypophyseal principle. Further data indicating the adrenal response to stress is conditioned by the protein content of the diet has recently been reported for rats exposed to muscular exercise, formalin and urethan (Constantinides, 1950). Handler and Bernheim (1950a) have presented additional data indicating that ACTH synthesis during stress is depressed in the protein-deficient rat. These workers demonstrated that diets low in protein were incapable of maintaining the elevated blood pressure induced by subtotal nephrectomy in the rat. Hypertension was promptly restored by the administration of adrenocorticotropin, although a similar dose of this substance was virtually without effect on the blood pressure of unoperated controls or partially nephrectomized, hypertensive rats ingesting a high protein ration. These findings indicate that the elaboration of ACTH is markedly reduced on a low-protein diet and that the reduction in blood pressure which is effected by feeding such a ration to partially nephrectomized rats is, in large measure, due to failure of ACTH synthesis. Further evidence was obtained by determining the percentage fall in the circulating eosinophil count 4 hours after epinephrine and ACTH administration (Handler and Bernheim, 1950a). It was observed that the decrease in eosinophil count elicited by epinephrine in rats fed a low protein diet was significantly less than that of animals similarly treated but fed a high-protein ration. After ACTH administration, however, the reduction in eosinophil count was equally marked in both dietary groups. Similar results were obtained both with unoperated and partially nephrectomized rats. Hence the ability of the adrenal cortex or the “target” cells of its secretions to respond t o tropic stimulation was not impaired in rats fed a low protein diet, but the elaboration of ACTH following the “stress ” of epinephrine administration was significantly reduced. Inasmuch as diets deficient in choline were equally effective in retarding ACTH secretion in the subtotally nephrectomized rat (Handler and Bernheim, 1950b), it may be that the effects of a low protein

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diet were due to a deficiency of labile methyl groups and not of protein per se. 1. V i t a m i n s a. Ascorbic Acid. There is much evidence that ascorbic acid is intimately related to adrenal cortical function. This vitamin is present in singularly high concentration in the adrenal cortex (Szent-Gyorgyi, 1928 ; Harris and Ray, 1933; Bourne, 1933; Giroud and Leblond, 1934), particularly in the zona fasciculata and reticularis (Glick and Biskind, 1936; Greep and Deane, 1949). Diets deficient in ascorbic acid result in adrenal hypertrophy in the guinea pig (LaMer and Campbell, 1920; Lockwood and Hartman, 1933; Quick, 1933; Nadel et aZ., 1950; Morikawa, 1920; McCarrison, 1921; Morgan and Simms, 1939; Schaffenburg et al., 1950; Hyman et al., 1950) and a depletion of the ascorbic acid content of this gland (Bessey et al., 1934; Siehrs and Miller, 1934; Winkler and Binder, 1939; Giroud et aZ., 1940b; Hyman et al., 1950). An increased cell size, enlarged vesicular nuclei and an increased number of cytoplasmic granules have been reported in the adrenal cortex of guinea pigs deficient in vitamin C (Hyman et al., 1950). These findings are similar qualitatively to those produced by ACTH stimulation and suggest the presence of an activated pituitary-adrenal system in the scorbutic animal (possibly resulting from the concomitant inanition). I n contrast to the above, involution of the adrenal cortex (as judged by the presence of small cells with shrunken dark nuclei and few, if any, granules) occurred in similarly treated animals injected with cortisone (Hyman et al., 1950), presumably through inhibition of ACTH secretion. Available data indicate that the corticosteroid content of the adrenals is markedly diminished in the scorbutic guinea pig (Giroud et al., 1939, 1940a) and that many of the effects of ascorbic acid deficiency can be partially counteracted with cortical extract (Giroud, 1940; Giroud and Ratsimamanga, 1940). More recently it has been demonstrated that cortisone (Schaffenburg et al., 1950; Hyman et al., 1950) and ACTH (Hyman et al., 1950) were similarly effective in counteracting certain of the manifestations of scurvy. Desoxycorticosterone, however, was without effect in this regard (Ratsimamanga and Giroud, 1944) and actually caused an accentuation of scorbutic symptoms (Schaffenburg et al., 1950), thus emphasizing an antagonism between cortisone and this steroid which has been demonstrated in other respects. The fact that the zona glomerulosa, where a desoxycorticosterone-like hormone is produced (Greep and Deane, 1947) is very poor in its content of vitamin C while the fasciculata and reticularis are richer (Glick and Biskind, 1936), would indicate that ascorbic acid may not be essential in the same degree for the production of these two types of cortical steroids.

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The above findings indicate that ascorbic acid deficiency impairs the formation of adrenal hormones of the cortisone type. Further evidence of a possible relationship between ascorbic acid and cortical function is the observation of Sayers and coworkers (Sayers and Sayers, 1946; Sayers et al., 1944a, 1946, 1948) that the ascorbic acid content of the adrenals was significantly reduced after injections of ACTH. A similar reduction was noted in animals subjected to hemorrhage, burns and other forms of stress (Long, 1947; Sayers and Sayers, 1948), due presumably to an increased elaboration of ACTH, since such effects did not occur in hypophysectomized animals, although significant depletion of ascorbic acid was observed in the adrenals of hypophysectomized animals injected with ACTH. The reduction in ascorbic acid levels noted above after ACTH injections or exposure to stress and the concomitant reduction in the cholesterol content of the adrenals (Sayers et al., 1943, 194413, 1946, 1948; Long, 1947) were associated with evidence of increased cortical hormone secretion (as judged by lymphopenia and increase in the liver glycogen of fasted animals 6 to 9 hours after ACTH administration or induction of stress). While these findings suggest that ascorbic acid is in some way involved in the production or secretion of cortical hormones, the mechanism involved is not clear. Lowenstein and Zwemer (1946) suggest that the cortical hormone is actually secreted by the adrenals in the form of a corticosteroid-ascorbate complex and claim to have isolated such a compound from the adrenal gland. This report has not been confirmed, but if correct, it suggests that the rapid and early depletion of ascorbic acid which occurs following ACTH administration or exposure to stress might represent the discharge of preformed hormone, which is followed by a slower depletion of cholesterol as new quantities of hormone are formed. There is evidence, however, to indicate that adrenal glands depleted of ascorbic acid may still synthesize and secrete corticosteroids. For example, Long (1947) has pointed out that if guinea pigs are placed on a scorbutic diet, the adrenal ascorbic acid is reduced in the course of 14-16 days to about 4% of the normal value while adrenal cholesterol has increased some 20% over the values found in pair-fed controls. The injection of 1mg. of ACTH into such animals is followed in 6 hours by the usual fall in adrenal cholesterol and the development of lymphopenia. No further loss, however, takes place in the ascorbic acid content of the adrenals since to all intents and purposes the glands were already depleted of this vitamin. It is evident from this experiment that in the guinea pig not only is the fall in adrenal cholesterol following ACTH administration independent of that in ascorbic acid, but also that it is still associated with evidence of increased cortical hormone secretion (as judged by the development of lymphopenia). More recently, Hyman et aE. (1950) have

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demonstrated an intense hypertrophy and increase in the secreting granules of the adrenals of scorbutic guinea pigs treated with ACTH. These findings strongly suggest that cortical hormones were produced in large quantities, in spite of the fact that the adrenals were virtually depleted of ascorbic acid. The maintenance of glycogen storage in the liver, adrenals, and muscles of scorbutic guinea pigs treated with ACTH or cortisone and the absence of demonstrable amounts of glycogen in comparable tissues of untreated scorbutic controls provides additional evidence of cortical hormone production in the C-deficient ACTH treated animal. It is difficult to reconcile these findings with the concept that ascorbic acid constitutes a limiting factor in the synthesis or secretion of corticosteroids by the adrenal cortex. Further data indicating an interrelationship between vitamin C and the adrenal cortex has recently been presented by ThCrien and coworkers and concerns the effects of large doses of ascorbic acid on cortical function during conditions of stress. It is known that requirements for cortical hormones are markedly increased under conditions of stress and that the adrenal hypertrophy occurring in such conditions is an aspect of the functional adaptation of the organism (Selye, 1937; Ingle, 1938a,b, 1939); in other words, that the gain in adrenal weight, under conditions of stress, is due to an hypertrophy of the cortex (presumably due to increased ACTH production) , with a corresponding increase in adrenal cortical hormone production, and consequently an increased resistance towards the damaging agent. It has also been shown that the enlargement of the adrenal cortex can be prevented (Ingle, 1938a,b; Selye, 1940) or even more that the gland may become atrophied (Selye and Dosne, 1942) when animals under stress are treated with sufficiently high doses of adrenal cortical hormones (presumably due to inhibition of ACTH secretion). Dugal and ThBrien (1949) have recently demonstrated that ascorbic acid when administered in large doses was equally effective (both in guinea pigs and rats) in preventing the typical enlargement of the adrenals that usually occurs on prolonged exposure to cold. In addition, large doses of ascorbic acid were also found to inhibit thymus atrophy and to counteract the hypertension that ordinarily develops following prolonged exposure to a low environmental temperature (ThCrien el at?.,1949). A t the same time resistance to cold was significantly increased as a result of ascorbic acid administration (Dugal and ThBrien, 1947, 1949). Similarly Bacchus and Toompas (1951) observed that large doses of ascorbic acid prevented the occurrence of eosinopenia that normally occurs 3 hours after epinephrine injection in the rat. The significance of these findings is not readily apparent. It may be that the ascorbic acid inhibited the formation or release of the additional quantities of ACTH normally secreted

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under conditions of stress (at least when cold was the damaging agent). It is also possible that the large doses of ascorbic acid facilitated the neutralization, inactivation or excretion of the adrenocorticotropic hormone or raised the threshold of cortical cells to ACTH stimulation. At any rate large doses of ascorbic acid do prevent manifestations of increased ACTH or cortical hormone production. Adrenal atrophy, however, did not occur, which would indicate that some ACTH secretion did take place (Dugal and ThBrien, 1949). Furthermore, since adrenal cortical hormones are essential for survival under conditions of low environmental temperature (Selye and Schenker, 1938), and since the animals receiving the large doses of ascorbic acid were more resistant to cold than those on a normal ration, it would appear that some synthesis and secretion of ACTH (and also corticosteroids) did occur. It is not unlikely that large doses of ascorbic acid improved the utilization of cortical hormones to the point where amounts normally secreted were sufficient to meet the increased requirement of the organism under stress. Under such conditions the need for additional ACTH was not present, and hence the stimulus for increased ACTH production was withheld. The suggestion that ascorbic acid improves corticosteroid utilization is not new. Giroud and coworkers (1940a,b, 1941a, 1941b) pointed out this relationship more than 10 years ago. The findings reported above have dealt with the effects of vitamin C on adjustment to stress. There is considerable data available on the reverse phase of this relationship, namely, the effects of stress on vitamin C requirements. This subject has recently been reviewed in considerable detail (see Ershoff, 1948a, 1951a, for an extensive list of pertinent references). It has been demonstrated that requirements for vitamin C are significantly increased during a number of stress conditions including the following: administration of toxic doses of 1-tyrosine, gold, lead, and arsenic compounds, benzene, phosphorus, trichloroethylene, T.N.T., barbiturates, anesthetics, sulfonamides, hydrazine, amphetamine, and various other drugs; infections, such as diphtheria, tuberculosis and others; hyperthyroidism; pyrexia; pregnancy; lactation; and others. Such “stress factors” may so increase the ascorbic acid requirement that a relative deficiency of this factor may result, although the intake is udequate by ordinary standards. Requirements for other nutrients may similarly be increased, and “ conditioned” deficiencies of these dietary factors may be precipitated (Jolliffe, 1942, 1943; Ershoff, 1948, 1951a). If such nutrients are essential for the maintenance of the pituitaryadrenal system, then impaired adjustment to stress or failure to maintain what Selye (1946) has termed the “stage of resistance” may result. It has been reported that the pituitary also contains a high concen tra-

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tion of ascorbic acid (Biskind and Glick, 1935; Giroud, 1938; King, 1939) and that a decrease in ascorbic acid content occurs in the pituitary of the female rabbit following copulation (Pincus, 1947). The latter finding, however, was not confirmed in more recent studies (Salhanick et al., 1949). An increase in the ascorbic acid concentration of the pituitary has been reported with increasing age in the rat (Salhanick et al., 1949). b. Pantothenic Acid. Evidence of a relation between pantothenic acid deficiency and the adrenal cortex has been accumulating since Morgan and Simms (1939) first noted that in the absence of the filtrate factors in the diet, certain definite changes occurred in the adrenal cortex of the rat. These observations have been confirmed and extended by numerous investigators (Daft and Sebrell, 1939; Nelson, 1939; Ashburn, 1940; Daft et al., 1940; Mills et aE., 1940; Morgan and Simms, 1940a, 1940b; Salmon and Engel, 1940; Mushett and Unna, 1941; Supplee et al., 1942; Ralli and Graef, 1944; Deane and McKibbin, 1946; and others). The following abnormalities have been reported in the adrenal cortex of rats deficient in pantothenic acid: hemosiderin deposition, fibrosis, “congestion,” hemorrhage, cellular atrophy, necrosis, and scarring. These alterations were limited to the inner zones, with no visible changes occurring in the zona glomerulosa. Adrenal hemorrhages were particularly pronounced in pantothenic acid-deficient rats after exposure to acute stress (Ralli, 1949; Skelton, 1950). Associated with the changes reported above a marked depletion occurred in the lipoid content of the cortex, which spared the glomerulosa and sharply delimited it from the remaining layers. The zona glomerulosa of pantothenic acid-deficient rats remained normal while the zona fasciculata lost its lipoid material rapidly and eventually completely (Ashburn, 1940; Deane and McKibbin, 1946; McQueeney et al., 1947). I n addition a marked depletion was observed in the ketosteroid content of the zonae reticularis and fasciculata (Deane and McKibbin, 1946). The changes reported above, however, are not specific for pantothenic acid deficiency. Similar alterations have been observed in animals subjected to a wide variety of “alarming stimuli” (Deanesly, 1931; Selye, 1937; Dalton et al., 1944; Popjak, 1944; Sayers et al., 1944). Moreover, rats deficient in pantothenic acid exhibit other characteristics listed by Selye as occurring in the advanced stages of the “alarm reaction,” such as lassitude, gastritis, and hemorrhagic thymuses and kidneys (Deane and McKibbin, 1946). These findings have led Deane and McKibbin (1946) to suggest that pantothenic acid deficiency acts as a n “alarming agent” for the rat. The increase in adrenal weight (when expressed in milligrams per 100 g. body weight), *the ‘cortical -1ipoid depletion, the thymic atrophy, and the involution of lymphoid tissues of pantothenic

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acid-deficient rats (Deane and McKibbin, 1946; McQueeney et al., 1947) are manifestations of increased ACTH and corticosterone production, such as might occur in response to any non-specific stress. A deficiency of pantothenic acid, however, is apparently too severe an “alarm ” stimulus for the organism to tolerate. The depletion of ketosteroids from the zona fasciculata indicates that the production of corticosterone was inadequate for the demand (Deanesly, 1931; Rokhlina, 1940). The adrenal stores of corticosterone become exhausted, and the pantothenic acid-deficient animal passes from what Selye (1946) has termed the “stage of resistance” to the “stage of exhaustion.” Unless pantothenic acid is administered and the severity of the stress condition ameliorated, such animals will ultimately succumb. Thus, although increased amounts of cortical hormone may be produced in the pantothenic aciddeficient organism, a relative deficiency of this hormone may nevertheless exist. The reduction in liver glycogen stores of pantothenic acid-deficient rats (Deane and McKibbin, 1946) also suggests a possible decrease in circulating corticosteroids. Apparently the initial response to pantothenic acid-deficiency, a t least in the rat, is an increased secretion of ACTH and adrenal corticosteroids. If the deficiency is sufficiently prolonged, however, adrenal cortical exhaustion supervenes and a terminal state of adrenal cortical insufficiency, either absolute or relative, occurs. One might, therefore, expect that certain manifestations of pantothenic acid deficiency are due not to a deficiency of this factor per se but rather to an insufficiency of cortical hormone. Unfortunately the physiological evidence indicative of impaired adrenal cortical function in such animals is not conclusive. On the basis of observed morphological differences, it has been suggested that hhe different layers of the adrenal cortex may have quite independent functions (Ashburn, 1940; Swann, 1940; Sarason, 1943). Expanding this concept, Dalton et al. (1944) and Deane and Greep (1946) inferred that the glomerulosa produces desoxycorticosterone-like hormones which affect electrolyte balance, while the fasciculata produces corticosteronelike hormones which are related to the process of gluconeogenesis. The degenerative changes, lipoid depletion and loss of ketosteroids in the zona reticularis and fasciculata of pantothenic acid-deficient rats and the apparent normalcy of the glomerulosa layer suggest that any cortical hormone deficiency which may be present in such animals is primarily one of the cortisone type. Furthermore, Dumm et al. (1949) found that the lymphopenia that normally occurs in rats following stress (swimming) or injections of ACTH was largely abolished (particularly in the stressed animals) in rats that were pantothenic acid-deficient. This suggests either (1) that the adrenal cortex was depleted of corticosterone-like

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hormones (or their precursors) and, therefore, could not respond to ACTH stimulation by an increased secretion of these hormones, (2) that the response of target cells in the adrenals to ACTH stimulation was impaired, or (3) that the target cells of the corticosterone-like hormones were refractory to stimulation. Since the sole function of ACTH so far as is known is to stimulate the production and secretion of hormones of the corticosterone type, and since the injection of corticosterone will produce lymphopenia similar to that observed in normal animals subjected to stress or administered ACTH (Dougherty and White, 1944), it seems likely that pantothenic acid deficiency in the rat may result in a deficiency (either relative or absolute) of corticosteroid-like hormones. No data are available on the effects of cortisone (or other corticosteroid-like hormones) on the resistance of pantothenic acid-deficient rats to conditions of stress. Such investigations, however, are strongly indicated. * Recent findings from this laboratory provide further data indicative of impaired pituitaryadrenal function in the pantothenic acid-deficient rat. Determinations of adrenal cholesterol, preceding and 4 and 8 hours after epinephrine administration, revealed no reduction in the cholesterol content of the adrenals of pantothenic acid-deficient rats after epinephrine administration in contrast to a reduction of 48% in the adrenal cholesterol of rats fed a similar ration supplemented with calcium pantothenate (Zabin and Ershoff, 1951).t Similarly rats deficient in pantothenic acid showed no reduction in the lymphocyte count 3 hours after epinephrine administration in contrast to the significant decrease noted in rats fed a complete diet (either ad libitum or a t a reduced caloric intake) or in rats fed a similar ration deficient in thiamine, riboflavin or pyridoxine (Ershoff and Parrott, 1951). I n contrast to Dumm et al. (1949), no impairment in lymphopenic response occurred in pantothenic acid-deficient rats injected with ACTH (Ershoff and Parrott, 1951). These findings suggest that inadequate synthesis or secretion of ACTH and not necessarily impairment in adrenal response may have been responsible, a t least in part, for

* Experiments conducted in this laboratory since the present manuscript was first submitted indicate that cortisone acetate administration significantly prolonged the survival time of pantothenic acid-deficient rats under conditions of cold stress. 100% of 32 rats depleted of pantothenic acid succumbed during the first 24 hours of cold exposure (average survival time 11.8 hours). Of 10 rats similarly treated but receiving 1 mg. of cortisone acetate daily (starting 6 days prior to cold exposure), the average survival time exceeded 100 hours. Inasmuch as the adrenal cholesterol level of pantothenic acid-deficient rats in the pre-injection series was less than one-third that of control rats fed a similar diet supplemenhd with calcium pantothenate, it is questionable to what extent failure of pantothenic acid-deficient rats to exhibit a further fall in adrenal cholesterol is indicative of impaired adrenocortical function.

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the manifestations of adrenal dysfunction in pantothenic acid-deficient rats indicated above. There is evidence that pantothenic acid deficiency may also impair the formation of hormones of the desoxycorticosterone type, despite the apparently normal appearance of the zona glomerulosa in these animals. Schaefer et al. (1942) observed that dogs deficient in pantothenic acid developed sudden prostration, gastrointestinal symptoms and a terminal picture consistent with severe dehydration; while Supplee et al. (1942) found that pantothenic acid-deficient rats consumed larger quantities of salt when on a self-selection salt intake than did similarly treated controls. Moreover, Gaunt el al. (1946) observed that rats deficient in pantothenic acid showed much less resistance to water intoxication than did normal pair-fed controls or deficient rats pretreated with either calcium pantothenate or desoxycorticosterone acetate. These findings are consistent with the premise that a deficiency of desoxycorticosterone-like hormones (either absolute or relative) may also occur in the pantothenic aciddeficient animal. Other workers, however, found no such evidence. Fouts et al. (1940), studying blood electrolytes in filtrate factor I1 deficiency in dogs, found no indication of adrenal insufficiency; while Wintrobe et al. (1943) observed no defect in sodium conservation in pantothenic acid-deficient pigs. Similar findings were obtained by McQueeney et al. (1947) in pantothenic acid-deficient rats, despite the fact that these animals were drastically restricted in sodium intake. It is questionable to what extent a deficiency of pantothenic acid will impair adrenal function in animals other than the rat. Adrenal atrophy, necrosis, and hemorrhage on pantothenic acid-deficient diets have been reported only for this species. No such changes were observed in pantothenic acid-deficient mice (Morris and Lippincott, 1941). Cortical lipoid depletion has been observed in the adrenals of pantothenic aciddeficient dogs (Deane and McKibbin, 1946). However, since food intake is reduced in pantothenic acid-deficient animals, and since caloric restriction per se is a potent activator of the pituitary-adrenal system, it is not clear whether pantothenic acid deficiency itself or the attendant reduction in calokic intake was the stimulus for cortical lipoid depletion. In the pantothenic acid-deficient rat, however, both the degree and speed of adrenal cortical lipoid depletion exceeded that which would have been expected from a comparable degree of simple inanition (Deane and McKibbin, 1946). Extreme food restriction involving actual loss of weight, or complete starvation, would apparently have been necessary to produce a similar exhaustion of the cortex (Blumenthal and Loeb, 1942; Whitehead, 1942). Moreover, the thymic atrophy of pantothenic aciddeficient rats was significantly greater than that of pair-fed controls

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(McQueeney et al., 1947); and further, pantothenic acid promoted an increase in the thymus weight of rats deficient in this vitamin even when food intake was kept constant (Ashburn et al., 1947). It appears, therefore, that pantothenic acid deficiency in the rat caused a greater degree of pituitary-adrenal stimulation than could be accounted for on the basis of reduced food intake alone. There is evidence that large doses of pantothenic acid may also condition response to stress. Ralli (1950) has reported that the addition of excess calcium pantothenate to a diet apparently adequate in this factor counteracted in part the increase in total number of white blood cells which occurred 4 to 8 hours after ACTH administration in both intact and adrenalectomized rats. The latter observation is of particular interest since, as Ralli (1950) points out, it raises the possibility that nutritional factors may modify the response to a tropic hormone (ACTH) even when its specific target gland is absent. c. Thiamine. Thiamine deficiency is a potent activator of the pituitary-adrenal system of the rat since it causes: (1) enlargement of the adrenal gland (Deane and Shaw, 1947; Skelton, 1950); ( 2 ) an initial increase and subsequent reduction in the steroid content of the fasciculata (which is the portion of the adrenal cortex most sensitive to ACTH stimulation) (Deane and Shaw, 1947); (3) a marked decrease in thymus weight (Deane and Shaw, 1947; Skelton, 1950); and (4) a reduction in the ascorbic acid content of the adrenal gland (Skelton, 1950). These responses are characteristic manifestations of non-specific stress and typical of the general adaptation syndrome (Selye, 1946). Stimulation of the adrenal cortex has also been reported in advanced thiamine deficiency in the dog (Goodsell, 1941a, 1941b) and other species as well including the monkey (McCarrison, 1922), mouse (Skutta, 1939), rabbit (Verzar and Peter, 1924) and man (Ohno, 1917). In this connection it is of particular interest that muscular exercise causes especially marked adrenal enlargement in rats having insufficient stores of thiamine (Beznak and Perjbs, 1935) and that exogenous administration of yeast extract prevents this hypertrophy (Perjbs, 1936). Inanition, however, is also a potent activator of the pituitary-adrenal system (Selye, 1939; Cameron and Carmichael, 1946; Boutwell et al., 1948; D’Angelo et al., 1948a, 1948b; and others). It is questionable, therefore, t o what extent the changes indicated above were due to a vitamin deficiency per se and to what extent they reflected the concomitant caloric restriction. Deane and Shaw (1947) reported that the effects of thiamine deficiency were more rapid and more severe than that of a comparable degree of inanition, during which the adrenals were stimulated but did not become exhausted so quickly; and Skelton (1950) observed that thiamine deficiency caused

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greater adrenal hypertrophy and thymus atrophy and a greater reductioii in adrenal ascorbic acid content than that occurring in paired weight controls. It may be concluded that thiamine deficiency (as well as pantothenic acid deficiency) caused a greater stimulation of the pituitaryadrenal system than could have been accounted for on the basis of reduced caloric intake alone. d. Choline. In 1939 Griffith and Wade reported acute involution of the thymus of choline-deficient rats and suggested that this phenomenon could be the result of a generalized adaptive reaction to stress (the “alarm reaction” of Selye). These findings were confirmed and extended by a number of investigators (Christensen, 1940; Engel and Salmon, 1941 ; Christensen and Griffith, 1942; and others). Changes have also been observed in the adrenal cortex of choline-deficient rats. Thus Olson and Deane (1949) observed changes both in adrenal weight and cytochemistry which paralleled comparable changes in kidney size and histology in choline-deficient rats. The kidneys of immature rats fed a cholinedeficient diet began to enlarge on the 4th or 5th day of feeding and attained a maximum size in respect t o per cent of total body weight on the 9th day. Congestion of the renal cortex and subscapular hemorrhage appeared on the 5th day and increased in proportion to the degree of kidney enlargement. After the 9th day the kidneys began to decrease in relative size and become pale and ischemic. By the 13th day the ratio of kidney weight to body weight was restored to virtually normal levels despite the continuance of the choline-deficient diet. With the onset of renal damage, food consumptiondeclined and the animal lost weight. The adrenals increased both in relative and absolute weight, reaching maximum values on the 9th day of feeding, while marked atrophy occurred in the thymus. After the 9th day the weights of both the thymus and the adrenals returned toward normal and by the 18th day had attained sizes proportionately comparable to those of choline-fed controls. Coincident with the increase in adrenal weight changes were observed histologically. From the 5th to the 9th day of feeding a marked enlargement occurred in both the fasciculata and glomerulosa layers of the cortex with a reduction in the size of lipid droplets in the former and virtually a complete disappearance of lipid in the latter. Subsequent to this period the fasciculata rapidly returned to normal. Recovery also occurred in the glomerulosa, but at a blower rate. By the 42nd day of deficiency both layers were restored to a normal appearance. Although considerable changes occur both in adrenal weight and microscopic appearance in choline-deficient rats, it would appear that these effects are not primary manifestations of choline deficiency. Olson and Deane (1949) point out that the changes in adrenal weight and

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histology paralleled those that occurred in the kidney during the development and resolution of the renal lesions. No abnormalities in adrenal cortical structure were found after kidney recovery despite the continuance of a choline-free diet. Furthermore, neither desoxycorticosterone acetate nor whole adrenal extract had any effect on the progress of the renal lesion. Olson and Deane suggest, therefore, that adrenal insufficiency was not the cause of kidney damage in choline-deficient rats, but rather that changes in the adrenal cortex were secondary to the renal changes. The increase in adrenal weight, enlargement of the fasciculata, reduction in the size of its lipid droplets and the accompanying atrophy of the thymus gland during the acute phase of choline deficiency in the rat all suggest that activation of the pituitary-adrenal system had occurred with an increased secretion of 11-oxycorticosteroids. The immediate recovery of the fasciculata and the thymus upon restoration of kidney circulation and normal food intake suggest, however, that the changes in the fasciculata were due t o the “alarming stimulus ” of general inanition and kidney damage and not to choline deficiency per se. The marked hyperactivity of the zona glomerulosa during the period of renal damage in cholinedeficient rats, a reaction not noted in other conditions of physiological stress, does not appear to be part of the general adaptation syndrome but apparently results from an altered electrolyte balance during renal failure (Olson and Deane, 1949). I n contrast to a presumably increased ACTH secretion in young rats during the acute phase of choline deficiency, recent findings indicate that ACTH secretion is markedly reduced in older animals fed diets deficient in this factor (Handler and Bernheim, 1950b). These workers demonstrated that adult rats fed choline-deficient diets were incapable of maintaining the elevated blood pressure induced by subtotal nephrectomy. Hypertension was promptly restored in these animals by the administration of ACTH, although this substance was virtually without effect on the blood pressure of choline-fed controls. The effects of ACTH administration, however, were temporary, for within 24 hours the blood pressure of choline-deficient rats injected with adrenocorticotropin had returned to essentially normal levels. These results suggest: (1) that ACTH secretion is impaired in choline-deficient rats, and (2) that the reduction in ACTH secretion (presumably by decreasing cortical hormone synthesis) was responsible for the failure of choline-deficient rats to maintain a hypertensive pressure. The decrease in the eosinophil count after epinephrine administration provides additional data suggesting that adrenocorticotropin secretion is impaired in {the choline-deficient rat. Speirs and Meyer (1949) have demonstrated that the percentage fall in the circulating eosinophil count 4 hours after epinephrine administration is a

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good index of adrenocorticotropic activity (epinephrine stimulates ACTH secretion which in turn stimulates cortical hormone secretion; the reduction in circulating eosinophil count occurs in response to the increased liberation of cortical hormones). Handler and Bernheim (1950b) found that the decrease in eosinophil count elicited by epinephrine in cholinedeficient rats was only half as great as that in choline-fed controls. The reduction in eosinophil count after ACTH administration, however, was equally marked in both groups. Hence it would appear that the ability of the adrenal cortex or the “target cells’’ of its secretions to respond t o tropic stimulation was not reduced in adult choline-deficient rats but that the synthesis or secretion of ACTH by the pituitary after epinephrine administration was impaired. e. Ribojlavin. There is evidence that riboflavin deficiency impairs pituitary-adrenal function in the rat. It has been demonstrated by a number of investigators that a normal fasted animal exposed t o low oxygen tension will increase its carbohydrate stores over the fasting values at sea level (Evans, 1934; Lewis et al., 1943; Long et al., 1940; Langley and Clarke, 1942). The source of this increased carbohydrate appears to be protein, and the stimulus to glyconeogenesis appears to be a n activated pituitary-adrenal system. Wickson and Morgan (1946) have pointed out, however, that rats deficient in riboflavin could not increase their liver glycogen levels, when exposed to low oxygen tension, to the same degree as normal animals. Furthermore, this inefficiency was not due to the lower food intake of the deficient animals. The injection of riboflavin, however, into deficient animals just before the exposure period began permitted glyconeogenesis to occur in a virtually normal manner. More recently Reade and Morgan (1949) observed that “cortin” was equally effective in restoring the carbohydrate-producing capacity of riboflavindeficient rats under conditions of anoxic anoxia. Injection of desoxycorticosterone, on the other hand, had little if any effect. It appears, therefore, that adrenal cortical hormone is essential for glyconeogenesis under conditions of anoxic anoxia, and that insufficient amounts of cortical hormone are produced by the riboflavin-deficient rat under these conditions for a normal glyconeogenetic response. Recent results from this laboratory also indicate the possibility of an impaired formation of cortical hormone in riboflavin-deficient rats under conditions of stress. It is well established that requirements for cortical hormone are markedly increased under conditions of low environmental temperature (Selye and Schenker, 1938; Kendall, 1941; Tyslowitz and Astwood, 1942) and that adrenalectomized or hypophsectomized rats fail to survive following prolonged exposure to cold. Resistance t o cold (as judged by failure of survival) is also impaired in riboflavin-deficient, rats

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(Ershoff, 1952a). Whether adrenal cortical hormones will prolong the survival time of such rats has not as yet been determined. It is not clear what factors are responsible for the impaired secretion of adrenal cortical hormones under conditions of anoxic anoxia and possibly other stresses as well. The defect might occur a t any one or more of the following levels: peripheral, pituitary or adrenal. A peripheral defect might manifest itself in an impaired stimulus to ACTH secretion following exposure to stress; a pituitary defect in impaired ACTH synthesis or liberation; and an adrenal defect in an impaired formation or secretion of adrenal cortical hormone. Ershoff and Parrott (1951) have observed that rats deficient in riboflavin show a normal reduction in lymphocyte count following ACTH administration, which indicates that riboflavin deficiency does not impair the ability of the adrenal cortex to respond to ACTH stimulation (since this reduction in lymphocyte count is presumably due to an increased elaboration of cortical hormone). The reduction in lymphocyte count (Ershoff and Parrott, 1951) after epinephrine administration was, however, significantly less than that observed in animals fed a similar ration supplemented with riboflavin. These findings suggest that the synthesis of ACTH or its secretion in response to stress is impaired in the riboflavin-deficient rat. Failure of cortical hormone production in riboflavin-deficient rats subjected to anoxic anoxia, and possibly other forms of stress, would appear to be due, therefore, to a defect a t the peripheral or pituitary level manifesting itself in an impaired ACTH secretion. Histologically the adrenals .of riboflavindeficient rats were found by Deane and Shaw (1947) to be virtually normal in appearance with no evidence of either stimulation or atrophy. Since caloric restriction per se is a potent activator of the pituitary-adrenal system, and since food intake is materially reduced in the riboflavindeficient rat, changes in the adrenal gland indicative of increased stimulation might have been expected in the latter animal. Failure of such changes to occur is consistent with the hypothesis that ACTH secretion is impaired in the riboflavin-deficient rat. $. Pyridoxine. Marked atrophy of the thymus has been reported both for rats (Stoerk and Zucker, 1944; Stoerk and Eisen, 1946; Deane and Shaw, 1947; Grhgoire, 1949; Agnew and Cook, 1949) and mice (Mirick and Leftwich, 1949) fed pyridoxine-deficient diets. The changes observed were greater than could have been accounted for on the basis of reduced caloric intake alone, A hypertrophy of the adrenals was reported by Grhgoire (1949) in rats fed a pyridoxine-deficient diet which was accentuated by the administration of desoxypyridoxine. Other investigators, however, found that pyridoxine deficiency had little if any effect on either the weight or histological appearance of the adrenals of the

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rat (Agnew and Cook, 1949; Deane and Shaw, 1947). It is questionable, therefore, whether the thymus atrophy of pyridoxine-deficient animals is caused by an increased secretion of adrenal cortical hormone. Recent findings from this laboratory indicate that rats deficient in pyridoxine failed to survive when exposed to a low environmental temperature, although survival was not appreciably impaired, if rats were adjusted to cold prior to the time they became pyridoxine-deficient (Ershoff, 1951b). It would appear from these findings that pyridoxine is essential for optimal adjustment to stress (cold), but that the effects of a deficiency are less pronounced once adjustment to stress has occurred. Inasmuch as requirements for adrenal cortical hormone(s) are greater during adjustment to stress than after adjustment has occurred, it would seem that pyridoxine deficiency may have impaired the formation or utilization of cortical hormone(s). Determinations of the leukocyte picture of pyridoxine-deficient rats preceding and 3 hours after epinephrine administration revealed no difference between that of deficient animals and of rats fed a similar diet supplemented with pyridoxine hydrochloride. Both groups showed a reversal in the differential count, an increase in total leukocytes, a marked increase in polymorphonuclears and a significant reduction in lymphocytes and eosinophils 3 hours after epinephrine administration (Ershoff and Parrott, 1951). These responses are indicative of an activated pituitary-adrenal system and are similar to those which occur in the normal animal after ACTH or cortisone administration or in response to non-specific stress (Dougherty and White, 1947; Speirs and Meyer, 1949). Evidently pyridoxine deficiency did not impair the ability of the pituitary-adrenal system to respond to acute stress. The activation of the adrenals which occurs in response to epinephrine administration, however, is based primarily if not entirely on the release of preformed ACTH. It is possible that the pyridoxinedeficient rat may have sufficient amounts of this hormone stored in its pituitary to effect a normal response to acute stress but that such rats may have an impaired ability to produce the increased amounts of ACTH required during chronic stress. Evidence in support of this hypothesis has recently been obtained in which it was observed that although no impairment in the lymphopenic response to epinephrine occurred in pyridoxine-deficient rats exposed to acute stress (a single injection of epinephrine), after 10 daily injections of epinephrine, the lymphopenic response to the last injection was significantly less in pyridoxine-deficient rats than in animals fed a similar diet supplemented with pyridoxine hydrochloride (Ershoff and Parrott, 1951). I n view of the increased requirement for thyrotropin (Brolin, 1946) and adrenocorticotropin (Sayers and Sayers, 1945) during prolonged exposure to cold, the failure of pyridoxine-deficient

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rats t o adjust to a low environmental temperature may result not from pyridoxine deficiency per se but rather from a deficient secretion of pituitary hormones. The role of pyridoxine in protein metabolism suggests the possibility that animals deficient in this vitamin may have an impaired ability to synthesize proteins, including adrenocorticotropin, which are required in increased amounts during adjustment to stress. g. Vitamin A . There is considerable data suggesting an interrelationship between vitamin A and pituitary-adrenal function. Carotene is present in moderate concentration in the adrenal glands of a number of animals (horse, ox, hog and guinea pig) but not the rat (Randoin and Netter, 1933). In the latter species (Davies and Moore, 1934; Von Querner, 1934, 1935; Popper and Greenberg, 1941) and in man (Popper, 1941, 1944), however, the adrenals contain large amounts of vitamin A. Fluorescence-microscopic examination of the adrenals of rats revealed that vitamin A was present in greatest concentration in the outer portion of the fascicular layer, whereas the glomerular layer contained none (Popper and Greenberg, 1941). The inner zone of the cortex contained little if any vitamin A. No vitamin A fluorescence was observed in the medulla. The adrenals of rats depleted of vitamin A showed no vitamin A fluorescence. The administration of carotene or vitamin A t o such animals, however, restored vitamin A fluorescence in these glands. The vitamin A content of the adrenals is dependent, however, on considerably more than the dietary intake of this vitamin. A reduction in the vitamin A content of this gland has been observed following experimental infection, hyperthermia, chloroform poisoning (McCoord, 1938), cold stress (McConnell, 1950), diabetes, jaundice, various infectious states (Patzelt, 1947) and other conditions of stress. A transient decrease in vitamin A content was noted in the adrenals of rats 6 hours after an LDm dose of X-irradiation. After 10 hours, however, the adrenals contained more vitamin A than untreated controls (Bennett et al., 1950). Data such as the above suggest that vitamin A participates in the formation or secretion of corticosteroids and raise the possibility of a relative cortical hormone deficiency resulting from vitamin A deficiency. That such may actually occur is indicated by the following observations: DeVall (1945) has reported that rats deficient in vitamin A show a n increased sensitivity to insulin. Sensitivity t o insulin is also increased in the adrenalectomized rat. Cortisone and other ll-oxygenated compounds, however, exert an anti-insulin effect (Grattan and Jensen, 1940). Hence a relative deficiency of ll-oxygenated corticosteroids may be responsible, a t least in part, for the increased insulin sensitivity of vitamin A-deficient rats. Additional data suggesting the possibility of cortical hormone insufficiency in the vitamin A-deficient rat has recently been presented from this laboratory.

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The survival time of rats deficient in vitamin A was found to be markedly reduced under conditions of low environmental temperature (Ershoff, 1950a, 1952b). Adrenalectomized rats also fail to survive exposure to cold (Selye and Schenker, 1938). Cortisone, however, will protect such animals against the stress of cold (Kendall, 1941). Whether it will exert a similarly protective effect in the vitamin A-deficient rat has yet to be determined. The need for further study of the effects of vitamin A deficiency on pituitary-adrenal function is obvious. A cytological and cytochemical investigation of the adrenals of vitamin Adeficient animals with techniques such as those employed by Greep and Deane (1949) are indicated as well as investigations on the effects of ACTH and other forms of stress on adrenal cholesterol and ascorbic acid content and blood lymphocyte and eosinophil levels in the vitamin A-deficient animal. * 3. Minerals

a. Sodium. Data on the effects of sodium restriction on pituitaryadrenal function are conflicting. Deane et al. (1948) observed a relative increase in adrenal but no effect on thymus weight in rats fed a sodiumdeficient ration. Histologically the adrenals showed no significant changes in the zona fasciculata but marked hypertrophy and hyperplasia of the glomerulosa with cytochemical changes indicative of increased cellular activity. The latter occurred both in normal and hypophysectomized rats, indicating they were not caused by pituitary stimulation and hence not directly related to the general adaptation syndrome. Danford et al. (1949, 1951), however, observed that a complete dietary deficiency of NaCl caused pituitary-adrenal changes similar to that observed with other forms of stress (increased urine volume, relative adrenal hypertrophy, thymus involution, and depletion of adrenal ascorbic acid). Differences in the composition of the basal diet or the age of animal employed may have been partly responsible for the diverse results. b. Potassium. Diets deficient in potassium activate the pituitaryadrenal system of the rat. Gardner et al. (1950) observed that rats fed a K-deficient diet gained weight slowly for 35 to 40 days after which time a gradual weight loss and anorexia became manifest. A t this time animals gave evidence of chronic alarm with increased production of corticosteroids as judged by a marked reduc,tion in the number of circulating eosinophils and increase in adrenal weight. A relative increase in adrenal weight and stimulatory changes in the zona fasciculata were also reported

* Recent findings from this laboratory (Ershoff and Parrott, 1951) indicate that vitamin A deficiency results in a virtually complete absence of eosinophils from the peripheral blood with virtually no other change from normal in respect to the total or differeptial leukocyte count.

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by Deane et al. (1948) in rats fed a potassium-deficient diet. However, caloric restriction may have contributed to the above results. Deane et al. (1948) also observed a reduction in size and atrophic changes in the zona glomerulosa of rats fed a potassium-deficient diet. These findings suggest that the production of desoxycorticosteroid-like hormone(s) may have been depressed in the potassium-deficient rat. This may in a relative manner have intensified the effect of the 11-17 oxycorticosteroids produced in the same gland. Excessive potassium administration has also been reported to stimulate the pituitary-adrenal system with effectssimilar to that produced by other forms of stress (Badinez and Croxatto, 1948a, 1948b; Jarpa and Croxatto, 1948). These findings differ from those reported by Deane et al. (1948) who observed that massive doses of KC1 had no significant effect on the zona fasciculata of the rat. The latter workers did find, however, that excessive doses of KC1 caused marked hypertrophy of the glomerulosa. Similar findings were also reported by Bacchus (1950). It would appear that changes in the glomerulosa occurring on sodium or potassium-depleted diets or following the administration of massive doses of potassium result from an altered sodium-potassium ratio and are not directly related to the general-adaptationgyndrome (Deane et al., 1948; Bacchus, 1950).

4. UnidentiJied Nutrients It appears that factors exist (presumably distinct from any of the known nutrients) which are required in increased amounts during conditions of stress. These “minor vitamins” are apparently dispensable under normal conditions, or their requirements are so small they may readily be met by the diet or through the synthetic activity of the intestinal flora or the animals’ own tissues. Certain drugs or other “stress factors” may, however, increase requirements for these substances to the extent that deficiencies occur, manifest by retarded growth or tissue pathology, and preventable by the administration in appropriate amounts of the missing nutrient. Whole liver is a potent source of such unknown nutrients. Thus whole liver or fractions thereof has been shown to counteract the deleterious effects of massive doses of strychnine, sulfanilamide, promin, atabrine, dinitrophenol, diethyl-stilbestrol, a-estradiol, thyroactive substances and other drugs (see Ershoff, 1948a, 1951a, for an extensive list of pertinent references). I n the case of a t least three of these drugs, thyroid (Ershoff, 1947b, 1948c, 1949, 1950d), promin (Ershoff and McWilliams, 1949), and atabrine (Ershoff, 1948b, 1950b), a protective factor is retained in the water-insoluble fraction of liver which is distinct from any of the known vitamins including vitamin €312. The protective effects of the

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liver factor are particularly marked in animals fed desiccated thyroid and other thyroactive substances. Thus, whole liver or its waterinsoluble fraction completely counteracted the growth retardation (Ershoff, 1947a, 1947b; Betheil et al., 1947; Ershoff and McWilliams, 1948) and gonadal inhibition (Ershoff, 1947a, 1950e, 1950f) of immature hyperthyroid rats fed a purified ration containing casein as the dietary protein and sucrose as the dietary carbohydrate. In addition it significantly prolonged the average length of survival of hyperthyroid rats on the above dietary regime (Ershoff, 1947a, 194713, 1948c; Betheil et al., 1947). Supplements of all the known vitamins (including vitamin BI2) either alone or in combination were, however, without significant effect (Ershoff , 1947b, 1950d). Desiccated and defatted kidney, crude aureomycin mash, and full fat soybean meal were also potent sources of a growth-promoting factor for the hyperthyroid rat (Ershoff, 1949b, 1950d). There is some question whether the factor which prolongs survival is identical with that which promotes growth or gonadal development. Yeast (Ershoff and Hershberg, 1945), whole liver (Ershoff, 1947a, 1947b), crude aureomycin mash, and dried penicillin mycelia (Ershoff, 1950g), for example, were all effective in prolonging survival when fed a t a 10% level to hyperthyroid rats whose diet contained casein as the protein and sucrose as the carbohydrate; however, yeast and dried penicillin mycelia in contrast to liver and aureomycin mash had no significant effect on gonadal development or rate of growth. Whole liver, on the other hand, when fed at a 2 % or 4% level in the diet had a pronounced effect on growth but no significant effect on length of survival (Ershoff, 1948~). These findings would seem t o indicate that prolonged survival and increased growth were not due to different concentrations of the same factor. The mechanisms whereby the “antitoxic factor (or factors) of liver” exerts its protective effect are unknown. Evidently this material in some manner enables the body to withstand the effects of a number of substances which would otherwise be toxic, but its modus operandi remains obscure. It has been suggested (Ershoff, 1951a) that the “antitoxic factor of liver” is essential for the maintenance of what Selye terms a “state of resistance” in the animal exposed t o stress and that a deficiency of this factor may result in a “state of exhaustion.” A marked stimulation of the pituitary-adrenal system occurred in all rats fed the various drugs indicated above. At least with the thyroactive drugs, promin and atabrine, the administration of the water-insoluble fraction of liver prevented the occurrence of a “state of exhaustion” and permitted the continuance of a “state of resistance.” Whether the protective effect occurred a t the pituitary, adrenal or peripheral level has not been deter-

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mined. The observation that both chorionic gonadotropin and the water-insoluble fraction of liver promoted ovarian development in hyperthyroid rats (Ershoff, 1950e) suggests that the liver factor mayfunction at the pituitarylevel.* Recent findings thatwhole liver (but not any of the known B vitamins) exerted a protective effect in immature rats fed massive doses of cortisone acetate (Ershoff , 1951c) suggests, however, that the liver factor@) may also function by protecting against the toxic effects of cortical hormone(s) produced in excess during conditions of stress. 5 . Miscellaneous Factors

Dietary factors other than those indicated above may also activate the pituitary-adrenal system. Chronic overfeeding (Ingle and Nezamis, 1947), thirsting (Nichols, 1949), excessive consumption of cabbage and water (McCarrison and Madava, 1932, 1933), rapeseed-containing diets (Kennedy and Purves, 1941), high casein and lactalbumin-containing rations (in which approximately 80% of the caloric value of the diet was derived from protein) (Ingle, 1945)-aIl these have been reported t o cause adrenal hypertrophy in the rat and other manifestations of the general adaptation syndrome. An increase in adrenal weight associated with a marked increase in the cholesterol constant of the cortex (mostly in the esterified form) has been reported in rats fed a diet containing 25% rape or turnip seed oil (Carroll, 1951). An increased cholesterol content of the adrenals has also been reported by Abelin (1948) in rats fed a diet containing 12yo“Speise oil” (a mixture of rape oil, peanut oil and poppyseed oil). Other fats and oils had little if any effect either on adrenal weight or cholesterol content (Abelin, 1948; Carroll, 1951). The increase in adrenal cholesterol of rats fed rape or turnip seed oil was considerably greater than that attained in response to stress (Carroll, 1951). What factors were responsible for this increase, what significance an increased cholesterol content might have on adrenal cortical function (cholesterol has been suggested as a precursor of adrenocortical hormones), what effect a high intake of the oil might have on adjustment t o stress, these are all problems in need of further investigation.

* There is evidence to indicate that somatotropin is required in increased amounts in animals exposed to various stressor agents (Selye, 1951). In view of the growthpromot,ing effect of the liver factor in animals receiving toxic doses of the various drugs listed above, experiments are indicated to determine to what extent the “antitoxic factor of liver” may be involved in the production of (or response to) somatotropin during conditions of stress. Available data indicate t h a t somatotropin will not promote a n increment in body weight in rats deficient in the “antitoxic factor” (Ershoff, 1951d).

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V. EFFECTS OF NUTRITIVE STATE ON RESPONSE TO PITUITARY HORMONES Considerable data are available indicating that somatotropin will not promote a weight increment in animals that are nutritionally deficient. Thus “growth hormone” failed to promote a gain in body weight in rats deficient in vitamin B1 (Simpson and Evans, 1944) , vitamin A (MargitayBecht and Wallner, 1937; Ershoff and Deuel, 1945), the essential fatty acids (Deuel et al., 1950) or the “antithyrotoxic factor of liver” (Ershoff, 1951d). Similarly “growth hormone” failed t o promote an increment in body weight in rats fed a low protein (6% casein) diet (Gordan et al., 1917, 1948). Supplementing the above diet with dl-methionine or increasing ’ of the ration resulted, however, in the casein content to a minimum of 12% a significant weight increment following “growth hormone” treatment. Chow and Greep (1948) have reported that the quality as well as the amount of dietary protein is also of importance in conditioning the response to somatotropin administration. These workers found that the gain in body weight of immature hypophysectomized rats treated with a growth-promoting fraction of the anterior pituitary was appreciably less when the source of dietary protein was soya protein than when it, was casein or lactalbumin. An impaired response to gonadotropin stimulation has also been reported for animals deficient in various nutrients, e.g. male and female rats deficient in vitamin BIZ (Chow and Barrows, 1950) or deficient in riboflavin or treated with a folic acid antagonist (Yadu and Meites, 1949). I n general, however, response to gonadotropin stimulation is not appreciably impaired in the nutritionally deficient animal. Thus neither vitamin A deficiency (Van Os, 1936; Mason, 1939a; Mayer and Goddard, 1951), essential fatty acid deficiency (Greenberg and Ershoff, 1951) , thiamine deficiency (Marrian and Parkes, 1929; Moore and Samuels, 1931), pyridoxine deficiency (Yadu and Meites, 1949), pantothenic acid deficiency (Figge and Allen, 1942; Yadu and Meites, 1949), protein restriction (Courrier and Raynaud, 1932 ; Samuels, 1950) nor reduction in total caloric intake (Marrian and Parkes, 1929; Mulinos et al., 1939; Moore and Samuels, 1931 ; and others) impaired the response to gonadotropin stimulation in the rat. The same appears to be true for other pituitary hormones. Thus Hyman et al. (1950) observed that a marked deficiency of vitamin C did not prevent the adrenal response to ACTH stimulation in the guinea pig despite the virtual absence of ascorbic acid in the adrenal gland. Evidence of cortical hormone production after ACTH stimulation has also been reported in man with symptoms of clinical scurvy (Treager et al., 1950). Increased cortical hormone production, as judged by a drop in the level of circulating lymphocytes in the blood after ACTH

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administration, has also been reported for rats deficient in thiamine, riboflavin and pantothenic acid (Ershoff and Parrott, 1951). An increased sensitivity to thyrotropin stimulation has been reported for guinea pigs deficient in ascorbic acid (Schulze, 1938). There is evidence that ascorbic acid may potentiate the effects of gonadotropin. Giedosz (1938) reported that the simultaneous administration of ascorbic acid and gonadotropin caused a greater stimulation of the ovaries and uteri of rabbits than resulted from either substance administered alone. Similar findings have been reported for the rat (Di Cio and Schteingart, 1942). Other nutrients may actually inhibit the effects of pituitary hormone administration. Thus Anderson and Evans (1937) observed that KI prevented the metabolic action of thyrotropic extracts in the guinea pig (presumably by preventing the discharge of thyroid hormone) although it did not interfere with the effect on the thyroid gland itself. Similar results have been .reported by Crepax (1948a). Large doses of KC1 have been found to prevent the increase in urinary nitrogen excretion which normally occurs following ACTH administration in the rat (Whitney and Bennett, 1950). Whether KCI inhibits the adrenal response to ACTH stimulation or whether it modifies the peripheral tissue response to increased cortical hormone production has not, however, been determined. Available data indicate that the composition of the experimental diet may also significantly affect the response to hypophysectomy, at least in the rat. Thus Shaw and Greep (1949) demonstrated that the length of survival of hypophysectomized rats maintained on a purified ration was significantly longer than that of similarly treated rats fed laboratory chow.

VI. EFFECTS OF PITUITARY HORMONES ON NUTRITIVE STATE The anterior pituitary hormones both directly and indirectly through the secretions of their target organs exert a profound effect on the utilization and metabolism of carbohydrate, protein and fat. This subject has been extensively reviewed (Samuels, 1947; Li and Evans, 1948; Barker, 1949; and others). I n addition to the effects noted above, the anterior pituitary, either directly or through the target organs of its secretions, markedly affects the utilization and metabolism of a number of specific dietary factors. The increased calcium retention of guinea pigs and rats treated with a growth promoting extract of the anterior pituitary (Antuitrin G) is a case in point (Krishman, 1942). The increased requirement for pantothenic acid following “growth hormone ” administration in the rat is another (Lotspeich, 1950). The decreased survival time and the earlier and more pronounced appearance of deficiency symptoms in vitamin A-deficient rats treated with “growth hormone” (Ershoff and

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Deuel, 1945) is a third example. Hyperthyroidism has been shown to increase body requirements for a number of essential nutrients including thiamine (Drill, 1938; Drill and Sherwood, 1938), pyridoxine (Drill and Overman, 1942), pantothenic acid (Drill and Overman, 1942), folic acid (Martin, 1947), vitamin Blz (Nichol et al., 1949; Emerson, 1949; Betheil and Lardy, 1949) and others (Drill, 1943; Ershoff, 1950a). An excessive secretion of thyrotropin if associated with a hyperthyroid state might be expected to increase body requirements for the above factors. A deficiency of thyroid hormone has been reported to impair the conversion of carotene to vitamin A (Wendt, 1935; Wohl and Feldman, 1939; Canadell and Voldecasas, 1947; Kelley and Day, 1948) and to decrease the absorption of carotene from the intestinal tract of the rat (Cama and Goodwin, 1949). A decreased secretion of thyrotropin sufficient to result in a hypothyroid state might be expected to cause changes similar to those indicated above. Thyroxine has been reported to play an important role in the conversion of pantothenic acid into coenzyme A (Minz and Cohen, 1949). As in the case of carotene it would appear that an adequate secretion of thyrotropin is essential for optimal utilization of this vitamin. The precipitation of a K deficiency (Elkinton et al., 1949) or the induction of hypoferremia (Hamilton el al., 1951) after ACTH administration are further examples of a pituitary hormone affecting (presumably through the adrenal cortex) the metabolism of a specific nutritional factor. A sustained increase in the urinary ascorbic acid excretion of 17 out of 23 patients administered ACTH (Beck et al., 1950; Johnson, 1950) and the occurrence of hemorrhagic manifestations presumably due to ascorbic acid deficiency in two other patients while receiving high doses of ACTH (Stefanini and Rosenthal, 1950) are additional examples of a pituitary hormone conditioning a nutritional disease.

VII. SUMMARY A number of interrelationships both specific and non-specific have been reported between various nutrients and endocrine glands. It has been demonstrated that the nutritional state (resulting from either an excess or deficiency of nutrient factors) and the composition of the diet fed may profoundly affect (I) the synthesis and secretion of hormones (2) the response of target organs and peripheral tissues thereto and (3) the metabolism and excretion of hormonal substances. * Conversely the endocrine

* Virtually no data are available on the effects of nutritional factors on the metabolism and excretion of pituitary hormones. The effect of nutritional state on estrogen inactivation, however, is an example of this type of interrelationship. The reader is referred to the reviews of Biskind (1946) and Hertz (1946, 1948) for a comprehensive discussion on this subject.

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glands exert a profound effect on the absorption, utilization and excretion of nutrients and body requirements for specific dietary factors. I n the present review a critical survey has been made of nutrition-endocrine interrelationships as they pertain t o the anterior pituitary and with special reference to their role in the general adaptation syndrome of the mammalian organism including man. REFERENCES

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Simpson, M. E., and Evans, H. M. 1944. Personal communication from Dr. H. M. Evans. Sinclair, H. M. 1948a. Brit. J. Nutrition 2, 161-170. Sinclair, H. M. 1948b. Vitamins and Hormones 6, 101-162. Singer, E. 1936. J. Physiol. 87, 287-290. Siperstein, D. M. 1921. Anat. Record 20, 355-381. Skelton, F. R. 1950. Proc. SOC.Exptl. Biol. Med. 73, 516-519. Skinner, J. T., Van Donk, E., and Steenbock, H. 1932. Am. J . Physiol. 101, 591597. Skutta, G. 1939. Verhandl. Anat. Gesellsch. 88, 87. Smith, P. E. 1930. Am. J. Anat. 46, 205-274. Speirs, R. A., and Meyer, R. K. 1949. Endocrinology 46, 403-429. Spence, A. W., and Scowen, E. F. 1935. Biochem. J. 29, 562. Starr, P. 1942. Internatl. Abstr. Surg. (Surg. Gynecol. Obstet.) 74, 309-322. Stefanini, M., and Rosenthal, M. C. 1950. Proc. SOC.Exptl. Biol. Med. 76,806-811. Stefko, W. 1931. Schweiz. med. Wochnschr. 61, 171. Stein, S. I. 1935. J . Nutrition 9, 611-619. Stephens, D. J. 1940. Endocrinology 26, 485-492. Stephens, D. J., and Allen, W. M. 1941. Endocrinology 28, 580-584. Stewart, C. A. 1918. J . Exptl. Zool. 26, 301. Stoerk, H. C., and Eisen, H. N. 1946. Proc. SOC.Exptl. Biol. Med. 62, 88-89. Stoerk, H. C., and Zucker, T. F. 1944. Proc. SOC.Exptl. Biol. Med. 66, 151-153. Stott, H. 1932. Ind. J. Med. Research 20, 147. Sturm, A., Smidt, W., and Beck, J. 1938. J. Endokrinol. 21, 1. Supplee, G. C., Bender, R. C., and Kahlenberg, 0. J. 1942. Endocrinology SO, 355-364. Sutton, D. C., and Ashworth, J. 1940. J . Lab. Ctin. Med. 26, 1188-1192. Sutton, T. S., and Brief, B. J. 1938. Endocrinology 23, 211-215. Sutton, T. S., and Brief, B. J. 1939. Endocrinology 26, 302-307. Sutton, T. S., Krauss, W. E., and Hansard, S. L. 1940. J . Dairy Sci. 23, 574. Swann, H. G. 1940. Physiol. Revs. 20, 493-521. Sydenham, A. 1946. Brit. Med. J. 2, 159. Szent-Gyorgyi, A. 1928. Biochem. J. 22, 1387. Talbot, N. B., Wood, M. S., Campbell, A. M., Christo, E., and Zygmuntowicz, A. S. 1951. Proc. 2nd Clinical ACTH Conf. 1,20-29. The Blakiston Co., New York. Telford, I. R., Emerson, G. A., and Evans, H. M. 1938. Proc. SOC.Exptl. Biol. Med. 38, 623-624. Terbruggen, A. 1937. Arch. Path. Anat. (Virchow’s) 298, 646-659. Teresa, S. I. 1937. Bull. biol. mdd. exptl. URSS 3, 175. ThBrien, M., Leblanc, Jr., and HBroux, 0. 1949. Can. J . Research E27, 349. Thompson, J. 1936. Endocrinology 20, 809-815. Treager, H. S., Gabuzda, G. J., Zamcheck, N., and Davidson, C. S. 1950. Proc. SOC. Expil. Biol. Med. 76, 517-520. Truscott, B. L., and Sadhu, D. P. 1948. Anat. Record 100,719-720. Tuchmann-Duplessis, H., and Aschkenasy-Lelu, P. 1948. Compt. rend. SOC. biol. 142, 472-474. Tyslowitz, R., and Astwood, E. B. 1942. Am. J . Physiol. 136, 22-31. Underhill, S. W. F. 1939. Chapter on “Vitamin Edeficiency and the endocrine glands” in Vitamin E-A Symposium. Soc. Chem. Ind., London. Van Os, P. M. 1936. Acta Brevia Neerland. Physiol. Pharmacol. Microbiol. 6, 151. Vim Wagenen, G. 1925. Anat. Record 29, 398.

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Hormone Assays in Obstetrics and Gynecology BY RUDI BORTH

AND

HUBERT

DE

WATTEVILLE

Clinique universitaire de gyndeologie et d’obstdtrique, Geneva, Switzerland CONTENTS

Page I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Methods.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 1. General Principles.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 2. Urine Assay vs. Blood Assay.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 a. Estrogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b. Progesterone and Pregnanediol. .... c. Testosterone and 17-Ketosteroids.. . d. Gonadotropins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 e. Conclusions.. .... .............................. 146 3. Excretion per Unit of Volume vs. Excretion per Unit of Time. . . . . . . . 147 4. Bioassay vs. Chemical Estimation.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148 5. Some Remarks on Individual Methods for Clinical Use ............................. .......................................... 150 c. Pregnanediol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150 d. 17-Ketosteroids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

e. Other Steroid Fractions ............. ............. 111. Menstrual Cycle and Menstrual Disorders. . . . . . . . . . . . . . . . . . . . . . . . . 1. Hormone Excretion during the Normal Menstrual Cycle.. . . . . . . . . a. Gonadotropins. . ......................................... b. Estrogens.. . . . . ........................................ c. Pregnanediol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d. 17-Ketosteroids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......... e. Conclusions.. ..... .............................. 2. Dysmenorrhea and Pr nsion . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Irregular and “Anovulatory ” Bleeding. . . . . . . . . . . . . . . . . . . . . . . . . . . . a. Regular Bleeding-Luteal Deficiency. . . . . . . . . . . . . . . . . . . . . . . . . . . .

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152 153 153

154 154 155 155 155

d. Conclusions.. . . . . . . . .

. . . . . . . . . . 158 Conclusions, . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . 159

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Page

V. Pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Hormone Levels in Normal Pregnancy.. . . . . . . . . . . . . . . . . . . . . . . . . . . .

Conclusions.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........................... 2. Diagnostic Tests for Pregnancy. . . . a. Chorionic Gonadotropin. . . . . . . . ....................... b. Pregnanediol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c. Estrogens. . . . . .................................. d. Conclusions.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Abortion and Premature Labor.. . . . . ........ ........... a. Chorionic Gonadotropin. . . . . . . . . . ......................... b. Estrogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c. Pregnanediol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Hydatidiform Mole and Chorioepithelioma. . . . . . . . . . . . . . . . . . . . . . 5. Toxemia . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Ectopic Pregnan .......................................... ..........................................

162 164 164 165 165 166 166 167 167 168

171 172 172

VII. Endocrine Disorders. . References. . . . . . . . .

............

. . . . . . . . . . . 175

I. INTRODUCTION This review is an attempt to summarize the pertinent information on hormone assays in women in such conditions as the gynecologist and the obstetrician are likely to encounter in clinical practice. It is intended to examine the evidence in favor of, or against, the use of hormone determinations as a tool that may help the physician to determine his diagnosis, to establish a prognosis, and to decide on the therapy. The indisputable value of hormone assays in metabolic studies and other basic research will not be discussed. The scope of the review is limited to the gonadotropic and the steroid hormones and their metabolites. Roughly, the available literature of the last ten years has been considered in the preparation of this report, but-as is the tradition in this series-no attempt has been made to give a complete bibliography or a historical survey. Although most of the original articles have been consulted, preference will be given to the citation of competent reviews and recent reports containing the older references. Some important points should be stressed at the outset. Morphological changes due to hormonal activity may occur or persist some time after the release or withdrawal of the hormones responsible. This time-lag may give rise to apparent discrepancies between the result of a hormone assay and the clinical and histologic findings noted at the same time. Moreover, it should be remembered that the level of hormones and hor-

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mone metabolites in body fluids does not depend exclusively on the rate of secretion. The latter may be the limiting factor more often than not, but numerous metabolic transformations, most of them of unknown nature, certainly intervene as the hormones pass from the site of elaboration through the bloodstream, liver, kidney, and target organs. The hormone levels found in the biological or chemical laboratory are mere symptoms which have to be integrated into the whole clinical picture of a case before they can be expected to become useful. They may be sometimes as equivocal as most other single clinical findings, and they may be equally open to different interpretations. It should be realized that this characteristic is a general biologic phenomenon and that it is not to the discredit of hormonal methods in particular. On the other hand, there may be a tendency to put too much faith on the reliability and clinical significance of hormone assays, inasmuch as one is impressed by the fact that many of these require considerably more skill and labor than most other clinical methods for routine examinations of blood and urine. The rational attitude is to regard hormone assays as one tool among others and to consider the information they furnish within this natural limitation.

11. METHODS

It has been stated repeatedly that the question of methods cannot be overemphasized. Different methods for the estimation of a particular urine or blood fraction may lead not only to numerical differences but also to widely divergent theoretical conclusions. Final agreement on a controversial subject may be reached once the exact significance of certain experimental conditions or technical details is fully realized. Two wellknown striking examples from the recent literature may serve to illustrate this point. The occurrence of unconjugated estrogen in urine a t the time of labor has been traced to the action of the glucuronidase present in blood clots and amniotic fluid (Clayton and Marrian, 1950). The observation of Smith et al. (1946) that stilbestrol treatment increases the “sodium pregnanediol glucuronidate ” fraction of pregnancy urine has been shown not to imply that pregnanediol itself, as determined by more specific methods, is increased by this treatment (Davis and Fugo, 1948; Seitchik, 1950; Sommerville et al., 1949; and others). 1. General Principles

In adopting any method for regular use, it is not enough to study the technique, i.e. the description of the procedure. Other points are equally important, a complete set of essential characteristics including the following items (Borth, 1952) :

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1. Technique 2. Reliability a. Specificity b. Accuracy c. Precision d. Sensitivity 3. Practicability a. Speed b. Expenditure c. Skill 4. Sources of error (The terms “accuracy” and “precision” are used here in the statistical sense (Cochran and Cox, 1950). The accuracy of a measurement signifies the closeness with which it approaches the true value. The term precision is concerned merely with the repeatability of the measurements as, for instance, in duplicate or triplicate determinations.) Specificity means that the substance or group of substances detected by a certain method should be well characterized by its chemical or biological properties. It does not necessarily mean that all the substances to be estimated are identified single chemical compounds, although this would be the ideal condition. Accuracy is usually determined by the percentage recovery of pure compounds added to a sample before analysis. This is the only way to measure accuracy, but Dobriner (1952) has rightly emphasized the approximation it implies, for instance with regard to hydrolyzing procedures, unless the composition of conjugates is fully known. Precision and sensitivity are easily ascertained, the former by repeated analyses of the same specimen, the latter by recovery experiments with decreasing amounts of pure compounds. The efficiency of a routine method depends on its practicability as much as on its reliability. How many determinations can be made per week and after how long are the results available (“speed”)? What is the cost per assay in terms of labor, animals, apparatus, and chemicals (“expenditure”) ? Does a technique require unusual skill or special knowledge or training? These questions have to be answered when the methods best adapted to particular purposes and facilities are to be chosen. Finally, possible sources of error should be made widely known in order to save much work and trouble. It is, for instance, most useful to know that cigarette smoke may interfere with the estimation of formaldehydogenic corticoids (Venning, 1952), or that concentrated sulfuric acid (C.P.)

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after dilution with half its volume of distilled water may fail to elicit the fluorescence reaction of pure estrone after standing one week at room temperature, while freshly prepared dilutions of the same batch still do. These considerations are not meant to imply that only the highest standards of reliability and practicability are acceptable. We are fully aware of the fact that in clinical laboratory work one has to compromise more often than not. It is of the utmost importance, however, that one realize the real nature and the limitations of the.methods actually employed. The set of standard characteristics set out in this section may be helpful in this respect. 2. Urine Assay us. Blood Assay

It seems that there is a tendency among physicians and biologists to regard hormone blood levels in general as more informative than excretion values, and to accept the latter only as approximations as long as suitable methods for blood assays are not available. Numerous remarks in the discussion sections of published papers are evidence of this tendency, and most laboratory workers will remember having been questioned occasionally about the existing possibilities for hormone analyses in blood, the desirability of the latter being stressed or assumed as self-evident. However, recent advances in this field as well as earlier findings partly invalidate this assumption, or show at least that it has to be carefully examined separately for each hormone or group of metabolites. a. Estrogens. Single high doses of estrogens (up to 24 mg. of estradiol) administered either by the intravenous or intramuscular route, disappear completely from the blood stream within a few hours (Hertz et al., 1949; Rakoff el al., 1944). Fourteen consecutive daily injections of 15 mg. estradiol benzoate in pregnancy fail to raise the estrogen blood level, which remained a t ca. 1-3 I.U./ml. as before the injections (Stroink and Muhlbock, 1948). On the other hand, continuous estrogen secretion in pregnancy maintains the blood concentration in terms of biological activity a t about & to & of the urinary concentration (Lax, 1949; Zondek and Black, 1947); 1.6-3.0 I.U./ml. serum is said to represent the normal range during the last trimester. b. Progesterone and Pregnanediol. Very small amounts of progesterone are found in urine only following its administration in huge doses (Ungar et al., 1951). Progesterone blood levels reported by different workers are extremely low. Butt et al. (1951) were unable to detect any blood progesterone in pregnancy with the notable exception of one woman suffering from infective hepatitis; the sensitivity of their polarographic method is given as 0.1 pg./ml. Haskins (1950) and Dobriner (cited by Butt et al., 1951), using spectroscopic methods with similar sensitivity,

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also reported failures. As an upper limit, these results agree well with the (mostly negative) findings of earlier workers (cf. Pearlman, 1948) and of Hoffrnann and von LBm (1948) who used ordinary or intra-uterine test methods on the rabbit. On the other hand, Forbes (1950, 195.1; intrauterine test on mice) reports blood levels of 0.5-2.0 pg./ml. in pregnancy and 1.7-5.2 pg./ml. during the second part of the menstrual cycle. Whereas most of these results are noteworthy for their contrast to the order of magnitude of pregnanediol excretion, Forbes’ figures are curious for another reason, namely, that they do not show any difference between pregnancy and the non-pregnant state. It is not known whether pregnanediol is present in blood or not. In the absence of a specific test for this compound, its estimation in small blood samples does not a t present seem feasible and would certainly involve the use of the elaborate fractionation procedures developed for the isolation of steroids from tissue extracts. c. Testosterone and 17-Ketosteroids. West et al. (1951a,b) have shown that, following the intravenous administration of testosterone, the blood level of this substance comes down to normal (below 20 pg./ml.) within one hour, whereas its metabolic effect is still notable several days later, and a secondary rise in 17-ketosteroid excretion seems to occur from the 4th to 8th days after injection. Storage in the tissues rather than the blood level must be the explanation for these observations. In this connection, i t is interesting to note the findings of Tschopp (1946) who determined the concentration of 7-methylbisdehydrodoisynolic acid (a very potent synthetic estrogen) in various organs after its administration to rats. The highest concentration was found in the intestine, about half as much in the liver, and only about one-sixth in the uterus. No extended study on the concentrations of androgens or androgen metabolites in the blood of untreated subjects has been reported (cf. Dorfman, 1948). d. Gonadotropins. The concentration of chorionic gonadotropin in normal pregnancy urine has been found to be, on an average, nearly exactly equal to (Loraine, 1950; prostatic weight increase in immature rats), or three times lower than (Gastineau et aZ., 1949; ovarian hyperemia in immature rats) the blood level in the same subject at the same time. During the menstrual cycle available data would seem t o suggest a ratio of similar order of magnitude. e. Conclusions. With the exception of estrogen and gonadotropin in pregnancy, the estimation of sex hormones in blood seems to be of little practical value. This is due (1) to the extremely small concentrations involved and the limitation of available blood volume its well as (2) to the fact that changes in blood level do not seem t o reflect

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very strongly the changes in secretion and utilization which can be inferred from physiological alterations and excretion values. It has long been known that no appreciable storage of hormones occurs in the secretory glands. One is tempted to speculate that the organism tends t o keep also the blood level of hormones (as well as the concentration of most other blood constituents) within low and narrow limits, which might conceivably be limits in solubility (cf. Bischoff and Pilhorn, 1948). 3. Excretion per Unit of Volume us. Excretion per Unit of Time

The vast majority of workers emphasize the absolute necessity of using complete 24-hour urine collections, and of expressing the results in mg. or units per 24 hours. This attitude is in keeping with present concepts of renal physiology. Several investigations have shown that the total amount of estrogen (Koller and Leuthardt, 1941), pregnanediol (Bachman et al., 1941; Henderson et al., 1949; Hoyt and Levine, 1950; Plotz and Darup, 1949; Stern, 1951), 17-ketosteroids (Bachman et al., 1941; Kassenaar et al., 1950; Oesting and Webster, 1938; Reymond, 1951) or chorionic gonadotropin (Hamburger, 1948; Pedersen-Bjergaard and Pedersen-Bjergaard, 1948; Votta, 1946) excreted during a certain period is independent of the corresponding volume of voided urine as well as of the time of day. Consequently, there is no justification for the widespread use of “early morning urine,” except as a matter of convenience. The strong negative correlation between urine volume and 17-ketosteroid concentration existing in younger subjects has been found by Reymond (1951) to disappear with increasing age ( r = +0.213, P > 0.05, in age group 51-80 years). Whereas this observation can perhaps be easily understood, three other reports must be cited that are definitely a t variance with the rest of the evidence. Devis (1949), reviewing his investigations with the method of Dingemanse et al. (1952), made the bald statement that “the 17-ketosteroid concentration in urine seems to be independent from the urine volume and does not decrease with increasing amounts of fluid.” Hollander et al. (1943) found high positive correlation (r = f0.72 - 0.99) between the daily volume of urine and the total excretion of androgens determined biologically in capon units per day whereas the concentration remained constant. McHenry et al. (1947) reported significant correlation ( r = 4-0.4930-0.7669) between daily volume of urine and tot,al output of 17-ketosteroids. The use of creatinine determinations as a check for the reliability of 24-hour collections (Cope, 1940; 0. W. Smith, 1942) has been repeatedly advocated. Presumably, too much practical value has been attributed to this device. Apart from the fact that the average creatinine excretion of an individual must be determined from “reliable” collections before it

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can be used asa check for reliability, it seems that th6daily creatinine output of different subjects may show variation coefficients between 11 and 35%. Moreover, it is evident from the preceding paragraphs that a numerical correction by more than 25 %-the tolerance limit given by Smith-can by no means be regarded as a substitute for a correct 24-hour collection.

4. Bioassay us. Chemical Estimation It should be realized that, strictly speaking, there is no such thing as bioassay of steroid sex hormones in urine. Although this statement may seem paradoxical, the evidence is well known. Estradiol, progesterone, and testosterone are the sex hormones in question. Practically no progesterone is excreted in urine, and its metabolites have no progestational activity. Testosterone, too, is not found in urine; its chief metaholites are androsterone, which possesses about of the androgenic activity of testosterone, and etiocholanolone which is inactive (Dorfman, 1948). Urinary estradiol is accompanied by variable amounts of estrone and estriol, and measurement of the comparative activities of the three estrogens depends, to a very large extent, upon the efficiency of fractionation procedures and the conditions of bioassay (Heard and Saffran, 1949; Pincus, 1948; Rosenmund, 1948). Thus, the bioassay of urine is concerned mainly or only with metabolites of the original hormones; these metabolites may, or may not, exhibit varying degrees of what one is tempted to call incidental biological activity, which in any case is appreciably less than the activity of the native hormone. I n bioassay, most of the methodological characteristics given in Section II,1 are strongly affected by differences in strain, living conditions and age of the test animals, and by several other factors. These points have been fully discussed in recent reviews on estrogens and progesterone (Pincus, 1948), androgens (Dorfman, 1948), and gonadotropins (Li and Evans, 1948; Evans and Simpson, 1950). Adequate experimental design and statistical analysis, and the use of reference standards (international standard preparations, if available) are essential conditions for satisfactory bioassays (Thayer, 1946). “The bald statement of so many rat units or mouse units in a preparation is inadequate unless the limits of variation, the specific test and the specific estrogen [or hormone] are included” (Pincus, 1948). Most chemical methods are cheaper, simpler, more rapid and of greater precision than biological tests which, in turn are often more specific and more sensitive. Refined chemical procedures should not be introduced into clinical practice without the assistance of a trained chemist. “The development of chemical methods depends upon the proof (a) that certain chemical structures with specific properties are

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typical for a hormone and/or its metabolites, and (b) that no other substances present in the extract under consideration show the same properties or interfere with the reaction employed” (de Watteville et al., 1949). Considering all the points and counterpoints, we have come to believe that the balance is strongly in favor of chemical estimation, provided that, by metabolism studies as well as clinical evidence, definite correlations have been jirmly established between the native hormones and the chemical substance or group of substances to be estimated. 5. Some Remarks on Individual Methods for Clinical Use

Complete and critical reviews on existing methods for bioassay and chemical estimation have been published recently by Dorfman (1948), Engel (1950), Evans and Simpson (1950), Li and Evans (1948), Loofbourow (1943), Pincus (1948), Thayer (1946), and no attempt will be made here to duplicate the work of these competent authors. The following remarks are offered merely as a short comment on some methods which seem to be suitable for, or have been frequently employed in, gynecological and obstetrical practice. a. Gonadotropins. Most of the current bioassay methods do not measure the specific activity of any one single gonadotropic factor alone. The organic changes used as end points in these tests (ovarian and uterine weight, vaginal estrus, etc.) are the result of complex interaction and synergism between the gonadotropic factors present in both extracts and test animals. The situation is further complicated by the fact that no international standard is available for either follicle-stimulating hormone (FSH) or interstitial cell-stimulating hormone (ICSH) from animal pituitary extracts, let alone for the similar (but not identical) activity of the principles present in human blood and urine outside of pregnancy. The separate estimation of the gonadotropic factors is highly desirable. In order to exclude the influence of the pituitary, hypophysectomized test-animals must be used. I n such animals, interstitial tissue repair and prostatic weight increase seem to be specific indices of the ICSH activity of injected extracts, whereas FSH activity can be estimated from the histological signs of follicular growth. Two interesting reports should be mentioned here. Nalbandow and Baum (1948) proposed the use of stilbestrol-treated male rats and chicks as a reliable substitute for hypophysectomized animals. Lloyd el al. (1949) claimed to be able to differentiate between “total gonadotropic” and “ICSH” activity in urine by using uterine weight and ovarian hyperemia in immature mice as the respective end points. The results

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of both these reports are suggestive and would bear further investigation. Efficient clinical methods for the quantitative bioassay of chorionic gonadotropin (CG) have been described by Albert and Berkson (1951 ; ovarian hyperemia in immature rats), by Loraine and Gaddum (1950; prostatic weight increase in immature rats), and by Delfs (1941; uterine weight increase in immature rats). Intravenous injection into the jugular vein, instead of the usual subcutaneous or intraperitoneal routes, has been recommended by Sturgis and Politou (1951), as this route avoids problems of absorption and permits a rigid control of the dose. b. Estrogens. The chemical estimation of urinary estrogens is essentially a problem of purification and separation, since relatively large amounts of interfering background material are present in the phenolic fraction. Satisfactory results are obtained with a modified Kober reaction (e.g., Jayle el al., 1949a; Stevenson and Marrian, 1947), unless the estrogen concentration in urine is below ca. 1.0 pg./ml. The much smaller amounts excreted in nonpregnant states necessitate the use of sensitive fluorimetric methods after further purification of the phenolic fraction (cf. Engel, 1950). Despite repeated claims to the contrary, we feel that, except for pregnancy urine, no reliable chemical method for clinical use has yet been presented, but recent preliminary reports and private communications from various sources suggest forthcoming developments in this field. A useful discussion of optimal hydrolyzing conditions has been published by Marrian and Bauld (1951), and Jayle et al. (1949a) have recommended the addition of phosphotungstic acid during hydrolysis in order to precipitate undesired pigments. e. Pregnanediol. Reasonably specific and rapid methods for clinical use include the procedures described by Guterman and Schroeder (1948) ; Jones et al. (1944); Rogers and Sturgis (1950), Sommerville et al. (1948), de Watteville el al. (1948). Less specific for pregnanediol, less sensitive, more laborious, and more susceptible to technical errors are Venning’s (1938) method which was used for most of the pioneer work, and Westphal’s (1944) modification which, however, is extremely sensitive. A qualitative precipitation test has been used with apparent success by Mack et al. (1949). When it is intended to work with the sulfuric acid color reaction for pregnanediol, it should be remembered that only highly purified fractions and reagents, together with the regular use of pure pregnanediol as a reference standard, can be expected t o yield reliable results. It has been shown that the colorimetric values of the pregnanediol fractions obtained in a chromatographic routine method (de Watteville et al., 1948), are practically identical with the weight of these fractions (Borth, 1952). d. 17-Ketosteroids. Recent advances include the preparation, hy

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Wilson and Carter (1947) and by Hamburger (1952), of stabilized potassium hydroxide solution for the Callow modification of the Zimmermann reaction, and the development of very rapid micromethods with specified reliability characteristics by Hamburger and Rasch (1948) and by Vestergaard (1951). Chromatographic separation procedures (Dingemanse et al., 1952; Zygmuntowicz et al., 1951) are extremely time-consuming, but it can hardly be denied t h a t the loss in speed may prove to be compensated by a gain in useful information. e. Other Steroid Fractions. Methods for the estimation of the hitherto somewhat neglected group of the total neutral steroid alcohols (of which pregnanediol is a member) have been developed (Engel et al., 1950; Tompsett and Oastler, 1948) and may prove to be useful in future studies. The same can be said of the estimation of either reducing or formaldehydogenic corticoids. The formaldehyde method seems to be more specific, but neither technique will achieve a satisfactory degree of accuracy unless the conditions of hydrolysis and extraction are thoroughly investigated (cf. Marrian, 1951). Jayle and his associates (Jayle, 1950, 1951a,b; Jayle et at., 1949b; Libert, 1950) have accumulated a tremendous wealth of data correlating various clinical conditions with the glucuronide content of urine fractions extractable into butanol at pH values varying from 2 to 13. The procedure has been called a “butylogram,” and its last step is the 1:3naphthalenediol color reaction for glucuronic acid (cf. Florkin and Crismer, 1940). The results are calculated in milligrams of steroid per 24 hours (pregnanediol equivalents). Twenty-four-hour collections or pooled successive night specimens are used. By far the greater part of the data deals with the fractions extracted a t pH 12-13 from the urine of pregnant and nonpregnant women. Based on clinical evidence, the excretion values as well as some of their ratios are interpreted as joint indices of adrenocortical and luteal functions. Since the mixture of pregnane derivatives excreted as glucuronides is said to reflect adrenocortical as well as luteal activity, the latter two cannot, and are not meant to be differentiated by this kind of analysis. More recently, metabolism studies have shown that the administration of progesterone increases the acetone-insoluble glucuronides extracted a t pH 13 (which, of course, correspond to Venning’s pregnanediol glucuronidate fraction), whereas the administration of testosterone increases the acetone-soluble glucuronides of the same fraction and, to a lesser degree, the fraction extracted a t pH 11. Cortisone metabolites enter the pH 11 and pH 6 fractions. It is felt that this approach to clinical problems as well as the great bulk of accumulated data requiring a special terminology, are beyond

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the scope of the present review. The reader is referred to the original articles.

111. MENSTRUAL CYCLEAND MENSTRUAL DISORDERS

I. Hormone Excretion during the Normal Menstrual Cycle The partly hypothetical, present concept of ovarian function may be briefly summarized as follows (Evans and Simpson, 1950; Freeman, 1950) : FSH stimulates follicular growth, synergized by small amounts of ICSH, to produce follicle ripening and estrogen secretion which, in turn, induces vaginal and endometrial proliferation. Rising estrogen secretion decreases the FSH output from the pituitary and releases larger quantities of ICSH which luteinize the ovary. Prolactin, released under the same stimulus, renders the corpus luteum functional; the latter begins to secrete progesterone, and maintains estrogen secretion. Both steroid hormones transform the proliferated endometrium into the secretory stage and gradually inhibit pituitary secretion until, in the absence of conception, involution of the corpus luteum occurs. The ensuing withdrawal of estrogen and progesterone causes the endometrium to disintegrate down to the basal layer, leading to a discharge of nonclotting blood, the menstrual flow. While the latter is still in progress, the ovarian cycle starts again. Ovulation depends upon the complex interaction of several factors, including the well-timed and well-graded secretion of FSH and ICSH (and, possibly, also of prolactin). There is general agreement about the fact that these rhythmic physiological changes occurring in healthy young women with normal variation in cycle length (Goldzieher et al., 1947) are more or less closely associated with changes in the excretion of the sex hormone metabolites. However, the degree of established correlation is not the same for all the factors involved. a. Gonadotropins. The most frequent observation seems to be one excretion maximum near midcycle, but several workers report one, two, or even more additional peaks not only during the second part of the cycle, but also sometimes altogether irregularly distributed throughout the whole cycle (Evans and Simpson, 1950; Joel, 1945; Pedersen-Bjergaard and Pedersen-Bjergaard, 1948 ; Pedersen-Bjergaard and TQnnesen, 1948). However, when looking at some of the published curves one gets the impression that perhaps not all the “peaks” are significantly above the range of daily variability. In this view, the finding of a multiplicity of peaks and the failure to detect any significant excretion maximum are one and the same, and would be expected in the absense of ovulation. On the other hand, the possibility of more than one ovulation occurring

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in one cycle has been suggested repeatedly (cf. Joel, 1945); it is certainly far less remote a possibility than the older school of gynecology would have it. I n discussing gonadotropin excretion during the cycle, it should not be overlooked that nearly all the published data (Evans and Simpson, 1950) have been obtained with methods which are not specific for any one of the three gonadotropic factors, and which, therefore, are not adequate in following their complex interplay. Preliminary results reported more recently suggest peak excretion of ICSH in midcycle (Lloyd et al., 1949; ovarian hyperemia) and of prolactin (180 I.U./24 hrs.) after mid-cycle (Coppedge and Segaloff, 1951;crop sac response). Further studies of this kind are urgently needed. The Farris test for ovulation (Farris, 1946) will be discussed in Section IV, 1. The findings of Corner et al. (1950) leave little doubt that ovulation is indeed correlated with a positive reaction, i.e., with the excretion of urinary material inducing ovarian hyperemia in immature rats. b. Estrogens. The normal excretion may vary considerably from day to day and from one cycle to another, but the generally accepted trend is that the values are low or zero a t the time of menstruation and that they gradually reach a peak (400-800 I.U./24 hrs.) somewhere shortly before mid-cycle; they drop before the onset of flow (Pedersen-Bjergaard and TGnnesen, 1948). Whether a secondary “peak” occurs during the second part of the cycle, or whether the excretion is maintained at moderately high but varying levels, does not seem to be a very relevant distinction. An average of 5000-10000 I.U. are excreted in one cycle (D’Amour, 1943). The evidence for a time-correlation between gonadotropin and estrogen peaks is by no means conclusive and only very slightly in favor of the view that the latter often precedes the former by one or two days (Evans and Simpson, 1950; Pedersen-Bjergaard and PedersenBjergaard, 1948; Pedersen-Bjergaard and Tgnnesen, 1948). Separate estimation of estradiol, estrone and estriol excretion may be expected to yield important information on clinical as well as research problems, but it seems that the earlier studies in this field should be repeated with the aid of improved techniques of fractionation (Heard and Saffran, 1949). A re-investigation of the effect of progesterone on estrogen metabolism (Smith et al., 1943) would be particularly interesting. c. Pregnanediol. Although the method of Venning (1938) has yielded very unsatisfactory results in the hands of several other workers (e.g., Hamblen et al., 1942) and has been replaced by more efficient techniques, it should be noted that most of the original findings of Venning and Browne (1937) have been confirmed in later investigations. Urinary pregnanediol (1-7 mg./24 hrs.) is present only during the second part

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of the menstrual cycle, and it disappears again 1-3 days before the onset of flow (Jones et al., 1949; McKelvey and Samuels, 1947; Plotz and Darup, 1950; Rogers and Sturgis, 1950; de Watteville, 1951). The quantities deriving from adrenal cortex precursors in healthy women are much too small to interfere with pregnanediol determinations in 24- or 48-hour collections of urine; this is true even after moderate stimulation of the adrenals by ACTH (de Watteville et al., 1951). The association of ovarian corpus luteum activity with pregnanediol excretion has been firmly established by histologic evidence (Brown and Bradbury, 1947; Buxton, 1940; Siegler and Bauer, 1943; de Watteville, 1949a, 1951). Daily excretion may vary considerably and does not seem to follow a definite pattern. Occasionally, even a rare zero 24-hour value has been seen in a sequence of normal levels (Buxton, 1940; Kaufman and Westphal, 1947; de Watteville, 1950a); the use of 48-hour urine collections is therefore indicated. The total excretion during the entire luteal phase is also variable; values of 14-45 mg. (Jones et al., 1949),5-24 mg. (Kaufmann and Westphal, 1947), 25-31 mg. (de Watteville, 1950a) have been reported. The possibility that the luteinized theca just prior to ovulation might secrete small amounts of progesterone (appearing as urinary pregnanediol) cannot be excluded, but the evidence presented so far in support of this hypothesis does not seem entirely convincing (Dibbelt, 1949; Plotz and Darup, 1950; Rogers and Sturgis, 1950; de Senarclens, 1948). d. 17-Ketosteroids. Cyclic variation of androgens or 17-ketosteroids during the menstrual cycle has not been observed (Devis, 1949; Dorfman, 1918; Furuhjelm, 1948b; Hamblen et al., 1939). These consistent findings are interesting with regard to the observation of Gaarenstroom and de Jongh (1916) that, in rats, ovarian androgens play a part in follicular maturation and ovulation. e. Conclusions. The normal menstrual cycle is characterized by rhythmic variation in gonadotropin and estrogen excretion and by the appearance of urinary pregnanediol in the luteal phase. Daily as well as individual variability is great, and whether the rhythm of a cycle is normal or abnormal w t h respect to estrogen and gonadotropin excretion could be established only by frequent or daily analyses. With regard to the gonadotropins, even such a tedious procedure could not be expected to yield satisfactory results, unless reliable clinical methods for the separate estimation of the three gonadotropic factors become available, and their interplay is better understood. On the other hand, the presence of pregnanediol can be readily ascertained by one or two determinations. Its appearance in urine depends entirely upon the hypophyseal and ovarian processes preceding corpus luteum formation.

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Therefore, the detection of pregnanediol gives implicit information that ovarian as well as pituitary gonadotropic functions are in order. The presence of urinary pregnanediol is not only an essential, but also a sufficient characteristic of the qualitatively normal menstrual cycle; varying degrees of luteal deficiency are, however, conceivable and will be discussed in Section 111, 3. 2. Dysmenorrhea and Premenstrual Tension

Various factors, most of them not of endocrine origin, may contribute to the occurrence of dysmenorrhea (submucous myoma, inflammation, hypoplasia, sympathetic neuritis, psychogenic factors; cf. Davis, 1938), and hormone assays would seem to be of little value. However, some hormonal relationship must be involved, since most authors insist that estrogen or androgen treatment early in the cycle suppresses ovulation and makes the next bleeding painless. On the other hand, luteal deficiency (cf. Section 111, 3) must also be assumed t o contribute. Endometrial biopsies in 150 cases of dysmenorrhea showed persistence of the proliferative phase in 42, and hyperplasia or atrophy in 11% of the cases (Marln Bonachera, 1951). Favorable results have been obtained in some cases by the administration of progesterone. A sound basis for the latter therapy in the individual case should always be established by endometrial biopsy or pregnanediol estimation. To suppress ovulation by estrogen or androgen treatment must be considered as an unphysiologic intervention, which cannot be repeated indefinitely. Therefore, the diagnostic selection of the patients with luteal deficiency, likely to benefit from progesterone therapy, serves a good purpose. The same approach may prove to be useful in cases with premenstrual tension or other periodic complaints associated with the rhythm of the menstrual cycle (megrim, acne, eczema). Morton (1950) has reported an estrogen-progesterone imbalance with a relative excess of estrogen due to diminished luteal activity in 29 patients complaining of premenstrual tension.

3. Irregular and " Anovulatory " Bleeding The usual symptomatic classification of ovarian disorders according to the type of bleeding (oligomenorrhea, hypermenorrhea, etc.) covers a variety of underlying causes. Some endocrine disorders may produce the same type of bleeding but various types of secretion (B&l&reand Simonnet, 1948). Causal therapy cannot be instituted unless one is able to determine correctly the etiologic factors. The question is, how far can hormone assays be useful in this respect? a. Regular Bleeding-Luteal Deficiency. Regular bleeding as in the

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normal menstrual cycle may occur from a proliferated endometrium in the absence of progestational changes. In such cases, both premenstrual endometrial biopsy and pregnanediol determinations reveal the absence of progesterone activity (e.g. Browne et at., 1947; de Watteville, 1951). The term “anovulatory cycle” is frequently used for this condition, but luted dejiciency or similar terms seem to express more closely the actual findings. In some cases, it is important to remember the distinction that urinary pregnanediol indicates the presence of progesterone in the body, whereas endometrial biopsy and vaginal smear reflect the response of the target organs. The latter may be absent in cases of reduced responsiveness, despite normal progesterone secretion. Occasionally, urinary pregnanediol may be found without endometrial response, despite the presence of an active corpus luteum as proved by ovarian biopsy (Plots and Darup, 1950; de Watteville, 1951). Such cases are rare, but they explain why some authors have advocated the combined use of more than one method (e.g., Burger and Roth, 1951; S6guy and Robey, 1948). For several reasons, however, urine analyses for pregnanediol must be considered as complementary or even superior to endometrial biopsy and other methods. Biopsies cannot be made in all patients, nor can they be frequently repeated ; moreover, proliferated and secretory tissue may occasionally be present at the same time (Bartelmez, 1931; Buxton, 1940). The evaluation of vaginal smears with regard t o progesterone activity is often equivocal and always difficult (Burger and Roth, 1951). Both these methods as well as basal temperature curves do not give quantitative information, whereas the duration and, to a lesser extent, also the degree of ovarian Iuteal function can be estimated from several pregnanediol determinations without any inconvenience greater than the collection of 24-hour urine specimens. This is especially valuable in view of the fact that, in health and disease, the hormonal pattern of a subject may vary considerably; for instance, normal and anovulatory cycles may alternate in otherwise perfectly healthy women. b. Shortened Cycles. Pregnanediol is not excreted before the 13th day of the cycle, regardless of the length of the cycle. This finding is in agreement with the histological evidence of Goecke (1942) who states that in shortened cycles the secretory phase of the endometrium is usually first seen between the 14th and 16th days. It seems that luteal deficiency under the form of a shortened life-time of the corpus luteum can be responsible for the occurrence of shortened cycles. Correlating estrogen and pregnanediol determinations with endometrial biopsies, vaginal smears, and basal temperature curves in women complaining of premenstrual tension, Morton (1950) found an estrogen-progesterone imbalance with a relative excess of estrogen due to diminished luteal activity. In 8 of the

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29 patients, the menstrual cycle was shortened. Apparently the higher frequency of bleeding in such cases is a consequence not of increased ovarian activity but rather of deficient luteal function. The vaginal smears of these women frequently give the impression that estrogenic activity also is reduced during the first part of the cycle. To substantiate this impression, estrogen assays by refined methods would be necessary. c. Irregular Bleeding. Hormone assays have shown also that most types of irregular bleeding are associated with luteal deficiency. With the one exception of irregular shedding of the endometrium due to persistent corpora lutea (oide infra), urinary pregnanediol is absent in the vast majority of patients with functional uterine bleeding. PedersenBjergaard and Tonnesen (1951) have studied gonadotropin and estrogen excretion in 222 cases of menstrual anomaly of various types (oligo- and polymenorrhea, hypo- and hypermenorrhea, mixed types). The estrogens were never increased, they were reduced in 23% of the cases (especially in hypo- and/or oligomenorrhea), and gonadotropin titers higher than normal were found in only 6%. The authors conclude that disturbances in gonadotropin and estrogen production play a subordinate part in these menstrual disorders. In metropathia haemorrhagica, on the other hand, prolonged and profuse bleeding from a hyperplastic endometrium is usually thought to be due to unopposed estrogenic hyperactivity of persistent follicles. Increased estrogen excretion has indeed been reported in such cases (BBcl2re and Simonnet, 1949; Furuhjelm, 1948a), but may not be an invariable finding (Mayer, 1950), since in experimental animals the prolonged administration of very small estrogen doses leads to extreme hyperplasia of the endometrium (cf. von Wattenwyl, 1944). It should be emphasized that bleeding may occur (a) when estrogen secretion is still high but nevertheless insufficient for the maintenance of the strongly proliferated tissue, or (b) when estrogen secretion decreases gradually as the follicle is becoming atretic. In analogy t o the persistent follicle, persistent corpus luteum has become a common term. This rare disturbance leads to the typical symptom of irregular shedding from a progestational endometrium. The presence of urinary pregnanediol at the time of bleeding is conclusive evidence for accepting delayed involution of the corpus luteum as the etiologic factor (McKelvey and Samuels, 1947; Plotz and Darup, 1950). d. Conclusions. With the one exception of irregular shedding from a progestational endometrium, “anovulatory ” and some shortened cycles, as well as various types of functional uterine bleeding, are ‘associated with luteal deficiency or complete absence of the luteal phase. This can be readily ascertained by pregnanediol determinations. Future studies and the use of more refined assay methods for the estrogens and gonado-

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tropins are needed. They may be expected to throw more light on the hormonal pattern in these menstrual disorders, thereby creating a sound basis for substitution therapy, instead of the mostly empiric rules used a t the present time.

4. Amenorrhea The usual clinical distinction between primary amenorrhea (in which menstruation has never occurred) and secondary amenorrhea (preceded by a sequence of periodic menstruations) is useful also with regard to hormonal considerations. In sexually mature women, a missed menstruation is usually the first sign of pregnancy; this condition will be discussed in Section V. In the rare cases of pseudopregnancy due to persistent corpus luteum, urinary pregnanediol is, of course, present (Guterman, 1945; McCormack, 1946; Plotz, 1949), and several authors have even reported positive Aschheim-Zondek or Friedman reactions indicating an increased excretion of CG or ICSH (cf. Plotz, 1949). Israel (1942), however, has rightly emphasized the difficulty of differentiating with certainty between pseudopregnancy and ectopic pregnancy or, we may add, early abortion. It seems that the diagnosis of true persistent corpus luteum is somewhat more likely if pregnanediol is found in the presence of a negative pregnancy test. Except for this rare condition, and in the absence of severe hyperfunction of the adrenal cortex (see Section VI), urinary pregnanediol is never found in secondary amenorrhea. Estrogen excretion has been reported to be low, or normal, or even high (BBclhre and Simonnet, 1949; Kaser, 1949; Mayer, 1950; PedersenBjergaard and Tgnnesen, 1951). In an extended study, PedersenBjergaard and Tdnnesen (1951) have found levels within the range of the normal menstrual cycle in one third, and reduced levels in two-thirds of their patients. This evaluation is based on the average values per subject per day. In following the daily output in a number of these patients, considerable variation, sometimes of cyclic nature, has been observed. The same applies to the gonadotropin excretion in these patients. Their average daily values were higher than normal in 16% and within the normal range of the cycle in 84%. Normal as well as increased levels are reported also by B6clhre and Simonnet (1949). In the five cases of Kiiser (1949), excretion was reduced. I n primary amenorrhea, apart from the fact that pregnanediol is also absent, the most consistent finding is low estrogen secretion, which may or may not be associated with increased gonadotropin levels (cf. Freeman, 1950; Lisser et aZ., 1947). In the already mentioned study by PedersenBjergaard and Tgnnesen (1951), only 19% of the patients showed estrogen and gonadotropin excretions within the normal range.

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These results are not as confusing as they may seem. In the presence of hypophyseal deficiency, stimulation of the ovary by FSH is lacking or reduced, and both gonadotropin and estrogen secretions fall below the normal level: this is amenorrhea of pituitary origin. If, on the other hand, estrogen is absent or reduced in spite of normal or increased gonadotropin excretion, it can be concluded that the amenorrhea is due to an ovarian failure to respond. The high levels of gonadotropin frequently found in this condition may be explained by uninhibited FSH secretion in the absence of estrogens, in analogy to the findings in menopausal women (see Section III,5). However, this simple correlation does not obtain in all cases. Presumably, the actual pattern is more complex and may pass through various stages as the disturbance develops. The observations of Pedersen-Bjergaard and Tfinnesen (1951) support the opinion that subliminal rhythmic variation in hormonal activity may occur and-while inadequate in maintaining cyclic bleeding-may cause various types of urinary hormone titers in the same patient, depending upon the time of analysis. These considerations, in addition to the possibility of reduced responsiveness of the endometrium, may account even for the occasional finding of high estrogen excretion. Conclusions. Amenorrhea results from an involved interplay of several factors, among which primary pituitary or primary ovarian failure can sometimes be recognized by means of estrogen and gonadotropin assays. However, the hormonal findings may vary not only from patient to patient, but also in the same patient according to duration and stage of the disturbance. Extreme caution is necessary in the interpretation of single hormone assays. With the exception of pseudopregnancy due t o persistent corpus luteum, no urinary pregnanediol is found in amenorrhea, provided that severe adrenocortical hyperfunction can be excluded. 5. Menopause

In physiologic as well as in artificial menopause, both a deficient estrogen secretion and an excess of gonadotropins have been definitely established (Heller et al., 1944a; Pedersen-Bjergaard and Tgnnesen, 1948; and many others). It should be noted, however, that the increase in gonadotropins does not appear in all cases, and that estrogen excretion even after surgical castration does not drop to zero but only to very low levels (10-20 I.U./24 hrs.). The latter fact suggests an extragenital (adrenocortical ?) source of estrogen. Both FSH and estrogen assays have been recommended as a check for the success of castration by X-rays (Nathanson et al., 1940). The appearance of menopausal symptoms (hot flushes, dizziness, etc.) has, of course, first been thought to be due to the withdrawal of estrogen.

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Later, the increasing production of gonadotropin has also been held responsible (Albright, 1936; Rust and Huber, 1940). The question does not seem to be settled, although Heller et al. (1944b) have shown that in surgical castrates the menopausal symptoms are alleviated by the administration of stilbestrol in doses too small t o depress gonadotropic excretion. It should not be overlooked that so-called menopausal symptoms may be caused by extragenital disorders (e.g., arteriosclerosis), tenhng t o appear in higher incidence a t the same age. In order t o avoid the useless or harmful administration of estrogens in such cases, the degree of estrogenic activity should be ascertained before starting a therapy. Until more accurate clinical routine methods for estrogen assay become available, the use of vaginal smears is the method of choice.

IV. STERILITY The cooperation of a multiplicity of biologic factors is required for conception to occur. Sterility, a problem of increasing importance in gynecology, may be due t o the deficiency or absence of any, or any combination, of these factors, most of which are beyond the scope of this review. Only two of the essential conditions will be considered: ovulation, and the formation of a functional corpus luteum, since both are concerned with hormonal regulation in the female. 1 . Time of Ovulation

It is, of course, not feasible to determine the time of ovulation by direct observation. Only indirect methods are available, which are based on the detection of correlated physiologic changes, e.g., in basal temperature or in vaginal cytology. The shortcomings of indirect methods are obvious, as the degree of correlation is subject to biologic variation. In order t o reduce the risk of errors, one should make it a habit t o use a combination of different methods (Sturgis, 1950), since i t is unlikely that all of them would be affected simultaneously by the same source of error. It seems that in some cases of sterility the most important problem is not t o detect the event of ovulation after it has occurred, but to predict it with a reasonable degree of certainty. According to our present knowledge as far as hormone assays are concerned (see Section 111, l ) , the only hope of achieving this would be by detecting the particular combination of gonadotropic factors upon which ovulation is thought t o depend. Several workers have claimed to be able t o do so. As early as 1934, Kursrok et al. (1934) were able t o correlate increased excretion of “FSH” with ovulation, and D’Amour (1943) came to the conclusion that the gonadotropin peak occurring in mid-cycle is the best indicator of ovulation time. The most recent development in this field is the test proposed by

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Farris (1946). Ovarian hyperemia in immature rats is said to be induced by the injection of 2 ml. of urine collected on 2-4 successive mornings prior to ovulation. Strictly standardized conditions and a single, tested, strain of animals must be used. The claim that a normal positive test indicates ovulation is based mainly on the incidence of conceptions resulting from intercourse or artificial insemination which have been timed according to the results of the test, and on the incidence of failures to conceive in the absence of positive tests or correct timing (Farris, 1916, 19-18; Murphy and Farris, 1947). Levin et al. (1949) have severely criticized the method and have questioned the validity of some of the reported results, apparently with some justification. On the other hand, increased amounts of hyperemia-producing material have been found to be escreted in mid-cycle, but extracts corresponding to at least & of 24-hour urine collections had to be injected to produce positive reactions; the mice used did not respond to unconcentrated urine (Lloyd et al., 1949). Riley et al. (1948) have produced ovarian hyperemia in rats with 2 ml. of urine collected on the 14th day of the cycle. Recently, Corner et al. (1950) have compared the Farris test with ovarian and endometrial histology in 39 women. The times of ovulation as estimated by the three methods agreed (mostly within zk 1 day) in 30 cases. The Farris test was negative in 3 cases in which no ovulation had taken place, but, it was positive in the remaining 6 cases in which early atresia of the follicle was seen a t the expected time of ovulation. No negative Farris test was obtained in the patients with proved ovulation. This distribution of the 39 cases has a probability of chance occurrence (Fisher, 1950) of P = 0.0092, and consequently, the correlation between the results of the Farris test and the histologic evidence must be considered as significant in this series. The authors draw special attention to the six discordant cases in which the histologic findings indicate “the existence of a special kind of ovarian crisis, characterized by the retrogression of a large follicle that has presumably developed normally up to a few days before ovulation should have taken place.” The applicability of the Farris test has to be further investigated. I t might well he that Farris is working with a strain of rats particularly sensitive to urinary gonadotropins; this possibility would explain also the high incidence of false positive reactions this worker has observed in an evaluation of the two-hour hyperemia test for pregnancy (Farris, 1944). 2. Control of the Luted Phase

The nidation of the fertilized ovum depends upon the presence of an endometrium in the secretory stage, and it cannot OCCUT unless progesterone is secreted in sufficient amounts. Luteal deficiency with “anovula-

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tory” or shortened cycles, as discussed in Section 111, 3, is rather frequently encountered in sterility cases. The routine use of pregnanediol determinations, in order to ascertain or exclude this condition, is indicated and has indeed been repeatedly recommended for the investigation of female sterility (Henderson et al., 1949; Mack el al., 1949; Sturgis, 1950; de Watteville, 1951). 3. Conclusions In female sterility, the usefulness of pregnanediol determinations as a control of the luteal phase is well established. The Farris test for the prediction of the time of ovulation requires further investigation, including an appraisal of sensitivity variation in different strains of test animals.

V. PREQNANCY The numerous morphologic and functional changes associated with pregnancy are by far the greatest alterations known to occur in normal physiology. There is abundant evidence that these changes are caused and regulated by the functional activity of the trophoblast. The chorionic villi secure the normal development of the growing fetus not only by feeding it from the maternal circulation, but also by influencing the maternal organism through endocrine stimuli, which result, inter a h , in the maintenance of the corpus luteum graviditatis, and in the growth and relaxation of the uterus. It is not surprising, therefore, that several complications of pregnancy originate from dysfunctions of the trophoblast. The fact that the chorionic villi produce CG is well established (Evans and Simpson, 1950), and there is sufficient evidence to show that the estrogens of pregnancy urine and blood are also of placental origin (Pearlman, 1948). The evidence suggests that this may also be true of progesterone, although this compound has not been isolated from placental tissue* (Pearlman, 1948; Pincus and Pearlman, 1943). It may, however, be mentioned that, contrary to our present concept, some workers have argued the hypothesis that the large amounts of steroids excreted in late pregnancy might be of adrenocortical origin (Devis, 1950; Jayle, 1951a; Rak, 1949). 1. Hormone Levels in Normal Pregnancy The earliest and most spectacular change is the steep rise of CG, beginning shortly before the date of the first missed menstruation (Evans and Simpson, 1950 ; Pedersen-Bjergaard and Pedersen-Bjergaard, 1948). Peak excretion of the order of 30,000-400,000 I.U./24 hrs. is reached * The isolation of progesterone from full term human placenta has been reported recently by H. A. Salhanick, M. W. Noall, M. X. Zarrow and L. T. Samuels (1952, Science 116,708-709),and by W.H. Pearlman and E. Cerceo (1952, J. Biol.Chern. 198, 79-82).

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between the 7th and 12th weeks. A t the same time, the concentration in serum attains levels of 200-600 I.U./ml. From about the 15th week onwards until term, fairly constant levels in urine and blood are maintained (about 5000-10,000 I.U.124 hrs., and 5-35 I.U./ml., respectively) (Albert and Berkson, 1951; Jones et al., 1944; Loraine and Gaddum, 1950). The average excretion may rise slightly with the approach of term (Loraine and Gaddum, 1950; Venning, 1946). In three pregnancies of two women, Pedersen-Bjergaard and Pedersen-Bjergaard (1948) have reported a second excretion maximum shortly before parturition. This rise in output was equally high but very much briefer than the increase in the second month-so brief, the authors state, that it might have been missed by the omission of merely one 24-hour urine. Both estrogen and pregnanediol excretion show an opposite trend, rising slowly from the values of the normal cycle to reach maximal output near term. At that time, the following normal estrogen levels have been reported: 1.6-3.0 I.U.per ml. of serum, 12-40 I.U. per ml. of urine (Zondek and Black, 1947; bioassay), 12-40 mg.124 hrs. (Venning, 1948; colorimetric estimation). More than 90% of the estrogens excreted in late pregnancy is estriol (e.g., Smith and Smith, 1941). The absolute values reported for pregnanediol vary somewhat according t o the method used. For the peak excretion in the last month, the following approximate averages and standard deviations have been calculated or estimated from published data or graphs (mg./24 hrs.): 79 f 22 (Venning, 1948); 73 f 12 (Davis and Fugo, 1947); 62 k 21 (Michie, personal communication); 50 k 12 (Plot2 and Darup, 1950); 49 k 12 (de Watteville, 1951); 22 f 22 (Kaufmann et al., 1951). It is interesting to note that the relative increase of pregnanediol in pregnancy is about tenfold, whereas that of the estrogens (in terms of weight) is about five hundred fold. Androgen, androsterone, and etiocholanolone excretions have been found t o decrease with advancing pregnancy, whereas a slight increase of the ketosteroids is seen when they are measured by means of the Zimmermann reaction; this is apparently due to the presence of large amounts (up to 40 mg./24 hrs.) of pregnanolone (Dobriner et al., 1948; Hain, 1939; Venning, 1946). The latter compound parallels the excretion of pregnanediol and contributes appreciably t o the “sodium pregnanediol glucuronidate” fraction obtained in the Venning method and similar techniques (Sutherland and Marrian, 1947). Burrows et aE. (1942) have tried to correlate 17-ketosteroid concentration of morning urine in early pregnancy with the sex of the fetus; despite the lack of statistical significance, their data seem suggestive, but no reinvestigation of the problem seems t o have been published. The output of glycogenic as well as formaldehydogenic corticoids is

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increased during the last trimester; the former show a smaller peak also at the time of the rise in CG (Tobian, 1949; Venning, 1946). After delivery of the placenta, CG, estrogens and pregnanediol drop immediately to lower values and disappear from the body fluids within a. few days. No consistent trend preceding the onset of labor in the individual case has been observed, although, on an average, pregnanediol excretion may decrease slightly during the last two weeks (Jayle, 1950; Smith et al., 1946; Venning, 1948; de Watteville, 1951). Conflicting reports on this question seem to reflect only the wide range of normal variation (Bachman, 1941 ; Giardinelli, 1949; Hain, 1942; Kaiser, 1951 ; Lyon, 1946; Mauzey, 1950; gstergaard, 1940; Pigeaud and Dubreuil, 1947). Conclusions. Early pregnancy is characterized by a tremendous transitory increase of CG and by the maintenance of luteal phase levels in estrogen and pregnanediol excretion. After the 15th week, CG remains at approximately constant lower levels, while estrogens, pregnanediol, and pregnanolone rise gradually until term. 2. Diagnostic Tests f o r Pregnancy The purpose of pregnancy tests is to decide whether amenorrhea is due to pregnancy or not. They differ from other hormone assays in that no graduated result is wanted: the answer must be yes or no, and equivocal or intermediary results are useless. The alternative nature of the diagnostic problem and its clinical and psychological importance are the reasons why only the highest standards of reliability are acceptable. Moreover, the diagnosis should be obtained very early, preferably a few days after the date of the missed menstruation. After three or four months of amenorrhea, the changes in the maternal organism are such that several diagnostic signs are available, and the right diagnosis can hardly be missed. The hormonal activity of the young trophoblast meets these requirements. The following brief remarks will be limited to certain aspects of the subject and are offered mainly for the sake of completeness. An excellent discussion of pregnancy tests has been published recently by Robbins (1950). References to the various methods can be found in the review by Evans and Simpson (1950). a. Chorionic Gonadotropin. It is evident from the facts given in Section V,l that the pregnancy tests based on the sharp rise in CG secretion are by far the most satisfactory. For most of them, accuracy is cited as between 96 and 100%. A great deal of confusion regarding their comparative merits could be avoided if the sensitivity levels actually

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tested were always given in LIT. per 24 hrs. or per milliliter of urine or serum, instead of the bald statement of positive or negative reactions. The following values of EDoo(dosage of hormone effective in inducing a response in 50% of the test animals) have been reported: corpus luteum formation in the rabbit, 5 I.U. (Albert, 1949) or 15 I.U. (Houssay, 1949) : ovarian hyperemia in the rat, 0.6-20 I.U., depending on strain, technique, and time of reading (Aschheim and Varangot, 1945; Albert and Berkson, 1951; Fried, 1949; Sturgis and Politou, 1951; Zondek and Sulman, 1947); discharge of spermatozoa in the toad Bufo arenarum Hensel, 22.5 !I.U. (Houssay, 1949); ovulation in the frog Xenopus laevis, 50-100 1.U. (Weisman et al., 1942). b. Pregnanediol. The main sources of error for the Guterman test and other techniques based on pregnanediol excretion are the occurrence of delayed menstruation, and the existence of a luteal cyst or a persistent corpus luteum, leading to false positive results; the latter is a very rare condition. It has been rightly emphasized that the presence of pregnanediol shortly after the date of the missed menstruation is suggestive of pregnancy only after a sequence of regular normal cycles (Guterman, 1945; Mack et al., 1949). If no pregnanediol is found, it is pract'ically certain that no intact pregnancy is present, provided that reliable methods and sufficiently large aliquots of 24-hour urine are used (cf. Sections 11, 3 and 11, 5). Obvious technical errors account for a t least some of the confusion in this field (Gene11 and Jensen, 1949; Kullander, 1948; Morrow and Benua, 1946; Reinhart and Barnes, 1946; cf. Guterman and Schroeder, 1948). With the exception of Kaiser (1950), Merivale (1948) and Swyer (1949) who used morning urine and/or the old qualitative Guterman technique, most other authors (even some of those using morning urine and crude methods) have accepted pregnanediol determinations as an aid in pregnancy diagnosis, although the inherent limitations of the method have also been stressed (Bradbury et al., 1950; Buxton, 1940; Guterman, 1945; Henderson et al., 1949; Jayle, 1950; McCormack, 1946; Mack et al., 1949; Pigeaud, 1951; %guy et al., 1950a; Semmons and McHenry, 1949; Soule and Yanow, 1949; Stern, 1951). c. Estrogens. Estrogen assays cannot be used for the early diagnosis of human pregnancy. This is due to technical difficulties (cf. Section 11, 5 ) as well as to the occurrence of secondary amenorrhea associated with normal or high estrogen output (cf. Section 111, 4). d. Conclusions. The detection of the steep rise in CG is the method of choice for the early diagnosis of pregnancy, especially since very rapid biologic methods have become available. It should be realized that several of the latter require some experience and the maintenance of rigidly standardized experimental conditions. Attention should be given

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to differences in sensitivity. Pregnanediol determinations are less reliable, although negative results practically exclude the presence of intact pregnancy. If adequate techniques are used, it appears that the frequency of occurrence of delayed menstruation is the limiting factor in correct positive diagnosis. 3. Abortion and Premature Labor

With the disturbance or cessation of trophoblast function, hormone secretion diminishes and hormone levels decrease. This always happens when premature interruption of pregnancy occurs, but it may happen for various reasons and at various times. Lethal factors, malformations of the uterus, mechanical manipulations, or contractions of general uterine origin may be the etiologic factor. In such cases trophoblast function may remain undisturbed for some time, the hormone levels may be high in spite of bleeding and contractions, and they may decrease only later when the ovum is already detached, dead, or expelled. Extreme cases of this kind have been described by Venning (1948) and by Zondek (1947). On the other hand, deficient activity of the trophoblast or ovarian failure may be the primary cause of the disorder. An early drop or consistent downward trend in hormone levels may occur even when clinical symptoms are still few or absent. In these cases, eventual miscarriage is apparently due to hormonal disturbances-progesterone withdrawal with insufficient uterine relaxation, estrogen deficiency with retarded growth and lack of hyperemia of the uterus, or CG deficiency with failure t o maintain the corpus luteum graviditatis and its steroid secretion. Hormone levels in pregnancy reflect, to a certain extent, the functional state of the trophoblast and, during the first weeks, of the ovary. Hormone assays can be used only to control the secretory activity at the time of assay and to follow its trend, which may or may not be indicative of the subsequent clinical development. Assumptions regarding the presence, fate, or absence of a living fetus are, strictly speaking, a matter of conjecture, although a high degree of correlation between clinical and laboratory findings may frequently permit one to draw valid conclusions. It should be realized that the purpose of hormone assays is not simply to confirm the clinical diagnosis of disturbed pregnancy, but rather to judge the severity of the condition and to assist in the choice of therapy. a. Chorionic Gonadotropin. The pregnancy tests may frequently remain positive despite deficient trophoblast activity, because the CG level, though decreased, may still be beyond the low threshold usually tested. The combined use of two tests with different sensitivities may serve as an approximation to quantitative bioassay (Eichenberger, 1948 ; Gerlach, 1941;Joel, 1946), but quantitative or semi-quantitative methods are, of course, more informative. From a series of 500 cases, Zondek et al.

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(1948) conclude that the rough titration of urinary CG (ovarian hyperemia in four rats injected with 4.0, 2.0, 1.0, and 0.2 ml. of urine, respectively) is of considerable diagnostic value in threatened and missed abortion. I n the last trimester, fetal death is practically certain if the concentration drops t o one-tenth of the normal level. The necessity of following the trend rather than of relying on a single assay is emphasized. Working with various methods, several other authors have come to similar conclusions (Bedoya and Jimhez, 1950; Behnken el al., 1948; Farris, 1950; Hinglais and Hinglais, 1950; Kaser, 1946; Baser and Eichenberger, 1949). b. Estrogens. Spielmann et al. (1933) have reported a sharp drop in blood estrogens in cases of missed abortion. Their favorable results do not seem t o have stimulated the application of their test by many other workers, although Zondek et al. (1948) have strongly recommended its routine use in cases of threatened abortion. Assays of urinary estrogens have not been widely employed either, although it has been reported that the excretion decreases rapidly in cases of inevitable abortion or fetal death (Kaser and Eichenberger, 1949; Leuthardt and Koller, 1944; Mayer, 1950). Further studies would be desirable. c. Pregnanediol. The clinical significance of pregnanediol excretion in disturbed pregnancy has been studied extensively, but it is still a highly controversial subject. The basic facts, however, are well established and emerge clearly from the following comparison of the average pregnanediol values found in various types of normal and disturbed pregnancy. In order t o make the observed values comparable and independent of the time trend, they must be calculated as percentages of the normal averages for the respective weeks of pregnancy. Thus the average value in normal pregnancy is 100% by definition, with a standard deviation of &43 (86 determinations). I n disturbed pregnancy, the figures are as follows: abortion accompanied by fever or caused by mechanical manipulations, 82 & 57 (36 determinations) ; threatened abortion and threatened premature labor with favorable outcome, 65 5 36 (126 determinations); spontaneous abortion, 44 & 43 (116 determinations). The average in the first of these groups is not significantly lower than in normal pregnancy (probability of chance occurrence of the difference, P > 0.05). All the other differences must be considered as highly significant (P < 0.001). These results are in good agreement with our concept of trophoblast activity in various types of abortion, as outlined in the introductory remarks t o this section. Furthermore, the great variability and the overlapping of the values make it abundantly clear that one single result in a particular case will be quite useless t o the clinician unless it is extremely low or zero. In the doubtful cases, the trend of excretion must be followed by serial determinations.

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Some reports have correlated decreasing or consistently low levels with a high incidence of inevitable abortion (Browne el al., 1939; Davis et al., 1942; Guterman and Tulsky, 1949; McCormack, 1946; Mack et al., 1949; Stern, 1951; Yanow et al., 1950). Others have failed to do so or stressed the exceptions (Cope, 1940; Hain, 1942; Kaufmann et al., 1951 ; Merivale, 1948; Qstergaard, 1940; Plota and Darup, 1949; Swyer, 1949). The question of prognostic value, though of scientific interest, seems to be somewhat academic from the clinical viewpoint. A true prognostic test would have to be 99.9-100% accurate if it were to be used to justify discontinuation of therapy and evacuation of the uterus. Nobody has yet claimed such a degree of accuracy. The truly essentiaJ clinical questions in the presence of uterine contractions, bleeding, or histories of repeated abortion, are: (1) Is the fetus living? (2) Which therapy shall be used? In both respects, pregnanediol determinations reflecting trophoblast activity, may be of considerable assistance. In many cases of fetal death, pregnanediol excretion decreases rapidly to very low levels or ceases altogether. Whether it is true or not that the administration of progesterone may be harmful in cases with high pregnanediol levels (Bender, 1948), there can be no doubt that there is no rational purpose in giving progesterone treatment to patients whose pregnanediol excretion is normal. The use of pregnanediol determinations as a guide to therapy has been advocated, directly or by implication, by several workers (Bender, 1948; Bradbury et al., 1950; Browne et al., 1946; Courtois and Balmary, 1949; Guterman, 1950; Jayle, 1950; Mayer and Levasseur, 1951; Pigeaud, 1951; Sdguy et al., 1950b; de Watteville, 1951). d . Conclusions. In cases of threatened abortion and premature labor, the functional state of the trophoblast may be controlled by serial hormone determinations. Although a wide range of normal variation exists, the results indicate frequently the severity of the disturbance. In the great majority of cases, fetal death is associated with decreasing or consistently low levels. If pregnanediol excretion is normal, progesterone treatment has no rational purpose. The subnormal values frequently seen in early pregnancy after repeated abortions may precede any clinical signs of abortion and justify preventive treatment. On the other hand, high normal levels warrant the assumption that pregnancy has developed normally up to the time of assay.

4. Hydatidiform Mole and Chorioepithelioma The prevailing view is that the proliferative degeneration of the chorionic villi known as hydatidiform mole is invariably associated with a greatly increased secretion of CG (Evans and Simpson, 1950). However,

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this widely accepted statement requires some qualification. Several workers have found the range in CG excretion to overlap considerably with normal pregnancy levels, and exceptional cases with very low titers have been described (Genell, 1946; Hamburger and Terkildsen, 1948; Payne, 1941; Richli, 1950). Even the appearance of CG in spinal fluid does not seem to be conclusive (Brody and Horowita, 1950). It seems that the diagnosis should be based mainly on clinical signs. The same wide range in CG excretion has been found in chorioepithelioma (same references), but in this case the situation is different, and CG assays are of high clinical value in order to detect malignant recurrence. After the expulsion or evacuation of a mole, CG assays should be made a t regular intervals for a period of at least two months. Even in uncomplicated cases, CG excretion may persist for some time, but a rising trend regardless of absolute levels, is strong evidence for the presence of malignant tissue growth, provided that a new pregnancy can be excluded (Hamburger and Terkildsen, 1948; Hinglais and Hinglais, 1950 ; Payne, 1941; Siegler et al., 1950). The earlier view that urinary pregnanediol is consistently absent or low in cases of hydatid mole and chorioepithelioma, has been disproved by the result of recent investigations (Pigeaud and Burthiault, 1951b; Plotz and Darup, 1950; Stern, 1951; de Watteville, 1951). If pregnanediol is found after the passage of a mole, frequent controls of the CG levels are mandatory (Pigeaud dnd Burthiault, 1951b). Estrogen excretion within the normal range has been reported in cases of hydatid mole (Hinglais and Hinglais, 1949; Payne, 1941). 6. Toxemia In hyperemesis gravidarum, sometimes called toxemia of early pregnancy, average CG levels have been reported to be higher than in normal pregnancy (Genell, 1946; Hinglais and Hinglais, 1950), and t o be correlated with the severity of the clinical signs (Schoeneck, 1942). The significance of this observation is open to discussion. A great number of investigators has contributed to the immense wealth of available information on the clinical, endocrine, and metabolic changes associated with toxemia of late pregnancy. Most of these changes must be assumed t o be secondary t o underlying causes which, however, have not been definitely established. Toxemia is known to occur in various clinical conditions, but never in the absence of placental tissue, and it is reasonable to relate the endocrine imbalance found in toxemia to placental dysfunction. The results of hormonal studies have been extensively reviewed by Smith and Smith (1948). Disregarding the subjects which are beyond the

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scope of the present report, it appears that the most consistent, though not invariable, aberrations from the normal are high levels of CG and decreased excretion of estrogens and pregnanediol in a large percentage of cases, associated with degeneration of the syncytial cells of the placenta. These abnormalities are underway prior to clinical manifestations. Based on these and other observations, the Smiths have advanced a stimulating theory of toxemia, including the concept that progesterone-deficient metabolism of the estrogens brings about a deficiency of oxidation products of the estrogens. These oxidation products are said to be responsible for the normal utilization of CG, by the placenta, for the secretion of the steroids of the latter. Thus, deficiency of the oxidation products leads to deficient utilization of CG and hence t o higher levels in blood and urine, and, in a vicious circle, steroid elaboration is further decreased. There is some evidence to suggest that increased CG production is indeed not the cause of the elevated titer. As a consequence of this theory, the preventative treatment with high doses of stilbestrol, acting as a substitute for the unknown oxidation products, has been introduced. The theory seemed t o be confirmed by the observation that the administration of estrogen t o pregnant women, and to castrated rabbits injected with progesterone, increases the excretion of the sodium pregnanediol glucuronidate fraction estimated by the Venning or similar methods (Davis and Fugo, 1948; Libert, 1950; Mayer and Levasseur, 1951; Smith et al., 1946). This increase seemed t o reflect a stimulation of progesterone secretion. However, the validity of this natural interpretation has become questionable since it has been shown that pregnanediol excretion, if estimated by methods more specific for this compound, is not affected or may even decrease under estrogen treatment (Davis and Fugo, 1948; Seitchik, 1950; Sommerville et al., 1949; de Watteville, 1949b). It seems that the assumption of an estrogen-induced shift in progesterone metabolism, rather than a change in secretory activity, would provide a satisfactory explanation of the experimental facts. I n a very careful study, Loraine and Matthew (1950) have recently re-investigated CG levels in blood and urine of toxemic patients; the results are given in terms of international units. The majority of their cases of severe pre-eclamptic toxemia (with urgent indication for interruption) were associated with readings of urinary and serum CG significantly higher than normal pregnancy levels. In the moderate and mild cases (without deterioration, or responding to treatment), the CG values were within the normal range. Renal clearance of CG was found to be significantly lower in a group of severe pre-eclamptic toxemia, but it was within the normal range in the mild and moderate groups; this observation

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was thought to be probably another index of the renal damage associated with severe toxemia (Loraine, 1950). An interesting correlation has been found to exist between the blood level of CG and the effect of tetraethylammonium bromide on the blood pressure. The drug causes a pronounced fall in blood-pressure in normal pregnant women and in the small number of toxemia patients with low CG titers. The drug has little or no such effect in cases of hypertensive toxemia associated with increased serum CG; it seems, therefore, that hypertension in these cases is of humoral origin (Govan et al., 1951). Eichenberger and Kaser (1949) have seen a decisive fall in urinary estrogens at the time of fetal death. Persistently low pregnanediol excretion in toxemia is frequently followed by stillbirth or the premature delivery of an underdeveloped child, regardless of the severity of clinical symptoms in the mother which may have regressed as a result of conservatory treatment. Extremely low pregnanediol values may be considered as an indication for prophylactic cesarean section in order to save the child from intra-uterine death, provided that its viability is ascertained by X-ray and clinical examination (Pigeaud and Burthiault, 1951a; de Watteville, 1950b, 1951). Recent reports from various laboratories suggest that the excretion of reducing and formaldehydogenic corticoids is increased in toxemia patients, especially in those with edema (Devis and Devis-van den Eeckoudt, 1950; Parviainen et al., 1950; Tobian, 1949). Such studies will certainly have to be repeated, when and if more accurate methods become available (cf. Section 11, 5). The high incidence of toxemia in pregnancy complicated by diabetes seems to be mainly responsible for the high fetal mortality as well as for the hormonal imbalance associated with this condition (Peel and Oakley, 1949). The concept of endocrine dysfunction in toxemia, as summarized in the preceding paragraphs, has in fact been derived partly from observations in diabetic pregnancies, leading to the same conclusions with respect to hormone assays as a guide to therapy with large doses of estrogen and/or progesterone (Smith and Smith, 1948; White and Hunt, 1943). Conclusions. Many cases of late pregnancy toxemia are associated with increased CG levels and reduced excretion of estrogens and pregnanediol. These changes often precede the onset of clinical symptoms and may be used as an indication for preventive treatment. Estrogen treatment results in reduced or unchanged excretion of pregnanediol and in increased output of other metabolites. Consistently low pregnanediol levels (in untreated cases) indicate that the fetus may suffer seriously from placental dysfunction, and prophylactic cesarean section may be justified.

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6. Ectopic Pregnancy

Although pregnancy tests are frequently used as an aid in differentiating tubal abortion from adnexal swelling of other origin (adnesitis, ovarian cyst), it is well known that the results are not conclusive. The reaction may still be positive in adnexitis after intra-uterine abortion, or it may be negative in spite of incomplete tubal abortion. According to Zondek et al. (1948), the combination of the estrus test for FSH with the ovarian hyperemia reaction may sometimes be useful, since only the latter becomes negative shortly after the death of the fetus. However, these workers emphasize rightly that a much less ambiguous diagnosis can be established in a matter of minutes by puncture of the Douglas pouch. Quantitative assays for urinary and serum CG (BQclBreand Mabileau, 1948; Ramos and Collazo, 1943) and for urinary pregnanediol (de Watteville, 1951) have shown that in ectopic pregnancy the levels are frequently lower than in intra-uterine pregnancy of the same age. This is due (1) to the fact that most cases are seen at a time when abortion is already underway, and (2) t o deficient development of the trophoblast in a mucous membrane poorly prepared for nidation. VI. TUMORS

It is obvious that the pathologic growth of glandular tissue should lead to changes in hormonal secretion. The diagnostic value of CG assays in chorioepithelioma after molar pregnancy has been discussed in Section V, 4; it should be noted in passing that, in the male, chorioepithelioma of testicular origin produces large amounts of CG (Evans and Simpson, 1950). Increased excretion of gonadotropins has sometimes been found in teratoma of the ovary, but assays seem t o be of rather limited significance (Geist and Spielmann, 1943; Paracchi and GalIico, 1947; Sorba, 1946). The same appears to be true for other ovarian tumors, since hormone levels are frequently within the upper normal range and are not necessarily correlated with the clinical course or the histologic findings. HOWever, after removal of a functional tumor, the excretion is reduced, and assays may serve as a check on recurrence (Pedersen, 1947). Various types of functional ovarian tumors are known (Freeman, 1950), such as granulosa cell tumor, thecoma, arrhenoblastoma, and adrenal rest tumor. The former two produce an excess of estrogen, resulting in early pubescence, disorders of menstruation, or uterine bleeding-depending on whether the patient is a child, an adult woman, or a post-menopausal woman. Arrhenoblastoma and adrenal rest tumor are masculinizing tumors, producing increased amounts of androgens,

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which are partly excreted as 17-ketosteroids. Mixed types may occur and may lead to divergent clinical symptoms in the same patient, presumably depending on the secretory activity of the various cell-types present (Shippel, 1950). Twombly (1946) has reported a case of masculinizing luteoma with high pregnanediol and 17-ketosteroid excretion, amenorrhea, and decidual transformation of the endometrium. The various steroids secreted by functional adrenocortical tumors may produce serious disorders within the genital sphere, such as amenorrhea, symptoms of masculinization, sexual precocity. According to Kepler et al. (1948), “the morphology of the tumor, the clinical picture, and the urinary excretion of steroids all vary materially. . . . In any one case, any one or several or all of the adrenal hormones might be produced in excessive amounts. Inferences drawn from analyses or bioassays of the urine regarding the character or the hormonal output of the tumor may be misleading and should not be accepted without reservation.” It may be mentioned that attempts have been made to correlate neoplastic disease in general with the occurrence of hormones or metabolites in urine. Dobriner (1948) and his associates have found that AQ-etiocholenoloneis highly significant but not wholly specific for cancer. Beard (1949) has claimed 95% correct diagnosis of malignancy; his test is based on the excretion of ether-alcohol soluble, spleenotropic, and gonadotropic principles tested on young female rats. ’

Conclusions

Hormone assays in cases of tumor growth are mainly of scientific interest. Even functional tumors of the ovary and the adrenals causing definite clinical manifestations may be associated with but slightly modified hormone levels. On the other hand, clinical symptoms suggesting a secreting tumor may be confirmed by decisively increased excretion values for gonadotropins, estrogens, 17-ketosteroids1 or pregnanediol. A rising trend after the removal of a functional tumor may be an early sign of recurrence.

VII. ENDOCRINE DISORDERS Endocrine disorders of extra-genital origin frequently lead to ovarian deficiency and amenorrhea, especially if the pituitary is involved. This condition, as well as disturbances due to tumor growth, have been treated in Sections 111, 4 and VI, respectively. In certain less severe psychic or physical syndromes it may be necessary to know whether glandular dysfunction is a contributory factor or not. I n such cases, normal secretion of ovarian hormones excludes dysfunction of the ovary as well as of the pituitary gland or at least of

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its gonadotropic component. Normal ovarian activity cannot be deduced from the occurrence of regular cyclic bleeding alone, which may be associated with luteal deficiency (cf. Section 111, 3). By means of premenstrual biopsy or pregnanediol estimation, this condition is indeed sometimes detected, for instance in patients with obesity. It is interesting in this connection that BQclhreet al. (1950) have reported estrogenic or gonadotropic hyperfunction in 56 of their 66 obesity cases. 17-Ketosteroid estimations, on the other hand, were found t o be of no diagnostic value in 53 women with obesity, whether concomitant with Cushing’s syndrome or not (Butt et al., 1950). It is certainly true that in many cases the disorder is not of hormonal origin. VIII. GENERALCONCLUSIONS I n several conditions encountered in gynecological and clinical practice, hormone assays furnish information of clinical significance, provided that certain limitations are kept in mind. Results should be evaluated only in close relation to the history and the clinical findings in the individual case. The exact significance of the methods used should be fully understood, and their reliability characteristics should be clearly defined. The assessment of normal variability, metabolic effects and other biologic factors may contribute appreciably t o a correct interpretation. Serial determinations in order to follow the trend are vastly superior to single assays. Blood-levels, except for chorionic gonadotropin and, perhaps, estrogens in pregnancy, must be considered a t best as very much less informative than excretion values. The latter should be calculated in weight or biologic (international) units per excretion time, and they should be obtained from complete twenty-four-hour collections. Reliable procedures are available for the bioassay of chorionic gonadotropin and estrogens, and for the chemical estimation of pregnanediol, 17-ketosteroids and large amounts of estrogens. The existing chemical methods for the determination of corticoids and of small amounts of estrogens require some further development before they can be put to regular clinical use. Separate assay of the different gonadotropic factors would be desirable. Gonadotropin assays are the method of choice for the diagnosis of early pregnancy and of chorioepithelioma. They reflect the functional activity of the trophoblast, and they sometimes permit the recognition of primary ovarian failure as the cause of amenorrhea. They may prove to be useful in determining the time of ovulation. Estrogen assays may be of aid in the diagnosis of secreting tumors and

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may serve as a controI after operation. Further investigation of their clinical value in pregnancy complications is desirable. Pregnanediol estimations are valuable in assessing the functional activity of the corpus luteum and the trophoblast in various disturbances of the menstrual cycle and in pregnancy. The occurrence of delayed menstruation may interfere with the pregnanediol pregnancy test and lead to false positive results. The clinical use of 17-ketosteroid determinations is limited to the detection of adrenocortical hyperfunction and certain secreting tumors of the ovary. Hormone assays are not infallible. It should be realized that in this respect they are not different from other methods of clinical diagnosis. REFERENCES

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Vestergaard, P. 1951. Acta Endocrinol. 8, 193-214. Votta, R. A, 1946. Obstet. y ginecol. latinearner. 4, 585-596. Wattenwyl, H. von. 1944. Tierexperimentelle Untersuchungen uber die Wirkung langdauernder Follikelhormonapplikation und die hormonale Tumorentstehung. Benno Schwabe, Basel, p. 65. Watteville, H. de. 1949a. Livre jubilaire offert au Professeur Gaston Cotte. Audin, Lyon, pp. 519-532. Watteville, H. de. 1949b. Bull. assoc. gynbcol. et obstdt. langue franc. 1, 452453. *Watteville, H.de. 1950a. GynCcol. et Obstbt. 49, 155-166. *Watteville, H. de. 1950b. Edinburgh Med. J . 67, 403-412. *Watteville, H. de. 1951. J. Clin. Endocrinol. 11, 251-266. Watteville, H. de, Borth, R., and Gsell, M. 1948. J. Clin. Endocrinol. 8, 982-992. *Watteville, H. de, Salinger, S., and Borth, R. 1949. Brit. Med. J . 2, 352-356. Watteville, H. de, Borth, R., Mach, R. S., and MUSSO,E. 1951. Acta Endocrinol. 8,319-326. Weisman, A. I., Snyder, A. F., and G a t e s , C. W. 1942. Endocrinology 31,323-325. West, C. D., Tyler, F. H., and Brown, H. 1951a. J . Clin. Endocrinol. 11, 833-842. West, C. D., Tyler, F. H., Brown, H., and Samuels, L. T. 1951b. J . Clin. Endocrinol. 11, 897-912. Westphal, U. 1944. Z.physiol. Chem. 281, 14-24. *White, P., and Hunt, H. 1943. J . Clin. Endocrinol. 3, 500-511. Wilson, H., and Carter, P. 1947. Endocrinology 41, 417-421. Yanow, M., Soule, S. D., and Meyerhardt, M. H. 1950. Am. J . Obstet. Gynecol. 69, 1160-1 163. Zondek, B. 1947. Lancet 1, 178-179. Zondek, B., and Black, R. 1947. J . Clin. Endocrinol. 6, 519-529. Zondek, B., and Sulman, F. 1947. J . Clin. Endocrinol. 7, 159-164. Zondek, B., Sulman, F., and Black, R. 1948. J . Am. Med. Assoc. 136, 965-969, 1107. Zygmuntowica, A. S., Wood, M., Christo, E., and Talbot, N. B. 1951. J . Clin. Endocrinol. 11, 578-596.

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Experimental Glycosuria : Its Production, Prevention, and Alleviation BY ROBERTSON F. OGILVIE Pathology Department, University of Edinburgh, Scotland CONTENTS

Page I. Introduction. . . . . . . . .................... 11. Insulin Insufficiency. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 1. Pancreatectomy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 2. Subtotal Pancreatectomy and (a) High Caloric Diet or (b) Bernard Puncture ............................... .. . . . . . . . 186 3. Intraperitoneal Glucose ........................................... 188 4. Administration of Insulin to (a) Subtotally Depancreatized and (b) Force-Fed Normal Animals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 5. Alloxan and Related Compounds.. . . . . . . . . . ..................... 190 6. Dehydroascorbic and Dehydroisoascorbic Aci . . . . . . . . . . . . . . . . . . . . . 195 7. Oxine and Dithizone.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196 . . . . . . . . . . . . . 196 111. Hormones ...........

6. Estrogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .........

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V. Glycogenolysis.. . . . . . . . . . . . . . . . . . . . .

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VI. Kidney . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . .

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I. INTRODUCTION The number of diabetic subjects in the United States and the United Kingdom has been estimated at about a million (Joslin, 1946c) and two hundred thousand (Lawrence, 1939), respectively, and thereby affords a measure of the enormity of the diabetic population throughout the world. Moreover, the number of diabetic individuals in the United States and presumably elsewhere, has increased rapidly during recent years. Thus, the first half of the last decade saw the diabetic population in the United States increase at the rate of about fifty thousand annually (Joslin, 1946a). From another angle, diabetes ranked twenty-seventh as a cause of death in the United States in 1900, but in 1943, excluding deaths from violence, it was placed seventh (Joslin, 1946b). Recently diabetes has also shown a distinct tendency to overtake tuberculosis and pneumonia as the commonest cause of death in the United States and so in this respect, may come to occupy fifth place before the lapse of many years (Joslin, 1946b). Indeed, the legitimate grouping of arteriosclerotic affections of the heart, kidneys, and brain, not as three diseases but as a single entity would advance diabetes to third place, after only arteriosclerosis and cancer (Joslin, 1946b). Finally, a similar recent increase in the mortality from diabetes has been observed in many other countries besides the United States (Joslin, 1946d). Such a widespread and marked increase in both the incidence of and mortality from diabetes is complex as regards explanation, but may probably be attributed in the main to the following factors (Joslin, 1946a): (a) the increase in the size of the population; (b) the increase in the average age of the population, so that more people are reaching the sixth decade, the period of most frequent onset of the disease; (c) the increase in the expectation of life of the diabetic subject to three or four times what it was at the beginning of the century; (d) an increase in the diagnosis of the disease; and (e) an increase in the medical consciousness of the population. These observations regarding the increasing impact of diabetes on the welfare of the nations and its probable reasons, serve to indicate the increasing urgency for a solution of the diabetic problem, both in respect to its causation and the discovery of a more fundamental therapy than is available a t present in insulin. The most outstanding advances in our knowledge of the disease etiologically and therapeutically, moreover, have emerged primarily in the laboratory, and the time accordingly seems opportune to review the experimental methods available in the production, prevention, and alleviation of glycosuria. The techniques available for producing glycosuria experimentally are

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extremely varied and any dogmatic classification thereof is impossible in that the mechanisms whereby different methods lead to the condition sometimes overlap considerably. Nevertheless, the possible avenues of approach may, as already suggested (Ingle, 1948a), be broadly separated into insulin insufficiency, hormones, diet, glycogenolysis and kidney. Accordingly each of these factors will be considered in turn, and appended by any procedures capable of preventing or alleviating the related glycosuric state. 11, INSULININSUFFICIENCY

A reduction in the requisite supply of insulin may be brought about by :1. Pancreatectomy This was first performed with a view to determining whether life was possible in the absence of the pancreas and so disclosed that a depancreatiaed dog becomes hungry and thirsty, passes excessive amounts of urine loaded with sugar and acetone, loses weight rapidly, and dies in less than four weeks (von Mering and Minkowski, 1890). A severe diabetes also characterizes the totally depancreatized cat (Homans, 1914), and man after complete removal of the pancreas shows a moderately marked glycosuria (Gaston, 1948). Such a well-developed diabetic reaction, however, is not produced by pancreatectomy in all species. Thus, only slight glycosuria is noted after removal of the pancreas in the monkey (Collip et al., 1937), goat and pig (Lukens, 1937, 1938), while the depancreatized duck is not significantly affected as regards blood sugar or sugar tolerance (Mirsky et al., 1941-42). The condition of the monkey, goat, pig, and duck following pancreatectomy accordingly resembles that described in the cat after removal of the pancreas and either the pituitary or adrenals (Long and Lukens, 1936; Lukens, 1937). Further, the dog requires a 90-95% resection of the pancreas for the induction of the diabetic state and is then more severely affected than after complete pancreatectomy (Dragstedt, 1943). Similarly, in man a case of subtotal extirpation of the pancreas is described as presenting a more intense diabetes than a case of total pancreatectomy (Priestley et al., 1944). These paradoxical variations in the effect of removal of the pancreas in the different species and of pancreatectomy as compared with subtotal pancreatectomy in the individual subject naturally raise problems regarding the control of carbohydrate metabolism in the different species and in the individual animal or man. Nevertheless, the discovery of the important role of the pancreas in the use of sugar (von Mering and Minkowski, 1890) has led to significant advances. Thus,

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the observed effect of ligation of the main duct of the pancreas in producing selective atrophy of the pancreatic acinar tissue without any incidental diabetic condition (Ssobolew, 1900; Schulze, 1900) points to the factor responsible for the prevention of glycosuria as a probable product of the pancreatic islets, while the truth of this surmise is proven by the possible extraction of a potent and relatively nontoxic variety of insulin from the canine pancreas following duct ligation (Banting and Best, 1921-22a and b). Previous hypophysectomy greatly ameliorates the diabetes following pancreatectomy in the toad (Houssay and Biasotti, 1930a), dog (Houssay and Biasotti, 1930b) and cat (Long and Lukens, 1936), while the cat responds to pancreatectomy with a similarly modified diabetes as a result of antecedent adrenalectomy (Long and Lukens, 1936). Thus, the depancreatized cat previously hypophysectomized or adrenalectomized shows a markedly decreased glucose, nitrogen, and acetone-body excretion and also an increased survival period as compared with the purely depancreatized individual, although carbohydrate tolerance is not significantly increased by antecedent hypophysectomy or adrenalectomy in the depancreatized cat (Long and Lukens, 1936). Removal or paralysis of the secretion of the adrenal medulla is without effect upon the results of a total pancreatectomy so that the consequences of adrenalectomy must be brought about by ablation of the cortical portion of the organ (Long and Lukens, 1936). Finally, the ameliorative influence of hypophysectomy and adrenalectomy in pancreatic diabetes was originally regarded as due to a diminished production of glucose and acetone bodies rather than t o the resumption of normal carbohydrate metabolism (Long and Lukens, 1936), and this view has certainly been substantiated by recently acquired knowledge regarding the function of the various hormones of the anterior hypophysis and adrenal cortex, as will be considered later. 6. Subtotal Pancreatectomy and ( a ) High Caloric Diet or (b) Bernard Puncture

These methods are considered together since their induction of the permanent diabetic state is probably effected through a common mechanism. Both were originally carried out in the dog (Allen, 1913a and e; 1922) and represent the earliest successful attempts at producing glycosuria in an animal with sufficient pancreatic islet tissue to prevent spontaneous diabetes. The glycosuria resulting from a high caloric diet in the dog is initially mild and can be prevented or stopped by reducing the diet or ligating the pancreatic duct, but is later severe and beyond the possibility of control by the foregoing procedures (Allen, 1913h,i,

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and j ; 1922). This incurable phase, moreover, is characterized by marked, widespread, hydropic degeneration of the pancreatic islets (Allen, 1913g; 1922), and the same condition of the insular apparatus is present in the permanent diabetes induced by a Bernard puncture (Allen, 1913f). Further, the hydropic state of the pancreatic islets is located wholly in the B-cells as distinct from the A-cells of the insular tissue (Homans, 1914). When the diabetes is uncontrolled, the vacuolation of the B-cells is first manifest within four to seven days of the onset, maximally developed in about four weeks, and followed by more or less complete disappearance of the B-cells in from six to eight weeks (Allen, 1922). The stage of extreme vacuolation probably lasts about a week (Allen, 1922). The hydropic state was originally regarded as reversible so long as the cell membrane and nucleus remain viable (Allen, 1922) and this belief has been convincingly substantiated by the resolution effected in the earlier stages of the condition by various methods (Lukens and Dohan, 1942; Lukens et al., 1943). Hydropic degeneration of the B-cells incidentally has been observed in the islets of human diabetic subjects (Weichselbaum and Stangl, 1901). It occurs in 4.5% of such cases and is described as occurring at all ages and most strikingly in acutely affected individuals (Warren, 1938). The diabetes induced by subtotal (95%) pancreatectomy in the rat is profoundly influenced by the thyroid gland. Thus, (a) simultaneous thyroidectomy and subtotal pancreatectomy prevents the eventual appearance of diabetes, but has no effect on the incipient or established diabetic state (Houssay et al., 1946); (b) the administration of thiouracil obviates the development of diabetes after subtotal pancreatectomy in 40% of animals (Houssay el al., 1946); (c) treatment with thiouracil has a curative effect in a certain number of cases of moderate diabetes induced by subtotal pancreatectomy and the pancreatic islets of these successfully treated animals show little or no evidence of damage to the B-cells, although no obvious hypertrophy or hyperplasia (Houssay, 1950); (d) the administration of powdered thyroid to subtotally depancreatized animals accelerates the appearance of diabetes during the first 15 days, but later causes the diabetic state to disappear gradually; untreated rats, on the other hand, are characterized by persistence of the diabetes or its appearance in all animals; the remains of the pancreas in the recovered rats exhibit enlargement of the original islets or formation of new islets (Houssay et al., 1946);.and (e) thyroid treatment of subtotally depancreatized rats causes complete disappearance of an incipient diabetes, whereas a diabetic state is eventually manifest in all the untreated controls (Houssay et al., 1946). The diabetes following subtotal pancreatectomy in the rat may thus be alleviated by both hypo-

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and hyperthyroidism. The beneficial effect of hypothyroidism has been correlated with the increase in the free sulfhydryl groups of the tissues, induced through thyroidectomy or the administration of thiouracil (Houssay, 1950), while hyperthyroidism in the form of thyroid administration is regarded as exerting its curative action by increasing the amount of pancreatic islet tissue (Houssay et al., 1946). The diabetes following subtotal (95%) pancreatectomy in the rat has an earlier appearance and greater frequency in males than in females (Foglia et al., 1947). It is also decreased and increased through the expediency of castration in males and females respectively, so that the incidence curves of the diabetes in male and female castrates are made t o approximate more than those of males and females with intact gonads (Foglia et al., 1947). At the same time, the incidence of the diabetes is decreased in castrated females by treatment with estrone, estradiol, stilbestrol, dienestrol, ethinylestradiol, ethinyltestosterone (Lewis et al., 1950) and phenocycline (Rodriguez, 1950) and also in castrated males given estrone and stilbestrol (Lewis et al., 1950), whereas the administration of testosterone and methyltestosterone to female castrates and of testosterone to male castrates markedly increases the incidence and severity of the diabetes (Lewis et al., 1950). The pancreatic islets of the rats treated with estrone, estradiol, stilbestrol and ethynilestradiol show marked hypertrophy and hyperplasia, while the insular tissue in the animals injected with testosterone and methyltestosterone is characterized by striking decrease in the B-cells, sclerosis and atrophy (Lewis et al., 1950). The sexual difference in the incidence of the diabetes obtaining in subtotally (95%) depancreatized rats is thus due to the protective and provocative actions of the ovaries and estrogens and of the testes and androgens, respectively. Moreover, the estrogens would appear t o achieve their beneficial effect mainly by reason of their pancreatropic action, whereas the induction of severe insular damage probably explains the deleterious action of the androgens. 3. Intraperitoneal Glucose

Repeated intraperitoneal injections of glucose-saline, sufficient to invoke prolonged hyperglycemia, induce marked hydropic degeneration of the B-cells of the pancreatic islets and permanent diabetes with acidosis in the normal cat (Dohan and Lukens, 1947a and b). The findings in sections (2) and (3) justify the following conclusions: (a) hydropic degeneration of the B-cells may be interpreted as evidence of hyperfunction and subsequent exhaustion in their endeavour to cope with the persistently raised blood sugar. As mentioned later, however, an extension and modification of this view is possible on the basis of

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recent observations in connection with the blood glutathione in alloxan diabetes; (b) with the A-cells remaining histologically normal the B-cells must secrete a hormone (insulin) of importance in the control of carbohydrate metabolism; (c) lysis and loss of the B-cells on an extensive scale means the conversion of an originally recoverable into an ultimately irrecoverable diabetes; and (d) a diabetes may be initiated by some extrapancreatic disturbance, but be finally maintained through B-cell degeneration and insulin insufficiency.

4. Administration of Insulin to ( a ) Subtotally Depanereatized and ( b ) Force-Fed Normal Animals Dogs subjected to a 30-75 % pancreatectomy develop glycosuria on the sudden withdrawal of insulin in the 25th week of treatment and also spontaneously between 20 and 40 weeks after the beginning of insulin therapy (Mirsky et al., 1942). In the latter instance the glycosuria continues for as long as 30 weeks after its appearance and is thus apparently permanent. Similarly the abrupt withdrawal of insulin from intact rats force-fed a high carbohydrate diet, results in temporary hyperglycemia and slight glycosuria (Ingle et al., 1944). Similar observations have been made in man. Thus, non-diabetic subjects given increasing amounts of insulin insufficient to produce hypoglycemia show a progressive diminution of sugar tolerance and also, on sudden discontinuance of the insulin, transitory hyperglycemia and glycosuria lasting up to 5 days (Clark et al., 1934-35). Again, the excision of an active adenoma of the pancreatic isIets leads to a marked diminution in sugar tolerance accompanied by slight glycosuria during the oral glucose test (Burtness et al., 1940-41). The basis of the glycosuria produced by these various experimental procedures in animals and man is probably the same in all and indicated by two observations: (a) the administration of insulin to intact rats effects a distinct decrease in the insulin content of the pancreas (Haist et al., 1940); and (b) the subtotally depancreatized dog subsequently made diabetic with insulin is characterized by a reduction in the number of pancreatic islets and corresponding atrophy of the remaining few (Mirsky et al., 1942). In other words, the glycosuria in the various above-mentioned circumstances is probably attributable to compensatory suppression of the function of the insular tissue. Sections 2-4 thus indicate that glycosuria may be induced by (a) hyperactivity with consequent hydropic degeneration of the B-cells of the pancreatic islets in an endeavour to cope with a persistently raised blood sugar; and (b) hypoactivity with consequent atrophy of the islets from compensatory suppression of their function.

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5. Alloxan and Related Compounds

Alloxan, the ureide of mesoxalic acid, produces necrosis of the pancreatic islets and subsequent diabetes. The insular damage was first observed in the rabbit (Dunn et al., 1943), while the diabetic state was soon thereafter reported for the rabbit (Bailey and Bailey, 19-13), dog (Brunschwig et al., 1943; Goldner and Gomori, 1943) and rat (Dunn and McLetchie, 1943). The diabetogenic dose of intravenous alloxan ranges in the various species between 40 and 600 mg./kg. in the rat and ape respectively (Bailey, 1949). After an injection of alloxan the blood sugar shows a characteristic triphasic reaction comprising a hypoglycemic interpolated between two hyperglycemic stages. The hypoglycemic phase may be so marked as to precipitate the animal into convulsions and is probably due to liberation of much preformed insulin from the necrotic islet tissue (Hughes et al., 1944). The hypoglycemic and convulsive states were, as a matter of fact, recognized as effects of alloxan for a considerable period before the discovery of its diabetogenic action (Jacobs, 1937-38; Wiener, 1899). At the same time, the primary and secondary hyperglycemic stages contrast in that they are respectively of transitory and of more sustained or even permanent nature (Goldner and Gomori, 1943; Kennedy and Lukens, 1944). The histological changes in the pancreatic islets immediately after an injection of alloxan are more or less identical in the rabbit, rat, and dog, and selectively limited to the B-cells (Duff, 1945). These elements show progressive degranulation and nuclear degeneration and final replacement by granular debris in 8-12 hours, depending on the dose. Such damage may affect the islets in part and then often only at the center, or in whole, and are followed by absorption of the debris within 3-5 days, with corresponding collapse of the insular structure. In contrast, the A-cells, although sometimes also degenerated, remain characteristically unaffected. The stage of permanent diabetes entails changes in the islets and small pancreatic ducts (Duff, 1945; Ogilvie, 1949). Thus, the insular phenomena include reduction in number and size, atrophy to groups of A-cells, and hydropic degeneration of many B-cells, while the small pancreatic ducts are characterized by hydrops of their lining epithelium. The first three insular changes clearly result from the acute necrotizing action of the compound. On the other hand, the hydropic condition of the B-cells is regarded as an effect of the prolonged hyperglycemia (Kennedy and Lukens, 1944) and the ductal hydrops remains an unexplained phenomenon. An increased sensitivity t o the diabetogenic action of alloxan may be effected by the administration of ascorbic acid (Levey and Suter, 1946;

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Lazarow, 1946), by starvation (Kass and Waisbren, 1945), or by a diet low in protein (Houssay and Martinez, 1947), high in fat (Houssay and Martinez, 1947), or deficient in methionine and cystine (Griffiths, 1948a). On the other hand, complete protection against the diabetogenic action of alloxan can be achieved by the immediately prior injection of cysteine, glutathione, thioglycollic acid, and dimercaptopropanol (BAL) (Lazarow, 1946, 1947); the increased sensitivity to the diabetogenic action of alloxan induced by starvation and a high fat diet is corrected by treatment with glucose (Kass and Waisbren, 1945) and thiouracil (Houssay and Martinez, 1947), respectively; and a decreased sensitivity to the diabetogenic action of alloxan may be brought about by the administration of cysteine or thiouracil and by thyroidectomy (Houssay, 1950). Now, ascorbic acid (Prunty and Vass, 1943), starvation (Hirano, 1934), and a methionine- and cystine-deficient diet (Griffiths, 1948a and b), each effects a reduction in the blood glutathione and the same may be true of a low protein and a high fat diet. Conversely, the injection of cysteine, glutathione, thioglycollic acid, and dimercaptopropan 01 infers an increase in the sulfhydryl content of the blood; glucose given during starvation induces a rise in the blood glutathione (Hirano, 1934); and the administration of cysteine and thiouracil and thyroidectomy lead to an increase in the sulfhydryl content of the tissues (Houssay, 1950). In other words, decrease and increase in the sulfhydryl content of the body infers increase and decrease in sensitivity to the diabetogenic action of alloxan, respectively, or, briefly, sensitivity to the diabetogenic action of alloxan varies inversely as the sulfhydryl content of the tissues. The mechanism whereby alloxan produces necrosis of the B-cells of the pancreatic islets and permanent diabetes, has been argued on the following lines. The intravenous administration of alloxan is accompanied by a rapid reduction in both the injected alloxan and the blood glutathione (Leech and Bailey, 1945). Such a concurrent decrease in these substances implies their immediate interaction and is therein explained by the affinity of alloxan for sulfhydryl groups (Labes and Freisburger, 1930; Purr, 1935). As a matter of fact, the interaction of alloxan and glutathione effects reduction of the alloxan to dialuric acid and the incidental formation of a new compound with an absorption spectrum maximum a t 305 mp (Lazarow, 1949a and b). Now, the blood glutathione is carried almost wholly by the red corpuscles, but all tissue cells contain a quota of the same material. Consequently, any alloxan not reduced by the sulfhydryl groups of the blood glutathione would be free to react with such groups in the tissue glutathione and even, as suggested (Lazarow, 1946), in the intracellular enzymes. Many of these enzymes require for their activity the presence of certain sulfhydryl

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groups (Barron and Singer, 1945; Singer and Barron, 1945) and oxidation to the disulfide form involves inactivation of the enzymes and consequently cell death. The necrosis of the B-cells of the pancreatic islets produced so selectively by alloxan presupposes, on the basis of the above theory, a selective attack by the compound on the enzymes of these cellular elements and here, by way of explaining such an attack, attention has been appositely drawn to the importance of an adequate cystinecysteine equilibrium in the B-cells in respect to their ability to synthesize both insulin and glutathione (Leech and Bailey, 1945; Lazarow, 1949a and b). Insulin is an unusual protein in that it contains as much as 12% cystine, while cysteine is a component of the glutathione molecule. The insulin and glutathione of the B-cells would thus appear to compete for possession of the cystine of the diet and might thereby lead, in view of insulin being the prime product of the B-cells, to a relative deficiency of glutathione in the B-cells as compared with other cells. Such a poverty of glutathione in the B-cells infers a corresponding lack of protective sulfhydryl groups and would thus facilitate an attack by intravenously injected alloxan, left over from reacting with the blood glutathione, on the sulfhydryl enzymes of the B-cells, with consequent cell death. According to this theory the selective necrotizing action of alloxan on the B-cells is thus due partly to the sulfhydryl-oxidizing property of alloxan and partly to the insulin-secreting function of the B-cells. Various homologues of alloxan and other related compounds, including N-methyl-, ethyl-, and propylalloxans, alloxantin, dimethylalloxantin, diethylalloxantin, and ciia!uric and methyldialuric acids, also possess diabetogenic activity (Bruckmann and Wertheimer, 1947). Temporary and permanent glycosuria are induced in methionine- and cystine-deficient rabbits by the intraperitoneal administration of one and two doses of uric acid respectively (Griffiths, 1948b, 1950). Now, a methionine- and cystine-deficient diet, as already mentioned, reduces the blood glutathione (Griffiths, 1948a and b). Further, uric acid may be synthesized from dialuric acid and urea by an enzyme contained in plasma or serum (Ascoli and Izar, 1909; Preti, 1909) and thus might possibly, through the action of the same enzyme under different conditions, give rise to dialuric acid. This substance, moreover, can be readily oxidized to alloxan (Archibald, 1945; Patterson et al., 1949). Again, intraperitoneal uric acid and intravenous alloxan elicit the same triphasic blood-sugar response in rabbits on methionine- and cystine-deficient and standard diets respectively, while similar pancreatic B-cell changes characterize uric acid and mild alloxan diabetes (Griffiths, 1948b, 1950). Accordingly, the basis of uric acid diabetes would seem to lie in the conversion of uric acid to alloxan, which then proceeds, in the presence of a

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deficiency of protective glutathione, t o damage the B-cells of the pancreatic islets. The relationship of the adrenocorticotropic hormone to carbohydrate and purine metabolism is mentioned later. Hypophysectomized rats given alloxan may or may not show a diabetic reaction. A resultant diabetes may even be produced in a majority of such animals, but is transitory or fluctuant in severity (Bailey et al., 1947). The blood sugar of animals not giving a diabetic response remains at more or less normal levels, although irregular fluctuations occur above and below the normal limits (Duff and Starr). At the same time, removal of the pituitary gland causes a marked decrease or even total disappearance of the glycosuria of alloxan-diabetic rats (Gaarenstroom, 1946-48). The fasting blood-sugar of such animals sinks below that of normal rats, but the administration of sugar is followed by a very marked rise in the blood sugar. Polyuria and increased output of nitrogen in the urine also persist after extirpation of the pituitary gland in these circumstances. Similarly, a marked reduction or complete disappearance of the signs of diabetes occurs in severely affected alloxan-diabetic rats after adrenalectomy (Janes and Friedgood, 1945). These results are naturally in line with the observation that hypophysectomy or adrenalectomy followed by pancreatectomy prevents or alleviates the diabetic condition which is ordinarily the sequel of removal of the pancreas (Houssay and Biasotti, 1930; Long and Lukens, 1936). Now, an improvement takes place in the persistent alloxan diabetes of rabbits when treated with anterior pituitary extract (APE) (Ogilvie, 1949). The recovery consists in replacement of a marked hyperglycemia and glycosuria by an almost or completely normal blood-sugar and urine, but is of only temporary duration. The pancreatic islets of both treated and control rabbits show reduction in number and size, atrophy to groups of A-cells, and hydropic degeneration of the B-cells, while the islets of the animals temporarily benefited by extract also exhibit regeneration, as evident in enlargement and budding and a suggestive growth of new islets from the ducts. Again, thiouracil has a curative effect in moderately severe cases of alloxan diabetes in the rat, although not in very profound examples of the condition (Houssay, 1950). Thus, three of eight and five of seven animals were relieved of their diabetic state after 26 and 39 days’ treatment respectively, so that the therapeutic value of thiouracil in this connection increases with the duration of the treatment. The pancreatic islets of the successfully treated rats show little or no abnormality of their B-cells, but incidentally exhibit no obvious evidence of hypertrophy or hyperplasia. Further, alloxan diabetes in the rat may sometimes be cured by treatment with a combination of estradiol and insulin (Houssay, 1951). Finally, the substitution of a high-fat for a normal diet causes disappearance of the glycosuria of alloxan-diabetic

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rats until resumption Qf the normal diet (Burn et al., 1944). This abrupt change to a high-fat diet is accompanied by some ketonuria, but no such phenomenon occurs with a gradual increase in the fat of the diet. Alternate periods of high-fat and normal diet, moreover, are paralleled by a gradual diminution in the glycosuria obtaining with successive periods of the normal diet. As mentioned later, an improvement also takes place in the metahypophyseal diabetes of dogs given a high-fat diet (Marks and Young, 1939). Alloxan diabetes is essentially due to insulin deficiency resulting from destruction of the B-cells primarily by the alloxan and secondarily by the hyperglycemia of the established diabetic state. The damage to the B-cells produced by a raised blood-sugar takes the form, it will be remembered, of hydropic degeneration (Dohan and Lukens, 1947a and b) and presumably subsequent necrosis and disappearance, and is therein probably achieved through the sequence (Lazarow, 1949b) of an increased demand for insulin, reduction in the glutathione of the B-cells, and exposure of the vital sulfhydryl enzymes of these elements t o natural inactivators. Accordingly, alleviation of alloxan diabetes will follow any procedure effecting an increase in the amount of available insulin or a withdrawal of anti-insulin factors. Increased insulin may in turn result from decrease and corresponding increase in the raised level of the blood sugar and glutathione of the B-cells respectively, or from an actual increase in the quantity of functioning islet tissue. Thus, the benefit t o alloxan diabetes from hypophysectomy (Gaarenstroom, 1946-48) and adrenalectomy (Janes and Friedgood, 1945) is probably mediated in part through removal of the anti-insulin secretions of the anterior pituitary gland and adrenal cortex (see pp. 196,201) and in part through an improved secretory capacity of the B-cells consequent on a reduced hyperglycemia. Similarly, a reduced blood sugar with increased function of the B-cells is also a likely factor in the amelioration of the alloxan-diabetic state induced by a high-fat diet (Burn et al., 1944) or by a combination of insulin and estradiol (Houssay, 1951). A high-fat diet brings about an increased sensitivity to the diabetogenic action of alloxan (Houssay and Martinez, 1947) and thereby infers a reduction in the' glutathione of the B-cells. However, such an effect might be overcome by the decreased demand for insulin incidental to the consumption of a high-fat as compared with a normal diet during the alloxan-diabetic state, more of the dietary cystine being consequently diverted to form protective glutathione. An increase in the glutathione content and consequent functional capacity of the B-cells also possibly explains the curative value of thiouracil in alloxan diabetes (Houssay, 1950), since treatment with this substance, as already mentioned, augments the free sulfhydryl groups of the tissues (Houssay,

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1950). On the other hand, the glutathione content of the tissues is reduced by various anterior pituitary preparations (Goss and Gregory, 1934-35; Ennor, 1939) so that the beneficial effect of crude APE in the alloxan-diabetic state (Ogilvie, 1949) is probably not dependent on any immediate sulfhydryl action. The pancreatic islet tissue is, nevertheless, substantially increased by treatment with APE (Ogilvie, 1949) and thus serves as a reasonable explanation of how this therapy alleviates alloxan diabetes. The mechanism of such a pancreotropic action on the part of APE remains obscure, but may in part take place through stimulation of the thyroid gland and ovaries, inasmuch as powdered thyroid (Houssay et aE., 1948) and various estrogens (Lewis et at., 1950) are known t o be capable of producing hypertrophy and hyperplasia of the pancreatic islets. Growth of the islets under the influence of APE would certainly appear not t o involve any incidental hyperglycemia (Ogilvie, 1944). The apparent paradox of the benefit of both hypophysectomy and APE in the treatment of alloxan diabetes is resolved in the observation that APE usually effects a primary increase and only secondary decrease in the severity of an existing alloxan diabetes by reason of its immediate diabetogenic and more delayed pancreotropie actions respectively (Ogilvie, 1949). Finally, a combination of insulin and estradiol probably alleviates alloxan diabetes partly through the effect of insulin in lowering the blood sugar and so improving the function of the B-cells and partly through the already-noted pancreotropic action of estradiol (Lewis et al., 1950). 6. Dehydroascorbic and Dehydroisoascorbic Acids

Both dehydroascorbic and dehydroisoascorbic acids are capable of inducing temporary and permanent diabetes according to the dosage (Patterson, 1949, 1950; Patterson and Lazarow, 1950). Dehydroascorbic-acid diabetes is characterized by (a) initiation through a triphasic blood-sugar reaction similar to that induced by alloxan (Patterson, 1949); (b) sensitivity to small amounts of insulin (Patterson, 1950); (c) pancreatic islet lesions corresponding with, although less necrotizing than, those found in alloxan diabetes (Patterson and Lazarow, 1950); and (d) prevention through the immediately prior intravenous administration of cysteine, glutathione, or 2,3-dimercaptopropanol, while the injection of any of these substances a few minutes after dehydroascorbic acid does not usually obviate the development of diabetes (Patterson and Lazarow, 1950). Dehydroascorbic acid, moreover, has certain structural features in common with alloxan (Patterson, 1950). Such observations accordingly indicate that dehydroascorbic acid may, as suggested for alloxan (Lazarow, 1947), exert its diabetogenic action through blocking an enzymatic sulfhydryl group in the B-cells of the pancreatic islets. This

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blockage, in view of the inability of sulfhydryl compounds to prevent diabetes on administration soon after dehydroascorbic acid, probably involves, not oxidation of the enzymatic sulfhydryl t o a disulfide linkage, but the formation of an irreversible combination between the sulfhydryl and dehydroascorbic acid (Patterson and Lazarow, 1950). Both ascorbic and dehydroascorbic acids act synergistically with alloxan in the induction of diabetes, dehydroascorbic acid being in this respect more effective than ascorbic acid (Patterson, 1950). Such effects have been interpreted in terms of oxidation of ascorbic to dehydroascorbic acid and reduction of the blood glutathione by dehydroascorbic acid with a consequent sparing of alloxan (Patterson, 1950). 7. Oxine and Dithizone

Oxine (8-hydroxyquinoline) and dithizone (diphenylthiocarbazone) have recently been accredited with diabetogenic properties (Kadota, 1950). Thus, intravenous oxine or dithizone result,s in permanent diabetes, while transitory glycosuria follows oral dithizone. The diabetes induced by the intravenous administration of both of these substances is immediately preceded by a brisk triphasic blood-sugar reaction similar to that obtaining with alloxan. Dithizone, however, produces a sharper blood-sugar response and ultimately more intense permanent diabetes than oxine. The first 24-36 hours after the administration of oxine and dithizone are characterized by selective necrosis of the B-cells of the pancreatic islets, while the islets show reduction in number, more or less atrophy to groups of A-cells, and hydrops of the remaining B-cells in the permanent diabetic phase. The amount of zinc in the islets, as determined by histochemical methods, is reduced or absent and scanty during the initial triglycemic and permanent diabetic phases respectively. This observation and their property of reacting strongly with zinc are the basis for the suggestion that oxine and dithizone lead to destruction of the islets and permanent diabetes through uniting with the zinc in the insular tissue (Kadota, 1950). 111. HORMONES 1. Anterior Pituitary Extract

Experimental proof of the significance of the pituitary gland in carbohydrate metabolism first accrued from the results of hypophysectomy in the dog (Houssayand Magenta, 1925), and thereby led tothe discovery of the effectiveness of a suitably prepared APE in inducing a diabetic condition in the intact animal (Evans et al., 1931-32; Baumann and Marine, 1931-32; Houssay et al., 1932-33). All species of animal, however, are not equally sensitive to the diabetogenic action of APE.

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Thus, the adult dog and cat on the one hand and the rat and mouse on the other are highly susceptible and resistant respectively, while the rabbit is intermediate in its response (Young, 1938, 1945; Ogilvie, 1944). The response of the dog to daily treatment with a diabetogenic APE proceeds in various stages (Young, 1936, 1937, 1939). Thus, a constant small dose produces glycosuria, ketonuria, and polyuria for a few days. Further periods of diabetes may be brought about by successivelyincreasing the amount of extract, while progressively more intense treatment with extract for 1+4 weeks ultimately results in a permanent diabetic state. Characteristically ushered in by a sharp ketosis, the condition so established persists indefinitely, tending to increase in severity with the passage of time. The transitory and permanent diabetic states obtaining with and after the administration of APE have been termed idiohypophyseal diabetes and metahypophyseal diabetes respectively (Young, 1948) , and this nomenclature is indeed apposite in that metahypophyseal diabetes differssubstantially from both idiohypophyseal diabetes and the diabetes of pancreatectomy. Thus, idiohypophyseal diabetes is characterized by relative insensitivity to the hypoglycemic action of injected insulin, nitrogen retention and increase in weight (Young, 1939, 1945), whereas none of these features is found in metahypophyseal diabetes (Marks and Young, 1939). Again, idiohypophyseal diabetes in the dog is manifest in degranulation and limited hydropic degeneration of the B-cells and reparative mitotic activity in the pancreatic islets, while more or less atrophy to groups of A-cells and hyalinization of individual B-cells or entireislets obtain in metahypophyseal diabetes (Richardson, 1939-40). The metahypophyseal diabetic differs from the depancreatized animal (a) in being able to survive indefinitely without insulin therapy provided it is given sufficient food, and (b) in requiring more insulin for the control of its glycosuria, despite the absence of any obvious insensitivity to the hypoglycemic action of insulin (Marks and Young, 1939). In conjunction with the more or less selective B-cell damage, the greater insulin need of the animal with metahypophyseal diabetes implies the secretion of an anti-insulin substance by the exocrine tissue or A-cells of the pancreas, and in this connection work on the glycogenolytic effect of certain commercial preparations of insulin (Shipley and Humel, 1945; Sutherland and Cori, 1948) has appositely led to the discovery of how extracts of the pancreas from various species contain a glycogenolytic factor of possible A-cell origin (Sutherland and de Duve, 1948). Finally, metahypophyseal diabetic dogs on a protein diet show a substantial glycosuria and ketonuria whereas a diminished glycosuria and ketonuria and an increased sugar tolerance are exhibited by such animals on a high-fat diet (Marks and Young, 1939). As noted above, these observations are paralleled by those

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in experiments involving the administration of a high-fat diet to alloxandiabetic rats (Burn et al., 1944). A diabetogenic APE may, as just indicated, be growth promoting. This phenomenon has been observed in the dog (Evans et al., 1931-32; Young, 1941b, 1944, 1945), cat (Young, 1945), rabbit (Ogilvie, 1944-46) and rat (Young, 1945), but varies with respect to the presence or absence of a concurrent diabetes according to the age and species of the animal. Thus, the puppy and kitten respond to daily diabetogenic APE merely by growing more rapidly than usual (Young, 1949). After some months of treatment the puppy may develop acromegalic features and usually in the end becomes diabetic, with incidental cessation of growth (Young, 1944, 1949). The kitten, however, fails to show diabetes, even though treatment is continued into adult life (Young, 1949). I n contrast, the administration of diabetogenic APE to the adult dog and cat, regularly induces accelerated growth for a few days and then a diabetes, on the advent of which the abnormal growth may continue in modified form or be replaced by loss of weight (Young, 1945). Again, the rat given similar treatment merely grows a t an abnormally rapid rate without ever showing diabetes (Young, 1945). The increase in weight obtaining with the administration of diabetogenic APE occurs on a diet previously just sufficient to maintain a more or less constant body weight (Ogilvie, 1944-46; Young, 1945) and is due to partial replacement of the oxidation of carbohydrate and protein by that of fat (Young, 1945), or, in other words, t o the induction in the non-fasting animal of the basic metabolic pattern found in the fasting state. Pituitary-induced growth, moreover, is accompanied by increase of the pancreatic islets (Richardson and Young, 1937-38; Young, 1944; Ogilvie, 1944-46, 1950) whereas degeneration of the islets characterizes metahypophyseal diabetes with loss of weight (Richardson, 193940). The effect of APE in leading to accelerated growth or diabetes would thus appear to depend respectively on high and low sensitivity on the part of the animal to the pancreotropic action of the extract. Such a conclusion is supported by the observation that the influence of APE in enhancing protein storage is mediated, a t least in part, through the secretion of insulin (Mirsky, 1939). Viewed from another angle, idiohypophyseal diabetes results from pituitaryinduced suppression of the oxidation of carbohydrate and protein under conditions that do not allow of the utilization of the conserved material for extra growth (Young, 1949). In agreement with this view, pregnant and lactating animals are not made diabetic by diabetogenic APE in doses of effective magnitude in the normal animal, thus resembling the young, growing specimen (Young, 1949). The synthesis of the additional materials required in pregnancy and lactation thus probably provides

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an alternative outlet for the utilization of the substances whose oxidation is depressed by the pituitary extract (Young, 1949). Although the outcome of the idiohypophyseal phase, metahypophyseal diabetes is, on the other hand, due distinctively to insulin insufficiency following more or less gross degeneration of the pancreatic islets. The persistent diabetes produced in partially depancreatized cats by APE has been treated with low diet, insulin or phlorizin (Lukens and Dohan, 1942; Lukens et al., 1943). In such a diabetes hydropic degeneration of the B-cells is the characteristic lesion of the pancreatic islets for the first 3 months, whereas after this period the conspicuous insular abnormality is atrophy of the islets from B-cell loss. Treatment of the diabetes in one or other of the above ways within the first 3 months results in morphological restoration and functional recovery of the islets and animals respectively, such as are maintained after the cessation of therapy, while no improvement of islets or animals follows the institution of treatment after 3 months. Animals that have recovered after the discontinuation of insulin can be made diabetic again by APE and may thereupon show a second remission on being suitably treated with insulin. The degeneration of the pancreatic islets produced by APE can also be obviated through the simultaneous administration of phlorizin. As already mentioned, hyperglycemia is regarded, on the basis of the foregoing observations, as probably playing a part in the genesis of the permanent diabetic state through inducing degeneration of the pancreatic islets (Lukens and Dohan, 1942; Lukens et al., 1943), while the degenerative influence of hyperglycemia has been suggested to lie in its increased demands for insulin, with consequent reduction in the glutathione of the B-cells and exposure of their sulfhydryl enzymes to natural inactivators (Lazarow, 1949b). 2. Growth Hormone Pure growth hormone was isolated from APE by two groups of workers (Li et al., 1945; Wilhelmi et al., 1948). It is a protein, probably consisting of a t least 340 amino acids in two chains (Li, 1947), and has been shown to be diabetogenic in intact (Cotes et al., 1949), partially depancreatized (Marx et al., 1943a; Houssay and Anderson, 1949), alloxan-diabetic (Bennett and Li, 1947; Russell, 1951) and anteriohypophysectomizeddepancreatized (Houssay and Anderson, 1949) animals. The partially depancreatized dog may actually be rendered permanently diabetic by suitable treatment with purified growth hormone (Houssay and Anderson, 1949). The diabetes so produced is regarded as similar, in respect of its mechanism, to that induced by crude growth-promoting APE; i.e., it is due to depressed oxidation of carbohydrate and protein in association with

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such a poverty of pancreatic insulin as to render the conserved materials incapable of being built up into and stored as new tissues (Cotes et al., 1949). The adrenocorticotropic hormone is also diabetogenic (Ingle et al., 1946a), but constitutes a very much smaller fraction of APE than the growth factor (Cotes et al., 1949). Further, both APE and purified growth hormone are diabetogenic in the intact cat (Cotes et al., 1949) whereas this animal shows no glycosuria on treatment with adrenocorticotropic hormone (Li, 1949-50). Again, ox anterior pituitary preparations made under widely differing conditions do not vary significantly as regards the ratio of their diabetogenic and growth-promoting activity (Cotes et al., 1949-50). The growth hormone may thus be regarded as almost wholly responsible for the diabetogenesis of crude APE; in other words, the diabetogenic principle and growth hormone of APE are probably identical substances. 3. Adrenocorticotropic Hormone ( A C T H )

ACTH produces hyperglycemia and glycosuria in man (Conn et al., 1948, 1949) and in rats force-fed a high carbohydrate diet (Ingle et al., 1946a), and also effects a marked increase in the glycosuria of alloxandiabetic rats (Bennett and Li, 1946). The augmented glycosuria observed in the latter investigation is incidentally much more consistent than that induced, as above mentioned, by pure growth hormone under similar conditions. The in vitro synthesis of glycogen by the diaphragms of normal rats treated with ACTH is reduced to about half the amount observed in controls under the influence of insulin, while in the absence of insulin the diaphragms of the treated rats actually show glycogenolysis (Li, 1949-50). ACTH also enhances both the glycosuria and urinary nitrogen excretion of alloxan-diabetic rats maintained on a carbohydratefree diet (Bennett and Laundrie, 1948). Again, the glycosuria produced by ACTH in man is accompanied by a reduced blood glutat,hione and an increased urinary uric acid excretion (Conn et al., 1948, 1949) and has accordingly been regarded, much along the lines suggested for alloxandiabetes (Section 11, 5 ) , as due to such a decreased and increased intracellular concentration of glutathione and purine metabolites respectively, as t o interfere with the enzymatic production of insulin by the B-cells of the pancreatic islets (Conn et al., 1948, 1949). On the grounds of these various observations, ACTH-induced glycosuria would thus appear t o depend on a complex mechanism involving gluconeogenesis and inhibition of both the production and effects of insulin. The concurrence of glycosuria and increased urinary uric acid excretion in man during treatment with ACTH (Conn et al., Zoc. cit.) is, moreover, of great interest as indicating a linkage between carbohydrate and purine metabolism, and so

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a basis for the we11 known association of diabetes and gout in the human subject (Thorn and Emerson, 1950). ACTH incidentally leads to increased urinary nitrogen excretion in man (Conn et al., 1948, 1949) and normal rats with proportionate weight loss (Gordan et al., 1946), and in keeping therewith retards the growth of normal rats (Evans et al., 1943; Becks et al., 1944a) and antagonizes the effects of the growth hormone in hypophysectomized rats (Marx et al., 194310;Becks et al., 1944b). Accordingly, ACTH may reasonably be regarded as possessing both diabetogenic and growth-inhibiting properties.

4. Adrenocortical Steroids Bilateral adrenalectomy greatly ameliorates the diabetes produced by pancreatectomy (Long, 1935-36; Long and Lukens, 1936) in the same way as hypophysectomy (Houssay and Biasotti, 1930a and b) and therein operates through removal of the cortex and not the medulla of the glands (Long, 1935-36). The diabetogenic influence attributed to the adrenal cortex on the basis of these observations was confirmed by the ultimately proven diabetogenic properties of corticosterone, 17-hydroxycorticosterone and 17-hydroxy-1l-dehydrocorticosterone (Kendall’s compound E or cortisone) (Ingle, 1941a; Ingle el al., 1945, 1946b). Each of the aforementioned agents leads, in rats force-fed a high carbohydrate diet, to hyperglycemia and glycosuria, while increased urinary excretion of sodium, chloride, potassium, and nitrogen and loss of weight are concomitant effects (Ingle et al., 1945). 17-Hydroxycorticosterone, however, is more potent in bringing about these various phenomena than corticosterone (Ingle et al., 1946b). Moreover, the glycosuria is highly resistant to insulin therapy, inasmuch as 1000 units of insulin may be insufficient to control even a moderate grade (Ingle et al., 1945). In this respect the condition is thus akin to idiohypophyseal diabetes, but contrasts with metahypophyseal diabetes and the diabetes produced by pancreatectomy. Adrenal steroid diabetes, it has been suggested, may be due to combined gluconeogenesis from protein, and perhaps fat, and to failure of utilization of some of the dietary carbohydrate (Ingle et al., 1945). Whether such inhibition of utilization, however, is due to direct interference with the action of insulin or to the negation of some other mechanism involved in carbohydrate metabolism remains for future decision. 6. Adrenaline Given subcutaneously, adrenaline induces hyperglycemia and glycosuria in animals (Blum, 1902), while man has also been reported as show-

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ing glycosuria in response to the therapeutic use of the compound (Garrod, 1912). These effects on the level of the sugar in the blood and urine are due to the stimulation by adrenaline of the terminal mechanism of the sympathetic nerves controlling glycogenolysis in the liver so that the hepatic glycogen is discharged as glucose into the circulation (Grollman and Slaughter, 1947a). Adrenaline also increases glycogenolysis in the muscles, and the lactic acid thus formed is discharged into the circulation and carried to the liver, where it is again deposited as glycogen (Cori and Cori, 1928). 6. Estrogens The’literature contains conflicting reports regarding the diabetogenic properties of the estrogens. Thus, the administration of stilbestrol to normal non-glycosuric rats on a constant high carbohydrate diet usually produces mild temporary hyperglycemia and glycosuria (Ingle, 194lb) . Further, stilbestrol, dihydrostilbestrol, estradiol, and equilin given to partially depancreatized, non-glycosuric or mildly glycosuric rats on a constant medium carbohydrate diet always induce severe hyperglycemia and glycosuria and sometimes also ketonuria; total diabetes actually ensues in a number of these animals during treatment with stilbestrol (Ingle, 1941b). Again, the administration of estradiol-17-carboethoxylate, estradiol benzoate, and a purified, non-crystalline estrogenic preparation from male urine to partially depancreatized, diabetic ferrets leads to increased glycosuria and acetonuria in most and all of the animals, respectively (Dolin et al., 1941). Several other observations have been made relative to stilbestrol (Ingle, 1941b). Thus, this substance is diabetogenic in very small dosage. Stilbestrol diabetes is transitory no matter whether induced by a constant or increasing amount of the material, while a measure of adaptation is also evident in the augmented diabetes of the stilbestrol-treated, partially depancreatized rat. The diabetes of stilbestrol administration is accompanied by an increased excretion of nonprotein nitrogen of insufficient magnitude to account for all the urinary glucose so that part of the dietary carbohydrat,e must be assumed to be excreted as a result of altered utilization or interconversion. Such a conclusion suggests, in view of the above-noted influence of the adrenocorticosteroids, that the diabetogenic action of stilbestrol and other estrogens is mediated through the adrenal cortex and is therein supported by the further capacity of these substances to bring about marked hypertrophy of the adrenal cortex. The last observation, however, is more or less neutralized by corroborative evidence since nonspecific damaging agents such as formaldehyde and carbon tetrachloride induce adrenocortical hypertrophy without having any effect on the

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diabetes of the partially depancreatized rat maintained on a high carbohydrate diet. Finally, both normal and partially depancreatized rats lose weight during stilbestrol-induced diabetes. On the other hand, the incidence of the diabetes following subtotal (95%) pancreatectomy in castrated rats is decreased by various estrogens (Lewis et al., 1950) and these substances also alleviate the diabetes and prolong the life of the pancreatectomized dog (Barnes et al., 1933) and monkey (Nelson and Overholser, 1936). Similarly, estradiol benzoate is accredited with significantly lowering the fasting blood sugar of a proportion of diabetic women after the menopause (Gessler et al., 1939), while estrogens are stated to reduce the insulin requirement of diabetic women before and after the menopause, especially in the former period (Spiegelman, 1940), and to enhance the sugar tolerance of diabetic women during and after the menopause (Cantilo, 1941). The antidiabetogenic effect of the estrogens in these investigations is attributed to their pancreotropic action (Houssay, 1951), or to their suppression of either the general secretory function of the anterior hypophysis (Barnes et al., 1933) or of its diabetogenic activity only (Nelson and Overholser, 1936; Gessler et al., 1939; Spiegelman, 1940; Cantilo, 1941). Finally, estrone, estriol, and stilbestrol have no influence on the intensity of diabetes in the pituitary-diabetic or insulin-treated, depancreatized dog (Young, 1941a), and the diabetes of women of menopausal age is not demonstrably affected by stilbestrol (Lawrence and Madders, 1941). According to the aforementioned reports estrogens may thus be diabetogenic or antidiabetogenic or without either such effect. These discrepancies have at present no satisfactory explanation, but may be due to variation in sensitivity of the different species and to differences in the experimental conditions. 7'. Androgens

Testosterone and methyltestosterone markedly increase the incidence and severity of the diabetes produced by subtotal (95%) pancreatectomy in castrated rats (Lewis et uZ., 1950). Again, massive doses of testosterone and methyltestosterone are weakly diabetogenic in partially depancreatized, non-glycosuric rats on a constant medium carbohydrate diet (Ingle, 1941b), while partially depancreatized, mildly glycosuric ferrets usually show a slightly increased diabetic condition in response to large amounts of testosterone propionate (Dolin et al., 1941). The androgens are obviously more weakly diabetogenic than the estrogens (Ingle, 1941b) and may so function by reason of their damaging effect on the pancreatic islets (Lewis et ul., 1950; Houssay, 1951).

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8. Thyroid Extract

Glycosuria can be produced in the subtotally depancreatized, nonglycosuric dog by the prolonged administration of bovine thyroid powder. This type of diabetes has the following characteristics (Houssay, 1944, 1946): It is predisposed to by sensitization of the animal with thyroid powder or APE, and may be observed only during treatment or persistently after cessation of feeding with thyroid. The temporary and permanent varieties of glycosuria so induced are termed thyroid and metathyroid diabetes, respectively. Thyroid diabetes entails increase in the intestinal absorption rate of glucose, decrease of hepatic, and later, of muscle glycogen, initially increased and finally decreased resistance to insulin, high basal metabolism, and increased liability to ketonaemia and ketonuria. Metathyroid diabetes, on the other hand, resembles the pancreatic variety in its striking hyperglycemia, glycosuria, and ketonuria, fatty liver, increased protein catabolism, marked and moderate decrease in hepatic and muscle glycogen respectively, high basal metabolism, reduced sugar tolerance, and average reactivity to insulin. Thyroid diabetes is characterized by degranulation and hydropic degeneration of the B-cells of the pancreatic islets, whereas metathyroid diabetes entails necrosis and disappearance of the B-cells, atrophy of the islets, and increased prominence of the insular connective tissue. The insular changes are apparently due to a combination of hyperglycemia, diminished pancreatic resistance, and toxic thyroid action. They are reversible and irreversible in thyroid and metathyroid diabetes respectively, and their irreversibility in metathyroid diabetes is accompanied by cessation of insulin secretion as indicated by the failure of the remains of the pancreas from cases of metathyroid diabetes to have any significant hypoglycemic effect on being grafted into the recently pancreatectomized, diabetic dog. Insulin is capable of protecting the islets from damage during the earlier stages of the diabetic condition only. Metathyroid diabetes may be induced after removal of the gonads, thyroid, and adrenal medulla, but cannot be invoked in the hypophysectomized or adrenalectomized dog since such an animal treated with thyroid soon dies in hypoglycemia. A fall of the blood sugar in metathyroid diabetes is also effected by extirpation of the liver. 9. Prolactin A preparation of prolactin containing 10-300/, of ACTH produces hyperglycemia, glycosuria, and polyuria always in the dog and usually in the cat, while 60% of batrachians respond to this material with an elevated blood sugar (Houssay and Anderson, 1949). The impure

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nature of the preparation raises the possibility that its diabetogenic action may be due not to the prolactin, but to the ACTH or to activation of the ACTH by prolactin, or to the additive effect of the two hormones or some other diabetogenic agent. However, the fact that the preparation is more potently diabetogenic than almost pure ACTH (Houssay and Anderson, 1949) suggests that prolactin is indeed possessed of diabetogenic properties.

10. Discussion of Mechanism of Hormonal Diabetes The above account of hormonal diabetes naturally leads to a consideration of the mechanism whereby the hormones induce glycosuria. The initial discoveries related thereto largely accrued from the brilliant work of one group of investigators and may be appreciated in the following summary (Colowick and Sutherland, 1942; Colowick and Kalckar, 1943; Price et aE., 1945; Cori, 1945-46; Price et al., 1946; Colowick et al., 1947). In order that it may enter in the metabolic pathways of the body, glucose must first be converted to glucose-6-phosphateJwhereupon it may in turn be changed to glycogen or fat or be oxidized according to the functional needs of the animal. Preliminary phosphorylation occurs a t the expense of adenosine triphosphate, thus: Glucose

+ adenosine triphosphate -+ Glucose-6-phosphate + adenosine diphosphate

Largely irreversible in nature, the formation of glucose-6-phosphate from glucose is catalyzed by hexokinase originally discovered in yeast (Meyerhof, 1927) and now known to be widely distributed in the animal body. The role of hexokinase in this reaction is strikingly inhibited in vitro by certain anterior pituitary extracts. Adrenocortical extract (ACE) has no such effect, but nevertheless in vitro greatly enhances the inhibitory influence of APE. In contrast, insulin removes the inhibition of hexokinase activity produced in vitro by APE alone or APE plus ACE, although the pancreatic hormone has not of itself any capacity to enhance the in vitro activity of the enzyme. These results naturally suggest a relation between the hexokinase-inhibiting and pituitary-diabetogenic factors. The extreme lability of the hexokinase-inhibiting substance (Cori, 1945-46), however, contrasts with the personally observed stability of the pituitary-diabetogenic factor for long periods at room temperature. Moreover, many anterior pituitary preparations of highly diabetogenic capacity in the intact adult cat, are without insulin-reversible hexokinase-inhibitory activity in vitro (Reid et al., 1948), while a similarly negative result accrues from the use of highly purified preparations of the growth, adrenotropic, and lactogenic hormones (Colowick

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et al., 1947), each of which, as above noted, is capable of inducing diabetes. At the same time, the administration to intact rats of APE, possessing high diabetogenic activity but no demonstrable insulin-reversible inhibitory action on hexokinase in vitro, so influences the muscle hexokinase activity as to render it capable of enhancement by the addition of insulin in vitro (Reid et al., 1948). Accordingly, the pituitary diabetogenic factor on being stored appears to undergo a rapid change whereby it is deprived of its insulin-reversible hexokinase-inhibiting capacity in vitro, but on injection in vivo apparently regains, presumably through a change of reverse order, its power to induce an inhibition of hexokinase activity reversible by insulin in vitro. The insulin-reversible hexokinase-inhibiting agent must thus be closely related to, even although not identical with, the pituitary-diabetogenic factor (Reid et al., 1948; Young, 1949). The preliminary treatment of the isolated rat diaphragm with diabetogenic APE abolishes the expected rise in glucose uptake on subsequent treatment of the diaphragm with an insulin-containing medium, but whether or not this is an effect on hexokinase remains to be determined (Ottaway and Smith, 1948; Young, 1949). The influence of APE and insulin on the part played by hexokinase in the phosphorylation of glucose, as above noted, justifies two inferences, viz. (a), the inhibitory effect of APE on the activity of the enzyme may reasonably be a factor in the diabetogenic action of the extract, and (b), the varied activities of insulin may all be related to its effect on a single enzymatic system. The intimate association between hormones and enzymes developed along the foregoing lines has thus clearly opened up a vast sphere, exploration of which must in due course lead to great advances in knowledge regarding the intermediary stages of carbohydrate metabolism.

IV. DIET

1. Starvation Dogs deprived of food for 2-3 days and then given sugar by injection into the mesenteric vein show slight glycosuria (Lehmann, 1874). Alimentary glycosuria is likewise very easily invoked in starved dogs (Hofmeister, 1890). The glucose tolerance of such animals is reduced from about 5 to 2 g. or less/kg., while about 5 g. of starch/kg. is sufficient to result in the excretion of sugar in the urine. The glycosuria produced by starch appears after 1-3 hours and is usually slight, but occasionally amounts to about one-third of the ingested material. Suitable undernutrition maintains a slight glycosuria for days or weeks, and the addition to the diet of abundant carbohydrate or a small amount of meat causes disappearance of the phenomenon.

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2. Overfeeding

The administration of an excess of various sugars produces glycosuria in dogs (Hofmeister, 1889). The same result is observed in normal rats force-fed a high carbohydrate diet, and in these circumstances lasts at most for 16 days prior to death (Ingle, 1946). Compared with untreated controls, such force-fed rats respond to insulin with a reduced, but not delayed glycosuria, a longer average survival period, and toleration of larger amounts of carbohydrate before death from overfeeding (Ingle and Nezamis, 1947). 3. Change A sudden shift from a high fat to an isocaloric, high carbohydrate diet consistently produces glycosuria in the force-fed normal rat (Ingle, 1948b). The glycosuria obtaining under these various dietary conditions manifestly results from the presentation of amounts of carbohydrate beyond the capacity of the body to assimilate by oxidation, storage, and conversion. V. GLYCOQENOLYSIS 1 . Nervous Damage

Hyperglycemia and glycosuria result from puncture of a certain area in the floor of the fourth ventricle (Bernard, 1858a, 1877b) and from decerebration (Mellanby, 1919-20). The glycosuria induced by both of these procedures appears after a brief space, rises to a maximum, and then subsides and disappears, lasting altogether a number of hours (Bernard, 1877b; Mellanby, 1919-20). Glycosuria is less marked and absent in animals subjected to puncture of the fourth ventricle after fasting (Bernard, 1877a) and ligation of the hepatic vessels (Bernard, 185813) respectively, while the hyperglycemia of decerebration is rendered less pronounced by antecedent bilateral splanchnectomy, or unilateral splanchnectomy and contralateral adrenalectomy (Evans et aZ., 1931), or bilateral adrenalectomy (Donhoffer and Macleod, 1932b). In conjunction with the known point of action of adrenaline (Grollman and Slaughter, 1947b), these observations infer that the hyperglycemia following puncture of the floor of the fourth ventricle and decerebration results from increased glycogenolysis in the liver produced through stimulation of the controlling sympathetic “receptive substance ” partly by impulses arriving via the splanchnic nerves from the site of damage and partly by adrenaline secreted in excess through splanchnic stimulation of the adrenal medullae. However, such an increased outpouring of adrenaline means a conversion of muscle glycogen through the inter-

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mediary stage of lactic acid into hepatic glycogen (Cori and Cori, 1928), accompanied as this is by the effect of decerebration in bringing about a decrease and increase in the amount of glycogen in the muscles and of lactic acid in the blood respectively (Donhoffer and Macleod, 1932a). At the same time, the total sugar calculable as arising from the glycogen in the liver and muscles following decerebration in animals with low initial glycogen, falls far short of what is necessary to induce the observed degree of associated hyperglycemia, thus suggesting gluconeogenesis as the mechanism of origin of at least some of the blood sugar (Donhoffer and Macleod, 1932a). Accordingly, the extra sugar appearing in the blood after decerebration and presumably, therefore, after puncture of the fourth ventricle and the administration of adrenaline, may be derived from three sources and in three ways respectively, via., the original liver glycogen by glycogenolysis, the muscle glycogen via the Cori cycle (Cori and Cori, 1928), and non-carbohydrate material by gluconeogenesis. 2. Pain and Emotion

Cats, on being bound to a board, tracheotomized (without anesthesia) and perhaps also catheterized by a suprapubic exposure of the urethra, show an abundant glycosuria in about half an hour (Boehm and Hoffmann, 1878). The blood sugar is slightly above normal so long as sugar is appearing in the urine, but returns to normal with the disappearance of the glycosuria. The possible causes of this “fesselungsdiabetes ” are tracheotomy, cooling, and pain, of which the first two may be readily eliminated. However, the inability to obviate pain is accompanied by a continuance of the glycosuria (Boehm and Hoffmann, 1878), while this phenomenon may also be induced in unbound animals by merely stimulating the sciatic nerves (Boehm and Hoffmann, 1878). Painful confinement is accordingly inferred from these observations to be in itself a sufficient explanation of the “fesselungsdiabetes ” (Boehm and Hoff mann, 1878). The probability of such glycosuria being mediated through the effect of pain on the adrenal medulla and thereby through increased glycogenolysis in the liver is supported by the way in which sensory stimulation, such as the application of a tetanizing current to the sciatic nerve, leads to an increased secretion of adrenaline (Cannon and Hoskins, 1911-12). Glycosuria may be observed in animals in which fright or rage is induced through being bound to a comfortable holder or through being caged and barked at by an active dog (Cannon et al., 1911-12). It is also sometimes observed in man after periods of acute nervous tension (Cannon, 1939a). Similarly, the condition of sham rage obtaining in some animals deprived of a cerebral cortex is attended by a hypergly-

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cemia, whereas no such phenomenon presents if decerebration fails to be followed by the pseudoaffective state (Bulatao and Cannon, 1925). On the other hand, adrenalectomized animals excited by being bound to a holder fail to show glycosuria (Cannon et al., 1911-12), while the hyperglycemia of the pseudoaff ective state neither rises high nor remains elevated, or may even fall, in the event of antecedent bilateral adrenalectomy or unilateral adrenalectomy and contralateral splanchnectomy (Bulatao and Cannon, 1925). Emotional hyperglycemia and glycosuria are thus probably mediated, like the glycosuria induced by pain, through increased secretion of adrenaline and consequent augmented glycogenolysis in the liver. Such a liberation of extra sugar into the blood stream a t a time when great muscular exertion is likeIy to be demanded of the animal is regarded as a highly interesting instance of biological adaptation (Cannon, 1939b). S. Asphyxia and Asphyxia2 Agents Asphyxia1 glycosuria was observed over 70 years ago (A. Reynoso, see Dastre, 1879b). It is produced by 10-12% carbon dioxide in the respired air, even although the percentage of oxygen be more than is present in atmospheric air, whereas a low percentage of oxygen unaccompanied by an excess of carbon dioxide never leads to glycosuria (Edie, 1906-07). The high percentage of carbon dioxide necessary to induce glycosuria also causes complete anesthesia in the dog and cat (Edie, 1906-07) so that carbon dioxide is no exception to the rule that all anesthetics produce glycosuria (Edie, 1906-07). Asphyxia depletes the hepatic glycogen (C. Bernard, see Dastre, 1879a) and removal of the liver is followed by hypoglycemia in asphyxiated animals (Macleod, 1908-09). Again, bilateral splanchnectomy inhibits the production of glycosuria by asphyxia (see Allen, 1 9 1 3 ~while ) ~ the latter phenomenon also effects an increased secretion of adrenaline (Cannon and Hoskins, 1911-12). The sequence of events leading to asphyxial glycosuria would thus appear to be excess of carbon dioxide in the respired air, stimulation of the splanchnic nerves via the central nervous system, increased secretion of adrenaline, and augmented glycogenolysis in the liver. Many chemical agents regularly or occasionally cause glycosuria and in this respect may often function through an asphyxia1 mechanism. A detailed list of such compounds, including carbon monoxide, chlgroform, digitalis, morphine, and veronal is available in a reliable monograph (Allen, 1913d). 4. Liver Damage The effect of injury to the liver in bringing about glycosuria through increased glycogenolysis in that organ has already been adequately

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reviewed (Allen, 1913b). Summarizing from this source, the factors capable of damaging the liver with these results are (a) poisons including (i) chemical agents, e.g. alcohol, chloroform, ammonia, and phosphorus, and (ii) animal products, e.g. hemolytic serum, lymph, saliva, and pancreatic juice; (b) local stasis and asphyxia as from temporary clamping of the hepatic veins; and (c) trauma such as ligation of the bile duct. 5. Trauma

Glycosuria may follow removal of the carotid body in the force-fed rat (Ingle, 1948~). In these circumstances it is mild and temporary and, being accompanied by respiratory distress, would appear to be mediated through glycogenolysis in the liver (Ingle, 1948~).

VI. KIDNEY Phlorizin Phloriain produces glycosuria on administration by the oral, subcutaneous, and intravenous routes (v. Mering, 1888,1889). The urinary sugar ranges in concentration from 5-15% or more and may be accompanied by acetone and P-hydroxybutyric acid so that the condition a t first sight closely resembles diabetes mellitus in man (Grollman and Slaughter, 1947~). The glycosuria, however, is paralleled by a normal (Deuel et al., 1927) or decreased blood sugar (v. Mering, 1889; Deuel et aZ., 1927). Furthermore, the injection of phlorizin into one renal artery is followed by the appearance of sugar in the urine from the corresponding kidney in two minutes and in the fluid from the opposite organ only after a few more minutes (Zuntz, 1895). The effect of phlorizin in leading to glycosuria is thus interpreted as due to a direct action on the kidney and, more exactly, to a selective inhibition of the tubular reabsorption of glucose (Cushny, 1917; Smith, 1937). The mechanism of this effect is suggested as lying in the inhibition by phlorizin of the phosphorylation of glucose within the tubular cells (Lundsgaard, 1933) and is therein supported by the reasonable similarity between the minimum concentrations of phlorizin required to bring about complete inhibition of glucose reabsorption in vivo and of glucose phosphorylation by kidney cortex extract in vitro (Beck, 1942). The fasting normal dog between the 4th and 13th day of starvation excretes 11 mg. N/kg./hr. (Schondorff, 1897) whereas the fasting phlorizinized dog between the 4th and 6th day of starvation puts out 37 mg. N and 133 mg. glucose/kg./hr. (Reilly et al., 1898). At the same time, the phlorizinized dog does not show any change in its D:N ratio after the ingestion of sufficient meat to double the nitrogen in the urine since

EXPERIMENTAL GLYCOSURIA

21 1

the urinary sugar is also equivalently increased (Reilly et al., 1898). The sugar appearing in the urine of the meat-fed phlorizinized animal must thus be derived solely by conversion from protein, while this material is also in all probability the main or even only source of the urinary sugar excreted by the fasting phlorizinized animal, a t least after depletion of the excess of sugar in the tissues. Now, the process of gluconeogenesis just described as obtaining in the fasting phlorizinized state has been investigated relative t o the secretions of the adrenal and thyroid glands (Wells and Kendall, 1940a and b; Wells and Chapman, 1940). Thus, the fasting phlorizinized rat shows a slight, moderate, and marked reduction in the urinary excretion of glucose and nitrogen after thyroidectomy, adrenalectomy, and hypophysectomy respectively. Hypophysectomy probably acts through subsequent deficiency of the thyroid and adrenals inasmuch as it effects more or less the same reduction in the urinary output of glucose and nitrogen as a combination of thyroidectomy and adrenalectomy. Thyroxine or thyrotropic hormone increases the excretion of glucose and nitrogen in the phlorizinized normal rat, while the phlorizinized, thyroidectomized animal eliminates normal amounts of glucose and nitrogen on treatment with thyroxine alone. At the same time, the simultaneous administration of cortisone and thyroxine or thyrotropic hormone brings about an excessive excretion of glucose and nitrogen in (a) hypophysectomized, (b) adrenalectomized-thyroidectomized, and (c) control animals. Both the adrenal and thyroid glands are regarded on the basis of these observations as influencing the rate of gluconeogenesis in the phlorizinized rat. REFERENCES

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Some Effects of Thyroxine and Iodinated Casein on Dairy Cows, and Their Practical Significance BY KENNETH L. BLAXTER The Hannah Dairy Research Institute, Kirkhill, A y r , Scotland CONTENTS

Page I. Introduction. . . . . . . . . . . . . . . . . . . . . . 11. The Biological Activity of Preparatio 1. Sources of Thyroxine for Dairy Cows.. . . . . . . . . . . . . . . . . . . . . . . 2. The Utilization of Thyroxine when Given Orally to Ruminants.. . . . . . 220 3. The Utilization of Thyroxine Present in Iodinated Proteins.. . . . . . . . . . 220 4. The Galactopoietic Potency of Iodinated Proteins in Relation to Their Thyroxine Content. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222 2. The Effect of Breed, Size, and Age of Cow.. . . . . . 3. The Effect of Stage of Lactation and the Yield of the Cow at the Com-

mencement of Treatment IV. Homeostatic Effects. . . . . . . . .

. . . . . . . . . . . 227 . . . . . . . . . . . 229

3. Nutritional Homeostasis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. The Effect on the Composition of the Milk.. . . . . . . . . . . . . . . . . . . . . . . . . . 1. Fat Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

232 234 234

4. Fat-Soluble Vitamins

6. Phosphatase and Phosphorylated Compounds. . . . . . . . . . . . . . . . . . . . . .

238 7. Iodine Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238 8. The Presence of Thyroxine in Milk. .... ..................... 239 9. The Suitability for Human Con ilk Produced by Cows Given Thyroxine or Iodinated VI. The Effect of Thyroxine and Iodina cow. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Energy and Protein Metabolism.. . . . . . . . . . . . . . . 2. Calcium and Phosphorus Metabolism. . . . . . . 3. Environmental Effects and Heat Regulation.. . . . . . . . . . . . . . . . . . . 242 217

218

KENNETH L. BLAXTER

Page VII. Secondary Effects.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243 1. Disease Resistance.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243 2. Reproductive Performance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243 3. Other Abnormalities.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244 4. Hyperthyroid Death.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245 VIII. Practical Conclusions. . . . . . . . . . . . . ... . . 245 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247

I. INTRODUCTION At the end of last century Hertoghe showed that when a cow was given dried thyroid gland in its diet, the daily milk yield rose by 15% (Hertoghe, 1896, 1899). Later reports of this work have placed the increase which Hertoghe observed as high as 36% (Lukacs, 1930). These observations are undoubtedly the first recorded instances of the use of any endocrine preparation in attempts to augment the productivity of farm livestock. Like so much of the scientific work carried out at that time, these early results were forgotten in the ensuing years and the role of the thyroid gland in milk secretion was not fully realized until the experiments of Graham in 1934. Graham showed that either thyroxine injection (1934b) or feeding dried thyroid gland (1934a) increased the milk and fat yield of dairy cows. These results were soon confirmed with both goats and cows (Jack and Bechdel, 1935; Folley and White, 1936; Herman et al., 1938), and it was realized by many of the workers concerned that stimulation of milk yield by the administration of thyroid hormone had a considerable potential value for the dairy industry. The primary consideration was that, unlike anterior pituitary extracts which show galactopoietic activity on injection only, thyroid extracts were also active when given by mouth to milking cows. The expense and scarcity of dried thyroid gland and of thyroxine preparations precluded any extension of these preliminary experiments to practical dairy farming at this time. In 1936 Ludwig and von Mutzenbecher made a preliminary announcement that they had isolated crystalline thyroxine from artificially iodinated proteins. This first announcement excited no comment, but when the complete and extended report of their experiments was published in 1939, it was soon realized that here was an inexpensive and apparently inexhaustible source of thyroxine which might well be used to increase the milk production of dairy cattle. In 1940, Bottomley & Folley in England, and Turner in America demonstrated that increases in milk yield could be obtained by feeding iodinated casein to goats. More recently, synthetic L-thyroxine, manufactured from L-tyrosine has provided a further inexpensive supply of thyroid active materials (Borrows et al., 1949; Clayton and Hems, 1950).

EFFECTS OF THYROXINE AND IODINATED CASEIN ON

cows

219

During the last ten years considerable attention has been given by animal husbandrymen throughout the world to the practical aspects of the use of iodinated proteins and to a lesser extent of L-thyroxine as galactopoietic agents. This review is concerned mainly with the results they have achieved. Some reference is made also to experimental hyperthyroidism in laboratory animals and non-lactating domesticated livestock, as well as t o experiments involving thyroidectomy and the administration of goitrogenic drugs. Major emphasis, however, is placed upon the dairy cow and goat. Previous reviews of this subject have been published by Reineke (1946), by Blaxter et al. (1949) and by Koller (1950).

11. THEBIOLOGICAL ACTIVITYOF PREPARATIONS 1. Sources of Thyroxine for Dairy

Cows

A large number of iodinated proteins have been prepared from which thyroxine has been isolated, or which have shown high potency in animal tests. A wide variety of starting materials has included casein, serum albumin, serum globulin, edestin, crude arachin, egg albumin, silk fibroin, and insulin (see Reineke, 1946; Roche et al., 1949). One report from India gives careful instructions to local farmers on how to prepare active materials on the farm, starting with separated cow’s milk (Das Gupta, 1946)! Only a few of these many preparations has been used with cows. At present iodinated caseins, marketed in the United States and continental Europe under proprietary names, appear to be the only ones in uae. In earlier work in Britain and America, low potency or even complete absence of activity of such preparations was common. Even a t the present time occasional reports of inactivity of preparations may be found (Opichal et al., 1949). The improvements made by the Missouri group (Reineke and Turner, 1942, 1945, 1946) in the methods of preparation of iodinated casein have largely removed these limitations, but, as will be shown subsequently, iodinated casein cannot be regarded as a highly standardized product of absolutely constant potency. A biological or chemical assay of thyroxine content is essential prior to the extended use of these materials. Synthetic L-thyroxine has many advantages over iodinated proteins as a source of thyroxine for dairy cattle (Bailey et al., 1949). Firstly, the necessity for bioassay is avoided; secondly the material is virtually tasteless, and the cow readily eats it when it is incorporated into rations. This is not so with many iodinated proteins. Lastly, the ingestion of L-thyroxine does not involve the consumption of large quantities of non-thyroxine iodine which with some iodinated proteins have proved sufficient t o cause signs of serious iodine poisoning.

220

KENNETH L. BLAXTER

Before dealing with the effects of these materials in cattle it is essential to discuss their potency in some detail. The ensuing sections deal with the utilization of thyroxine and iodinated protein by ruminants. 1. The Utilization of Thyroxine when Given Orally to Ruminants

Experiments with sheep have shown that it is necessary to give eight times as much Na DL-thyroxine by mouth as it is by subcutaneous injection in order to produce the same percentage loss of bodyweight (Turner and Reineke, 1946). Working with cows, Folley and his collaborators have similarly shown that the response in milk yield to 10 mg. Na DL-thyroxine given by subcutaneous injection is approximately equivalent to that obtained when 82 mg. Na L-thyroxine was given orally (Bailey et al., 1949). The relative biological potency of the sodium salts of the L- and the DL-isomers is approximately 2: 1, Na D-thyroxine having but negligible potency, and so it has been concluded that in the cow sixteen times as much thyroxine must be given orally as parenterally in order to elicit the same increase in milk production. Both these results, based on the ratio of parenteral to oral activity, measure the loss of activity undergone by thyroxine in reaching the systemic circulation by the oral route. Poor absorption from the digestive tract may not be the only reason for low oral potency, since decomposition in the rumen or other parts of the gut, or inactivation by the intestinal mucosa or the liver are not precluded. In the absence of direct experiments on these aspects, these values may only be described as “oral utilizations” of thyroxine. The oral utilization of thyroxine in the ruminant is thus probably in the region of 10% since large errors are involved in both the methods used to establish the ratio. This value is lower than those found with other species using similar methods. Thus Frieden et al. (1949) found that the oral activity of Na DL-thyroxine in the rat was 49% of its parenteral activity, while Monroe and Turner (194913) obtained a value of 45% for the same salt with chicks.

3. The Utilization of Thyroxine Present in Iodinated Proteins With sheep, iodinated casein is twenty to thirty-two times as effective in producing hyperthyroidism when injected as when given by mouth (Turner and Reineke, 1946). In view of the comparable data obtained with thyroxine these results suggest that there is probably about 50% to 70% less thyroxine absorbed from iodinated casein than there is from the racemic sodium salt. Similar experiments with rats are in agreement with this result. Thus Frieden et al. (1949) showed quite clearly that while thyroxine exerted 49 % of its parenteral potency when given to rats by mouth, orally administered iodinated casein revealed only 14% of its

EFFECTS OF THYROXINE AND IODINATED CASEIN ON COWS

221

parenteral activity. Such experiments do not involve any chemical analysis of iodinated proteins for thyroxine content and appear reliable estimates of its oral utilization. Experiments in which the activity of thyroxine has been compared with the activity of iodinated casein, when both are given by mouth, necessitate determinations of the thyroxine content of the iodinated casein if they are to be used to determine the relative utilization of the free hormone and the hormone present in the protein. Conventional analyses for the thyroxine content of iodinated proteins are not, however, reliable. The agreement between determinations of the acid-insoluble iodine content of iodinated proteins and their biological activity relative to thyroxine is so poor that it cannot in any way be assumed that thyroxine is the sole constituent of the acid-insoluble fraction (Deansley and Parkes, 1945a). In this respect it was pointed out by Pitt-Rivers and Randall (1945) that the iodination of tyrosine and the condensation of two molecules of diiodotyrosine thus formed to give thyroxine is likely to result in the formation of biologically inactive by-products containing acid-insoluble iodine. Similarly, tyrosine would not be the sole amino acid to be iodinated. For example, iodine would enter the imidazole ring of histidine (Pauly, 1910). Polarographic determinations of thyroxine in partially purified acid-insoluble fractions of iodinated proteins have, however, been in fairly good agreement with their thyroxine content calculated on the assumption that the whole of the iodine in these fractions is in fact thyroxine iodine (Simpson et aZ., 1947). These partially purified preparations contain less acid-insoluble iodine than comparable preparations analyzed by Harington and Randall’s method (1929). Modifications of the Blau butanol solubility procedure (1935) for the determination of thyroxine have given a fair agreement between biological potency and thyroxine content using a limited range of iodinated caseins (Reineke et al., 1945). Using the isotope dilution technique and labelled thyroxine prepared from radioactive iodinated casein (Courrier et al., 1949), it has been shown recently, however, that only 23% (range 18-29%) of the butanol-soluble iodine identified as thyroxine in the Reineke procedure, is in fact thyroxine (Reineke et d.,1950). The published analyses for the thyroxine content of iodinated caseins would appear therefore to be four or five times too high. With these limitations of chemical methods for the determination of the thyroxine content of iodinated proteins in mind, the experiments to determine the relative potency of thyroxine and of iodinated casein may be examined. Bailey et al. (1949) found that 15 g. of iodinated casein containing approximately 3 % of “thyroxine ” (butanol extract) was equivalent in galactopoietic potency in the cow to 60-78 mg. Na L-thy-

222

K E N N E T H 1,. BLAXTER

roxine. These figures imply that iodinated casein exerts only 13-17% of the potency which might be expected on the basis of its thyroxine content. Assuming that the analytical values are in fact four to five times too high, the potency of iodinated casein in the cow is probably about 70% of the value to be expected on the basis of its thyroxine content. This information obtained with cows is in reasonable agreement with that obtained with sheep (Turner and Reineke, 1945). As with the pure hormone, these results represent data on utilization rather than absorption. A more direct approach to the problem of the absorption of the thyroxine in iodinated proteins in the ruminant has been made by Monroe and Turner (1949a). A goat was given iodinated casein in its feed and both its feed and faeces assayed for thyroxine activity using the reversal of goiter in chicks as the assay method. The results showed that 78448% of the thyroxine activity given could not be recovered in the faeces and was presumed to be absorbed from the alimentary tract. These values are at least ten times the utilization rates calculated from the ratio of parenteral to oral potency in the same species, or the calculations that may be made on the basis of data of Bailey et al. discussed above. If the possibility of analytical error is excluded, they suggest that considerable destruction of the hormone occurs when it is given orally compared with parenterally. Experiments which throw light on this possible destruction are few. Inactivation by the rumen, microflora does not seem to be a major factor since direct introduction of iodinated casein into the abomasum, thus by-passing the rumen, does not result in greater biological activity (Turner and Reineke, 1946), nor has coating of particles of iodinated casein with materials resistant to chemical conditions in the rumen resulted in any increase in potency (Blaxter, 1945b; Turner and Reineke, 1946). Excretion of thyroxine in the bile of the ruminant (Monroe and Turner, 1948) would probably not account for this large difference between the single direct experiment on absorption and those on the oral utilization of iodinated casein.

4. The Galactopoietie Potencg

of Iodinated Proteins in Relation to Their Thyroxine Content The foregoing discussion has shown that the thyroxine of iodinated proteins is less effective than the sodium salt of the pure amino acid in stimulating milk yield and metabolism in ruminants. It is important in this respect to show that the galactopoietic activity of iodinated proteins does in fact reside in their thyroxine content. As well as reports that dried thyroid (Robinson, 1947a) or iodinated casein (Roche et al., 1948) increase lactational performance in women, it has been shown that Lugol’s iodine solution is also of value in the treatment of deficient breast feeding (Robinson, 194713). This report, which

EFFECTS OF THYROXINE AND IODINATED

CASEIN O N

cows

223

appears convincing, has not, however, been confirmed (Nicholson, 1948; Dean, 1950). As far as the cow is concerned, there is little doubt that the galactopoiesis observed when iodinated casein is given is in fact due to thyroxine. The changes in composition of the milk and in the metabolism of the cow given either material are highly comparable. The lower vitamin C content of the milk and the presence of sub-clinical iodism in COWS given iodinated casein, can be traced to the ingestion of larger quantities

l'ol

0 0 0

0.9

0

0.1 0

0 I

K

0

03

I

1

l

0.2 0.3 0.4

1

1

1

0.5

0.6

0.7

1

-

1

0.8 0.9

1

1.0

Activity measured with xenopus, standard 1.0

FIG. 1. The relative activity of preparations of iodinated proteins, showing the good agreement obtained between assays with milking cows and those with xenopus tadpoles.

of iodide by these animals, but, broadly speaking, the comparative responses suggest that thyroxine, is indeed the only substance involved. Mgllgaard (1947), however, has postulated that iodinated casein contains two hormones, one that increases the metabolism of the cow and is identical with thyroxine, and one that stimulates milk secretion without causing the general increase in cellular activity characteristic of the thyroid hormone. Poulsen (1949) has published experiments with lactating goats in which sodium hydroxide hydrolyzates of iodinated casein have been separated into two fractions. It is claimed that one of these fractions not only stimulates lactation by 50 to 150% without causing hyperthyroidism, but that the effects persist for 1-2 months after the cessation of treatment. These experiments, however, are not convincing. The most reliable evidence that the galactopoietic potency of iodi-

224

KENNETH L. BLAXTER

nated proteins resides in their thyroxine content is to be obtained from the data assembled by Deansley and Parkes (194513). It was found that the relative activity of a series of iodinated proteins in promoting metamorphosis in tadpoles coincided with their relative galactopoietic potency measured with cows. These data are shown graphically in Fig. 1. Thyroxine is not the only substance known to accelerate metamorphosis in Xenopus tadpoles, since diiodotyrosine exerts 0.5-1.0 % ' of the activity of thyroxine (Deansley and Parkes, 194513). It has been shown, however, by Deansley and Parkes, that their results could hardly be complicated by the amounts of diiodotyrosine present in iodinated proteins. It may be concluded, therefore, that the galactopoietic potency of iodinated proteins is in fact due solely to their thyroxine content.

111. THEEFFECTOF THYROXINE AND IODINATED PROTEINS ON MILK YIELD When a cow is given thyroid hormone every day there is generally an increase in milk yield. This increase is not immediate, the rise in yield following a sigmoid curve over a period of some 12-18 days. The rate of increase is greatest between the 4th and 7th days. Thereafter, yield is either maintained a t a maximum level for a week or more, or it slowly declines at a rate which may or may not be comparable to the rate a t which the milk yield of normal cows declines with the advance of lactation. All investigators are agreed that the maximal response is highly variable even under identical environmental conditions. The response of individuals in groups of cows given identical quantities of iodinated casein range from 0 to 50 % of the initial yield of the cow (Archibald, 1945). Similar variation has been observed with injected thyroxine (Hurst et al., 1940). The ensuing discussion deals with the factors thought to be responsible for such a wide variation of this initial response. Long term experiments are dealt with separately. 1. The Eflect of Dosage Level The marked variation in the biological activity of iodinated proteins makes it difficult to combine the results of investigators who have carried out experiments with different quantities of iodinated protein of unstated potencies. A number of experiments, however, have included more than one level of administration of the same sample of material, and for orally administered L-thyroxine a dosage response curve has been determined (Bailey et al., 1949). Blaxter (1943)concludedthat the percentageincrease in milk yield is in direct proportion to the amount of iodinated casein given, the range of dosage being 15-30 g. daily. Experiments in Holland, with a range of doses of iodinated casein, apparently of low potency (Schuurmans, 1949),also suggest a linear relationship over a considerable range of yield

EFFECTS OF THYROXINE AND IODINATED

CASEIN ON

cows

225

stimuIation (6-23 %). Blaxter’s results, together with those of Bailey et a2. (1949) with L-thyroxine, are shown in Fig. 2. The latter suggest that the dosage/response relationship is by no means linear and it might be inferred that the maximum possible response is in the region of 7 lb. milk/cow/day. Little information is available a t levels of stimulation above 30 g. iodinated casein or its approximate equivalent of 150 mg. Na L-thyroxine. It has been reported that 40 g. of iodinated casein given to a cow in which lactation had been induced with estrogens resulted in an increase in yield of 50% (Szumowski and Charenton, 1950), but this mg. I.Thyroxine

5

10

15

20

25

30

35

40

45

50

55

60

g. lodinated casein

FIQ. 2. The relation between the dosage of iodinated casein or of thyroxine and the response in milk yield of dairy cows. The triangles represent the results of Bailey et al. (1949) and the circles the results of Blaxter (1943).

provides no information regarding the response of the normal cow. Records of the changes in production which occurred when four cows were accidentally given 50-80 g. of a potent iodinated casein provide a little information. A mean increase in milk yield of 8.2 lb. daily was obtained from three cows given 53-61 g. iodinated casein and 12.6 lb. from one cow given 79 g. (Blaxter, 1946). These increases refer to the period from the 8th to the 12th day after treatment began, and thus are probably not maximal. As such they do not suggest an asymptote in the response curve a t approximately 7 lb./cow/day. Other less complete data also suggest an approximate proportionality between dosage and response over a fairly wide range of treatment. These results are not quoted in detail, and it suffices to say that responses ranged from lo%, when 10 g. was given, to 35-5575 when 25 g. was given (Lanik and Isajev, 1949; Moustgaard and Thorbek, 1949).

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KENNETH L. BLAXTER

Thomas et al. (1949), with a limited number of cows, could not detect differences in response to comparable levels of stimulation, and concluded that the individuality of the cow was as important a factor as dosage in determining the response. While individuality of the cow is clearly of considerable importance, it is nevertheless obvious that within a range of dosage resulting in increases in milk yield from 6 to a t least 40%, yield increases with increase of hormone level. At high levels of stimulation a diminution in response undoubtedly occurs, as instanced by the depression of yield observed by de Fremery (1936) when he injected a goat with an overdose of 15 mg. Na DL-thyroxine each day. Very few observations are available regarding responses to quantities of iodinated casein less than 10 g. per day (equivalent to about 50 mg. Na L-thyroxine by mouth or 5 mg. Na DL-thyroxine by subcutaneous injection). Positive responses have been obtained in this range by BaiIey et ab. (1949) and by Schuurmans (1949), but Reineke et al. (1944) failed to show increases in yield, though an increase in the fat content of the milk occurred when 5 g. of iodinated casein were given. German experiments with graded doses of iodinated casein from 2.3 to 10 g./day have also shown positive results, the response increasing progressively as dosage was increased (Richter, 1949; Zorn and Richter, 1949a,b). No quantitative information on the initial response to 0.625, 1.25, and 5 g. of iodinated casein is included in the papers of Swanson and Knodt (1948, 1949). Their work, together with other data referring to the effect of dosage level on responses measured over long periods of time, are discussed later. 2. The E$ect of Breed, Size and Age of Cow

Responses in milk yield have been obtained with a large number of different breeds of cow, ranging from the recognized dairy breeds of Britain and America to the Highland Spotted Cattle of Bavaria. Differences between breeds in their response could arise in two ways. Firstly there is the possibility of mild hypothyroidism of genetic origin in some breeds, more especially beef breeds, and secondly the mature size of cattle of different breeds varies from 700-1600 lb., so that a constant quantity of hormone would conceivably result in a greater stimulation in smaller cattle. In this respect, even if a constant dosage/kg. body weight was given, differences due to size might still be apparent since the requirement of hormone to give a constant level of stimulation would probably be proportional to'body surface rather than to body weight. From Table I, which is taken from the results of Blaxter (1946), there is some evidence that the smaller breeds produce greater responses to the same dose. Statistically, errors in assessing such differences are large. A more pronounced response by Ayrshires than by Holsteins (comparable

EFFECTS OF THYROXINE AND IODINATED

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227

to British Friesians) was also noted in Canada (Allen, DOW,et al., 1948). Significant differences between the responses of breeds of cows in most of the published experiments have, however, been masked by large variation in the response of individuals. It seems quite certain, however, that the response is no greater in the beef breeds than in the dairy breeds (Blaxter, 1946). TABLE I The Effect of Breed of Cow on the Response to a Constant Daily Dose of 20 g . of Iodinated Casein*

Breed Jersey Ayrshire Red Poll Dairy Shorthorn British Friesian

No. of cows

Increase in daily yield (1b.1

Increase (%)

Approximate weight of cow (1b.)

7 84 16 57 33

5.5 5.7 5.4 5.4 5.0

27.5 22.4 24.7 22.0 16.2

800 1000 I100 1250 1300

~~

* The results are for young cows: heifers and old COWS are excluded, and the responses are those obtained in the 3rd and 4th weeks of treatment.

The age of the cow has a decided effect on the response (Blaxter, 1946; Booth et al., 1947). Typical data from the British experiments are given in Table 11. The effect of age is understandable in view of the decline in both over-all metabolic activity and in the natural rate of secretion of the thyroid hormone with increasing age. TABLE I1 The Effect of Age of Cow on the Increase i n Yield Obtained i n the Srd and 4th Weeks of Treatment with Iodinated Casein Increase in yield Age of cow

No. of cows

lb./day

%

1st lactation 2nd or 3rd lactation 4th lactation and over

174 217 108

4.5 5.5 6.9

20.5 22.7 25.0

3. The Effect of Stage of Lactation and the Yield of the Cow at the Commencement of Treatment

Herman et al. (1938) showed quite clearly that the response to a Vtandard injection of thyroxine waq maximal during mid-lactation. Cows

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KENNETH L. BLAXTER

at the peak of their production as well as at the end of lactation, failed to respond at all; in fact, decreases in production were often observed a t these times. This has been confirmed (Ralston et al., 1940) and estended to iodinated proteins (Blaxter, 1943, 194513; Johansson and Korkman, 1946). Even a t the same stage of lactation, however, cows vary considerably in their normal yield. A good cow may easily give three or four times as much milk as a poor one when half her lactation is completed. The studies with iodinated casein have shown that both yield and lactation stage appear to be involved in determining the magnitude of the response. This is illustrated in Table I11 using data from the TABLE I11 The Mean Response (lb./day) of 104 Cows to 80 g. Iodinated Casein at Tioo Levels of Production and Two Siages of Lactation Mean daily yield before iodinated casein was given Mean initial number of weeks the cow had been in milk Between 10 and 20 Between 25 and 35

Between 10 and 20 lb.

Between 25 and 35 lb.

4 . 1 8 0.39 6.27 k 1.28

7.00 i 0 . 3 3 11.32 f 0 . 3 8

large scale British experiments (Blaxter, 1946). The very large increases in production that can be obtained from cows late in lactation and yet giving high yields is noteworthy. These two factors, which reflect the functional state of the mammary gland, alone account for the major part of the variation that has been observed in practical trials with thyroid hormone. Responses vary from less than 1 lb./day with poorly yielding heifers (cows in their first lactation) that have calved a few weeks only, t o over 12 Ib. with old cows capable of maintaining high yields late in lactation. As Thomas et al. (1949) state “It is not possible to make a good cow from a poor cow by administering thyroprotein.” These differences in response may be explained in terms of the number of alveolar cells capable of responding to stimulation and their state of activity. In early lactation the number of alveolar cells capable of response is maximal, and either they are functionally too highly active to respond or, more likely, increases in their secretory rate are limited by other factors, such as pressure relationships within the udder. Late in lactation involution of the udder commences and the number of secretory cells is much reduced. The metabolic rate of mammary tissue is known t o be correlated with the onset, development, and subsequent decline of

EFFECTS OF THYROXINE A N D IODINATED

CASEIN

ON

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229

lactation (Folley and French, 19498,b) and such a hypothesis seems reasonable. A number of experiments suggest, however, that thyroxine has an effect on the mammary gland distinct from the stimulation of the metabolism of existing cells. In the thyroidectomized cow, estrogen administration fails to induce mammary gland growth, but if the symptoms of myxoedema are relieved by thyroxine, mammary growth occurs (Petersen et al., 1941). In a normal virgin heifer, Dryendahl (1946) was able to induce mammary growth and lactation by administration of iodinated casein, but experiments using a virgin heifer with ovarian hypoplasia were unsuccessful. In later reports Dryendahl (1949) regards this mammary growth and induction of lactation as unique, since it was the only case observed among 19 animals of both sexes under comparable treatment. In experiments with non-pregnant, non-lactating cows in Australia, however, udder development and the production of “up t o 30 ml. of a milky secretion” followed thyroxine injections. Histologically the control cows all showed an advanced involution of the mammary lobule-alveolar system, but in the treated animals a proliferation of ducts without true alveolar development had occurred (McQuillan et al., 1948). These results suggest that thyroxine may not act solely by stimulating the metabolism of existing alveolar cells, and that under some conditions it can result in augmentation of the effects of estrogens and progesterone in promoting mammary growth.

IV. HOMEOSTATIC EFFECTS The above discussions have been concerned with the responses in milk yield obtained in relatively short periods-3 to 6 weeks at the most. They suggest that considerable increases in yield could be obtained by giving selected cows iodinated casein or thyroxine a t the stages of lactation when their milk yield is most sensitive to the effect of the hormone. They ignore, however, the consequences which follow the cessation of treatment, and provide no information regarding the maintenance of these responses for longer, and of course much more economically desirable, periods of time. 1. Evidence for a Decline in Response with Continued Treatment

When hormone treatment is stopped, after a lag of a few days, yield drops precipitously. It falls to levels that for several days are considerably below those to be expected had the cow received no treatment. Subsequently yield slowly rises until after a few weeks it has reached a level close to that of comparable control animals. A month or so after

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KENNETH L. BLAXTER

even a short experiment, any differences in yield discernible between control corns and cows that had been given the hormone are associated with large statistical errors. Experiments with small numbers of cows have thus often failed to show any pronounced effects on subsequent production of a short term of treatment. Experiments with larger numbers have, however, demonstrated conclusively that a slight depression of yield occurs. The British large-scale trials (Blaxter, 1946) place the depression at 0.3 lb./day, and it may be calculated that a cow given thyroxine for a period of 6 weeks fairly early in lactation and subsequently lactating for a further 30 weeks would give a total increase in production not of 250 lb. but of about 170 lb. Short periods of treatment involving temporary increases of 20-25 % thus involve only small (2-3 %) increases in total lactation yield. The length of the period of yield-depression following treatment is not known but has been estimated to be 140 days when long periods of treatment have been given (Thomas and Moore, 1948). In view of the small over-all increase in lactation yield to be obtained from temporary stimulation, attention has been given to the effects of long-term stimulation at different dosage levels. Most of the evidence suggests that long-term stimulation does not result in substantial increases in production. Thus Reece (1946) fed iodinated casein for 17 months and found that six of his cows produced more milk and three less; Moore (1946) found sharp declines in production after 4-8 weeks, and Booth and his associates showed the response to last only 6 to 15 weeks (1947). Leech (1950), in experiments with 59 cows, has shown that the milk yield response disappears after 10-12 weeks. The milk yields of control cows and treated cows then become indistinguishable, and, in fact, depressions of yield often become apparent. Several reasons have been suggested for this decline in response with continued treatment. An increased rate of senescence of aveolar cells during hyperthyroidism has been postulated, but has not been subjected to experimental study. An apparently increased senescence may, however, merely be a reflection of the two factors clearly involved in this decline, and which for convenience may be called “endocrine homeostasis ” and “nutritional homeostasis.” 2. Endocrine Homeostasis

It has long been known in laboratory animals that thyroxine administration depresses the activity of the animal’s own thyroid. This is due to a reduction of the secretion of thyrotropic hormone of the anterior pituitary. There is ample evidence that the same phenomenon occurs in cattle. In the Australian experiments (McQuillan et al., 1948) it was

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shown that as dosage of thyroxine was increased, pulse rates and presumably metabolism showed relatively little change, suggesting some type of homeostasis. Histological examination of the thyroids of these cows showed a significant depression of acinar cell height, and a reduction in their content of thyroxine (butanol-soluble) iodine. Even more spectacular was the complete absence of thyrotropic hormone activity in the pituitary glands of thyroxine treated animals, though those of control cows contained significant amounts. These results suggest a complete inhibition of thyrotropic hormone secretion and a marked reduction of the endogenous production of thyroxine. As Leech (1950) has pointed out, low doses of iodinated casein or thyroxine may be expected to result in proportional suppression of endogenous thyroxine production. The net result would thus be a complete cancellation of any initial effect of the exogenous thyroxine. There is ample evidence of such effects when small quantities of iodinated casein are given. Thus, in experiments lasting a year, iodinated casein given at levels of 0.625, 1.25, and 5.0 g./day failed to show any effect on total milk yield or milk composition, nor was there any effect on metabolic rate as judged by changes in body weight, body temperature, heart rate, or respiratory frequency (Swanson and Knodt, 1949). The highest of these doses would have been expected to give a small, but nevertheless perceptible, increase in yield, and, during the first four months of these trials, an initial stimulation did appear to be present. When feeding of iodinated protein was stopped, however, the depression of yield in the ensuing 35 days was greater for the cows given the hormone than for the controls. This is exactly what would be expected on the basis of pituitary inhibition by exogenous thyroxine. With higher doses Leech (1950) has shown that disappearance of the response occurs when both 15 g. and 20 g. of iodinated casein are given, but that with 25 g., although the response falls off, it is still maintained above that of the controls. This may be interpreted as a permanent increase in metabolic rate resulting from an excess of exogenous hormone over endogenous production. Few estimates of the endogenous production of thyroxine are available. In the cow the effect of thiourea, like that of thyroidectomy, is to depress established lactation. By determining the amount of thyroxine necessary to return yield to normal following the administration of thiourea, Schultze and Turner (1945) have shown that the cow secretes thyroxine equivalent to 10 mg. DL-thyroxine daily. As has previously been shown, this is approximately equivalent to the administration of 20 g. iodinated casein by mouth. Leech’s results, showing complete reversal of the effects of 15 g. and 20 g. of iodinated casein but only partial reversal of the effects of 25 g., suggest, therefore, that endocrine homeostasis will occur

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unless amounts of thyroxine are given in excess of the total secretion rate of the animal’s own thyroid. Unfortunately little is yet known regarding many of the time relationships of pituitary inhibition. At present it does not seem possible to increase yields significantly other than by short-term treatment in which endocrine homeostasis does not seem to be a major problem. 3. Nutritional Homeostasis

In a normal cow, sub-optimal nutrition with regard to most essential nutrients eventually gives rise to a decline in milk yield. Temporarily the cow is able t o utilize body reserves of nutrients to meet lactational demand, but eventually an adaptation of output of milk to input of nutrients takes place. This phenomenon of nutritional adaptation or nutritional homeostasis is very marked in the case of the energy metabolism of the cow. The cow can draw on body reserves of fat and protein for a limited period to sustain lactation, but eventually a new equilibrium is established at a lower body weight and milk yield. With cows treated with iodinated casein or thyroxine, similar adjustments presumably occur. Thus one conclusion arising from the British trials was that if there was any factor limiting the production of the cow, whether nutritional or managerial, then the response to the thyroxine would be small. This conclusion arose from short-term trials. In long-term trials it is extremely difficult to disentangle the endocrine homeostasis already discussed from any nutritional homeostasis of the type outlined above. When a cow is given iodinated casein, her metabolic rate, as judged by her pulse rate, increases. As with milk yield, the pulse rate declines after a few weeks t o levels which approach those of control cows (Moore, 1946; Hibbs and Krauss, 1947). Body weight has similarly been found to decline if the feed requirements of the cow, when under the influence of the hormone, were assessed a t the normal constant allowance t o cover basal metabolism and incidental activity (maintenance) , and a further variable allowance directly proportional to the milk produced. Nutritional homeostasis due to depletion of body reserves might thus be argued. Furthermore, when the allowance of food was increased by 25 %, milk production, pulse rate or body weight did not show the expected decline (Thomas et al., 1949). The interpretation of these results is complicated, however, by the fact that even in the normal cow pulse rate is related to feed intake and t o milk yield (Blaxter and Price, 1945). It was found that when pulse rate increased milk yield also increased, whether iodinated casein or extra food was given. These results have been confirmed (Thomas,

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1949; Thomas and Moore, 1951a). Similarly, in steers an increase in food intake increased pulse rate whether iodinated casein was given simultaneously or not (Sykes et al., 1948). The restoration or maintenance of a response in pulse rate or milk yield to iodinated casein by an increase in feeding level does not, therefore, necessarily imply that limitations of energy supply are the cause of the decline in response to the hormone. The decline in yield could still be due to pituitary inhibition, and the value of extra feed merely a reflection of its value in the normal animal. Unfortunately, there do not appear to be long-term experiments in which control animals have simultaneously been given 25 % more food. Comparison of lactation yields of animals given iodinated casein and 25 % more food with controls given 100% of their requirements is hardly valid. Nevertheless, studies on the efficiency of food utilization tend t o suggest that an explanation in terms of pituitary inhibition is correct. In short trials lasting 8 or 15 weeks Gardner and Millen (1950) found that the food intake of the control cows was almost exactly that to be expected on the basis of their requirements. With cows given iodinated casein, however, the calculated requirement was only 70% of the food taken in. This suggests a marked increase in heat loss due to an accelerated basal metabolism. In long-term experiments lasting 43 weeks, however, Thomas et al. (1949) could not show any differences in over-all efficiency. If an elevation of basal metabolic rate had been present throughout these latter trials it is difficult to account for such results. Pituitary homeostasis is undoubtedly involved, and the maintenance of the response probably reflects the increase in energy intake. Long-term experiments and the statistical study of published feeding trials (Jensen et al., 1942;Yates et al., 1942) all suggest that a 25% increase in total feed intake will result in an increase of 15-25% in the total yield of normal cows. Such an increase is comparable to that obtained in shortterm trials with 15 g. iodinated casein. If a 25% higher plane of nutrition merely maintains the response at this level, then there is no advantage to be gained by giving iodinated casein for such protracted periods. The above discussion is not intended to imply that short-term stimulation does not involve a depletion of body reserves, or that such depletion should not be made good. In much of the work in Britain, a fixed additional quantity of food, calculated to meet the increase in the cow’s maintenance requirement, was given. Similar fixed allowances have also been given in Canadian experiments. It is probable that such attempts to maintain body weight during stimulation prevent the cow from being left in a depleted state in which to continue her lactation when treatment is stopped.

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V. THE EFFECT ON

THE

COMPOSITION OF

THE

MILK

1. Fat Content

Most investigators agree that the fat content of the milk increases when iodinated casein or thyroxine is given, but there is considerable disagreement regarding the extent and duration of the increase. Some workers have demonstrated increases of as much as 2.0 units in the percentage of fat (Blaxter, 1943; Van Landingham et al., 1944), while in other experiments small or even negligible responses have been observed (Swanson, 1949; Hibbs and Krauss, 1947; Jar1 and Hyden, 1945; Leech, 1950; Zorn and Richter, 1949). A series of experiments summarized by Bartlett et al. (1949) show a decided difference in fat response between experiments conducted when cows were consuming pasture ad libitum and when they were given controlled amounts of indoor rations. The cows at pasture showed increases in fat content of 0.10 units of Percentage while with the cows indoors it was 0.46 units of fat percentage. The results of Hibbs and Krauss (1947) tend to suggest that a probable explanation of this phenomenon is t o be found in the difference in nutrient supply under the two systems of feeding. Complete starvation results in very large increases in the fat content of milk (Smith et al., 1938) and short periods of unclernutrition result in similar though less severe changes. Where the ration is controlled, as during winter feeding, the rise in fat, content might possibly be regarded as a “physiological under-nutrition ” in which increased metabolic requirement exceeds nutrient supply. Under ad-libitum feeding conditions such physiological under-nutrition is unlikely to occur. Studies on the composition of the milk fat tend to support this hypothesis. Both inanition (Smith and Dastur, 1938) and thyroxine injection (Smith and Dastur, 1940; Hilditch and Paul, 1936) result in an initial increase in the iodine number of the milk fat. Both starvation and thyroxine injection have similar effects on the plasma lipoids (Smith, 1938; Smith and Dastur, 1940). As with milk yield, the initial response in fat content disappears as the experiment progresses (Thomas et al., 1948; Reece, 1950), and where it occurs its duration has been placed at about 8 weeks. Presumably the factors responsible are the same as those concerned in the diminution of the response in milk yield. 2. Lactose and Protein

The content of non-fatty solids in the milk of stimulated cows shows only a slight increase even when corrections are made for the simultaneous change in fat content. This has been observed in most investigations (see

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Blaxter et al., 1949; Bartlett et al., 1949 for reviews). Many studies of the effect of thyroxine on lactose content have been made (Jones, 1935; Folley and White, 1936; Ralston et al., 1940; Smith and Dastur, 1940). Most workers concur that an increase does in fact take place and the magnitude has been assessed at approximately 10%. More recent studies (Chanda et al., 1952) have confirmed this work and have shown that hypothyroidism due to thiouracil administration causes the reverse effects. Earlier Grimmer (Grimmer, 1918; Grimmer and Paul, 1930) had shown that a decline in the lactose content of milk occurred in goats following thyroidectomy. To obtain osmotic equilibrium with an increased lactose content of the milk the milk chloride content falls slightly (Chanda et aE., 1952). The reverse change due to thyroidectomy was reported earlier by the German workers on this subject. The total nitrogen content of the milk produced by cows given iodinated casein is also variable. No change in the total N content was found by Van Landingham et al. (1944) or by Chanda and Owen (1951). Earlier results, however, have indicated a decline in total N content, albeit of rather small magnitude (Ralston et al., 1940; Smith and Dastur, 1940). Studies on the partition of the N containing fractions of milk have shown no significant change in many experiments (Hibbs and Krauss, 1946; Chanda and Owen, 1951). In others (Archibald, 1945; Booth et al., 1947) decreases in the casein content and small increases in globulin and albumin content have been observed. Large declines in the urea N of both blood and milk were noted also in the Wisconsin experiments (Booth et al., 1947). 3. Mineral Constituents other than Iodine Comparatively little work has been carried out on the mineral constituents of milk as affected by hyperthyroidism or hypothyroidism. The German studies with thyroidectomized goats (Grimmer, 1918; Grimmer and Paul, 1930; Gruter, 1932) may be complicated by simultaneous removal of a large part of the parathyroid tissue. A series of studies of mineral constituents has, however, recently been made by Chanda et al. (1952) in cows given either thyroxine or thiouracil. No change occurred in the calcium, magnesium, sodium, or potassium content of the milk with either treatment, but an increase in the total phosphorus content of the milk occurred when thyroxine was injected (Owen, 1948b; Chanda and Owen, 1951). Detailed studies showed that the increase in phosphorus content of the milk of the cows that received thyroxine was the result of an increase in ester and lipid phosphorus, partly at the expense of the inorganic fraction. Thiouracil affected the distribution in the opposite way.

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4. Fat-Soluble Vitamins No studies have been made on the effect of thyroxine on the amounts of vitamins D, E and K in milk, but vitamin A and carotene have been studied in detail. The method of expression of results influences the magnitude of the changes observed, since increases in vitamin A activity could result solely from increases in fat content without a change in the activity per gram of fat. In the following discussion changes in composition are referred to fat content. On this basis no change in carotene or vitamin A content was observed by Thompson (Thompson, 1945; Bartlett el al., 1949),either when thyroxine was injected or when iodinated proteins were given. In experiments by Hibbs and Krauss (1947) no change occurred in the fat content of the milk, or in the carotene or vitamin A content. Simultaneously, a slight decline in the blood level of vitamin A took place. Kemmerer et al. (1946) have not reported their results in detail, but state categorically that no change in either vitamin A or carotene content occurred. The absence of any change in the vitamin A or carotene content in the milk of the thyroidectomized goat, or the goat made hypothyroid with thiouracil, supports these results (Thompson et al., 1944-6; Smith et al., 1949). Many of these experiments were made with animals given diets containing highly adequate quantities of carotene. When rations containing no carotene were given, however, increases in the concentration of vitamin A in the milk fat occurred when thyroxine was injected and conversely thiouracil resulted in a decrease. The experiments were made with both cows and goats (Chanda and Owen, 1952). Further experiments showed that the apparent digestibility of dietary carotene was increased by thyroxine administration and depressed by thiouracil (Chanda et al., 1951a). Similar results had previously been observed in rats (Cama and Goodwin, 1949). These results might at first glance appear to be a t variance with the observations that calves given marginal levels of carotene in their diet together with iodinated casein show a depression of blood levels of both vitamin A and carotene. Correspondingly small increases of unknown significance occurred when thiouracil was given (Allen, Wise et al., 1948). Blood levels, however, are a notoriously unreliable basis on which to judge total rates of turnover. 6. Water-Soluble Vitamins

a. Ascorbic Acid. The effects of thyroxine and of iodinated proteins on the ascorbic acid content of milk are summarized in Table IV. Iodinated proteins result in a large fall in ascorbic acid content. Thyroxine has been shown by one group of workers to have but a negligible effect,

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while another group has found a decided depression. There is no apparent reason for the discrepancy. It is clear, however, that inorganic iodide alone has a depressing effect on the ascorbic acid content of the milk and so it may be concluded that, as far as iodinated protein administration is concerned, a major part of the depression is due to the large amounts of iodide ingested. The minute quantity of iodine arising from the metabolism of 10 mg. thyroxine could hardly be responsible for the effects observed by Chanda and his coworkers. TABLE IV Change i n the Ascorbic Acid Content of Milk of Cows Given Iodinated Casein, Thyroxine, or Potassium Iodide

Preparation Oral iodinated “ardein”6 Oral iodinated caseinb Oral iodinated caseinc Injected Dkthyroxine” Injected Dkthyroxined Oral L-thyroxine‘ Oral potassium iodide“ Oral potassium iodide! a

Decline in Increase in ’ ascorbic acid milk yield content (%)

(a)

+20 30 f 5 to +20 +25 13 + 6 . 3 lb./day (ca. 30%) Nil Nil

+ +

-30 -30 to -63 -33 -5 -14 to -34 No change

-26 -33

Blaxter 1945s; Bartlett et al., 1849.

* Booth ef al., 1949.

Van Landingham et al., 1944. Chanda ct al.. 1952. ‘ Bailey et al.. 1949; Thompson and Kon. 1949. I Brown et nl., 1941. 0

d

b. Vitamins of the B Complex. The total thiamine content of the milk, including free, phosphorylated, and protein-bound thiamine does not change appreciably during hyperthyroidism (Thompson, 1945; Bartlett et al., 1949; Kemmerer et al., 1946; Chanda and Owen, 1951). The contrary results of Hibbs and Krauss (1947) may be due to failure to determine the whole of the phosphorylated and protein-bound fractions. Riboflavin concentration has been shown not to change very much if anything a slight decrease being apparent (Thompson, 1945; Kemmerer et al., 1946; Hibbs and Krauss, 1947; Bartlett et al., 1949). It has been claimed that the concentration of niacin in milk doubles on feeding iodinated casein (Kemmerer et al., 1946). This has not, however, been confirmed (Chanda et al., 1951b).

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6. Phosphatase and Phosphorylated Compounds

It was shown by Folley and White in 1936 that thyroxine injection caused a large decrease in the phosphomonoesterase (phosphatase) titer of milk. This change was confirmed for iodinated protein stimulation by Blaxter (1945a), and is now well established. During early lactation the phosphatase content of the milk of cows is low and it increases gradually throughout lactation (Folley and Kay, 1936). The levels of phosphatase activity in the milk of cows given thyroxine are quite comparable t o those found in early lactation, and the ratios of free to phosphorylated compounds is entirely similar under the two conditions. Thus both during early lactation (Houston et al., 1940) and during thyroxine administration (Thompson, 1945; Bartlett et al., 1949; Chanda and Owen, 1951) the ratio of free to total thiamine decreases. The same relationship is seen in the marked increase in total ester phosphorus relative to inorganic phosphate in hyperthyroid cows (Chanda and Owen, 1951). Changes in the ratio of the ester substrate t o the free compound presumably take place for other phosphorylated compounds present in milk. There is evidence (Pons, 1943) based on urinary excretion, that thyroxine similarly affects the ratio of phosphorylated to free riboflavin. 7 . Iodine Content When iodinated proteins are given, the cow receives large quantities of iodine both as inorganic iodide and as organically combined iodine. Table V shows that a considerable increase in the iodine content of milk follows the ingestion of these materials, and that the concentration in the milk is approximately proportional to dosage. TABLE V The Effect of Iodinated Proteins on the Iodine Content of the Milk of Dairy Cows

Iodinated protein Normal value" Iodinated ardeinb Iodinated caseins Iodinated casein" Iodinated ardeinb 0

Bartlett d d . , 1949.

* Blarter, 1945a.

Additional iodine intake of cow (g./day)

Iodine content of milk (g./lOO ml.)

0.00 0.58 1 .oo 1.75 2.88

1.5 84 125 168 204

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8. The Presence of Thyroxine in Milk

Literature relating to the permeability of the mammary gland to thyroid hormone has been critically reviewed by Robertson (1945) who has concluded that many of the early clinical and experimental studies suggest a secretion of thyroxine in the milk. Robertson’s own careful observations with adults (1945) failed to demonstrate any effect of the ingestion of considerable amounts of milk from cows given iodinated casein. He nevertheless advised caution in the use of such milk for babies. Experiments with children, however, have failed to demonstrate any transmission of the hormone into the milk of the cow (Bruger and Silberbush, 1946). Similarly, experiments in which milk from cows receiving iodinated casein was given to rats have failed to show any of the metabolic effects normally associated with ingestion of the hormone (Reineke and Turner, 1944; Hibbs and Krauss, 1947). Monroe and Turner (1946), have studied this problem in a different way. Rats were given sufficient thiouracil in their diet to produce perceptible enlargement of the thyroids of their sucklings. Thyroxine was then injected into the mother in amounts equivalent to as much as 165% of the normal rate of secretion. No change in the enlarged thyroid glands of the sucklings occurred, although the thyroids of their mothers were considerably reduced. With mice, similar methods have given similar results (Hurst and Turner, 1948). Finally, studies with radioactive iodinated casein suggest that the excretion of iodine by the mammary gland is accounted for as inorganic iodide rather than as either diiodotyrosine or thyroxine (Courrier et al., 1949). 9. The Suitability for Human Consumption of Milk Produced by

Cows Given Thyroxine or Iodinated Proteins The changes in the composition of the milk due to the thyroid hormone are not in any way so severe as t o suggest that the milk is either unsafe or unsuitable for human consumption. If it contains any thyroxine a t all, the amounts are certainly not large enough t o result in the mildest of symptoms of hyperthyroidism-even with babies dependent on milk for the whole of their nutritional requirements. The increases in fat, lactose, and phosphorus content may be regarded as increases in the nutritive value of the milk. The only serious change that does occur is the marked fall in ascorbic acid content. Milk, however, is not regarded as a major source of this vitamin in the human diet, and the fall is not of great nutritional significance to man.

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KENNETH

L.

BLAXTER

VI. THE EFFECTS OF THYROXINE AND IODINATED PROTEINS ON METABOLISM OF THE Cow

THE

The amounts of thyroxine that have to be given to increase the milk yield of the cow necessarily result in an increase in her metabolism. In almost all experiments attempts have been made to follow these increases by recording pulse rate, body temperature and the frequency of the respiration. Without exception, all increases in milk yield have been associated with definite increases in these indices of the metabolic activity of the body as a whole. Their interpretation is not always simple, since the proport'ionality between these indices and metabolic rate are not known. What follows is concerned, therefore, with the results of those metabolic studies with hyperthyroid cattle and sheep which permit an unequivocal estimate of metabolic effects. 1. Energy and Protein Metabolism

The most complete studies of the energy metabolism of the hyperthyroid cow have been made in.Denmark (Thorbek et al., 1948; Moustgaard and Thorbek, 1949). Other studies have been made in America (Blaxter, 1948c, Mukherjee and Mitchell, 1951). Table VI presents TABLE V I The Effect of 26 g . lodinated Casein on the Heat Production of a Dairy Cow (Moustgaard and Thotbek, 1949) ~

Amount of iodinated casein given

None 25 g. Increase (%)

Metabolizable energy in ration (Cal.) 32,944 32,498

Heat produced (Cal.)

Milk produced (Cal.)

17,743 23,378 32

11,162 15,112 35

Fat and protein gained or last from the body (Cal.) +4,797 -5,854

the results of a typical Danish experiment. It will be seen that 25 g. iodinated casein increased the secretion of milk energy by 35% and simultaneously increased heat production by 32 %. This additional heat arose from the catabolism of the body tissues, and the cow lost body fat and protein. Replicates of this experiment showed excelIent agreement. With sheep, Blaxter (1948~)measured basal metabolism before and after the administration of various amounts of iodinated casein and showed that the increase in metabolism was proportional to dosage/kg. body weight raised to the power 0.73. On this basis, he calculated that 20 g. iodinated casein would be expected t o increase the

.

EFFECTS OF THYROXINE AND IODINATED

CASEIN ON

cows

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metabolism of the cow by nearly 30%, a figure that is in good agreement with the one obtained in the Danish experiments. Basing dosage on the equation which Blaxter deduced, Mukherjee and Mitchell (1951) found with growing bulls that a dose calculated to result in an increase of metabolism of 25% gave an increase of 30%. This is again in good agreement with expectation, and thus ruminants varying in size from the 30-kg. sheep to the 500-kg. cow probably all show the same response in metabolism when the dosage is the same per unit of metabolic body size, that is, body weight raised to the power 0.73. It appears highly probable that the increase in milk yield would also be proportional to dosage per unit of metabolic size in both the goat and cow. In any case there seems little doubt from the Danish results that metabolic rate and milk yield are, on the average, affected to the same extent. Such increases in metabolic rate have other effects on metabolism. Both Blaxter (1948a) and Mukherjee and Mitchell (1951) have shown that the endogenous nitrogen metabolism simultaneously increases. This entails a greater protein requirement on the part of the animal to maintain the integrity of its tissues. Similarly, if the cow loses body weight, her nitrogen balance becomes negative (Owen, 1948a), since not only fat but protein is catabolized. Owen has shown, however, that the drain on the protein reserves of the cow can be stopped by increasing the food intake, a result which is in agreement with practical trials in which larger quantities of food have been given. No experiments have yet been made to show that the declines in milk yield, pulse rate, and respiratory frequency which occur after prolonged treatment are in fact associated with a fall in metabolism. Pulse rate appears to be a fair index of metabolism in the sheep, steer, and calf (Blaxter and Wood, 1951), but in the cow prediction of metabolic rate from pulse rate is associated with large errors. Any calculations of the efficiency of utilization based on the metabolic results can only apply, therefore, to short-term treatment, and are not comparable to those presented when the efficiency of food utilization in long-term experiments was discussed. Two estimates of the nutrient cost of the extra milk produced have been made. Moustgaard and Thorbek (1949) found that the amount of energy needed to produce the extra milk was about three times that required to produce milk in the normal cow. Mukherjee and Mitchell (1951) similarly showed that the protein cost of the extra milk was 1.5 times the normal and the energy cost 2.5 times that which suffices in the untreated animal. Even these estimates may be too low since experiments with both sheep and cattle (Blaxter, 1948a; Owen, 1948a) show that the digestibility of the total food falls when the animal is made

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hyperthyroid. Nevertheless the estimates given above are clearly of great importance in assessing the economics of the use of iodinated proteins or thyroxine.

2. Calcium and Phosphorus Metabolism In metabolism trials with six cows, Owen (1948b) showed that thyroxine injection caused an increased loss of calcium from the body. Unlike the loss of nitrogen previously referred to, this loss was not prevented by increasing the supply of dietary energy. With sheep, Blaxter (1948a) also showed that iodinated casein caused a considerable depletion of body reserves of calcium; in fact, with high doses it was possible to remove 10% of the skeletal calcium in 24 days. This loss of calcium during hyperthyroidism has been followed radiologically in cattle and swine by Dryendahl (1949). Clinically, both species showed signs of rickets, and the X-ray photographs revealed considerable porosity of the bones, especially in the region between the diaphysis and epiphysis of the tibia. Control animals given identical rations were normal. Where such large losses of calcium occur, concomitant losses of phosphorus might be expected. With sheep, large losses did in fact take place and calculations showed that these losses could be accounted for quantitatively on the basis of catabolism of both the bone salt and the muscles. The larger proportion came from the bones (Blaxter, 1948a). With two cows, however, Owen (1948b) obtained the paradoxical result that phosphorus retention increased although t,he cows were in negative calcium balance. In these experiments the phosphorus intake of the cows was increased by 20-29% during the period when thyroxine was ingested, while simultaneously the calcium intake fell by 2-10%. Confirmation of this apparently anomalous observation is needed in experiments with larger numbers of animals. The results of these balance experiments and the studies of Dryendahl must be considered in relation to the annual cycle of mineral metabolism of the cow. During early lactation the cow’s bones tend to become osteoporotic, and calcium and phosphorus are replaced there in later lactation. The use of thyroxine in the middle or late phase of lactation would not only increase the annual loss, but would curtail the period during which calcium and phosphorus are normally stored to meet the demands of the next lactation. 3. Environmental E$ects and Heat Regulation

In normal cows, as the environmental temperature increases the frequency of the respiration increases. The cow does not have a highly developed sweating mechanism; in fact, it is doubtful whether there is

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any active secretion of water by the sweat glands. Her evaporative water loss at high temperatures is partially accounted for by a passive loss through the skin and partly from the respiratory passages. When stimulated by thyroxine, the CON has a greater amount of heat to lose and, if environmental temperature is concomitantly high, great distress occurs (Seath et al., 1944, 1945; Blaxter, 1945a; Booth et al., 1947; Gardner and Millen, 1950). Respiratory rates increase to very high levels, and often heat dissipation cannot keep pace with heat production, so the temperature of the body rises. In severely hyperthyroid sheep considerable increases in the respiratory rate, panting, paroxysmal coughing and partial prostration occur. These result eventually in lung edema and emphysema (Blaxter, 1948b). The role of the thyroid gland in temperature regulation in the bovine species has not been explored to any appreciable extent. VII. SECONDARY EFFECTS The possible effects of thyroxine on the length of life of the dairy cow have not been investigated to any great extent. The economic life of a dairy cow is approximately four lactations. She is disposed of for a variety of reasons: disease, low productivity, and failure to breed, Only rarely does she die of old age or diseases associated with senescence. A decrease in the length of milking life of the cow as the result of hormonal treatment would clearly have an effect on the economy of milk production, depending for its size on the relative cost of rearing the cow and its value on final sale. I . Disease Resistance There is very little information on the effect of iodinated casein feeding on the incidence of the communicable diseases of cattle. In large-scale experiments of short duration no effect on their incidence could be found (Blaxter, 1946). Similarly, in experiments lasting 4 years Crichton (1951) could not show any differences in the incidence of mastitis or tuberculosis in two experimental herds, one receiving iodinated casein and the other a control herd. 2. Reproductive Performance

Many difficulties are involved in the determination of effects of thyroxine treatment on reproductive performance. Very large numbers of animals have to be used, since the normal incidence of functional sterility is annually only 543%) and it affects some herds and cow families within herds considerably more than it does others. Experi-

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ments with small numbers of cows can rarely hope to demonstrate small changes in fertility. On the basis of experiments with seven cows, Van Landingham et al. (1947) stated that cows were more difficult to get with calf following the feeding of iodinated casein. The interpretation of such data is extremely difficult and cannot warrant the conclusion they drew. In other experiments with Jersey cows given iodinated casein throughout the larger part of their lactation the cows reproduced quite normally. Holstein cows, under the same conditions, however, failed or were slow to conceive. Some of these cows were closely relat,ed and the small numbers (total 20) again! make conclusions difficult (Thomas and Moore, 1951b). In Crichton’s experiments lasting for 4 years, neither the interval between calvings nor the number of services necessary for conception revealed any effect of either iodinated protein or L-thyroxine administration on reproductive performance (1951). There is thus no reliable evidence of any gross abnormality in the reproductive performance of the hyperthyroid dairy cow. At the same time there is insufficient evidence available to judge whether a small impairment does not in fact take place. A similar difficulty of interpretation arises from the statement of Thomas and Moore (1951b) that the mortality of calves born to cows given iodinated casein is greater than that of calves born to control cows. Calf mortality, while approximately 7% in most herds, shows wide variation with time and place, and a conclusion will be justified only when several hundreds of cattle are observed in carefully controlled experiments. 3. Other Abnormalities

Many other abnormalities occur in cattle given thyroid hormone. Irregularities of heart action, exophthalmos, increased nervousness, occasional diarrhea, and fine muscular tremors have been observed by many workers and their value in prognosis assessed. Their interpretation as gross abnormalities of behavior or function is not questioned, but it is very doubtful whether they are of economic significance. On an economic basis an abnormality is only of significanceif it curtails production or results in early death of the animal. Thus systolic murmurs are probably only indicative of the heart hypertrophy of the hyperthyroid ruminant (Blaxter, 194813, Dryendahl, 1949) and a diagnosis of permanent damage of economic importance does not necessarily follow. Electrocardiographic examinations of the hearts of cows have not thus far revealed any signs of permanent damage when thyroxine or iodinated casein has been given (Ralston et al., 1940; Moustgaard and Thorbek, 1949; Dryen-

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dahl, 1949; nlullick et aZ., 1948; Thorbek et al., 1948). Equally pertinent are the facts that firstly, no deaths of cattle which have been given iodinated casein or thyroxine a t any stage in their lifetime have ever been attributed to heart failure, and secondly, that the heart of the hyperthyroid bovine is not histologically abnormal (Dryendahl, 1949). Similar arguments may be adduced showing that most of the abnormalities of the mildly hyperthyroid cow have negligible economic significance. If, however, one considers the mental and physical state of many human patients with Graves disease, one may wonder whether the tachycardia, cardiac arrhythmia, and nervous tension of the dairy cow given iodinated casein or thyroxine should be considered solely on the basis of the economics of survival. It cannot be ignored that the deliberate induction of a metabolic abnormality in an animal raises questions concerning the humane treatment of farm livestock.

4. Hyperthyroid Death The above discussion has, in general, dealt with mild hyperthyroidism induced by the injection of about 10 mg. DL-thyroxine or the feeding of 20-25 g. of iodinated casein. No prolonged experiments with cattle have been made in which doses grossly exceeding the secretory rate of the animal’s own thyroid gland have been given. With sheep and goats, however, death has been caused by the administration of large amounts of the hormone (Blaxter, 194813; Owen, 1951). From the results with sheep, Blaxter concluded that doses which result in death were only two to three times those necessary to produce the same percentage increase in metabolism as had occurred in practical trials with milking cows. Two goats died when 10 mg. of DL-thyroxine was given. The metabolic body size of the goat is one-sixth that of the cow, and so this represents a very considerable (about six times) overdosage. Information on the toxicity of smaller amounts is not available. The postmortem findings in animals that have died as the result of the ingestion of large amounts of iodinated casein, or have been killed when receiving the hormone (Dryendahl, 1949), differ only in degree. Hypertrophy of the heart and adrenals is common, and where death has occurred, the lungs and right ventricle of the heart have clearly been unabIe to keep pace with the augmented metabolism of the body cells, The most spectacular aspect is the extreme emaciation and almost complete absence of fat in the carcasses.

CONCLUSIONS VIII. PRACTICAL At intervals during the course of the last ten years many opinions have been given regarding the practical potentialities of thyroxine and

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iodinated casein in stimulating milk production. They have varied between extremes of unequivocal acceptance and of wholesale condemnation. Throughout this period, however, informed opinion has uniformly advised a policy of caution, as instanced by the recommendation of the American Society of Dairy Science in 1947. At the present time, sufficient information has accrued to justify a less guarded definition of the practical possibilities. Broadly speaking, the use of these materials is justified provided that they result in the production of milk which is safe and nutritionally adequate for human beings, and that the increase in milk or fat yield is economically worthwhile. On the first aspect there is little doubt. The milk does not contain thyroxine, and on the whole the nutritive value of the milk is enhanced rather than impaired. The second aspect is more difficult to justify. It appears from the data that have been assembled that stimulation with small doses for long periods of time is certainly no more superior a method of increasing milk yield than an over-all increase in the plane of nutrition of the cow. Reduction of thyrotropic hormone secretion by the anterior pituitary effectively adjusts the total amount of thyroxine in the animal’s body to pretreatment levels, and, after approximately eight weeks, this homeostatic effect seems complete. Short-term stimulation a t favorable periods of the lactation can result in spectacular increases and equally spectacular decreases when treatment terminates. On the average, however, a net gain does occur which, calculated over the whole lactation, amounts to less than 3%. At present, economic considerations must be based on the profitability of this 3 % increase. The profitability of an increase in milk production depends on the price obtained for the increase less its cost of production. The cost of production includes a labor cost, housing costs, food costs, and a charge for depreciation-or appreciation-of the cow. If the food cost of the increase in production as the result of administration of thyroxine or iodinated protein is, as appears from the metabolism experiments, two to three times the normal amount, then treatment is hardly an economic possibility. This ignores, however, the seasonal oscillations of the cost of the cow’s feed supply. Thus, if thyroxine were used to produce milk a t the expense of body reserves of fat and protein when food is expensive and the replenishment of reserves allowed to take place when food is cheap and plentiful, a net profit could possibly occur (Blaxter, 1946). Similarly, increases in yield a t the expense of reserves may be economically feasible if the animal is sold a t the end of treatment and the losses of flesh do not appreciably affect the monetary value of the carcass (Graham, 1948). Other possible uses exist in which short-term use of

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iodinated protein or thyroxine could increase production and result in profit. In general, however, any profit will be small, as indeed will be the over-all increase in production. The future prospects of these materials for stimulating the milk production of cattle do not warrant optimistic enthusiasm. It is perhaps to the credit of the many investigators, however, that such a positive opinion can be given in the short space of ten years. The cow is not an easy experimental animal with which to deal, and her lactation cycle is very long compared with those of laboratory animals. Though the attempts to exploit these new sources of thyroid hormone t o augment the productivity of the dairy industry have not met with very great success, the results of these investigations have made a signal contribution to our knowledge of at least one aspect of lactational physiology. REFERENCES

Allen, C. E., DOW,D. S., Logan, V. S., and MacKenzie, C. D. 1948. Sci. Agr. 28, 349-356. Allen, R. S., Wise, G. H., and Jacobsen, N. L. 1948. J . Animal Sci. 7 , 538 Proc. Archibald, J. G. 1945. J. Dairy Sci. 28, 941-947. Bailey, G. L., Bartlett, S., and Folley, S. J. 1949. Nature 163, 800. Bartlett, S., Rowland, S. J., and Thompson, S. Y. 1949. Proc. 12th. Intern. Dairy Congr. 1, 102-109. Blau, N. F. 1935. J. B i d . Chem. 110,351-363. Blaxter, K. L. 1943. Nature 162, 751. Blaxter, K. L. 1945s. J. Endocrinol. 4, 237-265. Blaxter, K. L. 1945b. J. EndocrinoZ. 4, 266-299. Blaxter, K. L. 1946. J. Agr. Sci. 36, 117-150. Blaxter, K. L. 1948a. J. Agr. Sci. 38, 1-19. Blaxter, K. L. 1948b. J. Agr. Sci. 38, 20-27. Blaxter, K. L. 194%. J. Agr. Sci. 38, 207-215. Blaxter, I<. L., and Price, H. 1945. Vet. J. 101, 18-21, 39-48. Blarter, K. L., Reineke, E. P., Crampton, E. W., and Petersen, W. E. 1949. J. Animal Sci. 8, 307-352. Blaxter, K. L., and Wood, W. A. 1951. Brit. J . Nutrition 6, 29-55. Booth, A. N., Elvehjem, C. A., and Hart, E. B. 1947. J . Dairy Sci. 30, 443-455. Borrows, E. T., Clayton, J. C., and Hems, B. A. 1949. J. Chem. SOC.(Suppl.), 199204. Bottomley, A. C., and Folley, S. J. 1940. Ann. Rept. Natl. Znst. Research Dairying 34-35. Brown, W. C., Van Landingham, A. H., and Weakley, C. E. 1941. J. Dairy Sci. 24, 1035-1039. Bruger, M., and Silberbush, F. F. 1946. J . CZin. Endocrinol. 6 , 565-570. Cama, H. R., and Goodwin, T. W. 1949. Biochem. J. 46, 236-241. Chanda, R., Clapham, H. M., McNaught, M. L., and Owen, E. C. 1951a. Biochem. J . 60, 95-99. Chanda, R., Higginbottom, C., McNaught, M. L., and Owen, E. C. 1951b. Private communication.

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Jones, T. S. G. 1935. J. SOC.Chem. Ind. 64, 928. Kemmerer, A. R., Bolomey, R. A., Vavich, M. G., and Davis, R. N. 1946. Proe. SOC.Exptl. Biol. Med. 6S, 309-310. Koller, R. 1950. Tierdrztl. Monatschr. 37, 695-704, 768-778, 891-904. Lanik, J., and Isajev, F. 1949. Sbornik Ceskl. Akad. ZemSdClskl 22, 65-69. Leech, F. B. 1950. J . Endocrinol. 7, 42-53. Ludwig, W., and von Mutzenbecher, P. 1936. Z. physiol. Chem. 244, iv. Ludwig, W., and von Mutzenbecher, P. 1939. 2. physiol. Chem. 268, 195. Lukacs, J. 1930. Arch. Kinderheilk. 91, 9-13. McQuillan, M. T., Trikojus, V. M., Campbell, A. D., and Turner, A. W. 1948. Brit. J . Exptl. Path. 29, 93-106. MZllgaard, H. 1947. Oversigt over ForsZgslaboratoriets Arbejder, i 1946-7, p. 52. August Bang Commission, Copenhagen. Monroe, R. A., and Turner, C. W. 1946. Univ. Mo. Agr. Exptl. Sta. Research Bull. 403.

Monroe, R. A., and Turner, C. W. 1948. Am. J. Physiol. 164, 1-5, Monroe, R. A., and Turner, C. W. 1949a. Univ. Mo. Agr. Expt. Sta. Research Bull. 446.

Monroe, R. A,, and Turner, C. W. 1949b. Am. J . Physiol. 166, 381-386. Moore, L. A. 1946. J. Dairy Sci. 29, 532-533 Proc. Moustgaard, J., and Thorbek, G. 1949. 240 Beretning fra Fors@gslaboratoriet, 1-45. August Bang Commission, Copenhagen. Mukherjee, R., and Mitchell, H. H. 1951. J. Animal Sn'. 10, 149-162. Mullick, D. N., Alfredson, B. V., and Reineke, E. P. 1948. Am. J. Physiol. 162, 100-105. Nicholson, D. P. 1948. Brit. Med. J . (i) 1029-1030. Opichal, M., Chumchal, R., and Kopecky, 0. 1949. Sbornik Cesk.3. Akad. Zem& dglskd 21, 280-296. Owen, E. C. 1948a. Biochem. J. 45, 235-243. Owen, E. C. 1948b. Biochem. J. 43, 243-247. Owen, E. C. 1951. Unpublished observations. Pauly, H. 1910. See Reineke, 1946. Petersen, W. E., Spielman, A., Pomeroy, B. S., and Boyd, W. L. 1941. Proc. SOC. Exptl. Biol. Med. 46, 16-17. Pitt-Rivers, R., and Randall, S. S. 1945. J . Endocrinol. 4, 221-236. Ponz, F . 1043. Trab. Inst. Cajal Invest. biol. 1, 161-195. Poulsen, E. 1949. 240 Beretning fra Forsggslaboratoriet, 47-108. August Bang Commission, Copenhagen. Ralston, N. P., Cowsert, W. C., Ragsdale, A. C., Herman, H. A., and Turner, C. W. 1940. Univ. Mo. Agr. Expt. Sta. Research Bull. 817. Reece, R. P. 1946. J . Dairy Sci. 29, 533 Proc. Reece, R. P. 1947, J . Dairy Sn'. SO, 313-324. Reece, R. P. 1950. J. Dairy Sci. 33, 126-133. Reineke, E. P. 1946. Vitamins and Hormones 4, 207-253. Reineke, E. P., Herman, H. A., Turner, C. W., and Ragsdale, A. C. 1944. J. Animal Sci. 3, 439-440 Proc. Reineke, E. P., and Turner, C. W. 1942. Univ. Mo. Agr. Expt. Sta. Research Bull. S66.

Reineke, E. P., and Turner, C. W. 1944. J. Dairy Sci. 27, 793-805. Reineke, E. P., and Turner, C. W. 1945. J . Biol. Chem. 161, 613-619. Reineke, E. P., and Turner, C. W. 1946. J. Biol. Chem. 162, 369-375.

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Reineke, E. P., Turner, C. W., Kohler, G. O., Hoover, R. D., and Beezley, M. B. 1945. J . Biol. Chem. 161, 599-611. Reineke, E. P., Wallach, D. P., and Wolterink, L. F. 1950. J . Dairy Sci. 33,386-7. Richter, F. 1949. Der Tierziichter 1, 534-535. Robertson, J. D. 1945. J . Endocrinol. 4, 300-304. Robinson, M. 1947a. Lancet 263, 385-387. Robinson, M. 194713. Brit. Med. J . ii, 126-128. Roche, J., Deltour, G. H., Michel, R., and Mayer, S. 1949. Biochim. et Biophys. A d a 3, 658-74. Roche, J., Giraud, P., Coignet, J., Lafon, M., and Liardet, J. 1948. Compt. rend. SOC. biol. 142, 921-923. Schultze, A. B., and Turner, C. W. 1945. Univ. Mo. Agr. Expt. Sta. Research Bull. 392. Seath, D. M., Branton, C., and Groth, A. H. 1944. J . Dairy Sci. 27, 641-642. Seath, D. M., Branton, C., and Groth, A. H. 1945. J . Dairy Sci. 28, 509-517. Schuurmans, D. 1049. Landbouwkund. Tijdschr. 61, 586-603. Simpson, G. K., Johnston, A. G., and Traill, D. 1947. Biochen. J . 41, 181-184. Smith, J. A. B., and Dastur, N. N. 1938. Biochem. J . 32, 1868-1876. Smith, J. A. B., and Dastur, N. N . 1940. Biochem. J . 34, 1093-1107. Smith, J. A. B., Howat, G. R., and Ray, S. C. 1938. J . Dairy Research 9, 310-322. Smith, V. R., Niedermeier, R. P., and Schultz, L. H. 1949. J . Animal Sci. 7, 544 Proc. Swanson, E. \V. 1949. J . Dairy Sci. 32, 708 Proc. Swanson, R. G., and Knodt, C. B. 1948. J . Dairy Sn'. S1, 660 Proc. Swanson, R. G., and Knodt, C. B. 1949. J . Dairy Sci. 32, 257-264. Sykes, J. F., Wrenn, T. R., Moore, L. A., and Thomas, J. W. 1948. Am. J . Physiol. 163, 412-416. Szumowski, P., and Charenton, J. 1950. Rec. Med. Vet. 126, 479-497. Thomas, J. W. 1949. J. Dairy Sci. 32, 708-709 Proc. Thomas, J. W., and hloore, L. A. 1948. J . Dairy Sci. 31, 661 Proc. Thomas, J. W., and Moore, L. A. 1951. J. Dairy Sci. 34, 321-328. Thomas, J. W., and Moore, L. A. 1951b. J . Dairy Sci. 34, 507 Proc. Thomas, J. W., Moore, L. A., and Sykes, J. F. 1949. J . Dairy Sci. 32, 278-291. Thompson, S. Y. 1945. Ph.D. Thesis, University of Reading. Thompson, S. Y., and Kon, S. K. 1949. Ann. Rept. Nail. Znst. Research Dairying p. 55. Thompson, S. Y., Kon, S. K., and Cowie, A . T. 1944-6. Ann. Rept. Natl. Znst. Research Dairying p. 65. Thorbek, G., Hansen, I. G., and Moustgaard, J. 1948. J . Animal Sci. 7, 291-297. Turner, C. W. 1940. J. Dairy Sci. 23, 535-536 Proc. Turner, C. W., and Reineke, E. P. 1946. M o . Agr. Ezpt. Sla. Research Bull. 397. Van Landingham, A. H., Henderson, H. O., and Weakley, C. E., Jr. 1944. J. Dairy Sci. 27, 385-396. Van Landingham, A. H., Hyatt, G., Jr., Weakley, C. E., Jr., and Henderson, H. 0. 1947. J . Dairy Sci. SO, 576-577 Proc. Yates, F., Boyd, D. A., and Pettit, G. H. N. 1942. J . Agr. Sci. 32, 428. Zorn, W., and Richter, F. 1949a. Landw. Jahrb. Bayern l/2, 14-49. Zorn, W., and Richter, F. 194913. Proc. X I I Intern. Dairy Congr. 1, 110-113.

The Intermediary Metabolism of the Non-benzenoid Steroid Hormones* BY LEO T. SAMUELS AND CHARLES D. WEST University of Utah College of Medicine, Salt Lake City, Utah, and Sloan-Kettering Institute, New York, New York CONTENTS

page

I. Introduction. . . . . . . . . . . . . . . . .. 11. Intermediary Metabolism of the Androgens.. .......................... 1. Urinary Excretion after Administration of Androgens and Possible Intermediates.. ................................................ ......... 2. Metabolism of Androgens by Tissues. 111. Intermediary Metabolism of the Progesti ................... 1. Urinary Excretion after Administration of Progesterone.. . 2. Metabolism of Progesterone by Tissues. . . . . . . . . . . . . . . . . . IV. Intermediary Metabolism of Steroids of the Adrenal Cortex.. ............ 1. Urinary Excretion of Steroids of Pr 2. Metabolism of Cortical Steroids by ... V. General Conclusions. ........ References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

252 253 276

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I. INTRODUCTION Soon after the preparation of active lipide extracts having androgenic, adrenocortical, or progestational activity it was recognized that these compounds underwent changes in the body that led to decreased hormonal activity. In 1933 Buhler reported that only a small fraction of the androgenic activity of an extract prepared from urine could be recovered in the urine of a human male recipient. Ehrhardt and Hagena (1935) injected 30-50 Clauberg units of progestin into women and found no measurable amount excreted. Although Perla and Marmorston-Gottesman (1931) found small amounts of material in human urine that would protect adrenalectomized rats against histamine poisoning, there was no significant increase when active extracts were administered. The few studies made on human feces indicated that this was not a significant route of excretion in human beings compared with the urine. It was, * This work was supported in part by grants from the American Cancer Society upon recommendation of the Committee on Growth of the Nntional Research Council, the National Cancer Institute, United States Public Health Service, and Ciba Pharmaceutical Products, Inc. 25 1

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therefore, assumed that the hormonally active components of all of these extracts underwent metabolism in the mammalian organism. Endogenously produced hormones were metabolized as well as the injected extracts. In 1934 Gallagher and Koch pointed out differences in chemical character between their extracts from testes and androsterone, the active androgen which had been isolated by Butenandt (1931) from urine. The next year David el al. (1935) succeeded in identifying the substance in bull testes as a steroid, C19H2802,containing an a,/3 unsaturated ketone group and an alcohol. It was six times as active as the excretion product by the comb growth test used. In 1937 Venning and Browne showed that pregnanediol appeared in the urine during the second half of the menstrual cycle and in large quantities during pregnancy, times when progestational activity was present in the uterus. Since the same compound was excreted in the urine after the injection of progesterone it seemed highly probable that the endogenously formed pregnanediol represented metabolism of endogenous progesterone. Thus it was obvious that the animal organism, and particularly the human body, was a system in which the steroid hormones were not only synthesized but also further metabolized to less active or inactive products. What we have learned about the reactions involved in this metabolism and the locations where they occur is the province of this review. 11. INTERMEDIARY METABOLISM OF

THE

ANDROGENS

The androgens can originate from three endocrine glands. The major influence on the secondary sex characters of the normal male is the secretion of the testes. As mentioned before, David et al. (1935) isolated testosterone from bull testes. It has also been obtained from stallion testes (Tagmann et al., 1946), and the evidence of Prelog et al. (1947) indicates that hog testes probably contain this compound. It is the most active androgen that has been isolated, and no other has been found in these organs. While not conclusively proved, it is highly probable, therefore, that testosterone is the androgenic hormone of the testis in mammalia. While the adrenals will not maintain the male secondary sex characters under normal conditions, it has been shown that they account for a significant portion of the androgenic 17-ketosteroids in the urine of normal men, and for the larger part in normal women. In certain types of adrenal tumor in women and children the male secondary sex characters are markedly stimulated. Only very small amounts of androgenic steroids are found in adrenal extracts, and there is still considerable debate whether the major portion, .at least, of the excreted 17-ketosteroids of adrenal origin is not due to the metabolic action of some other tissue on

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the Csl steroids secreted by the adrenal gland (Dobriner et al., 1951a). Recently Gassner et al. (1951), however, have shown that androgenic material is found in adrenal-vein blood in greater concentration than in the general circulation, and that the chemical fraction in which this occurs is increased after the administration of ACTH. All of the evidence would indicate, however, that while the compounds are C19 steroids, they are weak androgens compared with testosterone. Burrill and Greene (1941a) have shown that the rat ovary has some androgenic activity, and Hill (1937a,b, and c) has demonstrated that when the ovary of the rat or mouse is transplanted into an exposed area like the ear, it produces relatively large amounts of androgen. Under normal conditions, however, the contribution of the ovaries must be very small; ovariectomy in women does not lower the excretion of 17-ketosteroids significantly. The metabolism of testosterone would, therefore, appear to be most important in the field of the androgens. 1. Urinary Excretion after Administration of Androgens and Possible Intermediates

One of the earliest and most widely used methods for studying the metabolism of any substance in the animal organism is to administer the compound and measure products in the excreta increased thereby. This has been effectively applied in the case of the androgens by Dorfman and coworkers and the group associated with Dobriner. On the basis of the early studies, which indicated that the urine was the major route of excretion of steroids in the human beings, major attention has been directed to this fluid. The method is open to a number of limitations, however. Since $he testes (McGee, 1927), adrenals (Reichstein, 1936), and ovaries (Hill, 1937a) have all been shown t o be endogenous sources of androgenic substances, there is always the question of the extent to which the endogenous metabolism has been changed by the administration of the exogenous steroid. This can partially be corrected by the elimination of one or more of these organs, but all three cannot be removed without serious secondary changes that could markedly affect the result. Another difficulty is that routes of excretion may differ in different species, and the selective character of the kidneys is introduced in all. A last objection is that the ordinary experiment involves the injection of large excesses of the particular compound and the routes of metabolism may be affected by the abnormal preponderance. With these limitations in mind, however, the method can give important data on probable pathways. Both Callow (1939) and Dorfman and Hamilton (1939) reported the isolation of androsterone (X) and 3a-etiocholanolone (IX)in increased

254

LEO T. SAMUELS AND CHARLES D. WEST

amounts following the administration of testosterone propionate to hypogonadal males. The administration of testosterone to normal women resulted in the excretion of the same metabolites with the possible addition of androstan-3a1l7p-diol (XIII) (Schiller et al., 1945). Dorfman and Fish (1940) were able to isolate isoandrosterone (XI) from the urine of a guinea pig after the administration of testosterone propionate. Later Dorfman (1941) obtained the same result in a hypogonadal man. The major products, however, were always androsterone and etiocholan-3cr-ol-17-one. Dobriner and Lieberman (1950) administered 90 mg. of testosterone daily for 45 days and were able to account for 50% of the injected material as an increase in urinary steroid excretion over control levels: 24% as androsterone, 19% etiocholanolone, 1% androstanedione (VIII) and etiocholanedione (VII), and 6 % as androstanediol (XIII) and etiocholanediol (XII). The metabolic pathway from testosterone to androsterone and etiocholan-3a-ol-17-one would require an oxidation of carbon 17 and a reduction of both the A4-double bond and the ketone on carbon 3. Since large amounts of both of these saturated isomers were found it would appear that the reduction of the double bond was not stericly directed. If the isoandrosterone found by Dorfman is actually formed from the injected testosterone the reduction of the ketone on carbon 3 cannot be completely stericly controlled. The small amounts found, however, and the fact that West et al. (1951a) did not find any significant amount of p-ketosteroids under conditions where endogenous metabolism of other steroids could not contribute significantly, make it probable that the increase represented some alteration of endogenous metabolism, and that reduction of the ketone was positionally specific. Gallagher et al. (1951) injected 100 mg. of deuterium-labelled testosterone into a normal male and analyzed the urine for 3 days thereafter. A t the end of this time the excretion of deuterium had reached insignificant levels. Since in previous studies on mice it had been demonstrated that no significant amounts of deuterium entered the metabolic pool, its presence in a steroid could be considered to indicate its origin from the injected hormone. After acid hydrolysis and isolation the following urinary steroids contained deuterium : A2-androsten-17-one1androstane-3, 17-dione, etiocholane-3,17-dione, androsterone, and etiocholan-3a-ol-17one. The Az-androstene-17-onewas an artifact formed from androsterone during the heating in acid solution. The excretion of androsterone accounted for 26% of the isotopic testosterone, etiocholanolone for 10%. The p-ketonic fraction did not contain a significant excess of isotope. This confirms the specificity of the reduction of the 3-ketone. It also,

INTERMEDIARY METABOLISM OF NON-BENZENOID STEROIDS

255

because of the presence of isotope in both possible saturated diketones, adds further support to the nondirectional character of the reduction of the double bond. The presence of the two diketones would also lead to the probability that the oxidation of the hydroxy group on carbon 17 occurred before the reduction on carbon 3, and even before the reduction of the double bond; otherwise, two oxidizing enzymes would be required instead of one. On this basis A4-andro~tene-3~17-dione (111) would be a key intermediate formed directly from testosterone. Dorfman and Hamilton (1940) administered this compound to a hypogonadal male and found increased amounts of both androsterone and etiocholan-3a-ol-17-one. Gallagher et al. (1951) administered deuteriumlabelled A4-androstene-3,17-dioneand recovered 43 % as androsterone and etiocholan-3a-ol-17-one in equal proportions. It, therefore, could well be an intermediate in testosterone metabolism even though it was not found in the urine after injections of the hormone. Other probable intermediates which have been tried by Dorfman and Hamilton (1940) are androstane-3,17-dione and androstane-3a,17fl-diol (XIII). I n both cases androsterone but no etiocholanolone was found in increased quantities. In the latter case small amounts of isoandrosterone were also found. When Gallagher el al. (1951) administered deuteroandrostanedione, 24 % was recovered as androsterone and none as etiocholanolone. A significant point in these studies is that in none did the recoveries account for all of the testosterone which was administered. The closest to complete recovery was in the experiments of West et al. (1951b) where a large amount of testosterone was delivered immediately into the circulation and in which the conversion to etiocholanolone and androsterone was very rapid. The question then arises: Are the two ketols further metabolized or is there an unknown alternative pathway for testosterone metabolism? Gallagher el al. (1951) tested this by injecting deuteriumlabelled androsterone :only androsterone was recovered and this accounted for only 25% of the administered material. No other products could be identified. He also injected isotopic etiocholan-3a-ol-l7-one and recovered only etiocholan-3a-ol-17-one, accounting for 60 % of the injected material. In view of the fact that acid hydrolysis of the conjugated 17-ketosteroids leads to considerable destruction, the latter recovery may represent most of the administered steroid, but the recovery after androsterone surely does not. The products of androsterone metabolism have not been identified in the urine: the saturated diols which are present would account for only a small portion of the missing steroid. In patients with adrenal tumors there is a marked increase in the excretion of dehydroisoandrosterone (11), present in small amounts in

256

LEO T. SAMUELS A N D CHARLES D. WEST

normal urine. Since it has not been found after injection of isotopic testosterone, or in experiments with intravenous injection of testosterone, it is thought that it represents an adrenal metabolite. Injections of dehydroisoandrosterone into humans led to an increased excretion of androsterone and etiocholanolone (Mason and Kepler, 1945a) as the major products. Since these have an opposite steric configuration on carbon 3, it would seem to indicate that a large proportion of the dehydroisoandrosterone was first converted into a diketone, probably A4-androstene-3,17-dione (111),before undergoing further reduction. Besides androsterone and etiocholan-3a-ol-17-one, small amounts of A6-androstene-3P,17a-diol(IV) and etiocholane-3a,l7~-diol (XII)were also isolated from human urine after injection. These are products of the reduction of the 17-ketone groups of the injected steroid and etiocholan-3a-ol-17-one respectively. Dehydroisoandrosterone would appear t o be reduced by the same enzyme which reduces the saturated 17-ketosteroids to 17-alcohols, as well as undergoing the reactions already indicated. In all cases most of the dehydroisoandrosterone, like testosterone, was not recovered as any known product. Probably after conversion to metabolites common to both hormones, further metabolism is similar to that of the testicular hormone. The conjugation of the steroids excreted in the urine is another metabolic process of importance. Funk et al. (1929) first called attention to the fact that larger amounts of androgenic material could be obtained from urine if it had been allowed to stand after acidification. Venning and coworkers (1942) isolated androsterone sulfate from the urine of a man with interstitial cell tumor. They found that hydrolysis of this compound by boiling for a short time with acid led to destruction of about 40% of the steroid. Munson et al. (1944) isolated dehydroisoandrosterone sulfate from the urine of normal man. West et al. (1951a) found that androsterone and etiocholanolone were excreted largely as the glucuronides after the intravenous injection of testosterone. The conjugates are quite soluble in water as the sodium salts, and apparently are readily filtered through the glomerulus (West et al., 1951b). Taking into consideration the information which has been obtained from studies of products in the urine, the metabolism of the androgens could take one or more of the paths illustrated in Fig. 1. The probable routes are indicated by heavier lines. 2. Metabolism of Androgens by Tissues

The urine studies tell nothing of where in the organism the apparent changes take place, nor do they distinguish between the possible sequences. Zondek (1934) was the first to explore the site of steroid metabolism.

INTERMEDIARY METABOLISM OF NON-BENZENOID STEROIDS

257

FIQ.1. Possible routes of metabolism of androgens as indicated by compounds excreted in the urine. Dotted lines indicate probable conversions which have not been established. Solid lines indicate that increased amounts of product have been found in the urine after injection of the compound from which arrow originates. Thickness of arrows is proportionate to number of previous steroids which have been shown to increase the urinary excretion of the product indicated.

258

LEO T. SAMUELS AND CHARLES D. WEST

His pioneer studies with estrogens implicated the liver as the major metabolic organ. He showed that the inactivation was enzymic, and claimed t o have prepared an acetone-dried powder which was active. In other experiments he also found that progesterone was inactivated by liver slices but not by liver brei. This centered attention on the liver as the important organ in the metabolism of the steroid hormones. Biskind and Mark (1939) first demonstrated the role of the liver in the metabolism of the androgens. They implanted pellets of testosterone in the spleens of immature or castrated rats. There was no increased development of the secondary sex organs as long as the spleen remained in situ; when, however, the spleen was transplanted so that its venous blood drained into the general circulation, the seminal vesicles and the prost,ate immediately underwent great enlargement. Burrill and Greene (1940) showed that the livers of rats would inactivate their own testicular androgen. When testes were implanted in the mesentery, the secondary sex organs atrophied; but if they were implanted intrarenally, no castration changes occurred. In similar experiments they found that transplants of the adrenal cortex were able to maintain some androgenic activity, even when the blood drained through the liver (Burrill and Greene, 1941b). They concluded, therefore, that the adrenal androgens must differ from those of the testes. Danby (1940) was the first t o demonstrate destruction in the isolated liver. She perfused livers with blood t o which testosterone had been added. The testosterone disappeared from the blood and the equivalent activity could not be extracted from the liver tissue. She also found that testosterone was destroyed when blood was perfused through a kidney. When she incubated the hormone with the pulp of either organ, however, she was unable to find any destruction. From the description it seems that the pulp mixture was simply set in an incubator for 6-24 hours without any provision for aeration. Clark and Kochakian (1944,1947) first succeeded in demonstrating an action of liver tissue on testosterone in vitro. When the hormone was incubated under aerobic conditions with rabbit liver slices, in addition to unchanged testosterone they were able to isolate a considerable amount of A4-androstene-3, 17-d;one, a small amount of trans-testosterone (XVI) and traces of some other compounds too small t o identify. Danby may have achieved a similar conversion. Since she depended on bioassay for evidence of metabolism, however, the difference in activity between a mixture of the two androgenic compounds, testosterone and A4-androstenedione, and testosterone alone may have been within the limits of error. Although the work of Clark and Kochakian gave specific qualitative

259

INTERMEDIARY METABOLISM OF NON-BENZENOID STEROIDS

information, it depended on actual isolation and identification by the methods of classical organic chemistry. This limited the experiments to grossly unphysiological concentrations of steroid and only gave approximations of quantitative change. Samuels (1947) developed analytical techniques that, while not distinguishing specific compounds, would permit the measurement of certain groups in the steroid molecule in microgram quantities; thus it was possible to use concentrations of steroid that would remain in solution in aqueous media and to maintain rat.ios of tissue to hormone that would more nearly approach physiological conditions. Under these circumstances Samuels el al. (1947) were able to demonstrate the enzymic destruction of the a,@ unsaturated ketonic system in ring A of testosterone by liver slices and minces, as measured by the decrease in the ultraviolet absorption band a t 240 mp, or by the Koenig reaction (Koenig el al., 1941). Material giving the Zimmermann reaction for 17-ketosteroids was also formed during the incubation. Oxygen was essential for the reaction t o continue under these conditions (Table I). Homogenates prepared by grinding with sand, disintegrating in a Potter type homogenizer, or by means of a Waring Blendor were inactive. TABLE I Inhibition of Hepatic Destruction of Testosterone by Nitrogen Atmosphere or by Boiling of Tissue Koenig reaction used for analysis (Koenig et al., 1941)

FIask Weight No. tissue

1 2

3 4

5 6 7

g1.075 1.000 1.010 0.890 0.985 1.150 1.100

Hormone used Atmos- Hormone Hormone Hormone per g. tissue phere added recovered lost per hour

02 02 0 2 0 2

Nz Nz NI

rg . 200 200 200 200 200 200 200

rg.

rg .

53.6 86.8 52.3 '175.0 189.9 165.2 200.0

146.4 113.2 147.7 25.0 10.1 34.8

rg./g./hr. 136.1 113.2 146.1 28.1 10.3 30.3

0

0

Tissue

Living Living Living Boiled Living Living Boiled

In view of the need for oxygen a search was carried out for compounds which might act as hydrogen donors or hydrogen acceptors in the system. Of a large number of compounds tried, only two significantly increased the destruction of the conjugated double bond system in ring A (Table 11). These were diphosphopyridine nucleotide (DPN) and citrate. The reaction was not the same in the presence of the two cofactors, however, for the production of ketone groups on carbon 17 was large in

260

LEO T. SAMUELS AND CHARLES D. WEST

TABLE I1 Effect of Various Biochemical Metabolites on Later from Fed and Fasting Rats Rats with fasted liver

Rats with fed liver Cofactor present Cofactor (molar concentrations) 0.05 Succinate 0 .005 Oxalate 0.001 Adenosine-3-phosphate 0.005 Hexose diphosphate 0.05 Pyruvate 0.05 Glutamate 0.001 Adenosine-5-phosphate 0.001 Calcium 0.05 Lactate 0.001 DPN 0.001 Citrate Per cent 0 . 1 Glucose

Cofactor absent

Cofactor present

Cofactor absent

a-8" 17-Kb

a+" 17-Kb a-8' 17-Kb a-p" 17-Kb (Values in micrograms per gram of tissue per hour)

107 103 115 143 119 119 128 143 50 209 269 125

0 0 0 0 0 0 0 0 0

70 0 0

105 125

0 0

126 125 125 127 125 111

0 0 0 0 0 0 0

111

17 41 39 49

197 235

38 61 61 51

112 0

49 49

0 0

12OC

In all incubation flasks 200 pg. of testosterone were introduced with approximately 0.7g. of liver mince and 25 ml. of buffer solution. The final concentrations of ions in the buffer solution were as follows: KCl 0.0056 M , MgClz 0.0021 M , NaCl 0.08 M, NazHPO,-NaH,POc buffer (pH 7.4) 0.04 M , glucose 0.1%. All flasks were incubated 1 hr. a t 38.5"C. The alpha-beta conjugated ketone groups destroyed were estimated using a testosterone standard; the 17-ketosteroids were estimated as androsterone. Alpha-beta conjugated ketone group deatroyed. 17-Ketosteroids formed. In the buffer used in this group of flanks the usual 0.1 75 glucose was not added. All other flasks had this concentration of glucose in the regular buffer. b

the presence of DPN while it was no greater than the controls without added cofactor in the case of citrate. The addition of both DPN and citrate t o the incubation medium led t o a greater effect on the destruction of the a,B unsaturated ketone system than with either alone, but to a formation of 17-ketones which was intermediate (Table 111, male rats). Two systems, therefore, appeared to be acting on ring A: one requiring DPN which also involved the oxidation of the alcohol group on carbon 17, and the other requiring citrate which did not involve oxidation on the other end of the molecule. After the discovery of the need for DPN, nicotinamide was added to

INTERMEDIARY METABOLISM OF NON-BENZENOID STEROIDS

261

all incubation media to inhibit the intracellular nucleotidases set ffee by cell damage. This increased the amount of activity without added cofactor and also made possible the preparation of active homogenates by either the Potter technique or by grinding with sand. Waring Blendor preparations were still inactive, indicating that the enzyme or enzymes were probably surface-denatured in the process. To explore the phylogenetic development of these systems, a study of the activity of the livers of a number of different species both with and without the two cofactors was carried out (Samuels et al., 1950) (see Table 111). I n the livers of all of the poikelothermic animals there was a slow destruction of the alpunsaturated ketone system without the formation of any 17-ketones; this was unaffected by the addition of either or both cofactors. In the birds and mammals, however, both the DPN and citrate-activated systems were present. Apparently in the poikelothermic forms the effect of temperature on production of the testicular hormones is adequate to regulate the sex cycle to the environment. The homeothermic animals, however, must make more complex adjustments. The temperature of the functional tissue is, therefore, sufficiently high for rapid production, but at the same time new methods of inactivation are developed so that the entire system is in a more dynamic and labile state. Studies of the relation between destruction of the alp unsaturated ketone system in ring A and the formation of 17-ketones were carried out. As seen in Fig. 2 the 17-ketones rise as the alpsystem decreases; they pass through a maximum, however, and then in turn decrease. This would indicate that they, too, are further metabolized to compounds that have neither group in their structure. We are apparently measuring the accumulation of an intermediate when we determine 17-ketosteroids. Since Clark and Kochakian had found A4-andro~tene-3~17-dione as a major product in their experiments, an incubation was continued to the point of maximum 17-ketone concentration and the alcoholic 17-ketones were separated from the non-alcoholic by means of the hemisuccinates. All of the 17-ketones were found in the alcoholic fraction. Apparently under the conditions of these experiments A4-androstene-3,17-dione did not accumulate to a measurable extent. Sweat et al. (1950) have described the concentration of the enzyem requiring DPN. The enzyme was in the supernatant after centrifugation for l hour a t 20,000 G, was not precipitated by dialysis against distilled water or by half saturation with ammonium sulfate, but was precipitated by complete saturation. When such a preparation was incubated with testosterone, the only reaction was the oxidation of the alcohol group on carbon-17 to a ketone (Table IV). A4-androstene-

h3

h h3

TABLE 111 Destr uction of Testosterone by Livers of Different Speciesn Buffer only

Animal

No. of livers combined

-

Original testostea,B .one con- destruccentration tion

Fish

6

rg. per flask 200

Frog

5

200

Turtle

5

200

Rattlesnake

2

200

Chicken

1

576

Steer

1

200

Rat fed, 0

4

288

Rat fed, 8

5

288

17-Ketosteroids formed

Buffer

+ DPN

a,B destruction

Per g zg. per g. a-per g. per hr.b per hr.6 per hr.6 0 64 56 (46-92) (30-70) 61 0 (54-69) 0 31 32 (22-38) (16-42) 39 0 66 (26-62) (43-89) 380 190 520 (350-406 (163-241 (485-588) 77 0 lo? (0-22? (0-29) 202 16 >367 (20 1-204 (11-20) ( >36& >367 72 195 261 (178-21 2 (55-88) (248-270)

Pg.

I7-Keto. steroids formed

Buffer

+ citrate

ff,B destruction

17-Keto steroids formed

Buffer

+ DPN + citrate

ff,B destruction

1FKetosteroids formed

o 5 u1

Pg. Per g per hr.b 0

ug. per g. Pg. Per g

per hr.* 62 (38-76)

per hr.b 0

rg. per g. per hr.b

ug. per g. per hr.*

2G 6

>

z

U

0

0

337 (312-359 25 1 (208-306 91 (87-97) 181 ( 144-216

41 (14-62) 40 (37-81) 634 (580-666) 74 (50-96) 304 (303-304) 316 (296-351)

E> FM

0

V’

0 135 (107-167 0

26 (21-30) 42 (31-53)

rj

980 (930-1015) 124 (106-143) >365 (>358->367 357 (352-362)

26 1 (236-292) 151 (144-158) 55 (17-76) 112 (98-124)

4

Rabbit

288

Guinea pig

288

Dog,

288

Monkey

288

Human

200

Human

288

Human

288

181 (170-1 93) 96 (52-149) 91 (78-104) 155 (146-162) 137 (106-196) 224 (223-226) 305 (294-316)

96 (86-105) 104 (98-111) 31 (28-37) 133 (122-148) 52 (40-68) 132 (118-145) 80 (66-94)

158 (150-167) 168 (150- 179) 154 (138-164) 210 (205-21 9) 164 183 466 (386-547)

97 (93-100 134 (102-1 74 100 (97-104 170 (147-1 90 153 270

36 255 (250-259) (33-42) 27 155 (152-160) (24-33) 31 117 (104-127) (28-34) 122 284 (282-287) (118-128

264 (260-266) 281 (256-298) 246 (239-254) 295 (284-306)

65 (63-66) 52 (36-65) 49 (44-52) 101 (90-112)

291

95 (86-104

312 (306-31 7)

224 (206-242)

J . Bwl. Chem. 183, 233 (1950). Micrograms of steroid per gm. of liver mince per hour

M

5

#

4

z+ r, m

183 (180-186

z

0

Except in the case of the fish and turtle, in which data from three incubations are combined in each case, each line represents a single incubation. The number of livers in all cases except the two mentioned represents the numbers which were minced together to furnish sufficient sample for the series. The numbers in parentheses indicate the range of values obtained from individual incubation flasks. Each flask contained 25 ml. of buffer and 0.50 to 1.00 gm. of liver mince. In most cases the results from three flasks have been averaged to give the mean value. Testosterone concentration is given because, over the range involved, destruction varies almost directly as substrate concentration. This must be considered in comparing the rates of destruction. b

i! z

-

a

e2

3Z $ Z

264

LEO T. SAMUELS AND CHARLES D. WEST

3,17-dione and unchanged testosterone were the only products which could be isolated, and more than 90% of the original hormone was accounted for in the two fractions. It would appear, then, that the first reaction for which DPN is the hydrogen acceptor is the oxidation at carbon 17; under the conditions used in the incubations with crude tissue, however, other enzyme systems act so readily on the A4-androstene-3,17-dione formed that the first intermediates to accumulate in measurable concentration are the 3-hydroxy-17-ketones. 200

W

0

J

a H

4

v)

/’

a I00 w a

)o

3 z 0

0

(20

60

TlME-

MIN.

FIG.2. Change in concentration of a# unsaturated ketone group and 17-ketone group with time. Incubation of testosterone with cockerel liver mince in Krehs phosphate buffer a t 37”. Unlabeled line represents amount of steroid unaccounted for by sum of other values.

Using their purified preparation, these workers studied the kinetics of the reaction. The Michaelis constant, K , = 3.3 X mole/l. Since in the equation:

EC

ki

kr

+ S%ECS-+ P + EC kr

+

K , = (k24- ks),then k~ = 3.0 X 104(kz ks) ki

One can conclude, therefore, that the hormone combines rapidly with the enzyme and cofactor, the complex then decomposing slowly into products at rate k , which the authors calculate to be approximately 1.0 X lo-*

INTERMEDIARY METABOLISM OF NON-BENZENOID STEROIDS

265

TABLE I V Enzymic Activily of Preparations from Steer Liver on Rings A and D of Testosterone*

Preparation

Conjugated system in ring A 17-Ketosteroids Enzyme destroyed formed (andros- solids (testosterone tenedione per per per flask) flask) flask

Centrifuged extract, 20,000 X g for 1 hr.: Before dialysis Dialyzed extract 0 . 5 Saturation with (NH4)&304: Supernatant Precipitate Complete saturation with (NH4)1S04: Supernatant Precipitate

reg.

Pi5

mg.

82 27

79 73

360

(11)

75

24.5

(6)

!(6)

(11)

0

(6)

56

All flasks contained 20 ml. of a buffer solution, pH 7.4, containing the following: KCl, 0.0056 M; MgC11, 0.0021 M ;NaC1, 0.08 M; NaaHPO4 - NaHzPO4, 0.04 M ; and extract equivalent to 2 g. of fresh liver tissue. The flasks were incubated a t 38" for 3 hr. after being filled with oxygen. The figures in parentheses are within the limits of experimental error and are therefore insignificant.

* From J , B i d . CAtm. 186. 76 (1950). set.-'. Under these conditions it is not surprising that A4-androstene3,17-dione does not accumulate in significant concentrations in the presence of other enzymes acting upon it. Methyltestosterone (XV) does not yield 17-ketosteroids in the presence of DPN, but ring A is acted upon by the citrate-activated system at approximately the same rate as is testosterone (Levedahl and Samuels, 1950). This is illustrated in Table V. The enzyme in the liver of the female rabbit which forms 17-ketosteroids is not a DPNactivated system ;consequently the destruction of a,B unsaturated ketone groups in either hormone is unaffected by DPN. Citrate increases the rate of disappearance in both cases. In the case of the rat, where DPN is the hydrogen acceptor for oxidation at carbon-17, the disappearance of the a,P conjugated system is accelerated by this cofactor in the case of testosterone but not in the case of methyltestosterone; the metabolism of both hormones, however, is affected by citrate. Apparently either the energy of the DPN reaction i s insufficient to split a carbon-to-carbon bond, or the methyl group at carbon 17 prevents the proper attachment

266

LEO T. SAMUELS AND CHARLES D. WEST

TABLE V Metabolism of Testosterone ( T ) and Methyltestosterone ( M T ) by Minced Liver Tissue of Various Species; InfEutnca of DPN and Citrate' Buffer Animal

Steroid

a,B Destruc-

tion

17-Ketoster- z,8 Destrucoid formed tion

+ DPN

Buffer

+ citrate

17-Ketoster. u,8 Destrucoid formed tion

17-Ketosteroid formed

~

Rat

T

Rat

nt T

Rabbit

T

Rabbit

MT

-

0.68 (0.62-0.75) 0.68 (0.56-0.82) 0.63 (0.59-0.67) 0.48 (0.42-0.55)

0.25 (0.19-0.31) 0 0.33 (0.3&0.37) 0

0.91 (0.86-0.94) 0.65 (0.42-0.97) 0.54 (0.52-0.58) 0.48 (0.33-0.56)

0.63 ( 0 . 5 O .76)

0 0.34 (0.32-0.35) 0

1.10 (1.03-1.22) 1.07 (0.88-1.22) 0.88

0.15 (0.11-0.18) 0

0.12

(0.86-0.90) (0.11-0.15) 0.78 (0.76-0.80)

0

The results are expressed as micromoles of steroid per gram of tissue per hour. The figures on the second lines represent the range of values obtained for individual incubation flasks. In all instances, three flasks were incubated; tissues from the same animala were used for both testosterone and methyltestosterone. Each flask eontsined approximately 0.5 g. of liver mince, 2.00 NM. of steroid. and 25 ml. of a buffer solution containing the following: NaCl, 0.08 M; KC1, 0.0056 M : MgCh. 0.0021 M; NasHPOd-NaHrPOd buffer (pH7.4) 0.04 M. The flssks were incubated in 0s for 1 hr. a t 38". * From J . B i d . Chem. 188. 857 (1950).

to the enzyme. The one carbon side chain does not, however, interfere with the energy relations in the citrate-activated reaction. ,If the failure to split the methyl group from methyltestosterone depended mainly on the inability to break the carbon-to-carbon bond after the enzyme-coenzyme-substrate complex was formed, this steroid should show competitive inhibition. As already pointed out, the kinetics of the system indicate that in the case of testosterone the complex is rapidly formed and relatively slowly split to the product. One would, therefore, expect inhibition to be readily demonstrable at total concentrations approaching that of testosterone where it ceases to be limiting in the reaction. This is between 2 and 3 pmo1/25 ml., the volume used in the incubation flasks. The results of such studies are shown in Table VI (Levedahl, 1949). No inhibition of 17-ketosteroid formation was demonstrable and i t seems probable, therefore, that the presence of the methyl group on carbon 17 has considerably reduced the ability of the steroid to bind with the oxidizing enzyme. The rate of destruction of the conjugated double bond system in Ring A of methyltestosterone at constant citrate concentration was studied by Levedahl (1949). As seen in Fig. 3, the reaction is first order with respect to steroid. No work on separation and purification of the citrate-activated system has thus far been reported.

INTERMEDIARY METABOLISM OF NON-BENZENOID STEROIDS

267

The state of nutrition of the rat has a pronounced effect on the ability of the liver to act upon testosterone. Since DPN and citrate play such important roles this is not surprising. The rapidity with which starvation influences the enzymic activity, however, was unexpected. When TABLE VI The Efect of Added Methyltestosterone on the Metabolism of Testosterone by Dog Liver Mince

Testosterone

Methyltestosterone

pmole 1

pmole 0

a-j.3 Conjugation

destroyed

17-Ketosteroids formed

moles/g. fir. 0.65 0.56 . 0.59

moles/g. /hr. 0.41 0.34

0.60

0.36

0.96 0.90

0.39 0.38

0.93

0.38

1.26 1.26

0.39

1.26

0.39

0.88 0.73

0 0

-

0.34

-

0.40

0.80 0

1

0.38 0.43

0 0

0.40 In each incubation sample 1 g. of dog liver mince was used with 25 ml. of buffer. DPN and niacinamide were added to all flasks in the ratio of 6 pmoles of DPN and 0.122 g. per pmole of steroid. All flasks were incubated for 1 hr. a t 37-38°C.

rats were fasted for 48 hours the rate at which the livers would destroy the C U , ~unsaturated ketone system in ring A was reduced t o less than half that of the fed littermates (Table VII). The addition of citrate increased the destruction proportionately. There was no effect on the rate of formation of 17-ketosteroids. This was low in both because

LEO T. SAMUELS AND CHARLES D. WEST

268

" I

1

I

I

I

30

I

I

I 90

60 MINUTES

I

I

1

120

FIG.3. Relation between the concentration of methyltestosterone and the rate of destruction of the conjugated double bond system in ring A. Temperature 37", concentration of citrate 0.001 M .

niacin had not been added to the buffer solution as an inhibitor of the nucleotidases. The addition of DPN seemed to have a greater effect in the livers of the fasted rats than in those of the fed animals. This was probably because the greater activity of the citrate-activated system in the fed rats reduced the substrate available to the DPN-activated system. TABLE V I I Eflect of .@ Hours' Starvation on the Metabolism of Testosterone by Rat Liver 31 irice Fasted rats 17-Ket. Destr. Formed Pg. r g. a,,¶

Cofactor None

0.001M Citrate

+ + +

13.5mg. D P N 0 . 0 4 M NAA 0.001 M Citrate 13.5 mg. D P N 0.04 M NAA

47 (39-54) 101 (88-108) 153 (145-1 62) 215 (174-240) ~

Fed rats

4 Destr. pg.

24 98 (18-31) (96-100) 22 184 (14-28) (166-197) 106 234 (106-107) (211-266) >273 69 (42-92) (>252->294) ~

All rats used in this experiment were mltle littermates.

Ratio 17-Ket. Formed Fed/ Fasted pg. a,D 17-Ket. 22 2.1 (11-27) 22 1.8 (13-27) 62 1.5 (62-62) 45 >1.3 (40-50)

0.9 1.0

0.6 0.65

INTERMEDIARY METABOLISM

OF NON-BENZENOID

2G9

STEROIDS

It is obvious that the rapid effect of fasting is not primarily due to the cessation of niacin intake. When a source of niacin is absent from the diet for a more prolonged period, however, it does influence the activity of the rat liver. Bryson et al. (1950) force-fed rats diets complete except for niacin, niacin and tryptophan, or tryptophan only. Since absorption of food was good, there was no general deficiency. When both niacin and tryptophan were absent for 2 weeks, the rate of destruction of the a,@ unsaturated ketone system was about half that of the controls

a: +@ Destruction

&Liver

Niacin

m=Urine Methvl-

4

FIG.4. The metabolism of testosterone by minced liver tissue of rats on diets deficient in either tryptophan, tryptophan and niacin, or niacin. Diet 1, complete; diet 2, tryptophan-niacin deficient; diet 3, tryptophan deficient; diet 4,niacin deficient.

on the complete diet; if either was absent alone, the rate was intermediate (Fig. 4). Biskind and Biskind (1943) reported that, within the limits of their method, the livers of thiamine-deficient rats were able to inactivate testosterone absorbed from splenic implants as well as normal controls. Similar results were obtained in vitro by Bryson et al. (1950). They found a small decrease in the activity of liver minces, but attributed this t o the semistarvation resulting from limited absorption from the gut. The effect was much less than in niacin-tryptophan deficiency. As Danby (1940) showed by perfusion, the kidney is also capable of

270

LEO T. SAMUELS A N D CHARLES D. WEST

TABLE VIII Incubation of Minced Kidneg Tissue with Testostercme and Added Cojactors* ~

Experiment No. Animal 1

Dog

a,p-Unsaturated steroid destroyed (as testosterone) Incubation media Mean value Buffer solution DPN buffer solution Citrate buffer solution D P N citrate buffer solution

+

+ +

2

3

Dog

Rsbbit

+

Buffer solution DPN buffer solution Citrate buffer solution DPN citrate buffer solution

+ + +

+

Buffer solution D P N +buffer solution Citrate buffer solution D P N citrate buffer solution

+ +

4

~~~

Guinea pig Buffer solution D P N buffer solution Citrate +buffer solution DPN citrate buffer solution

+

0.11 0.14

0.10- 0.12 0.07 0.06-0.08 0.13- 0.15 0.20 0.19-0.20

0.11

0.10- 0.13 0.08 0.07-0.09

0.17

0.16- 0.17 0.19 0.19-0.20

0.05 0.10

0.05- 0.06 0.07 0.064.07 0.09- 0.11 0.21 0.20-0.22

0.07

0.07-

0.08 0.05 0.04-0.05

0.11

0.10-

0.13 0.22 0.21-0.23

Insignificant -0.02-+0.04 0.12 0.11-4.14 Insignificant -0.03-+0.07 0.13 0.12-0.15 Insignificant -0.02-$0.05 0.03 0.03-0.04 Insignificant

-0.01-+O .08 0.04 0.04-0.05

Insignificant -0.06-+O .02 0.32 0.25-0.43 Insignificant -0.02- 0.09 0.80 0.76-0.85

+

+

17-Ketosteroids formed (as androstenedione) Mean Variation value Variation

+

Insignificant

0.01- 0.09 0.13 0.12-0.15

Insignificant

0.00-

0.04 0.50 0.47-0.53

The values are given in micromoles per gram per hour. While the means were not 0, they did not differ significantly from this value.

* From J . B i d . Chcm. 180, 829 (1961).

INTERMEDIARY METABOLISM OF NON-BENZENOID STEROIDS

271

acting on the steroid hormones. Kochakian and coworkers (1949) also found that incubation of testosterone with kidney tissue would bring about changes similar to those seen in the presence of liver tissue, but a t slower rates. West and Samuels (1951) have used the techniques developed by Samuels and coworkers to study the enzymes of kidney tissue. In the guinea pig and rabbit the only reaction that could be identified was the oxidation of the alcohol group on carbon 17 to a ketone, with DPN as the hydrogen acceptor; only in the dog kidney was there also significant reduction of the conjugated system in ring A (Table VIII). The concentration of the enzymes in kidney tissue was much lower than in liver, and one must conclude that it serves at most a secondary role in the intact organism. Samuels et al. (1947) have investigated a number of other tissues for evidence of enzymes which would act on testosterone; so far none has been found (Table IX). TABLE IX Metabolism of Testosterone bv Diferent Tissues* Testosterone introduced, Tissue

pg.

Rabbit uterus

200

Rat prostate

300

Rat seminal vesicle

300

Mouse mammary tumor

200

Testosterone recovered, pg. a-@-groups 17-Ketones Koenig Ultraviolet destroyed formed reaction (a,P) pgg./g./hr. pg./g./hr. 193 (190-197) 293 (292-295) 288 (275-30 1) 203 (196-210)

196

Insig.

Insig.

Insig.

Insig.

Insig.

Insig.

Insig.

Insig.

From Recent Progrerr in Hormone Reasarch 4, 79 (1949).

Schneider and Mason (1948a) incubated dehydroisoandrosterone succinate with rabbit liver slices under aerobic conditions. The products isolated were A5-androstene-3p,17p-diol (IV) in yields of 43-69 %, and AK-androstene-3p,16~,l7p-triol(XVIII), 2.4-8.9%. In addition, an unidentified a,p unsaturated ketone was formed in yields of about 1%. Thus the major reaction involved reduction of the 17-ketone group. When A6-androstene-3P,170-diol was incubated with liver slices, some dehydroisoandrosterone was formed ; this reaction is, therefore, reversible (Schneider and Mason, 1948b).

272

LEO T. SAMUELS AND CHARLES D. WEST

Under the conditions used, androsterone and etiocholan-3a-ol-17-one, the major steroids found in the urine after injection of dehydroisoandrosterone, were not found. Either the oxidation a t carbon 3 occurs in some other tissue or the necessary cofactors were not present. Schneider and Mason (1948a) also incubated androsterone and etiocholan-3a-ol-17-one hemisuccinates under conditions similar to those used in the studies just described. With androsterone, an average of 37% was recovered as crystalline material. The major portion was unchanged androsterone (about 20%). The major metabolite was the reduction product, androstane-3a1l7@-diol(XIII) (about 15%). Other compounds isolated were andro~tane-3~17-dione (VIII), isoandrosterone (XI), and two unidentified alcohols. Each of these represented about 0.5% of the incubated hormone, or about 5 mg. When etiocholan3a-ol-17-one was incubated, a much larger proportion of the steroid was recovered as metabolic products. The total recovery of crystalline material was 50.8%. Of this, only 8% was unchanged etiocholanolone. The major metabolite was etiocholane-3cu,l7~-diol(XIX), 32.8 %; next was etiocholane-3a, 17a-diol (XIX), 8.5 %. The last was etiocholane3,17-dione (VII), present to the extent of 1.5%. The routes of the two hormones appears to be the same, except that a larger proportion of androsterone is changed to unidentified products. It has already been pointed out that one of the important phases in the over-all metabolism of injected or endogenous androgens is the conjugation of the major portion of the 17-ketosteroids with glucuronic and sulfuric acids. This reaction has not been studied adequately in isolated systems. It is known, of course, that the liver contains enzymes which will carry on such conjugations with a number of other molecules but the steroids have not been so studied. Certain observations have been made in men suffering from hepatic disease. Several workers (Glass et al., 1940, 1944; Gilder and Hoagland, 1946) have reported that the average output of 17-ketosteroids is reduced in the presence of severe liver disease; the individual values, however, overlap the normal range. Cantarow et al. (1951) found that the average total output of 17-ketosteroids after a single injection of 200 mg. testosterone propionate in oil to patients with chronic liver disease was somewhat lower than in controls, and that the percentage of the excreted compounds present as the free steroid was significantly higher. They conclude that damage to the liver particularly interferes with the conjugation reaction. West et al. (1951b), using the intravenous injection of 150-200 mg. testosterone dissolved in serum albumin solution, found a marked decrease in the formation of 17-ketosteroids during the first hour whenever there

273

INTERMEDIARY METABOLISM OF NON-BENZENOID STEROIDS

TABLE X A Comparison of the Rate of Excretion of 17-Ketosteroids between Normal Subjects and Palienta with Liver Disease*

Subject

Diagnosis

CE CH CN CR

cv

DB DC DG DL DS

CG CL CM CP CQ

cs

CT

cu DM DO DR

cw

% Administered testosterone excreted as 17-IiS

Creatinine clearance in ml. of plasma per min.

1st hour

2nd hour

Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal

17.7 18.4 15.4 18.1 12.2 11.7 13.2 13.5 18.0 25.7

19.0 11.6 16.5 17.7 10.1 16.7 10.5 17.5 12.9 13.8

123 152 108 111 117 123 130

Normal averages

16.4

14.6

123

Cirrhosis Cirrhosis Cirrhosis Cirrhosis Cirrhosis Cirrhosis Cirrhosis Cirrhosis Hepatitis Cirrhosis Cirrhosis 1Ca. of liver and nephritis

4.1 8.1 0.2 6.1 2.6 6.9 1.9 5.2 11.3 4.4 4.3 0.7

8.9 10.4 3.5 6.9 2.7 3.2 5.5 8.1 10.8 6.8 0.9

Liver disease averages

4.6

6.6

99

Chronic glomerulonephritis

1.4

1.5

14

____

165 65 115 69 02 77 66

I41

~~

cz

*West, C. D.,

el al.

J.

C h . EndocrinoL 1Q51b, 11, 908.

was measurable liver damage (Table X). The 17-ketosteroids in both the blood and urine were largely in the form of conjugates. A comparison of the blood levels of testosterone as measured by the ultraviolet absorption due to the a,Punsaturated ketone group, and those of the 17-ketosteroids as measured by the Zimmermann reaction after hydrolysis indicated that there was some intermediate between testosterone and the androsterone isomers which accumulated in the individuals with liver disease; other-

274

LEO T. SAMUELS AND CHARLES D. WEST

wise, in the presence of normal kidney function the levels of 17-ketosteroids would not have risen while the testosterone levels fell (Fig. 5). The total amount of 17-ketosteroids which was excreted after the single intravenous dose of testosterone was much smaller in the cirrhotics than in the controls, indicating that a larger proportion of the injected hormone had been metabolized by some other route; there was no evidence of increased excretion of the hormone itself. West et al. (1951b) compared the renal clearance of the conjugated 17-ketosteroids with that of creatinine. For the normal subjects the average plasma clearance of 17-ketosteroids was 126 ml./minute; and of

_-*--- -x-

8200-

4 6

2

17-KETOSTEROIDS -TESTOSTERONE O-NORMALS. AVERAGE OF a CASES I-CIRRHOTICS, AVERAGE OF I C-5

100-

Boo-

a"

6

6040-

w L.l 20 I

0

20

I

,

I

!

40 60 80 100 MINUTES AFTER INJECTION

,

120

FIG. 5. Average levels of testosterone and total lirketosteroids in the blood following intravenous injection of testosterone into normal subjects and patients with liver damage.

creatinine, 123 ml./minute. In the cirrhotic patients the respective averages were 100 ml./minute and 99 ml./minute. The results indicate that the conjugates are filtered readily in the glomerulus and not reabsorbed or secreted. When the clearance of total testosterone was measured, however, it was very low. As Bischoff and Pilhorn (1918) first showed, however, testosterone and progesterone form complexes with serum albumin. Lumry et al. (1951) have demonstrated these are in true equilibrium with the free hormone dissolved in the serum. If the amount of free hormone in solution is calculated from the data on total testosterone in the blood of West's patients, assuming a level of 5 % ' albumin in the serum, it is found that, within the limits of error of the determinations, the free hormone is also filtered through the glomerulus and not reabsorbed. The reason that so little testosterone ever appears in the urine is that the greater portion is bound to protein and is, therefore, not filterable. If we now assemble the information gathered from the urine studies,

INTERMEDIARY

METABOLISM OF NON-BENZENOID

/ I

//

STEROIDS

275

Rapid?

J

HO"

Slow

H

FIG.6. Metabolism of testosterone and dehydroisoandrosterone in the liver as indicated by tissue studies.

the blood studies, and the in Vitro studies on enzyme systems, it would seem that the course of the metabolism of testosterone in the human being might be diagrammed as illustrated in Fig. 6. The net result is to maintain, under normal conditions, a constant low level of circulating active androgen with a very large turnover. The high rate of turnover makes possible the ready response of the organism to changes in pituitary hormone output or to a sudden increase in the local need of some target organ.

276

LEO T. SAMUELS AND CHARLES D. WEST

In the latter case rapid removal of the hormone in the target tissue will lower the rate of catabolism by mass action until a steady state is again reached. The metabolic systems in the liver and kidneys would seem to function largely as means of disposing of excess hormone. It has already been noted that among the tissues which show no significant metabolic effect on testosterone are some of the tissues inkhich its effect is most marked. In the studies of West et al. (1951b) on-the intravenous injection of testosterone the greater portion of the hormone had been metabolized and excreted before there was any measurable effect on nitrogen balance. Yet the metabolic effect from the single injection continued for several days after any hormone could be identified in the blood or any excess 17-ketosteroids appeared in the urine. On the fifth to eighth days after the injection, however, there appeared a small increase in excretion of 17-ketosteroids in all the cases that were followed. It was a t this time that the positive nitrogen balance decreased. A hypothesis which mould explain these phenomena assumes that the hormone does not affect the metabolism of the cell by actually entering into a reaction with substrate, but that it attaches to certain enzymes, perhaps in much the same way as it complexes with serum protein, and in this way affects the ability of the enzyme t o bring its appropriate substrates into a reactive state. In the intravenous studies the slight excess secretion after several days would then represent the turnover of the increased enzyme-steroid complex produced by mass action when the very high levels of testosterone were attained immediately after the injections. The material thus released from the target cell is carried in the blood and metabolized by the liver in the same way as the daily excess.

111. INTERMEDIARY METABOLISM OF THE PROGESTINS Like the androgens, substances having a progestational action on the uterus, the progestins, are found in a number of endocrine tissues. The two major sites are the corpus luteum of the ovary and the placenta. In addition, the adrenals and testes produce substances having progestational activity. The changes produced in the uterus by the corpus luteum were first clearly demonstrated by Loeb in 1907, but it was not until 1929 that Corner and Allen (1929) succeeded in preparing active extracts. In 1934 Allen and Wintersteiner, Butenandt and Westphal, and Slotta et al. all succeeded in crystallizing the active hormone, progesterone, from lipide extracts of copora lutea. Adler et al. (1934) prepared extracts of both human and cattle placentae that had progestin activity, but the

INTERMEDIARY METABOLISM OF NON-BENZENOID

STEROIDS

277

concentration was very low. Noall et al. (1952) have finally succeeded in isolating progesterone from normal human term placentae. Progesterone was isolated from adrenal extracts by Beall and Reichstein (1938). Callow and Parkes (1936) obtained progestational activity with testicular extracts, but again no progestin had been isolated. Ruzicka and Prelog (1943) recovered As-pregnen-3&01-20-one from hog testes, however, and Samuels et al. (1951) have shown that the interstitial cells of the testes contain an enzyme that will oxidize this compound to progesterone. Thus, while progesterone has been recovered from only two of the four tissues that show progestational activity, a third has all that is necessary for its synthesis. At the present time, therefore, it is the metabolism of this steroid that must be considered. Other compounds, however, may be found in the placenta which will be of equal importance. TABLE XI Urinary Steroids Related to Progesterone 1. Pregnane-3a, 20a diol (pregnandiol) 2. Allopregnane-3a120a, diol (allo-pregnan-diol) 3. Pregnane-3a-01 4. Allopregnane-3j3,20a-diol 5. Pregnane-3a-ol-20-0ne (epipregnanolone) 6. Allopregnane-3a-01-20~ne(epiallopregnanolone) 7. Allopregnane-3fl-ol-20-one 8. Pregnane-3,ILO-dione 9. Allopregnane-3, 20-dione 10. Pregnane-3j3, 2Oa-diol 11. As-Pregnene-3p, 2Oa-diol 12. Allopregnane-3j3, 16a, 2OO-triol 13. Allopregnenediol-3a16a-one-20 14. Pregnanediol-3a, 6a-one-20 15. 17-Isopregnanol-3a-one-20

* References 1. Marrian. 1929. 2. Butenandt, 1930. 3. Marker et al., 1937. 4. Marker, 1938. 5. Fieh et al., 1942. 6. Lieberman et ol., 1948. 7. Hartmann and Locher, 1935. 8. Marker and Lawaon. 19388. 9. Marker and Xamm, 1937. 10. Marker and Rohrmann, 19390. 11. Marker et al., 1937b. 12. Marker d d., 1938b. 13. Heard and McKay, 1939. 14. Mason and Kepler, 1945. 15. Hirschmann, 1943. 16. Hirschmann et al., 1948. 17. Lieberman sf al.. 19508.

278

LEO T. SAMUELS .4ND CHARLES D. WEST

1 . Urinary Excretion after Administration of Progesterone

The steroids listed in Table XI have all been isolated in increased amounts from pregnancy urines and therefore have been considered to be related to progesterone metabolism. If these do represent such metabolism it would seem that all of the various steps in the reduction of progesterone to the pregnanediols are found. The two possible saturated diketones are present, indicating that, as in the case of testosterone, the reduction is not stericly directed. Three of the four possible 3-hydroxy20-ones have been isolated but no 3-keto-20-hydroxy compounds have been found; apparently the ketone on carbon 3 is reduced first to yield both isomers. The four possible 2Oa-diols have been identified but no 20p-diols have been found; the reduction of the 20-keto group is apparently stericly specific. If large amounts of progesterone are being metabolized during pregnancy, this makes a beautiful sequential relation. Unfortunately it has not been possible to confirm the entire sequence by isolation of increased amounts of all these steroids after administration of progesterone. Only two members have been found : pregnan-3a-ol-20one and pregnane-3a,20a-diol. Dorfman and coworkers (1948) found that the ratio of the former to the latter in the urine of a man after oral administration of progesterone in large doses was 1: 10. A number of workers have noted the relatively constant recovery of pregnanediol after a given dose of progesterone in normal men and women. Sommerville and Marrian (1950a,b) recovered 9-16% of the injected progesterone as pregnanediol in nonpregnant women. During the 2628th weeks of pregnancy a significantly higher recovery, 35-45 %, was obtained. Since similar recoveries were obtained in the two conditions when pregnanediol itself was given it seems that the low recovery is probably due to metabolism of pregnanediol. The use of isotopes has not been so enlightening in the case of progesterone as in the case of testosterone. The two species which have been used for isotope studies, the mouse and rat, have never been demonstrated to excrete any known products of progesterone metabolism, either during pregnancy or after the administration of the hormone. Thus data from these species cannot readily be used for interpreting the situation in man and the other animals that excrete the reduced forms in the urine under these conditions. Riegel et al. (1950) used progesterone with C14in position 21. They found most of the isotope in the feces, but a considerable amount in the expired COs. The material in the feces was characterized as ketonic and non-alcoholic but was not progesterone. Gallagher et al. (1951), also using progesterone labelled in position 21 with C14,confirmed these findings. When this material was injected into mice, 9.1 % ’ of the

INTERMEDIARY

METABOLISM

OF NON-BENZENOID

279

STEROIDS

isotope was found in the expired carbon dioxide. Again the balance was largely in the feces. These results would indicate that the side chain is oxidized in these species, either before excretion into the gut or by organisms in the gastrointestinal tract. The rapid rate a t which progesterone can be metabolized by the human being has been illustrated by the studies of Sommerville and Bigler (1951). They infused three normal young men intravenously with the hormone dissolved in human serum albumin solution. The levels A

0.-QTOTAL #-a FREE W CONJUGATED SUBJECT J

\ \

\ \ \

/

U

K (3

0

I

I

0

I0

MINUTES

I 20

I

30

40.

50

6

FIG. 7. Levels of free and conjugated pregnanediol in the blood of a normal young man during and after the intravenous infusion of 30 mg.progesterone dissolved in 25 % human serum albumin solution.

of progesterone and pregnanediol were followed in both blood and urine. The infusions into the vein generally lasted 15-30 minutes. At the end of that time the levels of pregnanediol were already very high and the levels of progesterone were low. Progesterone was cleared from the blood so rapidly that within fifteen minutes after the infusion was stopped the concentration could not be measured; even the level of the pregnanediol fell from the first sample until the material disappeared. The curves of free and conjugated pregnanediol in the blood of one patient are given in Fig. 7. About half of the steroid was in the free state. This is in contrast to the situation after testosterone was administered under similar circumstances; the 17-ketosteroids formed were always

280

LEO T. SAMUELS AND CHARLES D. WEST

present solely as the conjugated forms within the limits of the methods. The levels of the Czlsteroids in the blood also fell more precipitously than did the 17-ketosteroids in the testosterone experiments. All of these findings point to a very rapid reduction of progesterone to pregnanediol, so rapid as to exceed the ability of the liver to conjugate the alcohol. 2. Metabolism of Progesterone by Tissues

The studies of urinary excretion have eliminated the sex organs and adrenals as major sites of the conversion of progesterone to pregnanediol. Hysterectomized women (Venning and Browne, 1940; Jones and TeLinde, 1941), normal men, and patients with Addison’s disease (Buxton and Westphal, 1939), ovariectomized hysterectomized rabbits (Hoffman and Browne, 1942), and castrated male rabbits (Hoffman, 1942)) all excrete pregnanediol when given progesterone. There is some evidence, however, that in women with intact uteri there is a gradual increase in the percentage of injected progesterone which appears as pregnanediol when the hormone is given continuously for periods of over a week (Sommerville and Marrian, 1950a). Since the endocrine and target organs did not seem to be the primary sites of this metabolic process, attention naturally turned to the liver. Zondek (1939) injected active corpus luteum extracts into mice and found that he could not recover the activity from the animal a short time after injection. He then tried incubation of the material with liver brei in the manner that had been so successful with estrogens; there was no inactivation. Engel (1946) confirmed this. While earlier work in intact animals was equivocal, recent studies of this type indicate that the liver does have an inactivating action. Selye (1941) found that the anesthetic action of progesterone was enhanced by partial hepatectomy. Dome (1944) reported that the antifibromatogenic action of progesterone was prevented if pellets of the hormone were implanted in the spleen rather than under the skin. Kochakian et al. (1944) found that pellets implanted in the mesentery did not produce progestational changes even though much more hormone was absorbed than was required as a subcutaneous dose (Table XII). Masson and Hoffman (1945) found that doses much larger than the subcutaneous dose were inactive when given by mouth unless the rabbits were partially hepatectomized; then progestational proliferation occurred. The evidence a t the end of 1945 was, therefore, strongly in favor of the liver as a site of inactivation in spite of the failures of the in vitro experiments. Samuels, in 1949, reported the destruction of the a,/3 unsaturated ketone group of progesterone by liver minces. This was measured by the disappearance of the strong absorption band a t 240 mp. When progester-

28 1

INTERMEDIARY METABOLISM OF NON-BENZENOID STEROIDS

one and testosterone were incubated together there was no depression of the rate of testosterone metabolism, and the progesterone also was normally metabolized. While it was not proved that the concentrations of the hormones were sufficient to saturate the enzyme systems, the lack of influence of either on the metabolism of the other was interpreted as suggestive evidence that the two hormones were metabolized by separate routes. TABLE XI1 The Effect of Various Tissues on the Activity of Progesterone" Site of implantation Subcutaneous Muscle Kidney Mesentery Mesenteryd

Endometrialb reaction

No. of rabbits 6 8

6 6 4

3 . 2 + (3+-4+) 3.2+ (3+-4t) 2 . 8 + (2+-3+) 0 0

Progesterone Relativec absorbed, mg. efficacy 3.0 2.6 5.1 4.7 20.2

1.1 1.2

0.5 0.0 0.0

From Am. J . Physiol. ill, 327 (1944). Corner Allen scale. * Relative efficacy equals endometrial response divided by milligrams progesterone absorbed. d Duration of experiment 14 days with 4 pellets (25k mg. each) implanted in each rabbit. 0

b

Wiswell and Samuels (1951) have investigated the in vitro metabolism of progesterone by rat and rabbit liver tissue in more detail. Unlike testosterone, the conjugated system in ring A of progesterone was destroyed a t normal rates in the virtual absence of oxygen (Table XIII). The reaction was accelerated by the presence of one of the tricarboxylic acids, but not by dicarboxylic acids or a number of sources of high energy phosphate including DPN (Table XIV). It seems probable that the influence of the tricarboxylic acids was due to their ability to form complexes with metal ions since cysteine and cyanide increased the metabolism of progesterone by liver tissue to a similar extent. TABLE XI11 Progesterone Incubated with Rabbit Liver Homogenate in NP

% ' Recovery Average Boiled control Phosphate buffer Buffer

+ citrate

92 86 90 71 64 67

Micromoles progesterone destroyed per gram of tissue

92 88.0

0.16

67.3

1.09

LEO T. SAMUELS AND CHARLES D. WEST

282

TABLE XIV Efect of Addition of Various Cofactors on the Metabolism of Progesterone by Liver Homogenate Progesterone destroyed pmole/g. tissue

Animal

Incubation medium

Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit

Buffer only Buffer citrate Buffer isocitrate Buffer aconitate Buffer succinate Buffer glutarate Buffer ATP AMP Buffer

0.32 1.iO 1.56 1.65 0.34 0.23 0.37 0.35

Rat Rat Rat Rat

Buffer only DPN Buffer Buffer citrate Buffer citrate

1.11

+ +

+ + + + +

+ + +

+ DPN

1.18 2.67 2.76

Since no oxygen was required, the reaction was probably reductive, but efforts to recover pregnanediol were unsuccessful. Methods of measurement, however, were relatively crude. Experiments with rabbit liver homogenate and progesterone labelled with C14a t position 21 gave the results seen in Table XV. All significant radioactivity which was not in unchanged progesterone was in the chromatographic fraction which would have contained pregnanediol or pregnanolone. In view of the failure to identify pregnanediol, it was thought that pregnanolone might be the product of the reaction, although the small amounts of diol might have been missed. It should be noted that, TABLE XV Per Cent Recovery of Radioactivity after Incubation of C~4-2f-Progesteronewith Rabbit Liver Homogenate and Citrate Chromatographic fraction

Boiled samples

Incubated samples

C D Hexane ethanol from partition of C Residue from extraction

0 0 0 0 0 0 96.2 9 0 . 5 89.9 1.2 1.5 2.3 1 . 4 1 . 7 I .8 0.6 0.8 0.8

0 0 0 0 0 0 7 8 . 8 79.1 80.1 11.9 15.0 14.6 1.5 1.5 1.7 0.6 0.8 0.5

Total

9 9 . 4 94.5 94.8

9 2 . 8 9 6 . 4 96.9

A B

+

METABOLISM OF NON-BENZENOID STEROIDS

INTERMEDIARY

283

contrary to the experiments of Riegel in intact mice, no significant amount of radioactivity was found in the COz. Grant and Marrian (1950) have studied the metabolism of pregnanediol by rat and rabbit liver. They prepared pregnane-3a,20ar-diol hemisuccinate and incubated this relatively water-soluble compound with liver slices or an acetone-dried rat liver powder. Pregnanediol disappeared during incubation and was not recoverable by acid hydrolysis. A B C

Exp. 1

D

E

II

Exp. 2

U

B C

c1 0

11

D E

25 50 75 100 Pregnanediol recovered (%)

FIG.8. Incubation of acetone-dried rat-liver powder with pregnanediol dihemisucrinate in Nz and 02-influence of methylene blue. A , pregnanediol recovered from controls; B, pregnanediol recovered after incubation in 0 2 ; C, pregnanediol recovered after incubation in N2; D,pregnanediol recovered after incubation in NI methylene blue; E, apparent pregnanediol recovered from blanks.

+

While oxygen was required, cyanide did not inhibit the system, so iron and copper enzymes were not involved. Methylene blue partially replaced oxygen in an anaerobic system (Fig. 8). The presence of this enzyme in human liver has not been studied, but it may account for the pregnanediol that disappears both in humans and in rabbits when the steroid is fed or injected. It would also explain the low recoveries of pregnanediol after administration of progesterone. From the in uitro evidence, therefore, it seems that the liver is active both in the reduction of progesterone to pregnanediol and in the oxidation of the latter steroid. Paschkis et al. (1951b) have studied the production of pregnanediol from progesterone in patients with severe liver disease. Intramuscular doses of 100 mg./day were given for 3-4 weeks. The

284

LEO T. SAMUELS AND CHARLES D. WEST

pregnanediol recovery was higher than in normal individuals. They conclude that the metabolism of pregnanediol is impaired more than its formation.

I

, ,

J

\

I

I I

H Clucuronides

1"'

Unknown

FIG. 9. Probable metabolic pathways of progesterone. Dotted lines indicate possible conversions not yet demonstrated. Heavy arrows indicate most probable pathway as indicated by tissue studies and urinary excretion after injection.

Summarizing all of the evidence, it would seem that progesterone is probably metabolized in the liver by a series of reductive steps t o pregnane3~u,20a-dioland possibly the three other 20a isomers. Part of the pregnanediol is conjugated with glucuronic acid and excreted in the urine. The balance is probably oxidized t o as yet unknown products (Fig. 9). The difference between species may largely be in the activity of the

INTERMEDIARY

METABOLISM OF NON-BENZENOID

STEROIDS

285

conjugating enzyme. In the human, as noted in the case of the 17-ketosteroids, this is very active. In the lower animals the rabbit may have an intermediate activity and the rat and mouse relatively low activities. Thus in the latter species the oxidizing enzymes have a much better opportunity to act on the pregnanediol formed from progesterone. This may also account, as in the androgens, for the greater excretion by way of the bile. IV. INTERMEDIARY METABOLISM OF STEROIDS OF

THE

ADRENAL CORTEX

I . Urinary Excretion of Xteroids of Probable Adrenal Origin

,

The urinary steroids that probably arise from precursors produced by the adrenal cortex are given in Table XVI. Of these, only 17-hydroxycorticosterone (Kendall’s compound F) and 17-hydroxy-1l-dehydrocorticosterone (Kendall’s compound El cortisone) have been isolated from the adrenal glands. A number have been identified only in the urine of individuals with adrenal tumors and may represent abnormal metabolites. There are certain relations within series that probably indicate the types of metabolism these compounds undergo in the body. First, the compounds fall into two groups: the C19 and the Cpl steroids. Apparently some Czl steroids can be metabolized to C19 compounds. Fukushima et a,?. (1951) found that when deuterium-labelled 17a-hydroxyprogesterone was given, some isotope appeared in the Cl9-17-ketosteroids. The C19 compounds can arise by metabolism of the CZIcompounds secreted by the glands; C19 steroids are also directly secreted since fractions that are androgenic also give the typical Zimmermann reaction for 17-ketosteroids (Gassner et al., 1951). When either cortisone or 17-hydroxycorticosteroneisadministered, the increase in 17-ketosteroids in the urine is irregular and at most represents about 57, of the injected hormone (Sprague et al., 1949). The same is true for corticosterone, 11-dehydrocorticosterone (Sprague et al., 1948), and 17-hydroxy-1l-desoxycorticosterone (Reichstein’s compound S). 11-Desoxycorticosteronehas no effect (Cuyler et al., 1942). On the other hand, according to Wolfson (1951), administration of an aqueous adrenal extract equivalent in glycogenic function to 100-150 mg. cortisone gave a marked increase in 17-ketosteroids; cortisone in similar amounts gave no significant increase over the control periods. Since adrenocorticotropic hormone produces large increases in 17-ketosteroid excretion, it seems probable that some compound other than the ones mentioned above is responsible for the 17-ketosteroids in the urine. The appearance of the As-compounds as 38 while the saturated compounds are all 3a is not only true for the C ~compounds; O it is also true for

286

LEO T. SAMUELS AND CHARLES D. WEST

TABLE XVI Urinary Steroids Probably Partially or Wholly Originating from the Precursors in Adrenal Corlex References* 1. As-Androsten-3 (p)-ol-17-one (dehydroisoandrosterone) (1-11) 2. Androstane-a(a), 11(p)-diol-17-one (11-hydroxyandrosterone) (10,12) 3. Androstane-3(a)-ol-lll 17-dione (11-ketoandrosterone) (13) 4. Etiocholane-3(a)-ol-ll, 17-dione (11-ketoetiocholanolone) (14) 5. Etiocholane-3(a), 1l(~)-diol-17-one(1l-hydroxy(12,13,15) etiocholanolone) 6. Etiocholane-3(a), 17(p)-diol-ll-one (11-ketoetiocholanediol) (13) 7. AQ-Androsten-3(a)-ol-17-one (13,16) 8. AQ-Etiocholen-3(a)-ol-l7-one (12) 9. 17-Hydroxycorticosterone (Compound F) (17,181 10. Pregnane-30, 1la, 17a, 21-tetrol-20-one (tetrahydro F) (31) 11. 17-Hydroxy-11-dehydrocorticosterone (Compound E) (18-20) (17,21,22,23) 12. Pregnane-3a, 17a, 21-triol-11, 20-dione (tetrahydro E) 13. 21-Desoxytetrahydro E (pregnane-3a, 17a-diol-11, 20-dione) (23) 14. Pregnane-3a-01-11, 20-dione (11-ketopregnanolone) (13) 15. Pregnane-3a, 2Oa-diol-11-one (I1-ketopregnandiol) (13124) 16. 17-Hydroxy-As-pregnenol-30-one-20 (25) 17. 17-Hydroxypregnanolone (ll,26) 18. 17-Hydroxypregnanediol (pregnanetriol) (10,28,32) 19. As-Pregnene-3p1 17~1,2Op-triol (29) 20. A6-Pregnene-3p, 17a, 2Oa-triol (29) (23) 21. As-l’regnene-3& 21-diol-20-one (21-hydroxypregnenolone) 22. Androsterone (1-4,6,7,11,30) 23. Etiocholanolone (1,3-7,10,20,23,28) 24. Pregnane-3a, 2Oa-diol (27) 25. As-Androstene-3fl, 17p-dipl (31) 26. A6.-Pregnene-3p,2Oa-diol (31) 27. As-Androstene-3j3, 1601, 17p-trio1 (10,33,34 Referenced 1. Butenandt and Dannenbsum. 1934. 2. Callow and Callow, 1938. 3. Callow and Callow, 1939. 4. Callow and Callow, 1940. 5. Hirschmann, 1940. 6. Wolfe et al., 1941. 7. Engel et al., 1941. 8. Pearlman. 1942, 9. Munson el al.. 1944. 10. Manon and Kepler, 1945. 11. Lieberman et al.. 1948. 12. Lieberman and Dobriner, 1948. 13. Lieberman st al., 1950. 14. Lieberman and Dobriner. 1948. 15. Dingemanse and Huh in’t Veld, 1949. 16. Miller el al., 1946. 17. Lieberman et al.. 1950.

18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.

Zafiaroni d ol., 1950. Schneider. 1950c. Mason. 1950.1 Schneider. 1950a. Schneider. 1950b. Dobriner et al.. 1951b. Maeon. 1948. Hirschmann and Hirschmann. 1947. Lieberman and Dobriner, 1945. Horwitt el 02.. 1944. Butler and Marrian. 1938. Hirachmann and Hirschmann. 1950b. Hirschmann, 1939. Hirschmann and Hirschmann, 1945. Butler and Marrian. 1937. Hirachmann, 1943. Marrian and Butler, 1944.

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287

the Czl steroids. This raises a serious question regarding the significance of the A6-steroids. One would expect that if both groups of compounds represented progressive steps in the reduction of the active alpunsaturated 3-ketones1 the configuration after reduction on C3 would be the same. Since it has been demonstrated (Samuels et al., 1951) that the adrenal cortex contains a highly active enzyme that will oxidize A6-3p-ols to A4-3-ketonq it seems more likely that these substances in the urine represent the escape of precursors of the active hormones than that the active compounds first undergo a reduction to the Ab-3p-ols in their catabolism followed by reoxidation before further reduction. Following ACTH administration the following urinary steroids have been isolated in increased amounts: cortisone, 17-hydroxycorticosterone, pregnane-3a,l7a1 21-triol-l1,20 dione, pregnane-3a, llP,17a, 21 tetrol-20one, pregnane3a, 17a-diol-11,20-dione, 21-hydroxypregnenolone, androsterone, etiocholanolone, pregnane-3a-ol-20-one, 1lp-hydroxyandrosterone, llp-hydroxyetiocholanolone, ll-ketoetiocholanolone, androstane-3,17dione and etio~holane-3~17-dione (Lieberman et aE., 1950b, 1951; Mason, 1950). When 1l-dehydrocorticosterone was given t o patients with Addison’s disease, Mason (1948) isolated pregnane-3a,20-diol-l l-one from the urine. This was not found after administration of cortisone. Patients receiving cortisone excrete cortisone, 17-hydroxycorticosterone, pregnane-3a, 17a,21-triol-11,20-dione,pregnane-3a,l7a, llp,21tetrol-2O-one1 pregnane-3a,l7a-diol-ll,20-dione,ll-ketoetiocholanolone, 11p-hydroxyandrosterone,and 11p-hydroxyetiocholanolone(Mason, 1950; Lieberman et al., 1950, 1951). There is no conversion to androsterone, etiocholanolone, or pregnanolone (Sprague et al., 1950; Dobriner et al., 1951a, 1951b). From these data it would appear that none of the oxygenated positions in the adrenal steroids except the primary alcohol at Cpl is reduced to a hydrogen-carbon bond. None of the injected 17-hydroxy steroids yielded 17-desoxy steroids and no ll-oxy steroids yielded 1l-desoxy steroids. Since the latter are products of adrenal metabolism, they probably arise from 1l-desoxy precursors. 2. Metabolism of Cortical Steroids by Tissues

Again, as in the case of the other endocrine organs, the studies of excretion prove that metabolism of the cortical hormones occurs, but give no clue regarding the region in which it takes place. Burrill and Greene (1942), using the technique which they had applied so successfully t o the other steroid hormones, implanted pellets of desoxycorticosterone in the mesentery and compared the effectiveness in maintaining the life of the

288

LEO T. SAMUELS AND CHARLES D. WEBT

adrenalectomized rat with that of similar pellets implanted subcutaneously. The animals with small intramesenteric pellets lost similar amounts of weight, and died a t about the same time as the untreated controls while the rats with subcutaneous pellets survived and gained weight. When pellets three times the original size were implanted in the mesentery, however, the adrenalectomiaed rats survived longer and showed some gain in weight. The authors conclude that the liver can inactivate desoxycorticosterone, but that the action is quantitatively more limited than in the case of androgens and estrogens. The role of the liver has been brought out by the work of Harding and Nelson (1951). They have studied the concentration of 17-hydroxycorticoids in the arterial and venous blood across various organs and tissues. As previously reported by Nelson et al. (1951), they found no significant difference aoross peripheral muscle tissue. The same was true for the kidney. But when they compared the levels in the arterial system and the hepatic vein, they found a drop of approximately 30 % at normal levels and as great as 60 % when concentrations were increased by intravenous injection of cortisone (Table XVII). While this result does TABLE XVII Levels of 1FHydroxycorticoids in Arterial and Hepatic Venous Blood of a Dog during and after Infusion with Cortisone Blood IPhydroxycorticoids Artery Hepatic vein Sample pg./100 ml. pg./IOO ml. 10 Before infusion 16 Infused 1mg. cortisone per min. 10 min. after infusion begun 76 55 17 min. after infusion begun 83 59 Infusion ended, total 25 mg. 1 min. after infusion ended 98 57 8 min. after infusion ended 90 42 22 min. after infusion ended 44 23 33 min. after infusion ended 32 10 148 min. after infusion ended 16 9

not distinguish between the liver and other viscera, the experience with other steroid hormones makes it highly probable that the metabolism is hepatic. Since the reaction used by these workers requires the presence of alcohol groups on CITand Czl as well as a ketone or alcohol group on CZo1 the disappearance could be due either to replacement of one of the oxygencontaining groups by hydrogen or to splitting off of the side chain. No analysis for metabolic products was carried out. Clark (1949) investigated the in vitro metabolism of cortisone by liver minces and liver brei. In small scale experiments he was able to demon-

INTERMEDIARY METABOLISM OF NON-BENZENOID STEROIDS

289

strate the disappearance of the ultraviolet absorption band due t o the C Y , ~unsaturated ketonic group in ring A. When large scale incubation was attempted with cortisone acetate, however, there was no destruction. Two possible reasons for this failure were apparent; first, the conditions for temperature control and oxygenation were inadequate; and second, cortical hormones do not circulate as esters. He then carried out an incubation on a large scale, using free cortisone and maintaining a more physiological environment. Approximately 50 % of the hormone could not be accounted for based on both analytical and isolation experiments. He was unable, however, to isolate any products. Paschkis et al. (1951a) incubated tissues with cortisone and then used the mouse liver-glycogen method of Venning to measure the degree of destruction. Boiled tissues were incubated with cortisone as controls. They found that 1 5 3 5 % of the glycogenic activity was lost during the period of the incubation, and that diaphragm, brain, and other tissues were as effective as liver. There was no change in the rate of destruction when tissues from stressed mice were used. As already obsel?ied, the relation of dehydroisoandrosterone to the adrenal cortex is not yet clear. If it is a secretory product of the gland, it must be secreted as rapidly as it is formed since it has not been isolated from glandular extracts. If it is a metabolic product, the precursor is unknown. If it represents leakage of an intermediate in synthesis, it must be utilized rapidly in the gland. It is certainly connected with adrenal function in some way. Its metabolism by tissues has already been considered as part of the problem of intermediary metabolism of androgens. In summary, it would appear that several steroids, both of the and the C19 groups, are secreted by the adrenals. As with other steroid hormones, they apparently undergo metabolic changes in the liver. The rate would seem to be less than with the other steroids. In addition there is some in vitro evidence of metabolism of cortisone by muscle and other tissues. The in vivo work would indicate that this is minor, however, compared with hepatic action. The changes, if the urinary steroids of similar structure are metabolic products, would seem, again, to be reductive in nature. This follows the general pattern which has been observed in the other non-benzenoid steroids. V. GENERALCONCLUSIONS From the foregoing discussion, certain generalizations seem worthy of consideration. First, the changes in the steroid hormones that lead to the relatively inactive metabolic products excreted in the urine are not primarily connected with hormonal action in the target organs; but are

290

LEO T. SAMUELS AND CHARLES D. WEST

largely, if not entirely, limited to the liver and kidneys. The liver plays by far the major role. It would appear that these processes are involved largely in regulating the blood level in relation t o varying production and need; they have, therefore, become more complex as the organism has become more independent of the external environment. The reactions involved in this metabolism of the non-benzenoid steroid hormones appear t o be largely reductive, particularly in ring A and on the side chain. Oxidative reactions involving DPN as hydrogen acceptor occur on carbon 17, and would appear to involve energy changes sufficient t o split off the side chain if both CI,and GOhave oxygen attached, but not otherwise. While reductive reactions predominate, the only position in which reduction t o a saturated hydrocarbon group has been established is in position 21. Once a diketone has been formed, as in the case of the conversion of or as in the original molecules testosterone to As-androstene-3,17-dione1 of the metabolic hormones of the adrenal cortex and progesterone, there appears to be a preferred sequence in the subsequent reductions. The double bond is reduced first, a stericly non-specific rbaction which results in the formation of both possible isomeric saturated diketones. Then the ketone in position 3 undergoes reduction. While in some over-all experiments and in the rather unphysiological incubations of Schneider and Mason small amounts of 3/3 compounds were isolated, it seems that this reduction is stericly directed to the trans or a structure. The ketone group a t the other end of the molecule (C17or Cz0)is the last to be reduced t o an alcohol. Again, as one might expect from a multiple attachment system, only one steric configuration seems to be formed: in the case of the 17-ketosteroidsJ the 17,/3-alcohols, and in the case of the C ~ketones, O the a configuration. The ease of conjugation with glucuronic or sulfuric acids seems to determine what metabolites of a given hormone will appear in the urine in largest quantity. Whereas the unconjugated steroid can form complexes with the serum proteins, particularly albumin, the conjugates are freely filtered through the glomerulus and thus are removed from the metabolic pool. The reasons for the difference in routes of excretion between some of the lower mammals and the human being need investigation, but it may lie in the relative facility with which the steroids are conjugated in the liver. The small amounts of A6-3P-hydroxy compounds that occur in the urine are thought t o be intermediates in synthesis that have found their way into the blood stream. When they are administered they always result in the excretion of the two possible isomeric 3a saturated compounds. This would require an intermediate oxidation of the alcohol

INTERMEDIARY METABOLISM OF NON-BENZENOID STEROIDS

291

group on carbon 3. Yet no such enzyme has been found in the liver or kidney, while very active enzymes of this type are found in all the endocrine tissue so far tested (Samuels et al., 1951). The oxidation may, therefore, take place in these tissues. It seems doubtful that the A6-3/3hydroxy compounds represent steps in the degradative metabolism of the active hormones. While some of the enzymes of the liver and kidneys seem to be relatively unspecific regarding steroid structure, other than that it be nonbenzenoid and the oxygen be present on both ends of the molecule, others, such as those reducing ring A, are specific at least between the CI9and steroids. Thus the enzyme reducing ring A in progesterone does not require oxygen while that reducing this ring in testosterone does; further, the two hormones seem not to show competitive inhibition. The metabolic systems of the non-benzenoid steroid hormones, therefore, while having certain general characteristics in common, are really complex. REFERENCES

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Munson, P. L., Gallagher, T. F., and Koch, F. C. 1944. J. Biol. Chem. 162, 67-77. Nelson, D. H., Samuels, L. T., Willardson, D. G., and Tyler, F. B. 1951. J . Clin. Endocrinol. 11, 1021-1029. Noall, M. W., Salhanick, H. A., and Zarrow, M. X. 1952. Federation Proc. 11,265. Paschkis, K. E., Cantarow, A., and Boyle, D. 1951a. Federation Proc. 10, 101. Paschkis, K. E., Cantarow, A., and Havens, W. P., Jr. 1951b. Federation Proc. 10, 101-102.

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Samuels, L. T., Sweat, M. L., Levedahl, B. H., Pottner, M. M., and Helmreich, M. L. 1950. J . Biol. Chem. 183, 231-239. Schiller, S., Dorfman, R. I., and Miller, M. 1945. Endocrinology 36, 355-363. Schneider, J. J. 1950a. Federation Proc. 9, 224-225. Schneider, J. J. 1950b. J. Biol. Chem. 183, 365-376. Schneider, J. J. 1950c. Science 111, 61. Schneider, J. J., and Mason, H. L. 1948a. J . Biol. Chem. 172, 771-782. Schneider, J. J., and Mason, H. L. 1948b. J . Biol. Chem. 176, 231-240. Selye, H. 1941. J. Pharmacol. Exptl. Therap. 71, 236-238. Slotta, K. H., Ruschig, H., and Fels, E. 1934. Ber. 67, 1270-1273. Sommerville, I. F., and Bigler, R. 1951. Unpublished work. Sommerville, I. F., and Marrian, G. F. 1950a. Biochem. J. 46, 285-289. Sommerville, I. F., and Marrian, G. F. 1950b. Biochem. J. 46, 290-296. Sprague, R. G., Gastineau, C. F., Mason, H. L., and Power, M. H. 1948. Am. J. Med. 4, 175-185. Sprague, R. G., Power, M. H., Mason, H. L., Albert, A., Mathieson, D. R., Hench, P. S., Kendall, E. C., Slocumb, C. H., and Polley, H. F. 1950. Arch. Internal Med. 86, 199-258. Sprague, R. G., Power, M. H., Mason, H. L., and Cluxton, H. E. 1949. J . Clin. Invest. 28, 812. Sweat, M. L., Samuels, L. T., and Lumry, R. 1950. J . Biol. Chem. 186, 75-84.

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The Influence of Corticoids on Enzymes of Carbohydrate Metabolism BY

F. VERZAR

The Physiological Laboratory, University of Basel, Switzerland CONTENTS

I. Introduction.. . .

............

........................ .........................

1. Blood Sugar.. ....

111.

IV.

V. VI. VII. VIII. IX. X. XI.

........................

Page 297 298 298

3. Gluconeogenesis from Protein. .................................... 300 4. Activities of Different Corticoids in Carbohydrate Metabolism.. ...... 300 5. Indirect Evidence on the Role of Phosphorylating Enzymes.. ........ 301 Experiments with Isolated Organs.. .................... 303 .................... 303 1. Glycogen Production after Adr 2. The Action of Corticoids.. ...... ............................ 305 3. Isolated Liver and Eviscerated Experiments.. . . . . . . . . . . . . . . . 307 4. Working Hypotheses for the Inhibitory Action of Corticoids on Glyco308 gen Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................................ 308 iration.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308 c. Competitive Inhibition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309 d. Actual . . . . . . . . . . . . .... . . . 309 . . . . . . . . . . . . . . . 309 Experiments on Enzymes. ............................. 309 1. Phosphorylase of Musc 2. Phosphorylaae of Liver.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Alkaline Phosphatase of Intestine, Kidney, and Other 0 4. Acid Phosphatase.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Histochemical Demonstration of Alkaline Phosphatase.. . . . 1. Intestine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316 2. Kidney . . . . . . . . . . . . . . . . . .................................. 317 Phosphoglucomutase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318 Hexokinase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319 Oxidsse Systems.. . . . . . . . . . . . . . . . . . . . . . . . . . . . Protein Enzymes.. .......................... Reactions between Carbohydrate and Potassium Metabolism in vitro.. . . 321 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 References.. .............. .................... 326

I. INTRODUCTION For a long time carbohydrate metabolism seemed to contain the key to the action of the adrenocortical hormones in cell metabolism. The 297

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VERZAR

present situation may be described briefly as follows: the action of the adrenal cortex is related to the central process of energy production of the cell, which is the degradation or transformation of glucose. How far this connection can be analyzed on the basis of the study of cell enzymes is the problem of this review.

11. GENERALCARBOHYDRATE METABOLISM It might be well first to recall some facts about the carbohydrate metabolism of the otherwise intact adrenalectomized animal, which proved to be the guiding lines for a more analytical approach.

i. Blood Sugar One of the longest-known facts in adrenal physiology is the decrease of resting blosd sugar after adrenalectomy or in Addison's disease, especially during crisis. This has been described in adrenalectomized dogs (Bierry and Malloizel, 1908; Porges, 1909; Rogoff and Stewart, 19?6), in cats (Swingle, 1927; Hartmann et al., 1927); and also in rats, monkeys, etc. (Britton et al., 1932, 1938). Adrenocortical extracts restored the blood sugar (Britton and Silvette, 1932) and adrenalectomized cats were kept alive with a normal blood sugar by desoxycorticosterone for months and years (Verzgr et al., 1941). For some time this was taken t o be one of the most characteristic signs of adrenocortical insufficiency. Kendall (1938) emphasized that adrenalectomized dogs kept alive with NaCl showed no decrease in blood sugar. Muscular work decreases the blood sugar of adrenalectomized animals considerably, even in an otherwise relatively well compensated state (after transplantation, Csik, 1930). Animals kept alive with sodium salts cannot stand heavy muscular work, or a fast, without serious hypoglycemia (Buell et al., 1938; Long et al., 1940, p. 342) which differs considerably from the hypoglycemia after insulin in that glucose injections have only very temporary effects and do not prolong life (Britton, 1938). Also the work performance of adrenalectomized rats shows only a temporary improvement after a glucose injection, and even continuous glucose infusion does not greatly increase the work performance (Ingle and Nezamis, 1948; Ingle et al., 1951). The low blood sugar thus seems to be only a secondary, and by itself a minor, part of the picture of adrenocortical insufficiency. 2. Glycogen Formation

Impaired glycogen formation was then thought to be the chief disturbance after adrenalectomy. It had also been known for a long time that adrenalectomized animals have low glycogen values in the liver and in

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299

the muscles, especially after a 24-hour fast. On this basis Britton and Silvette (1932b) advanced the theory that the carbohydrate metabolic disturbances are the chief metabolic changes after adrenalectomy. They, and others (Long et al., 1940; Holmes and Lehmann, 1940), were able to restore liver and muscle glycogen with adrenocortical extracts. Methods were worked out for the assay of the adrenocortical hormones based on glycogen formation in the liver (Reinecke and Iiendall, 1943; Olson et at., 1944), and the corticoids were differentiated as “carbohydrate active” if they increased the glycogen content of the liver in a 6-hour test in the rat or mouse. It was, however, shown on cats (Montigel and Verztir, 1943) and on rats (Wang et al., 1949) that desoxycorticosterone, which is inactive in short period tests, restores glycogen depots if given over a longer period. We shall refer later to certain differences of activity. While adrenalectomy decreases glycogen stores (especially in the fasting animal), it was shown by Evans (1936) that at a low oxygen pressure the glycogen content of the liver remains relatively high in normal, but not in adrenalectomized animals. In normal animals the adrenal cortex hypertrophies, and it was concluded that the greater glycogen storage is the result of increased adrenocortical activity. In confirmation of this, Fitzgerald (1938) obtained the following mean values for the liver glycogen contents of intact and adrenalectomized rats: intact, unstarved, 3.825%; intact, starved for 24 hr. at 760 mm., 0.147%; intact, starved for 21 hr. at 380 mm., 0.776%; adrenalectomized, starved for 24 hr., 0.044 % ; adrenalectomized, starved for 24 hr. at 380 mm., 0.087%. Similar observations were reported by Langley and Clarke (1942). A decrease in glycogen production after adrenalectomy from lactic acid (Nitzescu and Benetato, 1932; Britton and Silvette, 1934; Gobell, 1941) and from pyruvic acid (Fitzgerald, 1938; Buell et al., 1938; Keyes and Kelley, 1949) has been reported and this could be restored again by adrenocortical extracts. Since it was agreed that normal glycogen production is only possible in the presence of the hormones of the adrenal cortex, it was thought that this might be connected with an antagonism to insulin. The hyperglycemia and glycosuria of pancreatic diabetes do not occur in adrenalectomized animals, but can be restored with adrenocortical extracts or corticoids, as Hartmann and Brownell (1934) and Long and Lukens (1936) have shown. Such diabetogenic activity was found by Ingle (1941), not only with adrenocortical extracts, corticosterone and cortisone, but also with estradiol, diethylstilbestrol, and testosterone. Less pronounced diabetogenic activity was found also with desoxycorticosterone by Ingle (1948) using force-fed animals; while Wick (1949) found it to be

300

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inactive in this respect. The glucogenic activity of adrenocortical hormone is also found in the hypophysectomized animal, and the action must therefore be a direct one upon the tissues (Ingle, 1944).

3. Gluconeogenesis from Protein The work of Long et al. (1936) proved definitely that the nitrogen excretion of pancreatectomized animals shows a fall, parallel to the fall in glucose excretion, after adrenalectomy. Cortical extracts restored the glucose and nitrogen excretion, and the explanation was given that cortical hormone stimulates glucose production from protein. This led to the belief that the main action of the corticoids on carbohydrate metabolism is upon the glucose production from protein (see the review by Kendall, 1948). Long e2 al. (1940) compared the glycogen increase and the increase of N excretion after adrenocortical extract in normal, and Sprague (1940) in pancreatectomized, animals. However, Ingle and Thorn (1941) called attention to the fact that the increased N excretion after treatment with cortical hormone or ACTH is insufficient to account for the increase in glycogen, and on this basis Ingle (1942, 1949) stated that gluconeogenesis from protein is not the sole point of action of the corticoids in carbohydrate metabolism. Conclusive evidence was given by experiments on glucose production from amino acids. Engel et al. (1949) showed that the protein breakdown caused by the injection of an adrenocortical extract is completely suppressed if either glucose or a mixture of essential amino acids was simultaneously given by intravenous injection. Thus the adrenocortical hormone has no direct stimulatory action on the deamination of amino acids. It increases protein breakdown and glucose production from protein only if no other source of glucose is available. (Toxic doses of cortisone may act differently.)

4. Activities of Diferent Corticoids in Carbohydrate Metabolism The problem of whether different corticoids have different actions on carbohydrate metabolism is of importance from the point of view of later work.. Most of the experiments mentioned on glucose formation were made either with adrenocortical extracts or the 1 l-oxygenated corticoids. The latter only, showed stimulation of glycogen formation in the 6 hour tests of Reinecke and Olsen (see above). We called attention to the fact (Verzk et al., 1941; Montigel and VerzAr, 1943; Sass-Kortslk et al., 1949; Wang et al., 1949) that desoxycorticosterone also has a similar carbohydrate metabolic activity when given over a longer period. One group of adrenalectomized rats was kept on a mixed diet, and the animals were treated with desoxycorticosterone or with cortisone (1l-de-

CORTICOIDS AND ENZYMES OF CARBOHYDRATE METABOLISM

301

h y dr 0-17-h ydr oxycorticoster one). Five days after adrenalect omy the liver glycogen of untreated animals wasO.15% and muscle glycogen 0.34%. With daily doses of 2 mg. desoxycorticosterone for 10 to 20 days the values became 4.8%in liver, 0.44%in muscle; i.e., as high as in normal controls (4.8% in liver, 0.42% in muscle). In a second group the adrenalectomized animals were kept on a protein diet (meat) for a long period. After 15 days’ treatment with desoxycorticosterone, about as much glycogen was produced in the liver and muscle as after treatment with cortisone. In a third series, the animals were fasted for 24 hours on the 5th day after adrenalectomy. Then 1 g. glucose was given by stomach tube (50% solution), and 3 hours later liver and muscle glycogen was assayed. These were, as Table I shows, low in the untreated, but high in those animals which were treated either with desoxycorticosterone or with cortisone (Wang, 1950). TABLE I Glycogen Content after 1 g. Glucose Ingestion (Figures in parentheses denote the number of animals.) Glycogen % Liver Muscle Normal (5) Adrenalectomized: Untreated (5) Cortisone (0.5-2 mg./day for 3 days) (3) Cortisone (1 mg./day for 15 days) (3) Desoxycorticosterone (1 mg./day for 15 days) (2)

3.7-5.7

0.45-0.55

0.9-1.4 4.75-7 .O 2.8-5.0 3.3-4.7

0.37-0.45 0.56-0.59 0.44-0.69 0.47-0.55

All three series show that both cortisone and desoxycorticosterone have glycogenic activity, although the latter acts more slowly and only if the animals are continuously treated with it. The third series proves further that if glucose is given as substrate, glycogen production is also enhanced by these corticoids. Thus glycogenesis from glucose as well as from protein is stimulated. 5 . Indirect Evidence on the Role of Phosphorylating Enzymes

Summarizing the conclusions from the experimental evidence on the adrenalectomized, but otherwise intact animal, it may be said that the point at which the corticoids act is neither on blood sugar regulation, nor on glycogen production, whether in general or from protein. General experience points to some fundamental process in which the corticoids influence the utilization of glucose as a main source of energy production.

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F. V E R Z ~ R

An attempt was made originally (1933) to use the specific function of a single epithelial layer as the basis of further analyses. The absorbing epithelium of the small intestine is such a structure. This epithelium selectively absorbs glucose and galactose faster than other sugars, and it was suggested (VerzAr and McDougall, 1936) that this selective absorption was due to phosphorylation of the rapidly absorbed sugars, since poisons known to inhibit phosphorylation were also known to inhibit selective sugar absorption (Wilbrandt and Laszt, 1933; Lundsgaard, 1933), and since absorbed sugars had been detected in the epithelium as phosphoric esters (Laszt and Sullmann, 1935; Kjerulf-Jensen, 1942; Beck, 1942). The "phosphorylation theory " of selective sugar absorption was based on experiments with iodoacetate or phlorizin poisoning. According to Shapiro (1947) and Work and Work (1948) these substances, a t low concentration, inhibit the dehydrogenase systems that supply energy-rich phosphate bonds. Oxidoreductive phosphorylation might therefore be inhibited on the oxidoreductive side of the reaction. If the phosphate donor is not synthesized, phosphorylation itself becomes impossible. The specific inhibition of glucose and galactose absorption from the intestine has recently been analyzed by Bruckner (1951). He found that M inhibited completely phlorizin even a t a concentration of to the selective absorption of glucose, while atebrin and 2,4-dinitrophenol1 which inhibit oxidoreduction primarily (Loomis and Lipmann, 1948), showed no characteristic activity in similar concentrations. It is possible, and no contradiction, to suppose that phosphorylation might primarily be inhibited by the absence of the phosphate donors, but it is most clearly inhibited by phlorizin in otherwise nontoxic concentrations. Considerable support for this phosphorylation theory of selective absorption was provided when it was shown that the intestinal epithelium is the richest source of alkaline phosphatase in the body, and histochemical analysis showed that its highest concentration is in and next to the striated border of the epithelial cells. Further support to the theory is lent by the fact that the only other single epithelial layer with a similar epithelium with a striated border is in the kidney tubules, which also have a glucose reabsorbing activity (Lundsgaard, 1933), and this layer also contains large amounts of alkaline phosphatase, as can be shown chemically and histochemically. The activity of this epithelium is similarly inhibited by phlorizin, as indicated by the production of glycosuria. As shown by Cori and Cori (1927), Wilbrandt and Lengyel (1933) and more recently by Staehelin (1946), the rate of absorption of glucose is diminished after adrenalectomy to that of a non-selectively absorbed

CORTICOIDS AND ENZYMES

OF CARBOHYDRATE METABOLISM

303

sugar, such as xylose. While Deuel et al. (1937) and Marazzi (1940) were unable to obtain this result in adrenalectomized rats maintained on 1% NaC1, the observation was independently confirmed by Lundsgaard (1933) and by Soulairac (1946). Recently we confirmed it also on adrenalectomised rats on 1% NaCl after more than 50 days survival. (VeraBr and Sailer, 195213.) This problem cannot be discussed at length here. However, to summarize, it may be said that if phosphorylation is involved in the selective absorption of glucose and galactose, then the similar effects upon this selective absorption of iodoacetate and phlorizin on the one hand and of adrenalectomy on the other, suggest as a working hypothesis that the adrenals have some role in phosphorylating mechanisms. 111. EXPERIMENTS WITH ISOLATED ORGANS 1. Glycogen Production after Adrenalectomy

An attempt to identify the point of action has been made using the technique of the isolated muscle. The diaphragm of the rat was introduced by Gemmill (1940) for this type of research, and has proved to be an exceptionally good object because it is thin enough to allow quick diffusion into the undamaged tissue. From glucose (100 to 400 mg. per 100 ml.) at 38°C. in oxygenated Ringer’s solution (modified), glycogen is produced, and is much increased in amount by even one unit of insulin (VerzBr and Wenner, 1948a). Koepf et al. (1941) have shown that the diaphragm of the adrenalectomized rat also synthesizes glycogen from glucose in about the same quantities as the diaphragm of the normal animal, and they concluded that glycogen production from glucose is not the main disturbance aft.er adrenalectomy. This was confirmed by us (Verzdr and Wenner, 1947, 1948), but Mentha et al. (1948) in our laboratory showed in a large series of experiments that there is a small but statistically significant decrease in glycogen production after adrenalectomy. We have also used the diaphragm preparation to study the influence of work on glycogen metabolism. The diaphragm was divided into three parts: one served as control for the initial glycogen value, one for glycogen production during rest, and one for glycogen production during work. Direct stimulation with short tetani of 2 sec. (50 shock/sec.) was used. Work was measured by mechanical registration and summarization. The experiments were made on normal as well as on adrenalectomized rats. The glycogen content of the diaphragms of 139 normal rats (fasted 18 hours) was 365 mg. %, that of 64 adrenalectomized rats (5 days after adrenalectomy, untreated) 219 mg. %. There is a diminished carbohydrate metabolism in every phase of activity as shown in Table 11. In

304

F. V E R Z ~ R

TABLE I1 Glycogenesis, Glycogenolysis and Glycogen Consumption in Resting and Working Rat Diaphragm Normal

Adrenalectomieed

-

-

__

Glycogen mg. %

No. Change

con- Wor

sumed cm. dur-

lestWork hr.

Ringer solution With Glucose 100 mg. % With Glucose 400 mg. 5% With Glucose 100 $ 1 U. Insulin With Glucose 400 mg. % 1 U. Insulin

+

V __

mals

:t:k

-85 -245 +35 -145 $80 -110

160 180

-170 -90 -365 +I0

No. Vorl of :m.2 ani-

animals

--- I I1 I11 IV

I

of

---

190

27 32 41

25 23 10

- 4 0 -145 +45 -50 $60 -35

860

36

11

+90

38 _ _ $66 _~16 4 1 2 3 5 -

I I -40

-

106 96 96

22 36 28

10

150

32

6

28

11

9

10

In columns 4 and Q work is given in arbitrary units (crn.2 of registered work curve)

8 5

- __

-

Ringer’s solution without glucose (series I) glycogenolysis occurs during rest (columns 1 and 6) and is greatly increased if the muscle is stimulated (columns 2 and 7). If glucose is added (series I1 and 111) glycogen is produced during rest. If a t the same time the muscle is working, the glycogen is again broken down. This is even more obvious if insulin is also added (series IV and V). From the difference between the glycogen stored during rest and work, the glycogen used for work is calculated (columns 3 and 8). (The work performed was calculated in arbitrary units which are only of comparative value and are given in columns 4 and 9.) All glycogen values are significant, calculated for p and t values. The calculation of glycogen consumption is only an assumption. In all experimental conditions the diaphragms of the adrenalectomized animals produce or use less glycogen than the normal, the decrease being 33-50%. Also work (with one exception) was less after adrenalectomy. Thus, while it is true that the muscle produces glycogen also in adrenalectomized animals and also uses it for work, this is less than in normals in all groups. The diminished glycogen content of the diaphragm of adrenalectomized rats, and the decreased glycogen production from glucose, have also been described by Villee and Hastings (1949). They worked with

CORTICOIDS AND ENZYMES OF CARBOHYDRATE METABOLISM

305

glucose labelled with C14at C-3 and C-4, or labelled a t all C atoms. They explained their experiments by the theory, that both hexokinase activity and glucose oxidation are disturbed after adrenalectomy. VerzAr and Wenner (1948~)showed that the diaphragm is unable to produce glycogen from glucose-l-phosphate. Minced muscle (and liver, see Section 3 below), both from normal and from adrenalectomized rats, produces glycogen from glucose-l-phosphate in vitro. The inability of whole muscle to use glucose-l-phosphate is therefore caused by the inabilitly of this substance to diffuse into the muscle (Gemmill, 1941; Koepf et al., 1942) and obviously glucose-l-phosphate is produced inside the muscle fiber. 2. The Action of Corticoids

The action of corticoids on the glycogen metabolism of isolated diaphragm was studied by Verz&rand Wenner (1947, 1948a). Desoxycorticosterone, in concentrations of 5 to 10 mg./100 ml. of Ringer’s solution, completely inhibited glycogen formation from glucose with insulin, both in normal and adrenalectomized diaphragm. * This was unexpected since it seems contrary to the action which the corticoids are known t o have in the intact animal. The inhibition did not depend upon the presence of insulin, and thus is not simply an antagonism between corticoid and insulin. Mentha et al. (1948) confirmed this inhibition for the resting, and also for the working muscle. Desoxycorticosterone (5 mg./100 ml.) inhibits glycogen synthesis as well as glycogenolysis during rest and work, as the table on p. 306 shows. We thus came to the conclusion that desoxycorticosterone inhibits every phase of glycogen metabolism in the muscle, during rest and work, in both normal and adrenalectomized animals. We then repeated the experiment (Bosovic et al., 1949) using the muscles of the anterior abdominal wall of the mouse (it was not used for work experiments). An inhibitory action of 5 mg./100 ml. desoxycorticosterone on glycogen production was again seen. It was also shown that adrenalin (0.05 mg./100 ml.) increases glycogen breakdown, and this effect is summated with the inhibitory, or rather glycogenolytic, effect of the corticoids. It was concluded that desoxycorticosterone increases the glycolysis rather than the glycogenesis. This agrees with the observation that phosphorylase activity is increased by desoxycorticosterone, as will be shown on p. 311, a conclusion which was reached also by Schumann (1940).

* The effective concentration is probably lower in these and later experiments, since the solubility of DOC is even less according to Hayano et al. (1950b).

W 0

m

TABLE I11 Influence of Desozycorticosterone (6 mg. %) on Glywgen Consumption of Isolated Diaphragm Adrenaiectomiaed

Normal

I

Glycogen mg. % Series

1 1

Change

Solution

1

Rest

I

Work

ConBumption du& work

1 1

Work cm.* Rest

- DOC - DOC - DOC - DOC - - - ~

VI VII VIII

-85 -45 -245 -140 160 95 27 Ringer's With Glucose 100 mg % t +170 +15 -90 -115 260 I30 36 1 U.Insulin With Glucose 400 mg % 3 4-365 +165 +10 -25 355 f90 38 1 U. Insulin 1

2

3

4

5

6

7

-

DOC

work -

DOC

1

sumDtion during work

-

-40 -40 -145 -117 106

34

+90

35 8

1

r -4 M

DOC - DOC

-.__

26

~

Column. . . . . . . . . . . . . . . . . . . . . . .

I

Glycogen mg. %

77

22 39?

+20

-40

-60 1SO

80

32 34

+290 +65

4-80

-10 210

76

28 30

11

12

14

15

-9

lo

13

The data in columns 1. 3. 5. 7.9, 11. 13, 15 are identical with the corresponding data in Table I1 (with different column numbers). are also the same aa in Table 11. Other remarks nee under Table 11.

16

Number of animals

Emi

CORTICOIDS AND ENZYMES OF CARBOHYDRATE METABOLISM

307

The isolated diaphragm or the abdominal muscle takes up from Ringer’s solution about twice as much glucose as the quantity of glycogen produced in the same period (Krahl and Park, 1948; Perlmutter and Greep, 1948). Desoxycorticosterone also inhibits glucose uptake, as was shown with Leupin (1950a). In experiments with 5 to 10 mg desoxycorticosterone in solution with 200 mg. glucose and one unit insulin per 100 ml., the glycogen production of the rat diaphragm decreased from 272 to 56 mg. per 100 g. muscle. The glucose uptake decreased from 457 to 200 mg. Generally only about one half of the glucose uptake was inhibited: i.e., the quantity which would have been transformed to glycogen. This was seen also by Bartlett and MacKay (1949) in experiments with C1* labelled glucose. One might conclude that it is the glycogen metabolism which is inhibited, while the other part of the glucose may be used for basal metabolic purposes. The later experiments were combined with respiratory metabolic assay to investigate the fate of the consumed glucose. No change in glucose oxidation was found. The inhibitory action shown by desoxycorticosterone on glycogen formation is also exerted by other steroids (VerzBr and Wenner, 1947). Leupin, Voegtli and Verzar (1949) observed it with cortisone, though to a lesser extent than with desoxycorticosterone, while adrenocortical extract (Upjohn) had no inhibitory action. 3. Isolated Liver and Eviscerated Animal Experiments

The inhibition of glycogen metabolism by corticoids seems to occur in the liver also. It was found by Seckel (1940) that adrenocortical extract inhibits the glycogenolysis in liver slices of rats. He explained this as a direct action on cellular enzymes and compared his results wit.h those of Willstatter and Rohdewald (1936) and Przylecki (1935). With liver slices from rats and rabbits Chiu (1950) and Chiu and Needham (1950) observed an increase of “total carbohydrate ” formation from lactate, pyruvate, glucose, and alanine after the addition of adrenocortical extract or desoxycorticosterone. The results were conclusive only in certain cases. The inhibitory action of corticoids was also observed by Ingle (1948, 1949) on eviscerated and nephrectomized preparations of normal and adrenalectomized rats. A continuous intravenous glucose infusion was given. The addition of adrenocortical extract inhibited the rate of disappearance of glucose, and hyperglycemia developed. Ingle called this inhibition an “extrahepatic effect of cortin.” It is possible that a similar explanation can be given to the following observations of Ingle (1949) : The glucose excretion of depancreatized rats was decreased on treatment for several weeks with 1 and 2 mg. of desoxycorticosterone per day, but was

308

F.

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increased when the dose was raised to 10 mg. per day. This is interesting since it proves, firstly, an action of desoxycorticosterone on carbohydrate metabolism, and secondly, the opposite effects of small and large doses. It seems possible that the small doses may have produced their effect on the liver, while the large doses may have acted through extrahepatic tissues. Finally we should mention that an inhibitory action on carbohydrate metabolism can be seen in the intact normal animal also with large doses of desoxycorticosterone (10 to 20 mg./100 g.), which have an anesthetic effect on the rat (Selye, 1941a,b). Verzfir and Wang (1950) showed in such animals an inhibition of glycogen production in the liver and muscles with desoxycorticosterone, but cortisone was less active. Cholesterol, which has no anesthetic action, was found to be inactive.

4. Working Hypotheses for the Inhibitory Action of Corticoids on Glycogen Formation The inhibition of glycogen production by corticoids in isolated muscle and in the other cases mentioned above, might be explained in several different ways: a. Non-specific. The inhibition of glycogen production has been ascribed to a “ non-specific steroid reaction.” This supposition is based mainly on the fact, mentioned above, that different steroid hormones and related compounds have a similar action. However, cholesterol and other steroids have no such action. It seems possible that the cell metabolic activity of different non-corticoid steroids is similar. The difference in their activities may be due to the fact that in biological concentrations they act specifically on different organs, while basically there might be a similarity of action on cell enzymes. It is impressive that stilbesterol also has such inhibitory action, while the biologically inactive cholesterol has none (Verzfir and Wenner, 1948~). The question may also be raised as to the meaning of the term “nonspecific.” We considered solubility, surface activity and adsorption as explanations, but rejected them all. A simple mechanical covering of the tissue by the steroid, which would inhibit diffusion, does not seem plausible. b. Inhibition of Respiration. A fact which does not agree with this theory is the observation that the same corticoids also have an inhibitory action on the respiratory metabolism of brain and liver tissue (Tipton, 1939; Mune, 1935; Kaunitz and Selzer, 1938; Gordan and Elliott, 1947). An inhibition of the dehydrogenase enzyme system has been supposed, and it should be mentioned here that Hitchcock el aE. (1938) found that adrenal cortical extract reduced the oxygen consumption in muscular

CORTICOIDS AND ENZYMES OF CARBOHYDRATE METABOLISM

309

work (confirmed by Missuro et al., 1938). It seems probable that the inhibitory action of corticoids on respiratory metabolism is functionally related to their inhibition of glycogen metabolism. c. Competitive Inhibition. Another hypothesis is that the action of desoxycorticosterone etc. in vitro might be one of “competitive inhibition.” This supposition could only be maintained if a stimulatory action could be seen with small doses that would be reversed by large doses. It was not possible to prove this on isolated muscle, but it has been shown in the intact animal and on Ingle’s depancreatized, eviscerated preparation. d . Actual. Finally there is the experimental fact, that under certain circumstances (i.e. on isolated muscle) corticoids inhibit glycogen metabolism, while in the intact (or adrenalectomized) animal they stimulate it. It will be shown in the next chapter that phosphorylase activity is increased by desoxycorticosterone in the minced muscle i n vitro. Such an increase of phosphorylase means an increased glycogen breakdown, and if this occurs in the isolated diaphragm it will also give the observed effect of inhibition of glycogen production or rather of an “increase of glycogenolysis.” We do not think that anything more definite can be said at present. IV. EXPERIMENTS ON ENZYMES 1 . Phosphorylase of Muscle Experiments with minced tissues (“brei”) were made to test our working hypothesis that certain phosphorylating enzymes are influenced by the adrenal cortex. Up till then we had had only indirect evidence obtained from experiments on the function of a single epithelial layer (see p. 301); we therefore started to work on phosphorylase. Phosphorylase adds inorganic phosphate to glycogen, producing glucose-l-phosphate as the first product. of glycogenolysis. Schumann (1940) first showed that, after adrenalectomy, phosphorylase activity, i.e. the phosphorylation of glycogen by minced heart and skeletal muscle, was decreased. Montigel and VerzBr in a series of papers (1942, 1943) used the same method (Lohman’s procedure), as follows: 1% glycogen is added to minced muscle of rats, in Ringer’s solution, buffered a t pH 8.2. Addition of NaF inhibits the breakdown of the hexose-phosphate. The decrease of inorganic phosphate in the solution is measured after 15, 30, and 45 minutes. Since phosphorylation is accelerated a t 38”C., the experiments were performed a t 2OoC., where the differences could be demonstrated. The following figures are from a later paper of Montigel (1945, p. 47). (Table IV.)

310

F. V E R Z ~ R

TABLE IV Glycogen Phosphorylation with Minced Muscle

Series Minced muscle 1

2 3 4 5 6

Number of animals

Normal Adrenalectomized Adrenalectomized Adrenalectomized Adrenalectomized Adrenalectomized

% P decrease after: 30 min. 15 min.

Addition

-

9 34 30

0 . 1% DOC 2 % Progesterone 2 % Testosterone 2 % Estradiol

6

7 4

52.6 33.5 46.4 48.1 28.4 37.8

67.6 50.1 62.9 59.7 46.5 45.8

value for series 2: 3 was 7.1 for 15 min.. 7.6 for 30 min. Limit of significance 3.1. Addition: 0.1 ml of 0.1 % DOC Bolution per 0.5 g. muscle, or of 2 % testosterone etc.

f

1

%P

I

701

3

€4

i!E 20 10

10

20

30

40

Minutes

50

60

Minutes

FIG.1. Phosphorylase activity. Minced muscle of rats at 20°C. 1. Normal, 0 ;with 1 % DOC, in vitro X (No. 1098). 2. Adrenalectomized DOC treated, 0 ; with 1% DOC, in vitro X (No. 1). 3. Adrenalectomized adynamic, 0 ; with 0.1 % DOC in vitro, X (No. 1059). 4. Adrenalectomized adynamic, 0 ; with 0.1% DOC in uitro, X (No. 30). (After Montigel and Verzh, Helv. Physiol. Acta 1, 115, 1943.)

CORTICOIDS AND ENZYMES OF CARBOHYDRATE METABOLISM

31 1

The decrease of inorganic phosphate, i.e. the phosphorylation of glycogen, was 52.6% after 15 min. in normal and 33.5% in adrenalectomized animals. The values show a tendency to approach each other as time passes. As Fig. 1 show^, the difference between normal and adrenalectomized rat’s muscle is one of velocity, and ultimately similar values can be obtained. This may be the explanation of unsuccessful experiments of Helve (1943) and of Riesser (1945) and his pupil Smits (1945). Table V shows similar experiments of two other working groups: A similar decrease of glycogen phosphorylation was found in musTABLE V Glycogen Phosphorylation with Minced Muscle Minced muscle of rats Normal Adrenalectomized Normal Adrenalectomized

*

Author* Doetsch (1944) Doetsch (1944) Staehelin and Voegtli (1948) Staehelin and Voegtli (1948)

-

Number of animals

Per cent decrease of inorg. P after: 15 min. 30 min.

32 57 20

41.6 28.4 45 f 1 . 8

59.4 44.1 65 f 1 . 7

65

31 f0.9

52 5 1.0

-

For the difference between normal and adrenalectomized Doeteeh gives f 4.1 (15 rmn.), 4.5 (30 min.); Staehelin and Voegtli give f 7.0 and 6.6. The limit of significance is 3.1.

cles of eight adrenalectomized cats and two adrenalectomized dogs (Montigel and VerzAr, 1943). Staehelin and Voegtli (1948) made the following experiment: three adrenalectomized cats were kept alive by daily treatment with 5 mg. of desoxycorticosterone acetate. A piece of muscle was extirpated and tested for phosphorylase activity. The wound healed, and after a certain time the animals were brought into a crisis (adynamic) by discontinuing the desoxycorticosterone treatment. The phosphorylase activity of muscle was now significantly decreased. In cat No. I V first a piece of muscle was extirpated during a state of insufficiency, and the phosphorylation was compared with that of the same animal’s muscle after complete compensation with desoxycorticosterone acetate. The decreased phosphorylation during insufficiency became normal again after compensation. These experiments are shown in Fig. 2. Table I V shows also that desoxycorticosterone, the only available corticoid at that time, restored the decreased phosphorylation of the adrenalectomized animal’s muscle in vitro in quantities of 0.1 mg.% (see also Fig. 1). Progesterone acted in a similar way, but in 20 times higher concentration. No increase was seen in the phosphorylase activity of normal muscle.

F.

312

60

VERZLR

-

I1 60

I11

-

0

15

7

30

0

30

15

7

Mwter

Mlnutes

FIQ.2. Phosphorylase activity. Per cent P decrease in solution. The leg muscles of four adrenalectomized cats were used at different times. Ady-adynamic. (After Staehelin and Voegtli, Helu. Physiol. A d a 6, 317, 1948.)

%rFb-t a0

40

/----

20

/

n

J

15

/

30 0

7

Minutes

15 MLnUtet

M 0

7

$5

30

Mmlrta

FIG.3. Phosporylase activity. % P: decrease of inorganic P. A . Muscle; , 7 normal, and 7 adrenalectomized rats. B. Liver; , 7 normal, and 7 adrenalectomized rats. C. Muscle; ___ , 6 adynamic-rats, and ---*-; 6 adynamic rats with 120 mg. % cysteine. (After Staehelin and Voegtli, Helu. Physiol. Acta 6, 317, 1948.) -0-*-,

-0-*-,

CORTICOIDS AND ENZYMES OF CARBOHYDRATE METABOLISM

313

Cysteine and glutathione also increase the phosphorylase activity of adrenalectomized muscle (Fig. 3). This is of special interest since Williams and Watson (1940) found the same for kidney and bone phosphatase, and Cori et al. (1943) found that these substances also stimulated purified phosphorylase. 2. Phosphorylase of Liver The same method which was used for muscle was applied by Ostern et al. (1939) for liver phosphorylase, and was then used by us, with the difference that the NaF concentration was increased. After adrenalectomy a decrease in phosphorylase activity similar to that in muscle was observed by two independent working groups (see Table VI). TABLE VI Minced liver of rats Normal Adrenalectomized Normal Adrenalectomized

Decrease of inorg. P after: 15 min. 30 min.

Author Doetsch (1944) Doetsch (1944) Staehelin and Voegtli (1948) Staehelin and Voegtli (1948)

2 6 . 2 (18)* 7 . 8 (16) 22 (39) 12 (24)

3 3 . 8 (18) 15.0 (16) 35 (39) 24 (24)

* Figures in parentheam denote numbera of animals. 3. Alkaline Phosphatuse of Intestine, Kidney and Other Organs

Alkaline phosphatases, and especially alkaline phosphate-monoesterases, are characterized by a maximal activity a t pH 9 on p-glycerophosphate (Folley and Kay, 1936) or by the hydrolysis of phenylphosphate a t pH 9.5. Greenstein (1945) gave the following comparative values for different organs: Phosphatase activity: Skeletal muscle Liver Brain Spleen Lung Bone (adult) Kidney Intestinal mucosa

2 4 12 17 36 420 1072 2789

It is striking that the intestinal mucosa and the epithelium of the kidney contain phosphatase in such excessively high concentrations, and this supports the theory that the specific (selective) absorbing activity of these epithelial cells is connected with their alkaline phosphatase content.

3 14

F.

VERZAR

To test our assumption that certain phosphorylating enzymes are affected by adrenalectomy, Kutscher and Wiist (1941, 1942) studied the alkaline phosphatase of the intestinal epithelium and the kidney of the guinea pig after adrenalectomy. They found the activity (expressed in mg. P/g. tissue) t o be as shown in Table VII. TABLE VII Normal (10) * Adrenalectomized (10) Adrenalectomized, DCA treated (7)

-

Intestine 13.11 Intestine 5 . 1 6 Intestine 7.56

Kidney 4 . 9 7 Kidney 2 . 8 4 Kidney 5 . 4 9

* Figures in parentheses denote number of snimriln.

Folley and Greenbaum (1946) confirmed this decrease in the adrenalectomized rat and also stated that treatment with adrenocortical hormone restored it; 3 mg. per day of desoxycorticosterone acetate was more active than adrenocortical extract, 11-dehydrocorticosterone, or ll-dehydro-17-hydroxycorticosteronein similar doses. The alkaline phosphatase of the intestinal mucosa of the rat has been studied by us with Sailer (1952a,b). The scraped mucosa of the upper part of the small intestine was homogenized and a known quantity in 4 ml. a t pH 9.2 reacted on 2.5% B-glycerophosphate for 1hour a t 37°C. Figure 4 shows the phosphatase activity expressed in mg. P per 20 ml. mucosa. The decrease after adrenalectomy and the restoration with desoxycorticosterone acetate and cortisone is very obvious. Vail and Kochakian (1947) also found a decrease of alkaline phosphatase in the kidney of rats after adrenalectomy. They prevented the decrease with daily doses of 1mg. desoxycorticosterone acetate. Adrenal cortical extract had no effect, but 1% NaCl in the drinking water had. This inhibits the onset of adynamia, as is now known, by restoring for some time the blood circulation and improving the general condition of the animal. With the alkaline phosphatase of the liver, which has a very slight activity (see Greenstein), Kochakian and Bartlett (1948) had rather contradictory results. The activity increased after adrenalectomy and adrenal cortical extract; desoxycorticosterone acetate was ineffective and no parallelism with the glycogenesis was seen. This shows that in the liver the enzyme plays a different, and quantitatively unimportant, role, as judged by its low activity in contrast to that of phosphorylase. Kochakian (1945) found that the activity of the alkaline phosphatase of the kidney was decreased by testosterone, which produces kidney hypertrophy as do other physiologically inactive steroids. Testosterone increased the acid phosphatase activity. These reactions are certainly of a different character from those influenced by corticoids.

CORTICOIDS AND ENZYMES OF CARBOHYDRATE METABOLISM

315

The alkaline phosphatase in the epiphysis and diaphysis of bones of rats was studied by Williams and Watson (1940) and Wyman and Torn Suden (1945). The activity was increased by desoxycorticosterone acetate, while adrenocortical extract, corticosterone and 1l-dehydro-17hydroxycorticosterone inhibited it. It is not known whether this phosphatase is changed after adrenalectomy. ma.

40

9

30

-

-

w 2

-

1

-

I,

e

1" g

"

2 -10

1;

.:..

20-

b

-

c I

Normal

6 day

I

I

Adyoamic

Treated with DCA

I

Treate1 with artisone

I

Treatsd wiih adrenal cofiical W r K t

FIG. 4. Alkaline phosphatase of intestinal mucosa (after Verz&r, Sailer and Richterich, 1952. Helu. Physiol. Ada. 10, 231).

4. Acid Phosphatase According t o Knoevenagel (1940), the acid phosphatase of guinea pig muscle is not changed after adrenalectomy. The acid phosphatase of the kidney increases if this organ becomes hypertrophic after treatment with different steroids. The acid phosphatase is diffused throughout the cell plasma of the epithelial cells of the kidney tubules and is not localized in the cell border as is the alkaline phosphatase. Vail and Kochakian (1947) found no influence of steroid hormones on the acid phosphatase of the liver and kidney.

316

F.

VERZAR

V. HISTOCHEMICAL DEMONSTRATION OF ALKALINE PHOSPHATASE 1. Intestine

The localization of alkaline phosphatase in the intestinal epithelium can be demonstrated by the histochemical method of Gomori (1939) and Takamatsu (1939). It lies in great concentration in and just under the striated border of the cells (Bourne, 1944; Deane and Dempsey, 1945; Emmel, 1945; Verne and HBbert, 1948, 1949). The alkaline phosphatase on the striated border is completely isolated from the nucleus and the Golgi apparatus, which also contains some. It is especially rich in the duodenum and upper jejunum, diminishing towards the ileum. It is absent in the cells of the Lieberkuhn cryptes. It has been shown in the intestine of all vertebrates and is especially rich in birds and mammals. In the rabbit, guinea pig, dog, cat, and man the whole small intestine contains alkaline phosphatase in high concentration in and under the striated border of these cells (HBbert, 1950). Since we had pointed out the importance of the phosphatase of the intestinal epithelium for absorption processes, and had also shown the influence of the adrenal cortex on these processes, Verne and HBbert (1948) studied the influence of adrenalectomy on the histochemical reaction of the alkaline phosphatase of the intestine. They demonstrated that 2 to 3 days after adrenalectomy the alkaline phosphatase disappears. If the animals were treated with adrenocortical extracts (0.5 ml./day), it regenerated, or if treatment was started a t the time of adrenalectomy, it did not disappear at all. If one adrenal was transplanted after adrenalectomy, the phosphatase also reappeared again. The disappearance of the alkaline phosphatase after adrenalectomy was even more obvious in males, if they were castrated before the adrenalectomy. Testosterone, estradiol and progesterone produce only a limited regeneration of the phosphatase. Adrenalectomized rats kept alive with 1% NaCI, or only a single desoxycorticosterone acetate injection, showed no regeneration. Soulairac (1948), who confirmed Verne, claimed to have prevented the loss of alkaline phosphatase with riboflavin. The riboflavin relationships have already been reviewed by Morgan (1951). Stenram (1951) also confirmed the disappearance of alkaline phosphatase after adrenalectomy. His Conclusion, however, that it is the electrolyte disturbance which influences phosphatase activity, is not substantiated by his experiments. In the rat embryo this histological phosphatase reaction of the intestine is negative until birth, in the guinea pig until the seventh week, and in the human embryo it appears in the sixth month. Verne and HBbert

CORTICOIDS AND ENZYMES OF CARBOHYDRATE METABOLISM

317

(1949) note that the adrenal cortex shows signs of activity from the same period, and there seems to be a time-parallel between the beginning of the adrenal cortical activity and the appearance of alkaline phosphatase in the intestinal mucosa. They demonstrated this relation by the following experiment: adrenal cortical extract was injected on the fifteenth day of pregnancy in rats, when the embryos are about 20 mm. long, directly into some embryos. Twelve hours later the living fetuses were taken out and their intestine gave a strong reaction of alkaline phosphatase, while the non-injected fetuses showed no reaction. The same experiment was performed with tadpoles. A few hours after injecting adrenal cortical extract into them the alkaline phosphatase reaction became positive in the border of the intestinal epithelium. In connection with the experiments with phosphatase poisons, which we shall mention later, we refer to Emmel’s experiment, in which 0.001 M KCN caused the alkaline phosphatase of the cell body between the striated border and the Golgi region to disappear. Other phosphatases were influenced by somewhat different concentrations. Lagerstedt and Stenram (1950)remark that it seems difficult to differentiate between two phosphatases, one in the cuticular border and one in the Golgi region, which react in a different way to KCN. 8. Kidney

The findings with the alkaline phosphatase of the kidney were similar. This is localized in the epithelial cells of the convoluted tubuli, which are the only epithelial cells of a similar structure to that of the epithelium of the small intestine. The concentration of alkaline phosphatase in the kidney is second highest after that of the intestine, according to chemical estimations. It is localized in and under the striated border of the epithelial cells of the kidney, as in the intestine. These cells reabsorb glucose from the glomerular filtrate. In accordance with the chemical proof of a decreased activity of kidney phosphatase after adrenalectomy, TissiBres (1948)in our laboratory using the Gomori method, found a disappearance of this phosphatase in adrenalectomized rats. Curiously enough, he showed that it now appeared in the glomeruli, where it is not normally present. Kochakian et al. (1948)saw similar histological pictures after large doses of different steroids. These observations, however, hardly permit us to ascribe an active secretory function $0 the glomerulus. It is much more probable that this represents the accumulation of washed-out phosphatafe in highly colloidal form, kept back by the filter of the glomerulus. The problem requires to be reexamined. We may summarize by saying that histological analyses have produced the same result as enzyme estimations in tiasues, namely the disappear-

318

F.

VERZAR

ance of alkaline phosphatase in the intestinal epithelium and in the kidney tubular epithelium after adrenalectomy and its regeneration with adrenal cortical hormones. VI. PHOSPHOGLUCOMUTASE Conway and Hingerty (1946) have approached the problem of how phosphorylating enzymes are altered after adrenalectomy, by a complete analysis of the carbohydrate metabolites of the muscle of adrenalectomized animals, compared with those of normal muscle. They found a great change in concentration of the hexose esters. Glucose-l-phosphate increased by 34.3 %, while glucose-6-phosphate decreased by 67.9 %, and fructose-6-phosphate by 63.8 %, compared with normal contr 01s. There was no change in : total-P, acid soluble-P, adenosinetriphosphate, fructose-1,6-phosphate, triose phosphate, phosphoglyceric acid P, phosphopyruvic acid P, carnosine, anserine. They concluded from this observation that phosphorylating enzyme activities are disturbed by adrenalectomy, and expressed the opinion that it is the glucomutase which decreases. In normal muscle there is more than twice as much glucose-6-phosphate (Robison ester) as glucose-lphosphate (Cori ester). In adrenalectomized animals the relation is reversed. The equilibrium of the reaction Cori ester Robison ester in the normal is up to 94% to the right (Sutherland, Colowick and Cori, 1941). It can thus be concluded that the glucomutase activity decreases in the adrenalectomized animal. The following table of Conway and Hingerty (1946) contains these analyses, which seem to be of the greatest importance for the problem. TABLE VIII

+

(From Conway and Hingerty, 1946)

Normal mMlkg. Phosphocreatine-P Adenosine triphosphate-P Total hexosemonophosphate-P Glucose-6-phosphate-P Glucose-1-phosphate-P Fructose-6phosphate-P Fructose-1:6diphosphate-P Triosephosphate phosphoglyceric acid-P Phosphopyruvicacid-P

+

24.4 19.5 10.04 6.42 2.74 0.94 0.40

50.4 f0.8 f 0.87 f0.85 f 0.21 f 0.06 f 0.06

-

3.55 0.073 f 0.004

Adrenalectomized Change after mM/kg. adrenalectomy 27.7 19.9 5.67 2.06 3.68 0.34 0.35

10.4 f 0.5 f 0.46 f 0.16 f 0.19 f 0.03 f 0.05

3.55 0.077

-

-

-

Decrease Decrease Increase Decrease Decrease

-

CORTICOIDS AND ENZYMES OF CARBOHYDRATE METABOLISM

319

The theory that glucomutase is decreased was supported by Keyes and Kelley (1949) on the basis that adrenal cortical extracts increase plasma lactate and pyruvate and the glycogen content of the liver. It should be remembered that Viale, Neuschloss and Turcatti (1927) had already reported that after adrenalectomy the muscles contained less than half as much “lactacidogen ” (undifferentiated) as normal muscles. They explained their findings as due t o enzymatic disturbances. Houssay and Mazzocco (1927) also mentioned a decrease of this substance in the muscle of adrenalectomized animals. Ochoa and Grande (1932) found a reduction of as much as one third of the phosphocreatine in muscle after adrenalectomy, but Conway and Hingerty (1946) give in their table an increase of 13.501, in rat muscle after adrenalectomy.

VII. HEXOKINASE This enzyme was used by Cori (1946) in highly purified preparations. He showed that insulin increases the velocity of its action. It was also said that anterior pituitary extracts inhibit hexokinase, especially the so-called “diabetogenic anterior pituitary hormone.” Later it was stated that the ACTH is the hexokinase-antagonizing factor and that adrenal cortical extracts also inhibit the hexokinase system (Colowick et al., 1947). Smith and Young (1949), Stadie and Hangaard (1949) and Broh-Kahn and Mirsky (1947) were unable to confirm this. It may be necessary to postpone the discussion until more highly purified enzyme and hormone preparations are available. This is especially the case with the so-called crystalline alkaline phosphatase (Abul-Fad1 et al., 1949). VIII. OXIDASESYSTEMS The activity of phosphorylating enzymes is intimately related t o oxidoreductive processes (Meyerhof, 1935). It is therefore of interest that, in addition to demonstrations of the inhibitory action of corticoids on the glycogen production of isolated muscle, a number of observations have also been made of an inhibitory action of the same steroids on oxidase systems (see also the following review). According to Tipton (1941) liver slices of adrenalectomized animals oxidize pyruvate and lactate less than normal liver slices. Gordan and Elliott (1947) described the inhibition of glucose and succinate oxidation by desoxycorticosterone. Eisenberg et al. (1950) and Hayano et al. (1950) studied the a-amino acid oxidase systems. The action of D-amino acid oxidase on D-alanine was decreased by 82%, and tyrosinase was also strongly inhibited by desoxycorticosterone. Other enzymes, such as urease, ascorbic acid oxidase, lipase, and transaminase were much less inhibited, while many other enzymes were not influenced at all (see the

320

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review by Dorfman in this volume). Some related steroids had a much weaker inhibitory action. They concluded that flavine enzymes were suppressed by an “inhibition of the electron transfer system” and stated that the cytochrome-c system was not affected. Desoxycorticosterone does not react with the prosthetic group of the D-amino-odixase and tyrosinase, which is flavine adenosine dinucleotide (FAD). Its action is on the apoenzyme, with which a steroid-protein complex is formed. The latter could be precipitated with acetone and the apoenzyme was thus regenerated. This inhibition of flavine enzymes by desoxycorticosterone is a striking parallel to its inhibition of carbohydrate enzymes. It reminds one also of the repeated observations of a relation of the adrenal cortex to the action of B vitamins, (VerzBr et al., 1937; Pulver and VerzBr, 1939; Soulairac, 1948, etc.; but not confirmed by Nelson (1940), Ferrebee (1940), Clark (1941)) especially flavines and pantothenic acid (Dumm and Ralli, 1948).

IX. PROTEIN ENZYMES Since protein metabolism as a source of glucose is influenced by adrenal cortical activity, the changes which are observed on protein enzymes may also play a part in the relationship of adrenal cortical hormones to carbohydrate metabolism (see the review of Kochakian (1946)). Besides the D-amino acid oxidase it has also been shown that arginase activity in the liver decreases after adrenalectomy and is restored by 11-oxycorticoids (Fraenkel-Conrat et al., 1942, 1943). In normal animals ACTH increases it, by stimulating cortin production. Folley and Greenbaum (1946, 194813) also showed that the arginase activity of liver, kidney and mammary gland decreased after adrenalectomy and was restored by ll-dehydrocorticosterone, 1l-dehydro-17hydroxycorticosterone and also-unexpectedly-by desoxycorticosterone. Anorexia was not the cause of this decrease after adrenalectomy. Kochakian, in a series of papers between 1944 and 1951, showed that besides arginase, alanine deaminase and glutamic deaminase also decreased in the kidney but not in the liver after adrenalectomy. Desoxycorticosterone and also testosterone and estrogens restored the normal values. Adrenal cortical extract was found, in his earlier work, to restore the arginase of the kidney but not of the liver, but in 1951 with Robertson, the extract also restored arginase activity in the liver. Cortisone restores alkaline phosphatase in the adrenalectomized mouse, but much later than arginase. In tissue slices of kidney,’Jiminez-Diaz (1936) demonstrated a decrease of amino acid deamination in vitro in adrenalectomized animals. Russel and Wilhelmi (1941) confirmed the decrease of alanine deaminase and glutamic acid deaminase in the kidney, but found no decrease in the liver.

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In the kidney they were able to restore it with adrenal cortical extracts or with desoxycorticosterone. Koepf et al. (1941) found that the liver of adrenalectomized animals formed glucose from d-glutamate just as quickly as that of normals; thus deamination was not disturbed. Leupin (1950), using the rat’s surviving diaphragm in uitro in oxygenated Ringer’s solution containing glucose, was unable to find a change of NH, production in adrenalectomiaed animals; nor was it possible to influence this value by corticoids. There is probably no direct connection with other protein enzymes such as serum-peptidase, which increases after a single subcutaneous injection of adrenal cortical extract in the mouse. ACTH acts similarly (Uhite, 1947; Holman et al., 1947). This action was related to the breakdown of lymphoid tissue by these hormones, but as Engel et al. (1949) pointed out, serum aminopeptidase level varies with the general state rather than with adrenal cortical activity. Hyaluronidase, the “spreading factor,” is inhibited by cortisone and somewhat less by 11-dehydrocorticosterone (Opsahl, 1949). Shuman and Finestone (1950) injected hyaluronidase together with fluorescein in the skin. They showed that if ACTH production was stimulated by adrenaline, a persistence of fluorescein was seen, and explained this as due to inhibition of the action of hyaluronidase by a corticoid.

X. REACTIONS BETWEEN CARBOHYDRATE AND POTASSIUM METABOLISM IN VITRO The metaboIic changes in .the adrenalectomized animal are partly related to carbohydrate and protein metabolism and partly to disturbances of electrolyte metabolism. While some believe that these are effects of different adrenal cortical hormones, we called attention (1940, 1941) to the fact that the electrolyte changes observed might be connected with the changes in carbohydrate metabolism (see Wirz, 1950, 1951). Potassium metabolism shows many connections with carbohydrate metabolism. Some examples are the following: injection of adrenaline leads to glycogenolysis and potassium is increased in the blood plasma along with glucose; during the contraction of a muscle, with glycogenolysis and lactic acid formation, potasaium is also liberated; after an intravenous glucose injection a “hyperkaliemia” is seen together with the “hyperglycemia,” later this changes to a hypokaliemic phase (Somogyi and Verzhr, 1942). We therefore studied different in vitro reactions of glycogen metabolism together with changes of potassium. First (with Pulver, 1940a,b) glucose fermentation in a suspension of yeast cells (baker’s yeast) was studied. As Willstatter and Rohdewald (1937, 1940) had shown, the cells first

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take up the sugar and a polysaccharide (“glycogen”) is formed. After this first period of about 7 minutes, fermentation begins. The potassium content of the centrifuged outer fluid was assayed in short periods and the curve shown in Fig. 5 was obtained. This demonstrates how, in the first period of glycogen production in the cell, potassium moves together with the glucose from the solution into the cells. In the second period, when glucose ia-fermented, potassium leaves the me.%

GluFose

0

60

120

Minutes

FIG.5. Yeast cells (after Pulver and Verzir, Helv. Chim. Acta, 23, 1087, 1940).

cells again. It was therefore concluded that glycogen production from glucose leads to potassium uptake and glycogenolysis to potassium release, which agrees with the above-mentioned observations in mammals. The experiments have been repeated by Hevesy and Nielson (1941) on yeast and by Leibowitz and Kupermintz (1942) on Escherichiu coli. Similar changes are also known in erythrocytes (Danowski, 1941; Harris, 1941). Conway with Breen (1945) and with O’Malley (1946) explained the entrance of potassium as the result of an ionic exchange of H+ with K+ ions. They could replace K+ with NHI+. Muntz (1947) found that yeast extract converts glucose to hexose-monophosphate even in the absence of K+ or NH4+. If, however, these are present, the process continues and hexose diphosphate is produced. This would explain why Lasnitski and SzorCnyi (1935) found a stimulation of glucose fermentation by potassium and Farmer and Jones (1942) by NH4+. Boyer et al. (1943) found that K+ or NH4+is needed for the reaction 2-phospho-

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pyruvate adenosine diphosphate or adenosine triphosphate. However, Lardy and Ziegler (1945) also observed this reaction with dialyzed extracts of muscle without the addition of K+, and Muntz (1947) therefore believes that it is not proved that potassium is an essential factor for this reaction. He suggests that potassium may be needed for the activation of an enzyme for hexose diphosphate production. Pulver and VerzAr (1940a,b) found that active potassium uptake could be inhibited by monoiodoacetic acid, and this was confirmed by Rothstein and Enns (1946) on Escherichia coli. Orskov (1948) was able to inhibit potassium uptake in yeast with HCN as well as with monoiodoacetic Na

me.% K Glucose

150 -10

\.

- 100 I

--o

I

Minutes

FIQ.6. Leucocytes (after Pulver and Verzdr, Helu. Chim. Acta 24, 272, 1941).

acid, but not with NaF and urethane. He found that several substances, which the yeast cells can metabolize, also lead to potassium uptake, but generally only after a longer latency. Cowie et al. (1949) have studied the problem again with Escherichia coli and used isotope K42. Potassium could be replaced by rubidium. They came to the conclusion that potassium enters the cycle of glycogen production when fructose-1-6-diphosphate is produced from fructose-6phosphate. Potassium is then present as a di-potassium phosphate. They are not of the opinion that it is an ionic exchange and also do not believe that K+ is needed as an enzyme activator. Since 2Ii+ per mol glucose is taken up, this proportion is more likely to signify the production of a salt. Pulver (1941) repeated his experiments in our laboratory on leucocytes of the horse, which can be studied in vitro like yeast cells. Five hundred thousand per cubic millimeter were used a t 37°C. in Ringer-phosphate buffer with 200 mg. % glucose added. Figure 6 shows such anexperiment.

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A similar reaction was later demonstrated (Leupin and Ve,rz.Br, 1950) on the surviving diaphragm of rats (Fig. 7). In Ringer’s solution the muscle continuously releases potassium. This is stopped, and can often be reversed into an actual uptake of potassium, if 200 mg. glucose with 1 Glycogen mg.% K+ mg.5 400

-

350

-

300

-

0.52

-

0.51

-

0.50-

250

0.49

-

0.48

-

0.47

-

0.46

-

0.45

-

0.44

-

0.43

-

0.42

-

-

200

-

150

-

100 -

50

0

-

L 320 Glycogen in muscle with without glucose glucose 1 hour 1 hour

start

start

K in outer fluid with without glucose glucose 1 hour 1 hour

FIG.7. Rat muscle (4 diaphragms) in Ringer’s solution (after Leupin and Verzk, 1949. Helv. Physiol. Acta 8, C27).

to 200 units of insulin per 100 ml. is added and consequently large quantities of glycogen are produced. If, on the contrary, the glycogen production was inhibited (in the presence of glucose) by desoxycorticosterone (see p. 305),potassium was released from the muscle in the same way as in the absence of glucose. It seems to be certain that we are dealing here with a reaction that has a general cellular physiological importance. Potassium is present in

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muscle (and nerve) in a so-called “bound” form. Actually in muscle it seems to be in a complex with myosin (and perhaps also with glycogen) (Montigel, 1943) and is released during the excitatory process. Whatever the explanation may be of the potassium uptake during glycogen formation and its release during glycolysis, these experiments prove the intimate connection between the two processes, in vitro and also in vivo. These observations support the belief that there are not necessarily two different hormones which influence these two different cellular activities (carbohydrate and electrolyte metabolism)] but rather that one fundamental cellular metabolic process is influenced] which affects the metabolism both of electrolytes and of glucose. XI. SUMMARY The adrenal cortex undoubtedly plays an important role in carbohydrate metabolism. It was shown in the first parts that its action is specialized neither for glycogen production, for regu’ation of blood sugar, nor for glucose production from proteins. Apparently it is the production of glucose in general, from different sources, which is influenced by the adrenal cortex. It thus influences centrally the energy production of the cells. Whether glucose is used directly, stored as glycogen, or transformed to fat depends on the general metabolic situation. The puzzling inhibitory action of corticoids on glycogen production, which leads to a glycogenolysis in isolated muscle, may be the expression of a stimulation of phosphorylase activity, as has been shown directly on minced muscle. Or it may be a “competitive inhibition’’ of enzyme activity. Different corticoids show this action on carbohydrate metabolism i n vitro, and in vivo, in varying velocity and intensity. The similarity of the action of other steroids gives the impression that the effects are “nonspecific.” Such a possibility cannot be denied a priori. The cell metabolic activity of the sex hormones, however, is probably of a similar type to that of corticoids, with the difference that their action is directed to certain receptors. The metabolic activity of surviving tissues or isolated cells in vitro shows a connection between carbohydrate metabolism and electrolyte reactions. The fundamental cell metabolic activity, which the adrenal cortical hormones influence, may be such that it leads simultaneously to the different actions on protein, carbohydrate and electrolyte metabolism. So far the role of certain enzymes connected with phosphorylations, such as phosphorylase, hexokinase, and glucomutase, has been discussed in connection with the activity of the adrenal cortex. The alkaline phosphatase of the intestinal epithelium and the kidney tubular epi-

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Stenram, U. 1951. Acta Anatomica 12,316. Stetten, D.,Jr., Welt, I. D., Ingle, D. J., and Morley, E. H. 1951. J.Biol. Chem. 192, 817. Sutherland, E. W., Colowick, S. P., and Cori, C. F. 1941. J . Biol. Chem. 140, 309. Swingle, W.W. 1927. Am. J . Physiol. 79, 666. Takamatsu, H. 1939. Trans. SOC.Path. Japan 29, 492. Thaddea, S. 1936. Nebennierenrinde. Berlin. Tipton, S. R. 1939. Am. J.Physiol. 127, 710; 1941. 132, 14. TissiBres, A. 1948. Actu Anatomica 6, 224. Vail, V. N.,and Kochakian, C. D. 1947. Am. J.Physiol. 160,580. Verne, J., and HBbert, S. 1948. Compt. rend. soc. biol. 142, 300. Verne, J., and HBbert, S. 1949. Compt. rend. SOC. biol. 143, 201. VerzBr, F. 1939. Physiology of Adrenal Cortex. B. Schwabe, Basel. VerzSr, F. 1940. Verh. Schweiz. Naturf. Ges. Locarno, p. 200. VerzBr, F. 1941. Schweiz. med. Wochschr. 71, 878. Verzk, F. 1942. Schweiz. med. Wochschr. 72, 597. VerzBr, F. 1943. Muskelkontraktionstheorie. B. Schwabe, Basel. Verzar, F. 1950a. Schweiz. med. Wochschr. 80, 468. Verzk, F. 1950b. Bull. Acad. Firenze. VerzSr, F., Bucher, R., Somogyi, J. C. and Wirz, H. 1941. Helv. Med. A d a 7, Suppl. VI, 58. Verzk, F., Hiibner, H., and Laszt, L. 1937. Biochem. 2. 292, 152. VerzBr, F., and McDougall, E. J. 1936. Absorption from the Intestine. Longmans, Green, London. Verzbr, F., and Montigel, C. 1941. Schweiz. med. Wochschr. 71, 1382. VerzL, F., and Montigel, C. 1942a. Helv. Chim. Actu 26, 9. VerzBr, F.,and Montigel, C. 194215. Helu. Chim. Actu 26, 22. VerzL, F.,and Montigel, C. 1942c. Nature 149, 49. Verzbr, F.,and Sailer, E. 1952a. Helv. Physiol. Actu 10, 247. VerzBr, F.,Sailer, E., and Richterich, R. 1952b. Helu. Physiol. Actu 10, 231. Verzk, F., and Siillmann, H. 1937. Biochem. 2. 289, 323. Versbr, F.,and Wang, F. C. 1950. Nature 166, 114. Verz&r, F.,and Wenner, V. 1947. Bull. SOC. chim. biol. 29, 304. VerzBr, F., and Wenner, V. 1948a. Biochem. J. 42, 35. VerzBr, F.,and Wenner, V. 1948b. Biochem. J . 42, 42. Verzdr, F.,and Wenner, V. 1948c. Biochem. J. 42,48. Viale, G.,Neuschloss, S. M., and Turcatti, E. 1927. Compt. rend. SOC.biol. 97,266. Villee, C. A., and Hastings, A. B. 1949. J. Biol. Chem. 181, 131. Wang, F. C. 1950. Nature 166,277. Wang, F. C., and Verzdr, F. 1949. Am. J . Physiol. 169, 263. White, A. 1947/48. Haruey Lectures 45, 43. Wick, A. N. 1949. Proc. SOC.Exptl. Biol. Med. 71, 445. Wilbrandt, W., and Laszt, R. 1933. Biochem. 2. 269, 398-417. Wilbrandt, W., and Lengyel, L. 1933. Biochern. 2. 267,204. Williams, H. L., and Watson, E. M. 1940. J. BioL Chem. 136, 337. Willstatter, R., and Rohdewald, M. 1936. Enzymologia 1, 213. Willstatter, R., and Rohdewald, M. 1937. 2. physiol. Chem. 247, 115. Willstatter, R., and Rohdewald, M. 1940. Enzymologia 8, 1. Wirz, H. 1950. Helu. Physiol. Actu 8, 186;1951. Nature 167, 322. Work, Th. S.,and Work, E. 1948. The Basis of Chemotherapy. p. 65-86. Wyman, L. C.,and Turn Suden, C. 1945. Endocrinology 36, 340.

Steroids and Tissue Oxidation BY RALPH I. DORFMAN Worcester Foundation for Experimental Biology, Shrewsbury, Massachuselta CONTENTS

1. Introduction.. .......... .................................... 11. Steroids and Tissue-Enzyme Concentrations. . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Succinic Dehydrogenase and Succinoxidase. . . . . . . . . . . . . . . . . . . . . . . . . .

Page 331 332 333

5. Cholinesterase. . . . . . . . . . . . . . . . . . . . . . . . . . . .

6. Peptidase.. ......

10. Proline Oxidase.. .........................

Alkaline Phosphatase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Succinoxidase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Malic Oxidase.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Choline Acetylase.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Miscellaneous . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. In Vdro Effects on Metabolism.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Diaphragm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Liver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. 4. 5. 6.

360 360

362 362 362 364 364 366 366 368

I. INTRODUCTION Steroid-enzyme relationships have only recently been studied : the field is in its infancy and the possibilities many. A working hypothesis held by some current investigators consists of a system in which hormones exert their action by influencing enzyme systems. This influence may be exerted by: (1) changes in tissue-enzyme concentrations, (2) by the hormone functioning as a component of an enzyme system, (3) by the hormone accelerating or inhibiting an enzyme system, or (4) by direct or 331

332

RALPH I. DORFMAN

indirect effects on accelerators and/or inhibitors of enzyme systems. This review will be concerned with the various interrelationships that have been demonstrated or indicated between steroid hormones and =ENZYME

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FIQ. 1. Effect of castration, testosterone, and testosterone plus estradiol dipropionate on the weight and succinic dehydrogenase of rat prostate. Meyer and McShan (1950).

various aspects of tissue metabolism, especially from the point of view of steroid-enzyme relationships. 11. STEROIDS A N D TISSUE-ENZYME CONCENTRATIONS One pronounced influence of steroid hormones is their ability to modify:tissue-enzyme concentrations. These relationships will now be considered.

333

STEROIDS AND TISSUE OXIDATION

I . Succinic Dehydrogenase and Succinoxidase In the presence of a hydrogen acceptor, the enzyme succinic dehydrogenase oxidizes succinic acid to form fumaric acid. The succinoxidase

@ ENZYME ACTIVITY

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Fro. 2. Effect of castration, testosterone, and testosterone plus estradiol dipropionate on the weight and succinic dehydrogenase of rat seminal vesicles. Meyer and McShan (1950).

system consists of succinic dehydrogenase plus cytochrome oxidase. The concentration of this enzyme system in some tissues has been shown to be a function of steroid hormone concentration. The relationship between androgens with and without estrogens and succinic dehydrogenase concentrations in accessory tissue is illustrated in Figs. 1 and 2 (Davis et al., 1949). Evident in both seminal vesicles

334

RALPH I. DORFMAN

and prostate is the decreased concentration of enzyme after castration, which may be restored to normal by effective doses of testosterone or testosterone plus estradiol. On the other hand, Leonard (1950) has shown that the levels of this enzyme in the perineal musculature remain unchanged after castration of the rat and after the administration of androgens. Tipton et al. (1946)have observed slight decreases in succinoxidase of liver as a result of adrenalectomy. More recent studies by Wollman and Scow (1951) did not confirm this finding. After hypophysectomy, slightly elevated succinoxidase activity was found to occur proportionate to the elevated nitrogen content. Table I summarizes the data on observed changes in succinic dehydrogenase and succinoxidase content of tissues as influenced by steroid hormones. TABLE I Injluence of Steroid Hormones on Succinic Dehydrogenase ( S D ) and Suceinozidase (SO) in the Rat Condition Castration

+ +

Castration testosterone Castration testosterone estradiol dipropionate Castration Castration androgen Adrenalectom y

+

+

Gland

Change

Reference

Prostate and seminal Decrease in SD vesicles Prostate and seminal Increase in SD vesicIes Prostate and seminal Increase in SD vesicles

Davis et al. (1949)

Perineal musculature No change in SD

Leonard (1950)

Davis et al. (1940) Davis e l al. (1949)

Perineal musculature No change in SD Leonard (1950) Slight decrease in Tipton et al. (1946) Liver

so

Adrenalectomy

Liver

Hypophysectomy

Liver

No change in SO

Wollman and Scow (1951) Increase in SO pro- Wollman and Scow portional to in(1951) creased nitrogen

2. Arginase Arginase has the specific function of hydrolyzing arginine to ornithine and urea. Kochakian (1947) published a series of papers indicating a direct relationship between arginase concentration of liver and kidney and steroid hormones. A summary of the changes observed is recorded in Table 11.

335

STEROIDS AND TISSUE OXIDATION

TABLE I1 Influence of Steroid Hormones on Arginase Content of Tissues Condition of animal SDecies and treatment Mouse Castration Mouse testosteCastration rone propionate Rat Adrenalectorny & hypophysectomy Rat Normal, hypophysectomy, or adrenalectomy cortisone, corticosterone or ll-dehydrocorticosterone Rat Normal, hypophysectomy or adrenalectomy desoxycorticosterone Mouse Castrated cortisone or 11-dehydrocorticosterone Rat testosteCastration rone propionate Rat Hypophysectomy

+

Tissues studied Kidney Kidney

Change in arginase content Increase Increase

Reference Kochakian (1947) Kochakian (1 947)

Liver

Decrease

Fraenkel et al. (1943)

Liver

Increase

Fraenkel el al. (1943)

Liver

No change

Fraenkel et al. (1943)

Liver Kidney

Increase Increase

Kochakian and Robertson (1950)

No change Increase Decrease Decrease No change Increase

Kochakian and Robertson (1950) Kochakian and Robertson (1950) Kochakian and Robertson (1950)

Decrease Decrease No change No change Slight increase No change Slight increase No change Increase

Kochakian (1947) Kochakian (1947) Kochakian (1947) Kochakian (1947)

Increase Increase Increase Decrease

Kochakian and Clark (1942)

Increase

Marseli (1951)

Decrease None Small increase

Kochskian el al. (1948) Humm et al. (1948)

+

+

+

+

Rat

Rat Rat Rat Rat

Liver Kidney Liver Kidney Hypophysectomy (male) Liver testosterone propio- Kidney nate Adrenalectomy Liver Kidney Adrenalectomy saline Liver Kidney Kidney Adrenalectomy desoxycorticosterone Adrenalectomy Liver adrenal cortical extract Kidney

+

+ + +

+

Rat

Adrenalectomy Liver testosterone propionate Kidney adrenal cortical extract Rat Castrated male testos- Liver terone propionate Intestine Kidney Rst Castrate male f testos- Liver terone propionate Rat Normal male testoste- Liver rone propionate Hamster Castrated male testos- Kidney terone propionate Liver Guinea Castrated male Kidney methyl testosterone Liver Pig

+

+

+ + +

and Vail and Vail and Vail and Vail

Kochakian and Vail (1947)

Marseli (1951)

336

RALPH I. DORFMAN

Administration of androgens produces a profound increase in arginase concentration in the kidney, but no significant influence on liver or intestinal concentration. Castration in the mouse produced a slight increment in kidney arginase Concentration, mainly due t o the slight decrease in kidney weight. Most steroids that increase the size of the castrated mouse kidney also increase the arginase concentration of this tissue. A particularly interesting phenomenon is demonstrated in Fig. 3 by Kochakian’s original data (1945). As the dose of testosterone was increased, the size of the castrated mouse kidney increased approximately 100%, while the arginase concentration of the tissue increased some 600%. 1

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Fro. 3. The effect of the dose of testosterone on the arginase content of the kidney in the castrated mouse. Kochakian (1947).

Further, a maximum increment in tissue weight was attained at a dose of 2 mg. of testosterone for a 30-day period while the arginase concentration kept increasing up to a dose level of 18 mg. for the same period. After the administration of low concentrations of certain androgens to the castrated mouse, actual decreases in kidney enzyme concentration have been noted a t a time when the kidney is increasing rapidly in size. As the dose is further increased, the arginase concentration quickly increases. From Kochakian’s data dealing with the influence of androgens on mouse kidney arginase concentration, three categories of responses, depending upon the androgen administered, can be discerned (Table 111). Recent experiments by Kochakian (1951) showed that phlorizintreated rats on a 70% protein diet showed an increase in arginase proportionate to kidney weight. When androgen was administered to these animals, a further increment in kidney arginase was observed. Further, rats made diabetic with alloxan and having sclerotic kidneys responded to testosterone propionate with the typical kidney weight and arginase

337

STEROIDS AND TISSUE OXIDATION

TABLE I11 Qualitative Differences in Response of Mouse Kidney to Different Androgens Low dose effect Typical substance

Kidney weight

Change in arginase concentration

High dose effect Kidney weight

Testosterone

Fast max. incr. Initial decrease Leveling off

Methyltestosterone

Fast max. incr. Fast initial incr. (no decr.)

Androstanediol 3 (8),17(8)

Fast low max. Decrease incr.

Change in arginase concentration

Increase beyond tissue weight increase Leveling off Increase beyond tissue weight increase Leveling off at Prolonged low level decrease

increment. Cortisone produced an immediate increment in kidney arginase while the liver arginase increased after an initial delay. Fraenkel-Conrat et al. (1943) studied the influence of adrenalectomy and hypophysectomy on rat liver arginase concentrations. Adrenalectomized, hypophysectomized, or normal rats treated with corticosterone or 11-dehydrocorticosterone showed increased liver arginase concentration, while similar animals treated with desoxycorticosterone did not show these changes. Kochakian (1945) has shown that mice, in a state of under-nutrition, still show changes in arginase content of tissues similar to well-fed animals. Under these nutritive conditions, castration produced an increase in kidney-enzyme concentration which was further increased by the administration of adrogens such as testosterone and methyltestosterone. Table IV summarizes the influence of steroid hormones on kidney arginase concentration. Such androgens as testosterone and 17-methyltestosterone produced the most dramatic increases in enzyme concentrations. But others produced less significant increases, or, as in the case of isoandrosterone, a slight decrease. (3-Estradiol produced a moderate increase as did cortisone. Both progestational substances studied produced slight decreases. 3. Amino Acid Oxidase Clark et al. (1943) showed that castration in the mouse resulted in a decreased D-amino acid oxidase content of kidney tissue, but not of liver or

338

RALPH I. DORFMAN

TABLE IV Steroids and Kidney Arginase Hormonal activity Androgenic

~~~

~

~

Relative influence on arginase concentration

Steroid 17-Methyltestosterone Testosterone 17-Methylandrostanediol-3 (a), 17@)

Increase Very high activity

17@) Androstanediol-3 (a), Dehydroisoandrosterone

Increase Moderate activity

Androsterone Cistestosterone Androstanedione-3,17

Inactive

Isoandrosterone

Slight decrease

~~

Estrogenic

8-Estradiol

Increase, moderate activity

Progestational

Progesterone 17-Ethynyltestosterone

Slight decrease

Adrenal cortical

Cortisone

Increase

intestine. Adequate treatment of the castrated mouse with testosterone propionate restored the kidney oxidase content to normal or above normal levels. Jensen (1951) studied the role of the pituitary, adrenal, and thyroid glands in the amino acid oxidase (AAO) content of kidney and liver tissue. Their basic observations started with the finding that the administration of amino acids to normal rats caused a pronounced increase in the liver AAO content. These effects were absent in the adrenalectomized or hypophysectomized rat similarly treated. The findings by this group are illustrated in Table V. TABLE V Per Cent Increase in AAO after Amino Acid Injection

Tissue Liver Kidney

Minutes after injection Normals Adrenalectomized Hypophysectomized 30 90 30 90

50 38 89 7

0

-

4

-38 -

-9 59

29

STEROIDS AND TISSUE OXIDATION

339

The kidney AAO activity was promptly increased in normal animals in 30 minutes with the return to a normal level after 90 minutes. Adrenalectomized animals, on the other hand, showed no change at 30 minutes, but a significant increment at 90 minutes. Hypophysectomized animals also showed an increment in AAO at 90 minutes after amino acid administration. Suspecting that some adrenal factor controls the AAO activity of the liver, adrenalectomized or normal animals were treated with adrenal cortical extract. This treatment produced an increase in liver and kidney AAO activity (Jensen, 1951). Adrenalectomized animals showed a decrease in liver AAO while hypophysectomized animals showed an increase. Thyroidectomy resulted in a lowering of liver AAO. This has also been reported by Iilein (1939). Liver and kidney effects do not parallel each other. In adrenalectomized rat kidneys, the AAO level 4 hour after amino acid injection showed no change, but after 14 hours there was a 50% increase, in contrast to changes in the liver. In the hypophysectomized rat, the kidney showed a trend toward a change (*29%). Umbreit (1951) showed that adrenalectomy decreases the liver concentration of D-amino acid oxidase whereas kidney enzyme concentration is unchanged. Q. Cytochrome Oxidase Treatment of castrated rats by testosterone propionate results in an increased content of cytochrome oxidase in the seminal vesicles and prostate (Davis et al., 1949). 6. Cholinesterase

Cholinesterase hydrolyzes acetylcholine into choline and acetic acid. Everett and Sawyer (1946) studied the influence of gonadectomy and sex hormone replacement therapy on the cholinesterase content of blood in the rat. Table V I summarizes these data. Wattenyl et al. (1943) found that the serum cholinesterase of sexually mature male guinea pigs decreases after castration but may be brought back to normal by subcutaneous implantation of testosterone. This is in conflict with the results of Everett and Sawyer, who found an increase in serum cholinesterase after castration, and treatment of the castrated rat with androgen produced a decrease in enzyme concentration. Further work is needed to see if this discrepancy is due to species difference. 6. Peptidase

Although Holman et al. (1947) found an increased serum peptidase activity after adrenal cortical hormone administration to mice, Schwartz

340

RALPH I. DORFMAN

TABLE VI Influence of Steroid Hormones on cholinesterase Concentrations Species

Conditions

Gland

Blood serum Blood Castration male serum Castrated male or fe- Blood serum male plus estrogen Blood Castrated male plus serum testosterone propionate Blood Guinea pig Castration male serum Blood Castrated male plus testosterone propioserum nate

Rat

Ovariectomy

Change Decrease

Reference Everett and Sawyer (1 946)

Increase Increase Decrease Decrease

Wattenyl et al. (1943)

Increase to normal

and Engel (1949) were unable to demonstrate comparable effects in either the rat or mouse after cortical hormone or ACTH treatment. Johansen and Thygessen (1951) reported that the very high levels of serum peptidase activity of lupus erythematosis disseminatus were depressed by ACTH administration. These studies were carried out using alanylglycylglycine as a substrate for the tripeptidase and alanylglycine for dipeptidase activity. The authors interpreted their results to mean that ACTH inhibits the liberation of the enzyme from cellular elements. 7. Phosphatases This group of enzymes catalyzes a variety of reactions which are concerned with carbohydrate, nucleotide and phospholipide metabolism as well as bone formation. Influences of the steroid hormones on acid and alkaline phosphatases have been reported. Tables VII and VIII summarize representative samples of the experimental findings. Kochakian (1947) has made an extensive study of the influence of steroid hormones on the phosphatase content of the mouse kidney. Castration in the mouse does not affect the total amount of kidney alkaline phosphatase but increases the concentration. This is due to the fact that the mouse kidney suffers involution after castration. On the other hand, the acid phosphatases show a decrease which is proportional to the tissue mass. Thus, in the case of the acid phosphatases, no change in concentration is observed. Testosterone treatment of the castrated mouse results in an increase in

34 1

STEROIDS AND TISSUE OXIDATION

TABLE V I I Steroid Influences on Alkaline Phosphatase Concentrations Species

Condition, sex, and treatment

Tissues studied

Change

Reference

GLANDREMOVAL Rat

Castrated male

Kidney Liver

Rat

Castrated male

Seminal ves. Prostate

Mouse Ovariectomized female Rat Hypophysectomized male Mouse Castrated male

Uterus Vagina Liver Kidney Kidney Liver Intestine

Increase Slight increase Decrease Increase and decrease (8 days) Decrease Decrease Increase Decrease

Kochakian and Robertson (1950a) Stafford et al. (1949)

Harris and Cohen (1951) Kochakian and Robertson (1950a) Kochakian, 1947

ESTROQEN TREATMENT Mouse Ovariectomized female Estrogen Mouse Ovariectomized female Estrogen

Uterine glands, lumenal epith., Circulature layers of epith. Uterus

Increase

Atkinson (1947)

Increase

Harris and Cohen (1951)

Vagina

Increase

ADRENAL CORTICAL STEROID TREATMENT Rat

Rat Rat Rat Rat

Fasted, male ll-de- Liver hydrocorticosterone Kidney Male, adrenal corti- Femur, cal extract diaphyses epiphyses Male, cortisone Femur corticosterone diaphyses epiphyses Femur Male, amorphous fraction diaphyses epiphyses Femur Male, desoxycorticosterone diaphyses epiphyses

+

Increase Kochakian and Bartlett No change (1948) Decrease Decrease

Williams and Watson (1941a)

Decrease Decrease

Williams and Watson (1941a)

No change Williams and Watson No change (1941a) Increase Increase

William8 and Watson (1941a)

342

RALPH I. DORFMAN

TABLE VII.-(Continued) Species

Condition, sex, and treatment

Tissues studied

Change

Reference

ANDROGEN TREATMENT Mouse Castrated male, testosterone and other androgens Rat Hypophysectomized male, testosterone Rat Male, testosterone

Kidney

Decrease

Kochakian (1945)

Kidney

Increase

Bochakian and Robertson (1950a)

Liver Femur

No change Increase Williams and Watson (1941b)

PROGESTATIONAL Rat

Male, progesterone Mouse Ovariectomized female, 17-ethynyltestosterone Rat Castrated male, testosterone propionate

Femur

Increase

Kidney

Decrease

Williams and Watson (1941b) Kochakian (1945)

Seminal vesicles Prostate

Increase

Stafford et al. (1949)

Decrease (4 days) Increase (8days)

+

ESTROGEN PROQESTERONE Mouse Ovariectomized female

Uterus Vagina Kidney Liver Spleen

No change Cohen (1951) Decrease Increase Increase Cohen and Huseby (1951) Increase

kidney size and a striking decrease in the concentration of alkaline phosphatases. Actually the total alkaline phosphate is decreased. The total acid phosphatase content of the kidney is increased, but only a shade greater than the increase in kidney’ size. This results in only a slight increment in acid phosphatase content of the testosterone-stimulated kidney. Treatment of the normal mouse with androgens shows a picture of kidney phosphatase similar to that of the castrated animal. The phosphatases of liver and intestines are not influenced by androgens. In addition t o the study on the kidney of the mouse, there are data on the rat (Kochakian and Vail, 1947), the hamster, (Kochakian et al., 1948), and the guinea pig (Humm et al., 1948). In all these rodents, the influ-

343

STEROIDS AND TISSUE OXIDATION

TABLE VIIL Steroid Injluences on Acid Phosphatase Concentrations Species Mouse

Condition of animal and treatment

Mouse

Normal and castrated, testosterone and other androgens Castrated male

Rat

Castrated male Hypophysectomized male

Rat

Castrated male

Tissues studied

+

Reference

Kidney

Increase

Kochakian (1947)

Kidney Liver Intestine Kidney

No change

Kochakian (1947)

No change

Liver

Slight decrease Decrease Decrease

Kochakian and Robertson (1950a) Kochakian and Robertson (1950a)

Kidney Seminal vesicles Prostate

Hypophysectomized Liver male (testosterone) Kidney Seminal Rat Castrated male vesicles Testosterone propio- Prostate nate Liver Guinea Castration Kidney Pig Castration Prostatic Dog secretion testo- Prostatic Castration sterone propionate secretion Human Men, osseous metas- Serum tases in prostatic cancer castration or estrogen Human Men, osseous metas- Serum tases in prostatic cancer androgens Rat

Change

Stafford et al. (1949) Stafford et al. (1949)

Increase (4 days) Decrease (8 days) No change No change Increase

Kochakian and Robertson (1950a) Stafford et al. (1949)

Decrease (4 days) No change

Humm et al. (1948)

Decrease

Huggins et al. (1939)

Increase

Huggins et al. (1939)

Decrease

Huggins et al. (1941)

Increase

Huggins and Hodges (1941)

+

+

ence of androgens on phosphatase concentration of the kidney has been found to be qualitatively similar. The relationship of steroids to the concentration of phosphatases in male accessory tissues has been studied in some detail, especially with respect to the prostate. Kutscher and Wolberg (1935) reported a high

344

RALPH I. DORFMAN

content of acid phosphatase in human adult prostatic tissue. Gutman and Gutman (1938) confirmed the earlier finding and showed that the preputial gland had a similarly high content of enzyme in contrast to the relatively low concentration of enzyme in the testis, cowper’s gland, liver, and kidney. Huggins et al. (1939) showed that the acid phosphatase content of dog prostatic secretion was conditioned by androgens. The secretion of castrated dogs was significantly lower in acid phosphatase content than

-

SEMINAL VESICLE

I, 0

4

8

N NORYU CASTRATE T.P.-TESTOSTERONE PROPIONATE

\r-

0 OAY9

FIQ. 4. Influence of castration and castration plus testosterone propionate on acid phosphatase content of seminal vesicles and prostate. Meyer and McShan (1950).

that of normal dogs and the decrease could be reversed by adequate treatment with testosterone propionate. Gutman and Gutman (1939) obtained similar results in the monkey. Of particular interest is the change in serum acid phosphatase in individuals sufferingfrom osseus metastases of a malignant prostate. In such individuals, castration or treatment with estrogens produced a drop in acid phosphatase (Huggins et al., 1941). As might be expected, androgen treatment under the same conditions produced immediate rises in serum acid phosphatases (Huggins and Hodges, 1941). Figure 4 illustrates data of Stafford et al. (1949) on the effects of castration and testosterone propionate treatment on both the alkaline and acid phosphatases of rat seminal vesicle and prostate. These data

STEROIDS AND TISSUE OXIDATION

345

indicate that concentrations of both acid and alkaline phosphatases of seminal vesicles are decreased by castration as early as the fourth day postoperatively. The concentrations of both phosphatases of the seminal vesicles are restored to normal by testosterone propionate. In the prostate, a somewhat different situation is found, Although at 8 days postcastration the concentrations of both phosphatases are decreased, a t 4 days postoperatively a distinct increment in both acid and alkaline phosphatases was found. At 4 days the administration of testosterone propionate produced a decrease in acid and alkaline phosphatase as compared with the castrated uninjected control. The action of testosterone was reversed eight days postoperatively when the prostates of the operated animals were low in phosphates. At this time, the administration of the androgen produced a distinct increase in acid and alkaline phosphatase concentrations. 8. 8-Glucuronidase 8-Glucuronidase occurs in a number of organs throughout the body. The concentration is particularly high in liver and spleen. The enzyme hydrolyzes a variety of conjugated 0-glucuronides such as sodium menthol glucuronide, sodium borneol glucuronide, sodium pregnanediol glucuronide, and sodium estriol glucuronide. The estrogens have a specific effect on concentrations of this enzyme in the uterus. Fishman (1950, 1951) showed in a series of papers that ovariectomy in the mouse leads to a decreased uterine content of the enzyme which may be restored to normal by treatment with estrogens. Fishman suggested that this enzyme was concerned with the metabolism of estrogens in the uterus. Levvy et al. (1948) suggested that the increases in 0-glucuronidase concentrations are really correlated with increased cell proliferation. Harris and Cohen (1951) as well as Kerr et al. (1950) have confirmed and expanded Fishman’s findings. The former workers have shown that ovariectomy in the mouse is without influence on the 8-glucuronidase content of kidney, liver, spleen or vagina, but that the uterine enzyme is distinctly depressed. The uterine 8-glucuronidase content is increased by estrogens. Kerr et aE. (1949, 1950) observed an increment in 8-glucuronidase of liver in estrogen-treated ovariectomized mice. Harris and Cohen (1951), however, were unable to find this effect. Harris and Cohen (1951) studied esterase and 0-glucuronidase content of the tissues simultaneously. The most striking finding is an inverse relationship of esterase and p-glucuronidase content; an increase in one is reflected by a decrease in the second constituent. Progesterone administered along with estrone produced a decreased 8-glucuronidase content of vagina, kidney, liver, and spleen (Harris and

346

RALPH I. DORFMAN

Cohen, 1951). Kerr et al. (1950)reported that progesterone antagonizes the stimulating influence of estrone on the glucuronidase content of the uterus. Harris and Cohen (1951)found a similar effect, but the change was small. Blood serum p-glucuronidase content is related to steroid hormone levels. Estrogen and cortisone tend to increase the enzyme content while testosterone administered to women with cancer of the breast produced no changes. Cushing's disease and stress, both states in which increments in circulating corticoids of the cortisone type would be expected, showed increments in blood serum enzyme concentrations. Finally, in pregnancy, increased levels of the enzyme are found. Fishman et al. (1950) have shown that the serum P-glucuronidase drop postpartum is delayed if the mother receives oral stilbestrol. In all blood serum studies, the esterase content was again inversely proportional to the (3-glucuronidase content (Cohen, 1951). 9. Esterase Harris and Cohen (1951) have studied the influence of steroid hormones on the esterase content of tissues. Butyric acid liberation was employed and the histochemical method of Glick (1934) was modified and employed in the studies. As previously mentioned in the discussion of p-glucuronidase in tissues, an inverse relationship between the concentration of the latter enzyme and the concentration of esterase was found under diverse conditions. Thus ovariectomy in the mouse was followed by marked increases in esterase activity of both the uterus and vagina while the 8-glucuronidase concentration of these tissues decreased. Estrogen administration restored the esterase and j3-glucuronidase content of both the uterus and vagina of the ovariectomized mouse. The presence of the ovary influenced the responsivity of the uterus and vagina to estrogens. One microgram or ten micrograms of estrone administered to the ovariectomized mouse produced restorative changes in esterase activity of these tissues, but was' without influence on the enzymatic activity of these tissues in the intact mouse. Although ovariectomy produces a sharp decrease in uterine and vaginal esterase, no comparable change was found in the kidney, liver, or spleen. Estrone was without influence on the kidney, liver, and spleen concentration. The simultaneous administration of progesterone and estrone to ovariectomized mice resulted in an increased esterase content of the liver and spleen. Harris and Cohen (1951)have speculated on the possible direct relationship between lipide content of a tissue and its esterase content.

347

STEROIDS AND TISSUE OXIDATION

10. Proline Oxidase

Umbreit and Tonhazy (1951a) studied the influence of adrenalectomy on a variety of enzyme systems. In the adrenalectomized rat maintained on saline, no changes in liver concentrations of a variety of enzyme systems could be detected. These included a-ketoglutarate oxidase, oxaloacetic oxidase, succinic oxidase, lactic dehydrogenase, glutamic dehydrogenase, acetoacetic oxidase, octanoic acid oxidase, transaminase, and cholinesterase. The kidneys but not the livers of adrenalectomized rats showed a decreased ability to oxidize proline. Whereas the Qor for the homogenate from normal kidneys was 25 k 4, adrenalectomy decreased the Qo, to 10 f 2. The decrease in proline oxidase was not apparent for three days after adrenalectomy. Administration of cortisone (0.5 mg./day) restored the proline oxidase content to normal. Desoxycorticosterone was ineffective. In adrenalectomized rats, after the decreased content of proline oxidase content of kidney had been established, cortisone treatment had to be continued for 10 days to restore the enzyme content to normal. No in vitro effect of cortisone could be demonstrated. The change in proline oxidase content of kidney as a result of adrenalectomy is unique. Table IX Iists some Qo, d u e s . These data ilIusTABLE IX Praline Oxidase Content of Rat Kidney after Adrenalectomy and in Vitamin Deficiencies* Condition Normal Adrenalectomy Adrenalectomy plus cortisone Be deficiency Riboflavin deficiency Thiamine deficiency Pantothentic acid deficiency BIZdeficiency

Qo:

24

10 26 30 21 21

27 28

~

* Umbreit and Tonhazy, 19616. trate the dramatic change in enzyme content as a result of adrenalectomy. The fact that pantothenic acid and riboflavin deficiencies do not influence the concentration of proline oxidase is of particular interest since it is believed that in these two vitamin deficiencies adrenal insufficiency may be a complicating factor. Umbreit and Tonhazy (1951b) have also demonstrated that after

348

RALPH I. DORFMAN

adrenalectomy a number of substrates including a-ketoglutarate, fumarate, and oxalacetate are oxidized a t a reduced rate by kidney homogenates. The rate can be restored t o normal in vitro by the addition of adenylate or ATP. In vivo this defect may be corrected by cortisone treatment. 1 1 . Actomyosin and ATP-ase Actomyosin is considered to be the contractile “skeleton” of the striated muscle fibril. This material is present in the uterus and its concentration increases in the pregnant uterus. X-ray castration in humans resulted in a lowering in uterine actomyosin concentration (Csapo, 1948). Ovariectomy in the rabbit also results in a lowered uterine actomyosin content. Four days’ treatment with estrogen results in a fifteen fold increment in actomyosin content per uterus, an amount which exceeds that found during estrus. Ovariectomy also causes a decrease in ATP-ase activity. After 2 months, the amount may be decreased by a factor of 15. Within 12 hours after a single injection of estrogen, at a time when the actomyosin concentration is still low but increasing, the ATP-ase activity increases tenfold. After 4 days of treatment, the estrous level of ATP-ase is reached. 12. Choline Acetylase

The results of Torda and Wolff (1950) point to a control of acetylcholine synthesis in the brain by adrenal cortical secretions. Table X TABLE X Effect of Acetylcholine Synthesis of Hypophysectomy and Administration of A C T H to Hypophysectomized Rats*

Treatment

Sham operated Hypophysectomy Hypophysectomy and ACTH administration

Acetylcholine synthesized per 100 g. brain (net wt.) in 4 hours fig.

% -

14.7 k 0 . 3 8.21 f 0 . 6 13.40 f 0 . 3

100 56 91

* Torda and Wolff, 1950. indicates the results obtained with brain tissues from sham-operated, hypophysectomized, and hypophysectomized-ACTH-treated rats. Since the enzyme choline acetylase is sensitive t o inhibitors and

349

STEROIDS AND TISSUE OXIDATION

potentiating agents in cellular and extracellular fluids, it is not clear whether the change in acetylcholine synthesis is due to a direct change in enzyme concentration or whether regulatory factors may be quantitatively modified. This, as well as the direct action of adrenal cortical steroids, remains to be investigated.

111. In Vitro INFLUENCE ON OXIDATIVEMETABOLISM Gordan and Elliott (1947) reported that any of a group of steroids including desoxycorticosterone and testosterone could inhibit aerobic respiration of rat cerebral cortex homogenates. Desoxycorticosterone produced the most intense inhibition. The inhibitory action of the various steroids paralleled their anesthetic activity as described by Selye (1943). Subsequent studies by this group of workers (Eisenberg et al., 1949a) showed that the oxygen uptake of rat brains was increased by castration and that castration plus androgen treatment prevented the increment in brain oxygen uptake. These results are illustrated in Table XI. Further, testosterone added in vitro could inhibit the Qor of brain cells from normal, castrated, or castrated-testosterone-treated animals. Testosterone was added as a suspension at the level of 1mg. of testosterone per 2 ml. of solution or approximately 2 X 10-3 M . TABLE XI Respiration of Brain Cell Suspensions from Normal, Castrate, and Testosterone-treated Castrate Male Rats Effect of testosterone in vitro*

Type of rat Normal Castrate Castrate treated

No. rats

Qo, at 90 min. k SD

12 16 13

6 . 5 2 0.1 8 . 6 -1- 0 . 1 6.9 2 0.1

Final &or 180 Inhibition by Final Qo, 180 min. Tesmin. No terone added testosterone testosterone at 90 min. % 4.3 i O . 1 6.8 i O . 1 5.4 & 0.1

1.5 k O . 1 5.3 f O . l 2 . 6 +_ 0 . 1

65 22 52

* Eisenberg st ol.. 19498. Although castration had a profound influence on brain oxygen uptake, no such effect could be demonstrated in the liver, diaphragm, and levator ani muacle (Eisenberg et al., 1949b). However, castration did render the diaphragm muscle and liver tissue insensitive to the oxygen uptakeinhibiting action of testosterone. This phenomenon of insensitivity could be reversed by the in vivo treatment of the castrate rat with testosterone. The relative ability of various steroids to prevent the increase in

350

RALPH I. DORFMAN

brain oxygen uptake after castration is illustrated in Table XII. These rats were castrated a t 30 days of age. One milligram of steroid was administered daily for 7 days before the animals were decapitated at 60 days. TABLE XI1 Influence of Steroids on the Oxygen Uptake of Castrated Rat Brain Homogenate* &or

Type of rat

Steroid injected

pl./mg. dry wt./hr.

Normal male Castrated male Castrated male Castrated male Castrated male Castrated male Castrated male Castrated male Castrated male

0 0

5 . 4 f 0.1 7 . 3 f 0.1 6 . 5 zk 0 . 1 5 . 8 f 0.1 5.6 f 0.1 5.5 f 0.1 5.1 f 0.1 5.1 f 0.1 5 . 1 +0.1

Estradiol dipropionate ACTH (1.0 mg.-4 X daily) Methyl testosterone Testosterone propionate Testosterone Progesterone Testosterone cyclopentyl propionate

* Gordan d al., 19S1. The problem of the influence of steroids on the oxygen uptake of tissues was extended by Hayano et al. (1949). These investigators confirmed and extended certain aspects of Gordan's work. Liver, kidney, and brain tissues were studied as tissue slice preparations and as homogenates. Table XI11 summarizes the changes seen in rat tissues. These may be TABLE XI11 Steroids Causing Inhibition of Oxyven Uptake ~

~~

~

Concentration which produced inhibition of oxygen uptake*

Steroid ~

Liver slices molarity

Kidney slices molarity

Brain slices molarity

~

Dcsoxycorticosterone Deh ydroisoandrosterone Testosterone Methyl testosterone Progesterone ALAndrostenedione-3,17 Estrone Estradiol

* Haysno el ol., 1848.

1x 5x 2 x 1x 3 X

2 x 10-4 10-4 5 x lo-' 2 10-8 20 x 101 lo-' 10-8 10-5 Inactive at 3 X 10-8 Inactive at 3 x 10-8 3 X 10-8 Inactive a t 2 X lo-* Inactive a t 2 x 10-8 3 x 10-3 1 . 5 X 10-8 Inactive a t 2 X lo-* 10-4

10-4 10-8

2 2

x x x x

10-4

351

STEROIDS AND TISSUE OXIDATION

briefly indicated. Desoxycorticosterone, dehydroisoandrosterone, methyltestosterone, and testosterone consistently inhibited oxygen consumption at concentrations of the order of 2 X 10-3 M or less. No correlation of the magnitude of these inhibitions with the androgenic, protein anabolic, or renotropic activity was apparent. Modification of the steroid structure, on the other hand, appeared to determine in a small way the extent of the suppression. Those steroids containing keto groups were in general more inhibitory than those that only had hydroxyl functions. Hormonally inactive steroids were inactive with respect to their oxygen-inhibiting power. In the above experiments, desoxycorticosterone appeared to be the most active inhibitor. Accordingly, this steroid was selected for studies using a fractionated brain homogenate system. Table XIV illustrates TABLE XIV The InfEuence of Desoxycorticosterone on Ihe Oxygen Uptuke of Brain Hontogenate Fractions* Total pl. O2uptake Inhibition % per 90 minutes Supernatant and residue fractions Supernatant and residue fractions plus desoxycorticosterone Residue fraction Residue fraction plus desoxycorticosterone Supernatant fraction Supernatant fraction plus desoxycorticosterone

* Hayano et at..

120 49 95

59

41

57

42 33

22

1848.

these results. A homogenate of brain was prepared and tested in the presence of a boiled extract of liver and desoxycorticosterone. A strong suppression of oxygen uptake was noted. The homogenate was centrifuged and both the supernatant and residue fractions were studied for the desoxycorticosterone effect. The oxygen uptake of combined fractions was inhibited to the extent of 50%. The residue fraction alone was inhibited 57y0 while the supernatant alone was inhibited only 22%. Thus it appears that the insoluble particulate matter contains the enzyme system or systems on which the steroid desoxycorticosterone exerts its principal influence. The residue fraction was employed to study the effect of desoxycorticosterone on a variety of oxidizable substrates. These included hexoses, phophorylated intermediates, Krebs’ cycle components, and amino acids inhibited within 30 to 90%. These results pointed to the possi-

352

RALPH I. DORFMAN

bility that the site of steroid suppression was a reaction or reactions common to all oxidations; namely, those involving the transfer of hydrogen ions or electrons. A study of the cytochrome c-cytochrome oxidase reaction eliminated this system as the site of desoxycorticosterone action. A possible site for the inhibition is the flavoprotein entity of the electron transfer system. This aspect will be discussed in the following section. Kochakian (1951) in a recent communication has demonstrated that unlike most tissues, the respiration of primary Brown-Pearce epithelioma is not inhibited by testosterone (0.28 mM). Dirscherl and Hauptmann (1950) have reported that small amounts of androgens and estrogens stimulate anaerobic glycolysis and respiration of liver slices.

IV. STEROIDS INFLUENCING SPECIFICENZYME SYSTEMS 1 . n-Amino Acid Oxidase

The D-amino acid oxidase system is strongly inhibited by desoxycorticosterone and other steroids (Hayano et al., 1950). The characteristics of this inhibition have been studied in some detail. Figure 5

0

02 04 06 0.8 LO MG. DESOXYCORTICOSTERONE

FIQ. 5. The effect of desoxycorticosterone concentration on the activity of D-amino acid oxidase. Hayano et al. (1950).

illustrates the relationship between desoxycorticosterone concentration and extent of inhibition. Figure 6 is concerned with increasing concentrations of enzyme while the concentration of desoxycorticosterone remains constant. At low concentrations, complete inhibition could be demonstrated. Figure 7 presents the data on the influence of reaction time. In an incubation system containing both enzyme and inhibitor at zero time, some oxidation is apparent in the first 20 minutes. By 30

STEROIDS AND TISSUE OXIDATION

353

minutes, complete inhibition has been achieved. If the enzyme preparation is incubated for 5 to 10 minutes in the presence of desoxycorticosterone before the addition of the substrate, there is a complete suppression of activity which cannot be relieved by continued incubation. Figure 8 illustrates the influence of D-alanine concentration on the inhibition of the

0

a5

to

MG TISSUE N

~5

FIQ. 6. The effect of enzyme concentration on the inhibition of D-amino acid oxidase by desoxycorticosterone. Hayano et al. (1 950).

FIG.7. The effect of incubation time on the inhibition of D-amino acid oxidase by desoxycorticosterone. Hayano et al. (1950).

enzyme by desoxycorticosterone. This experiment was done to test the possibility that a competition of the substrate and desoxycorticosterone for the oxidase was the cause of the inhibition. When excess alanine was present at the start of the incubation, a protection of the enzyme against inhibition was seen, that is, at ten times the usual concentration of the amino acid a complete protection was noted. The addition

354

RALPH I. DORFMAN

of these large quantities of alanine 15 minutes after the start of incubation, after inhibition had been established, brought about no relieving of the inhibition. An experiment was carried out with excess isoalloxazine adenine dinucleotide (FAD) additions t o test the possibility of the inhibition being due to a competition of FAD and the steroid for the apoenzyme of the oxidase (Fig. 9). Here again additional FAD present a t the beginning of the incubation protected the enzyme from inactivation while additions after the start of the incubations brought no relief.

+

0

C

€

m

T

m

Ql 0.2 Q3 QQ FINAL MOLARlTY,OF DL-ALANINE

FIG.8. The effect of alanine concentration on the inhibition of n-amino acid oxidase by desoxycorticosterone. Curve A, alanine concentrations as indicated on the abscissa present in center compartment at zero time. Curve B, 0.04 M Dbalanine present a t zero time. Additional alanine to equal final molarities as indicated along the abscissa was tipped in from the side arm 15 minutes after the start of the incubation. O2uptake measurements were taken from the time of this addition. Hayano et at. (1950).

Thus it appeared that the inhibiting action of desoxycorticosterone on the enzyme system was not one of a substrate or prosthetic group competitive type. From the experiment illustrated in Fig. 10 where treatment of the steroid with FAD resulted in only a 30% depression of oxygen uptake while that with the apoenzyme was almost complete, it was concluded that the desoxycorticosterone reacts with the protein entity of the D-amino acid oxidase system. The steroid inhibition was not specific for substrate D-alanine alone since similar results were obtained with DL-isoleucine and DL-methionine. The action of the steroid on the enzyme is reversible. It is possible

STEROIDS AND TISSUE OXIDATION

355

to regenerate an active preparation by means of acetone precipitation of the enzyme. This treatment removes the desoxycorticosterone and regenerates a completely active enzyme.

*.I

I

/

t

15

30

45

TIME IN MINUTES

60

FIG.9. The effect of FAD on the inhibition of D-amino acid oxidase by desoxycorticosterone. Curve A, 0.0025 p M FAD present in the center compartment at zero time. Curve B, 0.015 pill of FAD was tipped in from the side arm after 30 minutes incubation. Curve C, no additionnl FAD. Hayano et al. (1950). CONTROLS

/

FIG.10. The effect of desoxycorticosterone on the coenzyme and apoenzyme of D-amino acid oxidase. Final substrate concentrations, m-alanine, p H 8.5, 0.4 M ; pyrophosphate buffer; pH 8.5,0.017M ; apoenzyme 1 mg.; FAD 0.025 p M . 1 mg. of desoxycorticosterone and FAD or apoenzyme were incubated 20 minutes at 38" before the addition of the other components of the complete system. Hayano et al. (1950).

Desoxycorticosterone was the most potent free steroid with respect to inhibition. The influence of a variety of steroids is described in Table XV. In the adrenal cortical series, no correlation could be found between

356

RALPH I. DORFMAN

TABLE XV Comparative Activity of Various Steroids on D-Amino Acid Oxidase* Inhibition produced by steroid Ratio Inhibition produced by 1 mg. DOC Mean inhibition of Mean desoxycorticosterone inhibition (1 mg.) run simulConc. % taneously % Ratio = Steroid mg. (Range) (Range) Steroid/DC C-21 STEROIDS 0.45 82 Progesterone 1.0 37 (3144) (68-97) 0.06 86 Ethynyltestosterone 1.o 5 (80-92) (3-6) 0.20 90 Desoxycorticosterone acetate 18 1.0 (11-25) (77-100) 0.38 Desoxycorticosterone glucoside 1.0 90 34 (83-97) (22-46) 0.20 1.0 14 Corticosterone 69 (3-16.3) (64-84) 0.19 1.0 16 11-Dehydrocorticosterone 84 0.0 17-Hydroxy-1I-Desoxycorticos- 1.o 0 65 terone (57-73) ( -9-8) 0.25 17-Hydroxycorticosterone 1.0 14 57 (13-14) 17-Hydroxy-1l-dehydrocorti0.40 87 1.0 35 costerone Allopregnanetriol-3(a),17(a),210.07 84 one-20 1.o 6 ANDROQENS 0.65 1.0 55 Sodium androsterone sulfate 85 (78-92) (52-57) 0.88 92 81 2.0 0.65 55 Sodium dehydroisoandrosterone 1.0 85 (51-58) sulfate (78-92) 0.94 2.0 86 92 0.13 1.0 10 Testosterone 77 (49-100) (-2-18) 0.12 10 1.0 86 Androsterone (2-20) (80-92) 0.16 75 1.0 12 Dehydroisoandrosterone (5-18) (64-80) 0.13 71 1.o 9 (6-11) (49-92) 0.00 86 1.o 0 Androstanediol-3(a), 17(B) (-54) (80-92)

357

STEROIDS AND TISSUE OXIDATION

TABLE XV.-(Continued)

Steroid

Mean inhibition by Mean desoxycorticosterone inhibition ( 1 mg.) run simulConc. % taneously % Ratio = mg. (Range) (Range) Steroid/DC

ANDROGENS As-Androstenediol-3(@), 17(@) Methyltestosterone 17(a)-Hydroxyprogesterone

1 .o

7 (2-12) 1.0 -8 ( - 16-2) 1 .o 3 (2-3)

92 (92-92) 90 (77-100) 92 (92-92)

0.08 -0.08 0.03

ESTROGENS Sodium estrone sulfate

0.1 0.2 0.5

1.0 2.0 Sodium estradiol sulfate

Sodium equilen sulfate

Estrone

1.0

2.0 1.0 2.0 1 .o 1.0

18 28 58 96 (96-96) 98 86 (76-95) 97 86 100

6

( - 1-12)

-2 ( -8-5)

Equilen

1.o

4 (3-5)

80 80 80 80 (80-80)

0.22 0.35 0.72 1.20

80

1.23 1.00

86 (80-92) 80 64 64 65 (49-80) 65 (49-80) 86 (80-92)

1.21 1.34 1.56 0.09 -0.03 0.05

MISCELLANEOUS Cholesterol

1.0

Pregnanediol-3(a),20(a)

1.0

21-Chloroprogesterone

1 .o

AcPregnenediol-17,20-one-3 20,2 1-Epoxy-A'-pregnenediolone-3 Sodium cholesterol sulfate

1.0 1.0 1.o

2.0

* Hayano and Dorfman. 1951.

16 (4-27) -8 (-5--10) 6 ( - 12-22) 20 12(11-13) 5 (2-8) 18

75 (49-100)

70 (49-92) 93 (83-100) 92 79 (77--80) 72 (64-80) 64

0.21 -0.11 0.07 0.22 0.15 0.04 0.28

358

RALPH I. DORFMAN

activity and polarity. Thus, 17-hydroxycorticosterone, the most polar in this series, yielded an inhibition ratio of 0.25 (see Table XV). Hormonally-inactive steroids such as cholesterol, 20,21-epoxy-Aepregnenoione-3, pregnanediol-3a,2Oa and A4-pregnenedione-17,20-one-3had no inhibitory activity. Even the sodium cholesterol sulfate was inactive. This is in contrast to the finding that such steroids as estrone, estradiol, and dehydroisoandrosterone, although completely inactive, on possessing modest activity as the free compound show high activity as the sodium sulfate ester.

FIG.11. The effect of prior incubation of testosterone and desoxycorticosterone with D-amino acid oxidase before the addition of substrates. Desoxycorticosterone 1 mg., testosterone 1 mg. Alanine was tipped in from the side arm at the times indicated on the abscissa. Hayano et at. (1950).

Figure 11 illustrates another point of interest. Substances like testosterone produce only a slight inhibition (19%) when the system is complete a t zero time as compared with 53 % inhibition if a preincubation period of 30 minutes a t 38°C. is permitted. 2. a-Glycerophosphate Dehydrogenase

Hochster and Quastel (1951) have studied the influence of a variety of steroids on the anaerobic and aerobic oxidation of a-glycerophosphate. The enzyme preparations were derived from both yeast (Tables XVI, XVII) and rat liver (Table XVIII). Essentially steroids have an in vitro inhibitory action on the a-glycerophosphate dehydrogenase system. 3-ketosteroids possess the highest inhibitory activity while 17- and 20-ketosteroids possess activity but a t a reduced level. Brain homogenates are capable of neutralizing the steroid inhibition of the yeast dehydrogenase system.

359

STEROIDS AND TISSUE OXIDATION

TABLE XVI Inhibitory Effects of Steroids and Quinones on the Anaerobic Oxidation of a-Glycerophosphate from Yeast, with Ferricyanide-Manganese Dioxide as the Hydrogen Acceptor (2 mg. of steroid in 3.2 ml.) *

Steroid A4-Androstenedione-3,17 Androstanedione-3,17 Testosterone Dehy droisoandrosterone As-Androstenediol-3 (@),17(8) Progesterone Pregnenolone Desoxycorticosterone acetate Estrone Estradiol-17(@) Cholic acid Dehydrocholic acid As-3-Oxycholenic acid Cholesterol A*-Cholestenone-3 7-Ketocholesterol acetate Diethylstilbestrol quinone 2-Methyl-l14-naphthoquinone

* Hochster and Quastel.

Per cent inhibition 27°C. 37°C. 0.009 M 0.037 M Substrate Substrate 84 79 86 46 0 80 20 45 0 13 35 17 54 0 5

0 98 44

82 73 79 61 14 79 42 38 21 38 56 26 55 0 0 2 89 71

1951.

TABLE XVII Effect of Steroids and Diethylstilbeslrol on Aerobic Oxidation of a-Glycerophosphate by Yeast Catalyzed by Methylene Blue (2 mg.of steroid in 3.2 ml) * Steroid A4-Androstenedione-3,17 As-Androstenediol-3 ( p ) , 17(@) Dehydroisoandrosterone Testosterone Progesterone Pregnenolone Cholesterol Diethylstilbestrol Hoahster and Quastel, 1951.

Per cent inhibition 60 minutes 120 minutes 48 0 0 52 51 0 0 100

58

0 5 46 71 0 0 90

360

RALPH I. DORFMAN

TABLE XVIII Efects of Steroids and Diethylstilbestrol on the Anaerobic Ozidution of a-Glycerophosphate by a Rat Liver Homogenate in the Presence of DPN, Nicotinamide and Ferricyanide and Manganese Dioxide as the Terminal Hydrogen (2 mg. of steroid in 3.2 ml)* Per cent inhibition 60 minutes

Steroid A4-Androstenedione-3,17 As-Androstendiol-3(P),17(8) Dehydroisoandrosterone Testosterone Progesterone Pregnenolone Cholesterol Diethylatilbestrol

43 9 22 48 70 25 0 42

* Hochster and Quastel, 1951.

3. Alkaline Phosphatase Aldman et al. (1951) have shown that certain phosphate esters of estrogenic steroids inhibit the action of rabbit kidney alkaline phosphatase. The enzyme preparation was a highly purified material which was active toward phenyl phosphate, a-glycerophosphate and glucose-6phosphate. Table XIX illustrates the comparative inhibitory activities of various estrogenic phosphates. The diphosphate ester of estradiol was the most potent. The inhibition is independent of the substrate employed, is non-competitive, and is reversible. TABLE XIX Inhibition of Kidney Alkaline Phosphulase by some Derivatives of Estrogenic Hormones (Acetate, carbonate-borate buffer, pH 9.3 16 min. incubated a t 37". Substrate 0.004 M-phenyl phosphate. Enzyme protein concentration 0.81 pg./ml.) * Inhibitor Estradiol diphosphate Estradiol 3-phosphate Estradiol 17-phosphate Estrone glucoside

Concentration M

x x 1x 2x I 2

10-6 10-4 10-4 10-4

Per cent inhibition 38 5

19 0

Aldman e4 el., 1961.

4. Succinoxidase Meyer and McShan (1950) have reviewed various aspects of the in uitro inhibition of the succinoxidase system. Diethylstilbestrol

361

STEROIDS AND TISSUE OXIDATION

effectively inhibits this enzyme system in brain, adrenal, pituitary, lutein, kidney, and heart tissue. At concentrations in the range of 2X M , diethylstilbestrol gave complete inhibition. Table XX lists a variety of compounds studied as to their inhibitory effect on the TABLE XX Effect of Various Compounds on the Succinoxidase System of Rut Liver* Per cent inhibition at final cone. of compound X 10-4 M:0.5 1.0 2.0 4.0

Compound Sodium estrone sulfate Premarin

NATURAL ESTROGENS

-

19 10

27 17

-

78 77 77

-

-

-

6 0

16 1

42

0 1

1 8

2 0

0 96

3 97

-

SYNTHETIC ESTROGENS

Diethylstilbestrol Hexestrol Dienestrol Sodium 3,4 diphenylhexane-p hydroxy p‘ oxyacetate Disodium 3,4-diphenylhexane-p’-dioxyacetate ANDROGENS Sodium androsterone sulfate Sodium dehydroisoandrosteronesulfate INACTJYE PHENOLS Phenol Hydroquinone 91

-

-

-

-

2

* Meyer and McShan, 1950. succinoxidase system of rat liver. Essentially, the amount of inhibition observed is dependent upon the number of phenol groups present, although pure phenol had no influence on the enzyme. The two androgens studied, which have no phenolic groups, were inactive. Some non-phenolic, non-hormonally active compounds have been shown to M ) . The possess inhibitory activity at low concentrations (1 X inhibitory nonphenolic compounds include l-keto-l,2,3,4-tetrahydrophenanthrene and dianisalacetone. Meyer and McShan (1946) found that diethylstilbestrol is more effective than malonate in inhibiting the succinoxidase system and that the cytochrome c was in the reduced state after being in contact with this synthetic estrogen. On the basis of these observations, these workers suspected that the phenolic groups of the estrogens combined with the active centers of the oxidase and in this manner inhibited the enzyme. When cytochrome c was replaced with brilliant cresyl blue, no inhibition was noted. Thus it appears that the major part of the

362

RALPH I. DORFMAN

inhibition is through cytochrome oxidase and only a minor part is due to a direct action on succinic dehydrogenase. Case and Dickens (1948) studied a number of compounds on the succinoxidase system of rat liver homogenates. They found that 1 (4'-hydroxyphenyl)-2-phenylethaneinhibits the complete succinoxidase system as well as cytochrome oxidase and succinic dehydrogenase. Of particular interest is the inhibitory action of 4,,4'-dihydroxystilbene. This compound inhibited the complete succinoxidase system but was inactive with respect to either cytochrome oxidase or succinic dehydrogenase. These results have been confirmed (Meyer and McShan, 1950). 6. Malic Oxidase

The influence of various synthetic estrogens and androgens on the malic oxidase system is similar to that described for the succinoxidase system. Diethylstilbestrol, hexestrol, and dienestrol inhibit this system M . Sodium androsterone sulfate was at concentrations of 1 X inactive (Meyer and McShan, 1950). The inhibition of malic oxidase is due mainly to the action on cytochrome oxidase. 6. Choline Acetytase

Torda and Wolff (1950) have studied the influence of steroids on the synthesis of acetylcholine by minced frog brain. Cholesterol had no in vitro influence, but testosterone, dehydroisoandrosterone, methyl testosterone, and A'-androstene-3,17-dione depressed the synthesis. 7. Miscellaneous

Hayano et al. (1950) studied the influence of desoxycorticosterone on a variety of enzyme systems as illustrated in Table XXI. Desoxycorticosterone was chosen as the steroid representative since it had the greatest inhibitory activity toward various tissues and D-amino acid oxidase. In addition to D-amino acid oxidase, tyrosinase was strongly inhibited while urease, ascorbic acid oxidase, lipase, and transaminase were partially inhibited. On the other hand, the activity of glutaminase, the decarboxylation of pyruvate by whole yeast cells, and the hydrolysis of denatured hemoglobin by trypsin were increased in the presence of desoxycorticosterone. Dirscherl and Knuchel(l950) have reported the activation of enolase, hexokinase and carboxylase by androgens and estrogens. Opsahl (1950) has described the in vitro inhibition of hyaluronidase by adrenal cortical steroids. Other biologically active steroids such as progesterone and testosterone were practically without influence.

363

STEROIDS AND TISSUE OXIDATION

TABLE X X I The Influence of Desoxyeortieosterone on Enzyme Systems* (1 mg. of steroid per 3.0 ml. of solution)

Enzyme D-Amino acid oxidase Tyrosinase Urease Ascorbic acid oxidase Lipase

Source Substrate Aqueous extract of pig DbAlanine kidney acetone powder Aqueous extract of po- Tyrosine tato peeling Urea Commercial Aqueous extract of squash Commercial

Ascorbic acid Butyrin

Transaminase

Saline extract of rabbit Glutamic and heart muscle oxalacetic acids Ribonuclease Crystalline enzyme Yeast nucleic acid Succinic dehydrogenase Rat liver homogenate Succinic acid Arginase Trypsin Cytochrome oxidase Cytochrome oxidase IcAmino acid oxidase ATP-ase Protease Jackbean carboxylrtse Amylase

Purified preparation from beef liver Commercial

Arginine

Brain homogenate residue Rat liver hornogenate

Phenylenediamine Ascorbic acid

Casein

Venom from Agkistrodon mokasen Rat liver homogenate

tleucine

Rhozyme-DX (Rohm & Haas) Jackbean meal

Gelatin

ATP

Pyruvic acid Starch

Pepsin

Rhozyme-DX (Rohm & Haas) Commercial

Acid Phosphatase

Polidase-S (Schwarz)

Carboxylase

* Hayano and Dorfman, 1961.

Hemoglobin

Change in activity range % -83 (-49 to -100) -98 (-95 to - 100) -39 (-27 to -50) -28 ( - 2 3 t 0 -32) -27 (-25 to -28) -21 ( -14 to -28) -12 (-6 to -17) -10 ( - 7 t o -12) -9 (-6 to -12) -9 (-3 to -15) -9 (-7 to -11)

4-6 (4-3 to +8) -6 ( - 5 to -6) -4 (-2 to -5) -4 (-4 to -4) -4 (-9 to f l ) -2 (-4 to 0) 0 ( - 5 to + 5 ) f 2 ( + I to +3)

364

RALPH I. DORFMAN

TABLE XX1.-(Continued) Change in activity range, Enzyme Xanthine oxidase Glutaminase Trypsin Yeast carboxylase

Source Liver xanthine oxidase

Substrate

%

+12

Xanthine

Rat and rabbit kidney Glutarnine homogenates Commercial Denatured hemoglobin Bakers’ yeast whole Pyruvic acid cells

( + l o to +13) 26 (12 to +36)

+73 (+44 to +124)

+I10 (+lo6 to +116)

V. I n Vitro EFFECTS ON METABOLISM 1. Diaphragm Verzdr and Wenner (1948a) have reported that desoxycorticosterone increased the glycogen breakdown of surviving rat diaphragm and can counteract the stimulating effect of insulin on this tissue. The technic is essentially that previously used by Gemmill and Hamman (1941), who showed that insulin stimulated glycogen production in the surviving rat diaphragm. In another paper (VeraL and Wenner, 1948b), the specificity of desoxycorticosterone was studied for this reaction. A variety of steroids and related compounds were studied (Table XXII). In addition to desoxycorticosterone, progesterone, As-pregnenediol3(8),2l-one-3, testosterone, and stilbestrol showed high inhibitory activity. 17-Hydroxycorticosterone, the adrenal cortical steroid possessing the highest in vivo activity on carbohydrate metabolism, was practically inactive. The finding of Verz4r’s group that desoxycorticosterone inhibits glycogenesis from glucose and abolishes insulin stimulation in the isolated rat diaphragm has been confirmed using isotopic glucose (Bartlett and MacKay, 1949; Bartlett et al., 1949). Leupin and Verzk (1950) studied the simultaneous glycogen formation and glucose uptake by isolated diaphragm muscle of normal and adrenalectomized rats. These experiments were done in the presence of glucose and insulin. A significant decrease (80%) in glycogen formation and a smaller decrease (57%) in glucose uptake was produced by 5 mg./ 100 ml. desoxycorticosterone. Cortisone produced the same effect but was significantly less active. In another study (Leupin, 1950) an unsuccessful attempt was made to demonstrate changes in deamination in

STEROIDS AND TISSUE OXIDATION

365

TABLE XXII The in Vitro Effect of Various Steroids and Related Substances on the Glycogen Content of Rat Diaphragm in the Presence of Glucose and Insulin* Change in glycogen content after Concentration steroid addition % Steroid mg./100 ml. C-21 STEROIDS Desoxycorticosterone 1 -5 5 -36 -50 10 Corticosterone 5 -15 1 -3 17-Hydroxycorticosterone 5 -9 10 20 11-Dehydrocorticosterone Progesterone 1 -23 5 -45 10 -42 ' 10 -46 17-Hydroxyprogesterone Ab-Pregnendiol-3(8),2 l-one-3 1 - 11 5 -36 10 -48 5 - 14 Pregnandiol-3 (a),12(8)-one-20 10 -30 Allopregnanepentol-3(p),11(B), 17(cr),20,21 10 -11 Desoxycorticosterone glucoside 10 -8 10 -39 21-Acetoxypregnenolone C-19 STEROIDS 1 -37 Testosterone 5 - 19 10 -46 Androstanediol-3(p),17(?) 5 -18 10 -25 5 -9 AK-Androstenediol-3(B), 17(@) 10 -7 C-18 STEROIDS Estradiol 5 -3 10 -23 MISCELLANEOUS COMPOUNDS Bisdehydrodoisynolic acid 5 0 10 -26 10 +4 3 (8)-Hydroxyetiocholenic acid Stilbestrol 1 - 19 5 -42 10 -51 Oleic acid (Na salt) 5 +7 10 +7 ~~

* VerzBr and Wenner, 1Q48b.

366

RALPH I. DORFMAN

surviving rat diaphragm under the influence of added corticoids. The system contained glucose and insulin similar to the previous studies in which glycogen production was measured. This work is discussed in more detail by Verzdr in the preceding review in this volume. 2. Liver

Chiu and Needham (1950) studied the in vitro influence of adrenal cortical extracts using the system of Seckel (1940). This latter worker showed that the addition of adrenal cortical extract to liver slices caused a decrease in glycogen disappearance. Chiu and Needham (1950) and Chiu (1950) confirmed and extended the finding of Seckel (1940) that adrenal cortical extracts added in vitro cause a diminution of glycogen breakdown in liver slices. This was shown for rat and rabbit tissues. Oxygen is required for this inhibition. It appears that the active materials affect the synthesis and not the breakdown of glycogen. The extract also prevented in part the disappearance of carbohydrate. The adrenal cortical extract also increased the synthesis of carbohydrate. No effect was discernible on the ATP levels of the tissue slices during carbohydrate synthesis in oxygen. In the second communication, Chiu (1950) showed that 11-desoxycorticosterone, 11-dehydrocorticosterone, and cortisone were all effective in replacing the adrenal cortical extract. In all experiments with these steroids there was greater increase in carbohydrate content or less carbohydrate disappearance in the presence of the steroid. The steroids had no significant effect on oxygen uptake. Preliminary experiments indicated a slightly increased concentration of nonprotein nitrogen in the presence of pure steroids or adrenal cortical extracts.

VI. CONCLUSIONS Data have been presented on the influence of steroid hormones on tissue metabolism with emphasis on the relationship of the steroids t o specific enzyme systems. In the introduction, it was mentioned that a group of investigators have been attempting to answer the questions: Do steroids influence tissue metabolism by their influence on enzyme systems? If such is the case, what is the nature of these influences? In the introduction we set out the possibilities that: (a) Steroid hormones may influence enzyme concentrations; (b) Steroid hormones may function as components of an enzyme system; (c) Steroid hormones as such may accelerate or inhibit enzyme systems; and (d) Steroid hormones may directly or indirectly influence accelerators and/or inhibitors of enzyme systems.

STEROIDS AND TISSUE OXIDATION

367

Possibility (a) has been answered in the affirmative. Steroid hormones can influence enzyme concentrations. Many examples have already been cited in the text. It is most likely that increased concentrations of certain steroid hormones distinctly influence enzyme concentrations in tissues. Arginase is an outstanding example. In addition to arginase concentration changes, amino acid oxidase, cholinesterase, phosphatases, P-glucuronidase, esterase, proline oxidase, ATP-ase, and choline acetylase concentrations are in part controlled by steroid hormones. In some cases, more evidence is needed to establish the fact that actual enzyme concentration changes are involved rather than influences on accelerators and inhibitors. An intriguing aspect of enzyme concentrations being modified by steroid hormones is the element of tissue specificity. Although no discernible difference has been detected between proline oxidase from liver and the same enzyme from kidney, only the kidney enzyme concentration decreases in the absence of adrenal cortical steroids. In a similar manner, adrenalectomy causes a decreased concentration in liver D-amino acid oxidase, but no enzyme concentration change in the kidney (Umbreit, 1951). Such specificity cannot be explained at this time, and points to fertile fields of future investigations. Returning to our set of logical possibilities, let us consider (b): Do steroid hormones function as components of enzyme systems? No answer can be given to this question. However, since protein steroid complexes are indicated from the work of Roberts and Szego (1946), we can only say that a model of an enzyme can be visualized with the steroid being the prosthetic group. A working hypothesis of this may prove to be a fruitful line of research. Do steroid hormones as such accelerate or inhibit enzyme reactions? Here there appear to be model experiments; the influence of desoxycorticosterone on the D-amino acid oxidase (Hayano et al., 1950), and the influence of ketosteroids, particularly the 3-ketosteroids, on the cu-glycerophosphate dehydrogenase system (Hochster and Quastel, 1951). In both instances the action is one of inhibition. The D-amino acid oxidase inhibition was studied in some detail. High concentrations of the substrate D-alanine protected the enzyme from inhibition almost completely, but did not relieve the inhibition once it had been established. The inhibition was found to result from a reaction of the steroid with the apoenzyme of D-amino acid oxidase. The inhibition was reversed by an acetone precipitation treatment of the inhibited enzyme with the recovery of essentially all the original enzyme activity. These examples of steroid hormones exerting inhibitory actions on enzyme systems do not necessarily imply that these restraints take place

368

RALPH I. DORFMAN

in the intact organism. They only point the way toward a possibility. Further facts are needed. Finally, we ask the question: Do steroid hormones directly or indirectly influence acceleration and/or inhibitors of enzyme systems? The answer to this question is linked with our first question: Do steroid hormones influence enzyme concentrations? It is quite likely that in some instances the change in enzyme concentration is only apparent-the real change may be due to influences on cofactors, inhibitors, or accelerators. An indication of such a possibility is seen in the recent work of Umbreit and Tonhazy (1951b). After adrenalectomy in the rat there is an increase in factors causing reduction in enzyme activities. A number of substrates such as a-ketoglutarate, fumarate, and oxaloacetate are oxidized at a reduced rate. The destructive factors causing the lowering in rate of oxidation can be counteracted by increasing the adenylate additions to such homogenates. The destructive factors can be controlled also by treatment of the adrenalectomized rat with cortisone. Again, it may be said that steroid-enzyme relationships have only recently been studied: the field is in its infancy and the possibilities many. REFERENCES

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Fraenkel-Conrat, H., Simpson, M. E., and Evans, H. M. 1943a. J. Biol. Chem. 147, 99-108. Fraenkel-Conrat, H., Simpson, M. E., and Evans, H. M. 1943b. Am. J . Physiol. 138, 439-449. Gemmill, C. L., and Hamman, L. 1941. Bull. Johns Hopkins Hospital 68, 50-57. Glick, D. 1934. Z . physiol. Chem. 223, 252. Gordan, G. S., Bentenick, R. C., and Eisenberg, G. 1951. Ann. N.Y. Acad. Sci. (in press). Gordan, G. S., and Elliott, W. H. 1947. Endocrinology 41, 517-518. Gutman, A. B., and Gutman, E. B. 1938. Proc. SOC.Exptl. Biol. Med. 39,529-532. Gutman, A. B., and Gutman, E. B. 1939. Proc. SOC.Exptl. Biol. Med. 41, 277-281. Harris, R. S., and Cohen, S. L. 1951. Endocrinology 48, 264-272. Hayano, M., and Dorfman, R. I. 1951. Ann. N.Y. Acad. Sci. 64, 608-618. Hayano, M., Dorfman, R. I., and Yamada, E. Y. 1950. J. Biol. Chem. 188,603--614. Hayano, M., Schiller, S., and Dorfman, R. I. 1949. Endocrinology 46, 387-391. Hochster, R. M., and Quastel, J. H. 1951. Ann. N . Y . Acad. Sn'. 64, 626-635. Holman, H. R., White, A., and Fruton, J. S. 1947. Proc. SOC.Exptl. Biol. Med. 66, 196-199. Huggins, C., and Hodges, C. V. 1941. Cancer Research 1, 293-297. Huggins, C., Masina, M. H., Eichelberger, L., and Wharton, J. D. 1939. J. Exptl. Med. 70, 543. Huggins, C., Scott, W. W., and Hodges, C. V. 1941. J. Urol. 46, 997-1007. Humm, J. H., Kochakian, C. D., and Bartlett, M. N. 1948. Am. J. Physiol. 166, 251-254. Jensen, H. & Gray, J. L. 1951. Ann. N . Y . Acad. Sci. 64, 619-625.1 Johansen, A., and Thygessen, J. E. 1951. Scand. J. Clin. Lab. Invest. 3, 66-70. Kerr, L. M. H., Campbell, J. G., and Levvy, G. A. 1949. Biochem. J . 44,487-494. Kerr, L. M. H., Campbell, J. G., and Levvy, G. A. 1950. Biochem. J. 46, 278-284. Klein, J. R. 1939. J. Biol. Chem. 128, 659. Kochakian, C. D. 1945. J. Biol. Chem. 161, 115-125. Kochakian, C. D. 1947. Recent Progress i n Hormone Research 1, 177-216. Kochakian, C. D. 1951. Ann. N. Y. Acad. Sci. 64, 534. Kochakian, C. D., and Bartlett, M. N. 1948. J . Biol. Chem. 176, 243-247. Kochakian, C. D., Bartlett, M. N., and Gongtira, M. N. 1948. Am. J. Physiol. 163, 210-214. Kochakian, C. D., and Clark, L. C., Jr. 1942. J. Biol. Chem. 143, 795-796. Kochakian, C. D., and Robertson, E. 1950a. Arch. Biochem. 29, 114-123. Kochakian, C. D., and Robertson, E. 1950b. Federation Proc. 9, 191. Kochakian, C. D., and Vail, V. N. 1947. J . Biol. Chem. 169, 1-6. Kutscher, W., and Wolberg, H. 1935. 2. physiol. Chem. 236, 237-240. Leonard, S. L. 1950. Endocrinology 47, 260-264. Leupin, E. 1950. Biochem. J. 46, 567-568. Leupin, E., and VereAr, F. 1950. Biochem. J. 46, 562-566. Levvy, G. A., Kerr, L. M. H., and Campbell, J. G. 1948. Biochem. J. 42,462-468. Meyer, R. K., and McShan, W. H. 1950. Recent Progress in Hormone Research 6, 464515. McShan, W. H., and Meyer, R. K. 1946. Arch. Biochem. 9, 165-173. McShan, W. H., Meyer, R. K., and Erway, F. 1947. Arch. Biochem. 16, 99-110. Opsahl, J. 1950. Josiah Macy, Jr. Foundation Conference on the Adrenal Cortex. Vol. 2, pp. 115-163.

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Roberts, S., and Szego, C. M. 1946. Endocrinology 38, 183-187. Schwartz, T. B., and Engel, F. L. 1949. J . Biol. Chem. 180, 1047-1052. Seckel, J. P. G. 1940. Endocrinology 20, 97-101. Selye, H. 1943. Encyclopedia of Endocrinology. A. W. T. Franks, Montreal, VOl. IV, p. 19. Stafford, R. O., Rubinstein, I. M., and Meyer, R. K. 1949. Proc. Soc. Ezpll. B i d . Med. 71, 353-357. Tipton, S. R. 1944. Endocrinology 84, 181-186. Tipton, S.R., Leath, M. J., Tipton, I. H., and Nixon, W. L. 1946. Am. J. Physiol. 146,693-698. Torda, C., and Wolff, H. G. 1950. Am. J . Physiol. 161, 534-539. Umbreit, W. W. 1951. Ann. N.Y. Acad. Sci. 64,569-574. Umbreit, W. W., and Tonhazy, N. E. 1951a. J . B i d . Chem. 191, 249-256. Umbreit, W. W., and Tonhazy, N. E. 1951b. J . B i d . Chem. 191, 257-261. VerzBr, F., and Wenner, V. 1948a. Biochem. J. 42, 3 5 4 1 . VerzBr, F., and Wenner, V. 194813. Biochem. J. 42,48-51. Wattenyl, H., Bessinger, A., Maritz, A., and Zeller, E. A. 1943. Helu. Chim. Actu 20, 2063-2070. Williams, H. L., and Watson, E. M. 1941a. Endocrinology 29, 250-257. Williams, H. L., and Watson, E. M. 1941b. Endocrinology 29, 258-266. Wollman, S. H., and Scow, R. 0. 1951. Endocrinology 49, 105-109.

Author Index A Abelin, I., 125, 129 Abinzano, C., 64, 65 Abt, A. F., 55 (see Kagan), 66 Abul-Fadl, A. & 319, I., 326 Atlamstone, F. B., 96, 129 Adler, A. A., 276, 291 Adlersberg, D., 48, 49, 62, 65 Adolph, W. H., 35, 42 Agnew, L. R. C., 119, 120, 129 Agnoli, R., 93, 129 Agrawala, I. P., 84, 136 Alam, M., 26, 42 Albanese, A. A., 96, 97, 129, 133 Albert, A., 146 (see Gastineau), 150, 163, 165, 115, 177,287 (see Sprague), 294 Albright, F., 83 (see Klinefelter), 134, 100, 175 Alrayaga, R., 114 (see Wintrobe), 140 Aldnian, B., 360, 368 Alexander, L., 30, 31, 42 Alfredson, B. V., 245 (see Mullick), 249 Allen, C. E., 227, 236, 247 Allen, E., 88, 126, 132 Allen, F. €I.,Jr., 49 (see May), 50, 54, 66 Allen, F. M., 186, 187, 209, 210, 211 Allen, J. G., 190 (see Brunschwig), 212 Allen, R. S., 247 Allen, M7.RI., 81, 139, 276, 291, 292 Altschule, M. D., 49, 66 Anderson, E., 193 (see Marx), 204, 205,

Argetsinger, H. L., 98 (see Sharpless), 138 Aring, C. D., 6, 7, 8, 9, 42 Argonz, J., 64, 66 Arons, P., 91, 129 Arvy, L., 95, 129 Aschheim, S., 165, 175 Aschkenasy-Lelu, P., 05 (see Arvy), 101, 129, 139

Ashburn, L. L., 111 (see McQueeney), 112, 114, 115, 129, 135 Ashenbrucker, H., 128 (see Hamilton), 133

Ashworth, J., 83, 139 Ascoli, M., 192, 211 Asley, C., 154, 177 Astwood, E. B., 94, 99 (see Grew), 118, 129, 1SS, 1S9

Atkinson, W. D., 341, 368 AubeI, C. E., 91 (see Hughes), 134 Azevedo, M. D., 75, 76

B

Babcock, S. H., Jr., 111 (see Daft), 130 Bacchus, H., 109, 123, 129 Bacesco, M., 70, 75, 77 Bachman, C., 147, 164, 176 Badinez, O., 123, 129 Biilz, E., 4, 10, 16, 17, 18, 42 Bailey, C. C., 190, 191, 192, 193, 211, 214 Bailey, G. L., 219, 220, 221, 222, 224, 225, 226, 237, 247 $13, 214 Bailey, 0. T., 190, 193 (see B., C. C.),211 Anderson, E. N., 84, 127, 129, 130 Baillie, J., 73, 76 Anderson, J. A., 299 (see Buell), 326 Balassa, G., 102, 129 Anderson, L. A. P., 41, 42 Baldwin, W. H., 70, 77 Anthony, E. K., 98 (see Sharpless), 138 Balmary, J., 168, 176 Antopol, W., 9, 42 Banta, A. M., 73, 76, 77 Antoshkiw, T., 55, 65 Banting, F. G., 186, 211 Aposhian, A. V., 271 (see Kochakian), Baptist, M., 153 (see Hamblen), 154, 168 293 (see Davis), 176, 177 Archibald, J. G., 224, 235, 247 Barker, S. B., 127, 129 Archibald, R. M., 192, 911 Barnes, A. C., 165, 179 371

372

AUTHOR INDEX

Bentenick, R. C., 350 (see Gordan), 369 Barnes, B., 49, 66 Barnes, B. O., 203, 211 Benua, R. S., 165 (see Morrow), 179 Barrie, M. M. O., 92, 94, 129 Bercovite, Z., 49, 66 Barron, E. S. G., 192, 211, 216, S68 Berg, C. P., 129 Barrows, L., 126, 130 Berger, H. hf., 49 (see Kramer), 55, 66 Barry, M. C., 254 (see Gallagher), 255, Berkson, J., 150, 163, 165, 176 Berkwitz, N. J., 29, 42 278, 292 Berman, H., 122 (see Gardner), 232 Bartelmez, G. W., 156, 176 Bartlett, G. H., 299 (see Wick), 307, 526, Bern, H. A., 64, 66 Bernard, C., 207, 209, 21.2 364, S68 Bernheim, F., 106, 117, 118, 133 Bartlett, J. W., 224 (see Hurst), 248 Bartlett, M. N., 314, 317, 328, 335 (see Berry L. J., 99 (see Williams), 140 Kochakian), 341, 342, 369 Bertrand, I., 39 (see Guillain), 43 Bartlett, S., 219 (see Bailey), 220, 221, Besman, L., 48 (see Sobel), 49, 51, 52, 222, 224 (see Bailey), 225, 226, 234, 53, 55, 67 Bessey, 0. A., 107, 129 235,236,237,238,247 Bartoli, A., 103, 129 Bessinger, A., 339 (see Wattenyl), S70 Best, C. H., 186, 189 (see Haist), 211, 213 Bassett, L., 85 (see Selye), 138 Bauer, D., 154, 180 Betheil, J. J., 124, 128, 129 Bauld, W. S., 150, 179 Beznak, A., 115,129 Baum, G. J., 149,179 Biasotti, A., 186, 193, 196 (see Houssay), Bauman, C. A., 63, 66 201, 213 Baumann, E. J., 102 (see Marine), 136, Biddulph, C., 92, 93, 94, 95, 129 196, 111 Biely, J., 55 (see Halpern), 66 Beall, D., 277, 291 Bierry, H., 298, 326 Bean, MT. B., 6 (see Aring), 7, 9, 42 Bietti, A., 34, 42 Beard, H. H., 173, 176 Biggart, J. H., 4.2 Bechdel, S. I., 218, 248 Bigler, R., 279, 294 Beck, J., 102 (see Sturm), 139 Billingsley, P. R., 26, 44 Beck, J. C., 128, 129 Binder, A., 107, 140 Beck, L. V., 210, 212, 302, 526 Bird, 0. D., 50 (see Lemley), 66 Bisceglie, V., 93, 189 Becks, H., 201, 212 Bischoff, F., 147, 176,274, 291 BBclBre, C., 155, 157, 158, 172, 174, 176 Bedoya, J. M., 167, 176 Bishop, K. S., 81, 87, 95, 132 Beerstecher, E., 99 (see Williams), 140 Biskind, G. R., 107, 111, 129, lS2, 258, Beerstecher, E., Jr., 69 (see Williams), 269, 291 Biskind, M. S., 80, 82, 128, 129, 269, 291 72, 74, 75, 76, 77 Black, R., 145, 163, 166 (see Zondek), Beezley, M. B., 221 (see Reinecke), 249 167, 172, 181 Behnken, E. W., 167,176 Blackfan, K. D., 49 (see May), 50, 54, Bellon, M. T., 110 (see Giroud), 132 57, 66, 66 Bendall, J. R., 326 Bender, L., 29, 42 Blau, N. F., 221, 247 Bender, R. C., 111 (see Supplee), 114, 139 Blaxter, K. L., 217, 219, 222, 224, 225, Bender, S., 168, 176 226, 227, 228, 230, 232, 234, 235, 237, 238, 240, 241, 242, 243, 244, Benetato, G., 299, 329 Bennett, L. L., 103, 126 (see Gordan), 245, 246, 247 127, 133, 199,200, 201 (see Gordan), Blum, F., 201, 212 Blumenthal, H. T., 114, 129 212,213 Bodansky, O., 49 (see Lewis), 51, 53, 55, Bennett, L, R., 121, 129 Bennett, V. C., 121 (see B., I,.R.), 229 62, 64, 66, 66

373

AUTHOR INDEX Bodechtel, G., 28, 42 Boehm, R., 208, 212 Boelter, M. D. D., 89, 101, 129 Bolomey, R. A., 236 (see Kemmerer), 237, 249 Bontius, J., 18, 4.2 Booth, A. N., 227,230,235,237,243,247 Borell, U., 102, 129 Borradaiie, L. A., 76 Borrows, E. T., 218, 247 Borth, R., 141, 143, 148 (see Watteville), 150, 154, 176, 181 Bottomley, A. C., 218, 247 Bourne, G., 107, 129, 316, 326 Boutwell, R. K., 81, 85, 115, 130 Bowman, K. M., 33 (see Jolliffe), 40, 43 Boyd, D. A., 233 (see Yatea), 260 Boyd, W. L., 229 (see Petersen), 249 Boyer, P. D., 89, 100, 101, 130, 322, 326 Boyle, D., 289, 294 Bozovic, L., 305, 326 Bradbury, J. T., 154, 165, 168, 176 Brain, W. R., 48 Brand, M. A., 103 (see Sherwood), 138 Branton, C., 243 (see Seath), ,9960 Breen, J., 326 Breeze, B., 50, 54, 66 Brief, B. J., 90, 139 Britton, S. W., 298, 299, 326 Brody, H., 169, 176 Brody, S., 102, 137 Broh-Kahn, R., 319, 326 Brolin, S. E., 120, 130 Brown, G. E., 23, &? Brown, H., 146 (see West), 181, 255 (see West), 256, 272, 273, 274, 276, 296 Brown, L. A., 73, 76 Brown, M. R., 9, 42 Brown, P., 161 (see Riley), 180 Brown, R. A., 50 (see Lemley), 66 Brown, W. C., 237, 247 Brown, W. E., 154, 165, 168 (see Bradbury), 176 Browne, J. S. L., 153, 156, 168, 176,180, 256 (see Venning), 280, 293, 2996 Brownell, K. A., 298 (see Hartman), 299, 327 Broiek, J., 83 (see Keys), 134 Bruce, R. A., 278 (see Kochakian), 280, 28 1, 293

Briickmann, G., 192, 812 Briickner, J., 302, 326 Bruger, M., 239, 247 Brunschwig, A., 190, 212 Brush, M. K. ,81 (see Boutwell), 85, 115, 130 Bryson, M. J., 269, 291 Buchanan, J. M., 326 Bucher, R., 321 (see Verzdr), 330 Buhler, F. C., 251, 291 Buell, M. V., 299, 326 Bulatao, E., 209, 212 Burack, E., 5, 46 Burger, H., 156, 176 Burgess, R. C., 11, 36 Burn, J. H., 194, 198, 218 Burr, G. O., 99, 130, IS2 Burr, M. M., 89, 1SU Burrill, M. W., 81, 87, 88, 103, 130, 131, 253, 258, 287, 291 Burrows, H., 163, 176 Burthiault, H., 169, 171, 179 Burtness, H. I., 189, 212 Burton, R. B., 286 (see Zaffaroni), 896 Buschke, W., 96, 129 Busnel, R. G., 74, '76, 77 Butenandt, A., 252, 276, 277, 286, 891 Butler, G. C., 286, 291, 293 Butler, R. E., 34, 44 Butt, W. R., 145, 174, 176 Buxton, C. L., 154, 156, 161 (seeLevin), 165,176, in, 280, 291 C

Cahill, W. M., 96 (see Maun), 196, 136 Calbert, C. E., 126 (see Deuel), 130 Callow, N. H., 286, 891 Callow, R. K., 253, 286, 291 Cama, H. R., 128, 130, 236, 8.47' Cameron, A. T., 86, 115, 130 Camien, M. N., 70, 76 Campbell, A. C. P., 31, 42 Campbell, A. D., 229 (see MeQuillan), 230,249 Campbell, A. M., 105 (see Talbot), 139 Campbell, H. L., 107, 134 Campbell, J., 189 (see Haist), d l 3 Campbell, J. G., 345 (see Levvy), 346 (see Kerr), 369

3 74

AUTHOR INDEX

Canadell, J. M., 128, 130 Cannon, W. B., 208, 209, 212 Cantarow, A., 84 (see Gomez Mont), 133, 1-15 (see Rakoff), 179, 212, 283 (see Paschkis), 289, 691, 294 Cantilo, E., 203, 212 Carlson, W. E., 30 (see Green), 43 Carmichael, E. A., 6, 43 Carmichael, J., 86, 115, 130 Carroll, K. K., 125, 130 Carter, P., 151, 181 Cartwright, G. E., 128 (see Hamilton), 133

Cary, C. A., 89 (see Hartman), 133 Case, E. M., 362, 368 Castle, W. B., 6 (see Gildea), 43 Cerceo, E., 162, 294 Chamberlain, 6 Chanda, R., 235, 236, 237, 238, 247, 248 Chandler, R. E., 160 (see Heller), 177 Channell, G. D., 23 (see Ungley), 46 Chapman, A., 211, 216 Chardon, M., 19, 42 Charenton, J., 225, 250 Charipper, H. A., 85 (see D'Angelo), 86, 87, 99, 100, 115, 130 Cheng, C. P., 104 (see Sayers), 137 Chesney, A. M., 99, 140 Chesney, J., 49, 50, 66 Chinn, A. B., 6, 46 Chiu, C. Y., 307, 326, 366, 368 Chow, B. F., 126, 130 Christensen, K., 116, 130 Christensen, R. A., 346 (see Fishman), 368

Christo, E., 105 (see Talbot), 139, 151 (see Zygrnuntowicz), 181 Churnchal, R., 219 (see Opichal), 249 Chung, A. W., 55 (see Morales), 66 Church, C. F., 30, 42 Cienfugos, S., 50, 66 Clapham, H. M., 236 (see Chanda), 247 Clark, B. B., 189, 212 Clark, D. H., 288, 291 Clark, E., 5, 46 Clark L. C., Jr., 258, 261, 292, 335, 337, 368, 369

Clark, W. G., 320, 326 Clarke, C. A., 35, 39, 42 Clarke, G. L., 72, 73, 76, 76

Clarke, R. W., 118, 134, 299, 328 Clausen, S. W., 49, 65 Clayton, B. E., 143, 170 (see Somrnerville), 176, 180 Clayton, J. C., 218 (see Borrows), 247, $48

Cleckley, H. M., 33, 42, 46 Cluxton, H. E., 285 (see Sprague), 294 Coates, C. W., 165 (see Weisman), 181 Cobb, S., 29 (see Strauss), 44 Cochran, W. G., 144, 176 Cohen, G. N., 128, 136 Cohen, I., 70, 73, 74, 77 Cohen, S. L., 341, 342, 345, 346, 368, 369 Cohlan, S. Q., 55 (see Lewis), 66 Coignet, J., 222 (see Roche), 260 Colbert, C. N., 9 (see Jolliffe), 29, 43 Collazo, A. G., 172, 179 Collip, J. B., 81, 84, 129, 130, 138, 185, 612

Collonge, S., 70 (see Fontaine), 74, 76 Colonge, R., 89, 130 Colowick, S. P., 205, 212, 215, 318, 319, 326, 369

Comfort, M. W., 185 (see Priestley), 215 Conn, J. W., 200, 201, 21.2 Constantinides, P., 106, 130 Conway, E. J., 318, 319, 326 Cook, C. D., 122 (see Gardner), 133 Cook, R., 119, 120, 169 Cooper, H. J., 72 (see Stuart), 73, 75, 77

Cope, C. L., 147, 168, 176 Coppedge, R. L., 153, 176 Corcoran, A. C., 107 (see Schaffenburg), 138

Cori, C. F., 197, 202, 205 (see Price), 208, 612, 216, 302, 313, 318, 326, 329 Cori, G. T., 202, 205 (see Colowick), 208, 112, 216, 302, 318, 319 (see Colowick), 326 Corner, G. W., 153, 161, 176, 27G, 29.2 Corner, G. W., Jr., 153 (see C., G. W.), 161, 176 Cotes, P. M., 199, 200, 212 Courrier, R., 95, 126, 130, 239, 248 Courtois, J., 168, 176 Coward, K. H., 87, 89, 130 Cowie, A. T., 236 (see Thompson), 250 Cowie, D. P., 323, 326

AUTHOR INDEX

Cowsert, W. C., 228 (see Ralston), 235, 244, 249 Cox, G. M., 144, 176 Craig, W. McK., 23, 42 Crampton, E. W., 219 (see Blaxter), 235, 247

Cravens, W. W., 128 (see Nichol), 136 Creelman, M., 160 (see Kurzrok), l Y 8 Crepax, P., 102, 127, 130 CrBpy, O., 150 (see Jayle), 151, 178 Creutzfeldt, H. G., 28, 42 Crichton, J. A,, 243, 244, 248 Crismer, R., 151, 176 Crooks, H. M., Jr., 277 (see Marker), 293 Cross, N., 326 Crotti, A., 99, 130 Croxatto, H., 123, 129, 134 Cruickshank, E. K., 1, 19, 21, 24, 27, 31, 32, 42 Csapo, A,, 348, 368 Csik, L., 93, 130, 298, 326 Culver, P. J., 49 (see Jones), 65 Curtis, L. E., 158 (see Lisser), 178 Cushny, A. R., 210, 212 Cuyler, mi. K., 153 (see Hamblen), 154, 168 (see Davis), 176, 177, 285, 292

D Daft, F. S., 89 111, 112 (see McQueeney), 114, 115, 129, 130, 135 Dalton, A. J., 111, 112, 130 Dam, H., 48, 66 D’Amour, F. E., 153, 160, 176 Danby, M., 258, 269, 292 Danford, H. G., 122, 150 Danford, P. A., 122, 130 D’Angelo, S. A., 84, 85, 86, 87, 115, 130 Daniels, A. L., 100, 130 Danish, A., 57 (see Klopp), 65 Dannenbaum, H., 286,291 Danowski, T. S., 322, 326 Darup, E., 147, 154, 156, 157, 163, 168, 169, lY9 Das Gupta, K., 219, 248 Dastre, 209, 212 Dastur, N. N., 234, 235, 250 Daus, M. A., 277, 293 David, K., 252, 292 Davidson, C. S., 126 (see Treager), 139

375

Davidson, D. M., 54, 56, 58, 59, 66 Davies, A. W., 121, 130 Davis, A. A,, 155, 176 Davis, C. D., 168, 176 Davis, J. S., 333, 334, 339, 368 Davis, M. E., 143, 163, 170, 176 Davis, R. M . , 96 (see Mann), 136, 136 Davis, R. N., 236 (see Kemmerer), 237, 249

Day, H. G., 128, 13.4 Dean, R. F. A., 223, 2.48 Deane, H. W., 107, 111, 112, 114, 115, 116, 117, 119, 120, 122, 123, 130, 138, 136, 316, 396 Deanesly, R., 111, 112, 130 Deansley, R., 221, 224, 248 DeGrandprC, 105 (see Henriques), 130, 133 DeHaas, J. H., 59, 65 de Johngh, S. E., 154, 177 Delfs, E., 150, 154, 163 (see Jones), 176, 178

Deltour, G. H., 219 (see Roche), 239 (see Courrier), 24.3, 250 Dempsey, E., 316, 326 Denny-Brown, D., 14, 16, 19, 33, 34, 38, 39,42 Deuel, H. J., 210, 212, 303, 326 Deuel, H. J., Jr., 59 (see Hrubetz), 65, 126, 128, 130, 131 Deutsch, H., 103, 131 DeVaal, 0. M., 121, 151 Devis, R., 147, 154, 162, 171, 176 Devis-van den Eeckoudt, hl., 171, 176 Diakov, F. A., 93, 131 Dibbelt, L., 154, 176 Di Cio, A. V., 127, 131 Dickens, F., 362, 368 Diczafalusy, E., 360 (see Aldman), 368 Dietrich, L. S., 128 (see Nichol), 136 Dill, D. B., 308 (see Missuro), 328 Dillman, L. M., 103 (see Reed), 157 Dingemanse, E., 147, 151, 176, 252 (see David), 286, 292 Dirscherl, W., 352, 362, 368 Dju, M. J., 48, 63, 66 Dobriner, K., 144, 145, 163, 173, 176, 253, 254, 255 (see Gallagher), 277 (see Lieberman), 277, 278, 285 (see Fukushima), 286, 287, 992, 293

AUTHOR INDEX

376

Doetsch, R., 311, 313, 327 Dohan, F. C., 187,188,194,199,212,214 D o h , G., 202, 203, 212 Domhoffer, C., 207, 208, 212 Dontigny, P., 105 (see DeGrandprC), 130, 131 Dorfman, R. I., 145 (see Ungar), 146, 148, 149, 154, 176, 180,253,254,255,277 (see Fish), 278, 286 (see Miller, Howitt), 292, 293, 294, 305 (see Hayano), 319, 320, 327, 331, 350, 351 (see Hayano), 352,353,354, 355, 357, 358, 362, 363, 367, 369 Dorrance, S. S., 118 (see Lewin), 136 Dosne, C., 85 (see Selye), 109, 238, 280, 292 Doughherty, T. F., 85, 113, 120, 131 DOW,D. S., 227 (see Allen), 247 Dragstedt, L. R., 185, 212 Drill, V. A., 80, 81, 87, 88, 128, 131 Drummer, G. D., 49 (see Jones), 66 Drummond, J. C., 92, 93, 131 Dryden, L. P., 89 (see Hartman), 133 Dryendahl, S., 229, 242, 244, 245, 248 Dubin, A., 49 (see Popper), 56, 66 Dubois, M., 83 (see Zimmer), 140 Dubreuil, P., 164, 179 Duchateau, G., 70 (see Camien), 76 Duchateau-Bosson, G., 74, 76 Diirck, H., 5, 42 Duff, G. L., 190, 193, 815 Dugal, L. P., 109, 110, 191 Dumm, M. E., 112, 131, 320, 327 Dunham, M., 253 (see Dobriner), 287, 298 Dunn, J. S., 190, 213 de Duve, C., 197,216 Dyniewicz, H. A., 49 (see Popper), 56,66

E Eakin, R. E., 69 (see Williams), 77 Ecker, A. D., 29, 42 Eden, E., 49, 66 Edie, E. S., 209, 215 Edmonson, H. A., 272 (see Glass), 292 Edwards, H. T., 308 (see Missuro), 328 Eftman, H., 341 (see Atkinson), 368 Ehman, B., 253 (see Dobriner), 286, 287, ,9998

Ehrhardt, K., 251, 292 Eichelberger, L., 343 (see Huggins), 344, 369 Eichenberger, E., 166, 167, 171, 176, 178 Eijkman, C., 5, 42 Eisen, H. N., 119, 139 Eisenberg, E., 319, 327 Eisenberg, G., 349,350 (see Gordan), 368, 369 Ekbom, K. A., 23, 4.9 Elgart, S., 189 (see Mirsky), 214 Elkinton, J. R., 128, 131 Elliott, H. w., 308, 319, S27, 349, 368, 369 Elmer, A. w., 102, 131 Elvehjem, C. A., 101 (see Kernrnerer), 111 (see Mills), 114 (see Schaefer), 128 (see Nichol), 134, 136, 138, 227 (see Booth), 230, 235, 237, 243, 247 Emerson, G. A,, 89, 94 (see Telford), 128, 131, 139 Emerson, K., 201, 216 Emge, M. K., 145 (see Hertz), 177 Emmel, V., 316, 317, 327 Emmet, A. D., 50 (see Lemley), 66 Engel, E., 49 (see Sobel), 54,59,60,61,67 Engel, F. L., 300, 321, $27, 340, 369 Engel, L. L., 149, 150, 151, 176,286, 892 Engel, P., 280, ,992 Engel, R. w., 6, 43, 111, 116, 137 Engle, E. T., 90,131, 161 (see Levin), 178 English, M. M., 128 (see Beck), 129 Ennor, A. H., 195, 813 Enns, L. H., 323, 329 Ermster, L., 63, 66 Ershoff, B. H., 79, 80, 82, 93, 100, 103, 110, 113, 119, 120, 122, 123, 124, 125, 126, 127, 128, 131, 132, l3SJl4O Erway, F., 362 (see McShan), 369 Escamilla, R. F., 158 (see Lisser), 178 Esh, G. C., 62, 66 Ettlinger, M. G., 99 (see Greer), 133 Evans, C. A., 30 (see Green), 43 Evans, C. L., 210, 213 Evans, G., 117, 152, 299, 397 Evans, H. M., 81, 84 (see Li), 87, 88, 89 (see Nelson), 90, 91, 93, 94 (see Telford), 95, 126, 127, 129, 132, 132, 133,134,136, 196,139, 148, 149, 152, 153,162,164,168, in,176,178, 198,

377

AUTHOR INDEX

199 (see Li, Marx), 201, 212, 213, 214, 320 (see Fraenkel-Conrat), 3.27, 335, 337, 369 Evans, J. P., 6 (see Aring), 7, 9, 42 Evans, J. S., 189 (see Ingle), 201, 213 Everett, J., 339, 368 Everson, G. J., 100, 130

F Fairclough, M., 59 (see Hrubetz), 66 Farmer, S. N., 327 Farney, J. P., 159 (see Heller), 177 Farris, E. J., 153, 161 (see Corner), 167,176, ir9 Faulkner, R., 111 (see McQueeney), 112, 114, 115, 129, f36 Fein, H. D., 9 (see Jolliffe), 30, 31, 33,40,

@

Feldman, J., 103 (see Bartoli), 129 Feldman, J. B., 128, 140 Fellers, C. R., 70 (see Lubitz), 75, 76 Fellinger, K., 102, 13.2 Fels, E., 276 (see Slotta), 294 Ferrebee, B., 320, 3.27 Fieser, L. F., 277 (see Lieberman), 286 (see Wolfe), 293, 196 Figge, F. H. J., 88, 126, 132' Filer, L. J., 49, 62 (see Wright), 65, 67 Findlay, G. M., 5, 4.9 Finestone, A. J., 321, 329 Fish, W. R., 254, 277, 286 (see Horwitt), 292, 293 Fisher, L. R., 75, 76 Fisher, R. A., 161, 1?6 Fishman, J. B., 199 (see Wilhelmi), 216 Fishman, W. H., 345, 346, 368 Fitzgerald, O., 299, 827 Flach, M., 95, 138 Florkin, M., 69, 70 (see Camien), 76, 151, 176 Fluhma.nn, C. F., 91, 132 Foldi, M., 828 Foglia, V. G., 187 (see Houssay), 188 (see Lewis), 195, 203, ,213, 214 Folley, S. J., 218, 219 (see Bailey), 220, 221, 222, 224 (see Bailey), 225, 229, 235, 237, 238, 247, 248, 313, 314, 320, 3.27 Follis, R. H., Jr., 114 (see Wintrobe), f40

Fong, C. T. O., 199 (see Marx), 214 Fontaine, M., 70, 74, 76 Forbes, F. A., 35, 43 Forbes, T. R., 146, 176, 177 Fouts, P. J., 114, 132 Fox, R. R., 337 (see Clark), 368 Fraenkel-Conrat, 320, 3.27, 335, 337, 369 Franseen, C. C., 193, dl1 Frank, R. T., 147 (see Hollander), 167 (see Spielmann), 178, 180 Frazer, A. C., 53, 66 Free, A. A., 248 Freeman, H., 152, 158, 172, 177 Freisburger, H., 191, 214 de Fremery, P., 226, 248, 276 (see Adler), 291 French, T. H., 229,248 Freud, J., 252 (see David), 992 Fried, P. H., 165, 177 Frieden, E., 220, 248 Friedgood, C. E., 193, 214 Friedgood, H. B., 286 (see Wolfe), 296 Friedman, G. S., 96, 132 Friedman, M. H., 96, 132 Fruton, J. S., 321 (see Holman), 827, 339, 569 Fry, E. G., 85 (see Long), 118, 135, 137, 298, 299, 300, 3.28 Fugo, N. w., 143, 163, 170, f76 Fukui, T., 126 (see Deuel), 130 Fukushima, D. K., 254 (see Gallagher) 255, 277 (see Lieberman), 278, 285, 286, 287, 292, 293 Fumarola, G., 41, 4.3 Funk, C., 256, 89.2 Furuhjelm, M., 154, 157, 177

G Gaarenstroom, J. H., 154, 177, 193, 194, 813 Gabe, M., 92 (see Arvy), 102, 1.29, 13.2 Gabuzda, G. J., 126 (see Treager), 139 Gaddum, J. H., 150, 163, 178 Gagel, O., 28, 42 Gallagher, T. F., 252, 254, 255, 256 (see Munson), 278, 285 (see Fukushima), 286, 29.2, 294 Gallaher, P. D., 64, 66 Gallico, E., 172, 179

378

AUTHOR INDEX

Galtsoff, P., 70, 76 Gamper, E., 28, 43 Gardner, K. E., 233, 243, 248 Gardner, L. I., 122, 132 Garrod, A. E., 202, 21s Gassner, F. X., 253, 285, 292 Gastineau, C. F., 146, 177, 285 (see Sprague), 294 Gaston, E. A., 185, 215 Gaunt, R., 114, 132, 202 (see D o h ) , 203, 212 Geduldig, R., 49 (see Sobel), 54, 59, 60, 61, 67 van Geertruyden, J., 56 (see Kowalewski), 65 Geeslin, L. E., 33 (see Cleckley), 42 Geiger, E., 97, 132 Geist, S. H., 172, 177 Gelfan, S., 103, 152 Geller, F. C., 92, 93, IS2 Gellis, S. S., 72, 73, 76, 76 Gemmill, C. L., 303,305, 321 (see Koepf), S27, 528, 364,369 Genell, S., 165, 169, 177 Gerlach, w., 166, 177 Gessler, C. J., 203, 815 Giardinelli, M., 164, 177 Gibson, R. B., 189 (see Clark), 212 Giedosz, B., 101, 102 (see Elmer), 127, 1SIl1S.2 Gierhake, E., 92, 152 Gildea, E. F., 6, 43 Gilder, H., 272, 296 Gill, J. E., 346 (see Fishman), 568 Giraud, P., 222 (see Roehe), 260 Giroud, A., 107, 110, 111, 152, 157 Gittleman, I. F., 50, 66 Glass, S. J., 272, 292 Glick, D., 9,42,107, 1 11, 129,132,346,369 Goddard, J. W., 91, 126, 136 Godfrey, L., 128 (see Elkinton), 131 Gobell, O., 299, 327 Goecke, H., 156, 177 Goettsch, M., 30, 44 Goff, B. L., 49 (see Clausen), 65 Goldberg, M. B., 158 (see Lisser), 178 Goldberger, M. A., 167 (see Spielmann), 180

Goldner, M. G., 190 (see Brunschwig), 21 2, 21 3

Goldthorpe, H. C., 269 (see Bryson), 291 Goldzieher, J. W., 152, 177 Golla, Y. M. L., 105, 132 Golub, S. J., 82, 131 Gomez Mont, F., 84, I S 3 Gomori, G., 190 (see Brunschwig), 212, 215, 316, 327 Gong6ra, M. N., 335 (see Kochakian), 342, 569 Goodsell, J. E., 115, 133 Goodwin, T. W., 128, 160, 236, 247 Goormaghtigh, N., 102, 103, 133 Gopalan, C., 22, 27, .@ Gordan, G. S., 126, 135, 201, 215, 308, 319, 326, 349, 350, 568, S69 Gordon, A. S., 85 (see D'Angelo), 86, 87, 99, 100, 115, 130 Gordon, S. M., 49 (see Kramer), 55, 66 GOSS,H., 81, 95, 155, 195, 215 Gottfried, S. P., 49 (see Kramer), 50, 51 (see Sobel), 52, 53, 55, 66, 67 Gough, N., 150 (see Sommerville), 180, 293 Govan, A. D. T., 171, 177 Grachus, T., 121 (see Bennett), 1 8 Graef, I., 111, 167 Graham, W. R., Jr., 218, 227 (see Herman), 246, 248 Grande, F., 319, 329 Grande Covian, F., 19, 22, 26, 34, 38, 39,

43 Grant, J. I<., 283, 292 Grattan, J. F., 121, 153 Graves, P. R., 40,43 Gray, J. L., 338 (see Jensen), 339, B(iM Gray, L. A., 165 (see Bradhiiry), 168, 175 Grayman, I., 185 (see Rlirsky), 189, 214 Greaves, A. V., 35 (see Adolph), /t? Green, A., 313, 326 Green, H. N., 91 Green, R. G., 30, 43 Greenbaum, A. L., 314, 320, 327 Greenberg, D. M., 89, 101, 129 Greenberg, R., 121, IS7 Greenberg, S. M., 126 (see Deuel), 126, 130, 15.3

Greene, R. R., 103,150,253,258,287,291 Greenfield, J. G., 6, 4s Greenstein, J. P., 313, 314, 327 Greep, R. O., 94, 107, 112, 122, 123, 126,

AUTHOR INDEX

127, 129, 130, 233, 138, 307, 329 Greer, M. A., 99, 153 Gregoir, C., 119, 133 Gregory, P. W., 195, 213 Gribets, D., 49, 56, 66 Grierson, J., 19, 43 Griesbach, W. E., 100, 133 Griffith, W. H., 116, 130, 133 Griffiths, hi., 191, 192, 213 Grimmer, U'., 235, 248 Griswold G., 83 (see Klinefelter), 134 Groen, J., 89, 138 Grollman, A., 202, 207, 210, 213 Groth, A. H., 243 (see Seath), 2bU Gruber, C. J., 128 (see Hamilton), 133 Grubbs, R. C., 308 (see Hitchcock), 327 Griiter, 235, 248 Gsell, M., 150 (see W'atteville), 181 GuBgan, Y., 174 (see BBcBre), 175 Guilbert, H. R., 81, 90, 95, 133 Guillain, G., 39, 43 Gunn, F. D., 298, 327 Guterman, H. S., 150, 158, 165, 168, 177 Gutman, A. B., 344, 569 Gutman, E. B., 344, 369

Hackney, J. W., 128 (see Beck), 129 Hagena, A., 251, 296 Hain, A. M., 163, 164, 168, 177 Haist, R. E., 189, 213 Hale, F., 61, 65 Hallman, L. F., 303 (see Deuel), 326 Halpern, C . R., 55, 65 Halsted, J, A., 203 (see Gessler), 213 Halverson, A. W., 99, 133 Hamblen, E. C., 152 (see Goldzieher), 153, 154, 168 (see Davis), 176, 177, 285 (see Cuyler), 692 Hamburger, C., 91, fS3, 147,151,169,177 Hamilton, J. B., 253, 255, 292 Hamilton, L. D., 128, 133 Hamman, L., 364, 369 Handler, P., 106, 117, 118, 133 Handovsky, H., 102, 103, 133 Hangaard, N., 319, 329 Hanley, B. J., 59 (see Hrubetz), 66 Hansard, S. L., 90 (see Sutton), 139

379

Hansen, I. G., 240 (see Thorbek), 245,260 Hansen, L. P., 145 (see Rakoff), 179 Harding, B., 288, 292 Hardy, F., 55 (see Halpern), 65 Harington, C. R., 221, 248 Hariton, L. B., 286 (see Lieberman), 287, 293 Harper, R. hf., 72, 74, 75, 76 Harper, W. F., 171 (see Govan), 177 Harris, J. E., 322, 327 Harris, L. J., 107, 133 Harris, P. I,., 56, 57, 65, 80, 100 (see Hickman), 133 Harris, R. S., 341, 345, 346, 369 Harrison, G. F., 19, 21, 25, 27, 43 Harrison, H. C., 327 Harrison, H. E., 327 Harrow, B., 256 (see Funk), 292 Hart, E. B., 99 (see Halverson), 101 (see Waddell), 101 (see Kemmerer), 133, 134, 227 (see Booth), 230, 235, 237, 243, 247 Hart, G. I€., 90, 133 Hartman, A. M., 89, 133 Hartmann, F. A., 107, 135, 298, 299, 308 (see Hitchcock), 327 Hartmann, M., 277, 292 Hartmann, W. E., 298, 387 Hartogh-Katz, S. L., 147 (see Dingemanse), 151, 176 Hartop, W. L., Jr., 278 (see Riegel), 294 Haskins, A. L., 145, 280 (see Kochakian), 291, ,993 Hastings, A. B., 304, 326, 330 Hauptmann, K. H., 352, 368 Havens, W. P., Jr., 272 (see Cantarow), 277 (see Paschkis), 291, 294 Hawker, R. W., 101, I33 Hay, E. C., 105 (see Dontigny), 231, 133 Hayano, M., 305, 319, 327, 350, 351, 352, 353, 354, 355, 357, 358, 362, 363, 367, 369 Heard, R. D. H., 148, 153, 177, 277, 292 HBbert, S., 316, 327, 330 Heller, C. G., 81, 82, 83, 135, 159, 177 Helmer, 0. M., 114 (see Fouts), 132 Helmreich, M. L., 261 (see Samuels), 263, 274 (see Lumry), 277, 287, 291, 293, 294

Helve, O., 311, 327

380

AUTHOR INDEX

Hem, M., 248 Hems, B. A., 218 (see Borrows), 247, 248 Hench, P. S., 287 (see Sprague), 894 Henderson, H. O., 235 (see Van Landingham), 237, 244, 660 Henderson, J., 147, 162, 165, 177 Henkin, A. E., 152 (see Goldzieher), 177 Henriques, 0. B., 105, 1S3 Henriques, S. B., 105 (see H., 0. B.), 133 Henroitin, E., 56 (see Kowalewski), 66 Henry, J. S., 156 (see Browne), 168, 175 Henry, K. M., 62, 66, 75, 76 Henschel, A., 83 (see Keys), 134 Hepburn, H. H., 17, 18, 43 Herbert, D., 313 (see Ostern), 389 Herman, H. A., 218, 226 (see Reinecke), 227, 228 (see Ralston), 235, 244, 248, 249

HBroux, O., 109 (see ThBrien), 1S9 Herrin, R. C., 122 (see Danford), 130 Hershberg, D., 124, 131 Hertoghe, E., 218, 248 Hertz, R., 80, 82, 128, 133, 145, 177 Hertz, S., 84, 99 (see Williams), 133 Hesser, F. P., 49 (see Popper), 56, 66 Hevesy, G., 322, 927 Hewitt, J., 171 (see Govnn), 177 Hibbard, J. S., 98, 1S3 Hibbs, J. W., 62 (see Esh), 65, 232, 234, 235, 236, 237, 239, 248 Hickman, K. C. D., 80, 100, 233 Higginbottom, C., 237 (see Chanda), 247 Hilditch, T. P., 234, 848 Hill, B. R., 277 (see Lieberman), 286, 293 Hill, R. T., 91, 96, 13.9, 253, 292 Hingerty, D., 318, 319, 326 Hinglais, H., 167, 169, 177 Hinglais, M., 167, 169, 177 Hirano, Y., 191, 213 Hirschmann, F. B., 277 (see H., H.), 286, 292, 293

Hirschmann, H., 277, 286, 292, 293 E r s t , D. V., 285 (see Cuyler), 288 Hitchcock, F. A., 308, 326 Hoagland C. L., 272, 298 Hobbs, H. E., 35, /tS Hochstadt, O., 102, 132 Hochster, R. M., 358, 359, 360, 367, 369 Hodges, C. V., 343 (see Huggins), 344, 369

Hogberg, B., 360 (see Aldman), 368 Hoffman, M. M., 256 (see Venning), 280, 293, 294, 295

Hoffmann, F., 146, 178 Hoffmann, F. A., 208, 212 Hofmeister, F., 206, 207, 213 Hollander, F., 147, 178 Holman, H. R., 321, 327, 339, 369 Holmes, N., 326 Holmgren, H., 102, 129 Holt, L. E., Jr., 55 (see Morales), 66, 97 (see Albanese), 129, 133 Homans, J., 187, 213 Homes, E. G., 299, 313 (see Ostern), 527, s29

Hoover, R. D., 221 (see Reinecke), %4H Hopper, B. K., 50, 67 Hopson, E. hl., 100, 138 Hori, 33, 43 Horn, H. W., 303 (see Koepf), 305, 321, 328

Horowitz, E. A., 169, 175 Horton, B. T., 23, 42 Horwitt, B. N., 286, 298, 293 Hoskins, R. G., 208, 212 Houssay, B. A., 165, 178, 186, 187, 188, 191, 193, 194, 195, 196, 199, 201, 203, 204, 205, 213, 319, 327 Houston, J. S., 238, 248 Howard, A,, 41 (see Anderson), 4.2 Howat, G. R., 234 (see Smith), 660 Howe, P. R., 90, 91, 1.60 Hoyt, R. E., 147, 178 Hrubetz, M. C., 59, 65 Huber, F., 160, 180 Hubner, H., 320 (see VeraBr), 827, 630 Hiimel, E. J., 197, 215 Huggins, C., 63 (see Scott), 67 Huggins, C. S., 343, 344, 568, S69 Hughes, E. C., 149 (see Lloyd), 153, 161, 167 (see Behnken), 176, 178 Hughes, H., 190, 213 Hughes, J. S., 91, 134 Huis in’t Veld, L., 147 (see Kassenaar), 178

Huis in’t Veld, L. G., 147 (see Dingemanse), 151, 176, 286, 692 Humm, J. H., 335, 342, 343, 369 Humphreys, S., 114 (see Wintrobe), 140 Hundhausen, E., 102, 138

AUTHOR INDEX

Hundhausen, G., 88, 95, 102, 134, 138 Hunt, A. D., Jr., 128 (see Elkinton), 131 Hunt, H., 171, 181 Hurst, V., 224, 239, 248 Hurwitz, D., 143 (see Smith), 164, 170, 180

Huseby, R. A., 342, 368 Hyams, M. N., 64, 66 Hyatt, G., Jr., 246 (see Van Landingham), 260 Hyden, S., 234, 248 Hyman, G. A., 107, 108, 126, 134

I Igaravidez, P. G., 17 (see Riddeli), 44 Ingle, D. J., 85, 105, 109, 125, 134, 185, 189,200,201,202,203,207, 210, 813, 214, 298, 299, 300, 307, 309, 327, 328, 3.29 Isajev, F., 225, 249 Israel, S. L., 158, 178 Itoh, M., 101, 103, 13.4 Izar, G., 192, 211

J Jack, E. L., 218, 248 Jackson, C. A., 19, 43 Jackson, C. M., 81, 83, 84, 134 Jacobiius, H., 28, 43 Jacobs, E. C., 83, 134 Jacobs, F. A., 299 (see Olson), 329 Jacobs, H. R., 190, 214 Jacobsen, N. L., 247 Jacobsohn, K. P., 75, 76 Janes, R. G., 193, 214 Jarl, F., 234, $48 Jarpa, S., 123, 194 Jayle, M. F., 150, 151, 162, 164, 165, 168, 178 Jensen, C. C., 165, i77 Jensen, E., 233, 248 Jensen, H., 121, 193, 338, 339, 369 Jimenez Garcia, F., 19, 22 (see Grande), 26, 34, 38, 39, 43 Jimbnez, T. V., 167, 176 Jiminez-Diaz, C., 320, 628 Joel, C . A., 92, 134, 152, 153, 166, 178 Joffe, P. M., 9 (see Jolliffe), 29, 43

381

Johansen, A., 340, 369 Johansson, I., 228, 248 Johnson, L. G., 128, 134 Johnson, R. M., 63, 66 Johnston, A. G., 221 (see Simpson), 260 Johnston, M. W., 200 (see Conn), 201, 21.2

Jolliffe, N., 9, 29, 30, 31, 33, 40, 43, 80,

ioo,iio, 134

Jones, Jones, Jones, Jones,

B. F., 111 (see Dalton), 112, 130 C. M., 49, 65 D. A., 327 G. E. S., 150, 154, 163, 178, 280,

293

Jones, T., S. G., 235, 248 Jordan, D. A., 55 (see Kagan), 66 Joseph, S., 202 (see D o h ) , 203, 212 Josephson, E. S., 107 (see Nadel), 136 J o s h , E. P., 184, 214 Judas, O., 150 (see Jayle), 178 Jukes, T. H., 111 (see Daft), 130 Julesz, M., 101, 134

K Kadota, I., 196, 214 Kiiser, O., 158, 167, 171, 178 Kagan, B. M., 55, 66 Kagawa, S., 33, 34, 43 Kahlenberg, 0. J., 111 (see Supplee), 114, 139 Kahn, R. H., 64, 66 Kaiser, R., 164, 165, 178 Kajdi, C., 97 (see Holt), 133 Kalckar, H. M., 205, 21% Kallas, H., 91, 134 Kaltwinkel, E. E., 6 (see Gildea), 43 Kamm, O., 277 (see Marker), 293 Kann, S., 49 (see Adlersberg), 62, 66 Kanoff, A., 49, 56, 66 Kant, F., 28, 43 Kass, E. H., 191, 214 Kassenaar, A., 147, 178 Katzin, B., 85 (see Long), 118, 136, 298, 299, 300, 328 Kaufmann, C., 154, 163, 168, 1% Kaunitz, H., 92 (see P'An), 136, 308, 328 Kay, H. D., 238, 2@, 313, 327 Kelley, B., 128, 134 Kelley, V. C., 299, 319, 928

382

AUTHOR INDEX

Kelsey, F. D., 194 (see Burn), 198, 212 Kemmerer, A. R., 101, 134, 236, 237, 249 Kendall, E. C., 118, 122, 1-94, 211, 216, 287 (see Sprague), 294,298,299,300, 328,329 Kennedy, T. H., 99, 125, 134 Kennedy, W. B., 190, 214 Kepler, E. J., 173, 178,256,277,286,293, 294 Kerb, H., 72, 76 Kern, C. J., 55, 65 Kerr, L. M. H., 345 (see Levvy), 346, 669 Keutmann, E., 286 (see Zaffaroni), 295 Keyes, G. H., 299, 319, 328 Keys, A., 83, 134 Kimura, O., 5, 8, 43 King, A., 35, 46 King, C. G., 107 (see Bessey), 111, 129, 134 King, E. J., 319 (see Abul-Fadl), 326 Kirkman, I. J., 160 (see Kurzrok), 178 Kissin, B., 63, 66 Kittinger, G. W., 278 (see Riegel), 294 Kjerulf-Jensen, K., 302, 328 Klein, J. R., 339, 369 Klein, J. W., 233 (see Jensen), 248 Klempner, E., 147 (see Hollander), 178 Kline, R. F., 298 (see Britton), 326 Klinefelter, H. F., 83, 134 Klopp, C. T., 57, 66 Knodt, C. B., 226, 231, 250 Knorrich, F. W., 72, 73, Y6 Knoevenagel, C., 315, 328 Knuchel, W., 362, 368 Koch, F. C., 252, 256 (see Munson), 286, 292, 294 Kochakian, C. D., 258, 261, 271, 278, 280, 281, 292, 293, 314, 315, 317, 328, 330, 334, 335, 336, 337, 340, 341, 342, 343, 352, 368, 369 Koehler, A. E., 189 (see Burtness), 612 Koenig, V. L., 259, 293 Koepf, G. F., 118 (see Lewis), 135, 303, 305, 321, 328 Kornyey, S., 43 Kohler, G. O., 221 (see Reinecke), 249 Koller, R., 219, 249 Koller, T., 147, 167, 178

Kon, 15.. K., 62 (see Henry), 65, 70, 75, 76, 236 (see Thompson), 237, 238 (see Houston), 248, A50 Koneff, A. A., 92, 134 Kopecky, O., 219 (see Opichal), 248 Kopp, L. J., 299 (see Olson), 329 Korenberg, M., 185 (see hlirsky), 214 Korkman, N., 228, 248 Kowalewski, K., 56, 65 Krahl, M. E., 307, 328 Kramer, B., 48 (see Polskin, Sobel), 49. 50, 51 (see Natelson), 52, 53, 54, 55, 56, 57, 59, 60, 61, 65, 66, 67 Krause, M., 33, 43 Krauss, W. E., 62 (see Esh), 65, 90 (see Sutton), 139, 232, 234, 235,236, 237, 239, 248 Krishman, K. N., 127, 134 Kriss, B., 147 (see Hollander), 118 Krizenecky, J., 93, 231 Krogh, A., 72, 76 Kruse, H. D., 80, 134 Kuilman, J., 38, 43 Kuizenga, M. H., 200 (see Ingle), 201, 213, 214

Kullander, S., 165, 178 Kupermintz, N., 322, 628 Kurzrok, R., 81 (see Mulinos), 126, 136, 160, 178 Kutscher, W., 314, 328, 343, 369

L Labes, R., 191, 214 Lafon, M., 222 (see Roche), 260 Lagerstedt, S., 317, 328 Lake, G. C., 5, 45 Lam, L. yon, 146, 178 LaMer, V. K., 107, 134 Landor, J. V., 16, 21, 26, 34, 39, 43 Langley, L. L., 118, 134, 299, 328 Lanik, J., 225, 249 Laqueur, E., 252 (see David), 292 Lardy, H. A., 124 (see Betheil), 128, 129, 322 (see Boyer), 323, 326, 328 Lasater, M. B., 274 (see Lumry), 277 (see Samuels), 287, 291, 293, 294 Lasnitczki, A., 322, 328 Laszt, L., 302, 320 (see VerzBr), 328, 330 Laundrie, B., 200, 212

AUTHOR INDEX

Lawney, J. C., 35 (see Adolph), 42 Lawrence, R. D., 184, 203, 214 Lawson, E. J., 277 (see Marker), 293 Lax, H., 145, 178 Lazarow, A., 191, 192, 194, 195, 196, 199, 214, 215 Leath, M. J., 334 (see Tipton), 3YO Leblanc, Jr., 109 (see Thkrien), 139 Leblond, C. P., 107, 132 LeCompte, P. M., 193 (see Bailey), 211 Leduc, J., 105 (see DeGrandprB), 130 Lee, M. 0. A., 84, 134 Leech, F. B., 230, 231, 234, 249 Leech, R. S., 191, 192, 214 Leekley, D., 147 (see Bachman), 175 Leftwich, W. B., 119, 136 Lehmann, H., 299 (see Homes), 326 Lehmann, W. L., 206, 214 Leibowitz, J., 322, 328 Leitner, 2. A., 57, 64, 66 Lejwa, A., 256 (see Funk), 292 Lemley, J. M., 50, 66 Lengyel, L., 302, 830 Lennox, B., 31, 32, 45 Leonard, S. L., 91, 135, 334, 369 Lepkovsky, S., 89 (see Okey, Evans), 114 (see Fouts), 132, 136 Lereboullet, J., 39 (see Guillain), 43 Leupin, E., 305 (see Bozovic), 307, 321, 324, 326, 328, 364, 369 Leuthardt, F., 147, 167, lY8 Levasseur, C., 168, 170, iY9 Levedahl, B. H., 261 (see Samuels), 263, 265, 266, 293, 294 Levey, S., 190, 192 (see Patterson), 214, 215

Levin, L., 161, 178 Levine, H., 98, 136 Levine, M. G., 147, lY8 Levvy, G. A., 345, 346 (see Kerr), 369 Lewis, H. L., 108 (see Sayers), 138 Lewis, J. M., 49, 51, 53, 55 (see Morales), 62, 66 Lewis, J. T., 188, 195, 203, 214 Lewis, R. A., 118, 135, 286 (see Engel), 292

Lewis, T., 23, 26, 43 Lewis, T. H. C., 194 (see Burn), 198, 212

Li, C. H., 84, 105 (see Ingle), 126 (see

383

Gordan), 127, 133, 134, 135, 148, 149, 178, 199, 200, 201 (see Gordan, Evans, Becks), 212, 213, 214, 327 Liang, T. Y., 108 (see Sayers), 138 Liardet, J., 222 (see Roche), 250 Lieberman, S., 163 (see Dobriner), lY6, 252 (see Prelog), 253 (see Dobriner), 254, 277, 286, 287, 892, 293, 294 Libert, O., 178 Lichtblau, J., 51 (see Sobel), 53, 54, 55, 61 Lienhardt, H. E., 91 (see Hughes), 234 Lilienfeld, M. C . C., 49 (see Lewis), 51, 53, 55, 62, 66 Liling, M., 114 (see Gaunt), 132 Lindberg, O., 63 (see Ernster), 65 Lipman, F., 302, 328 Lippincott, S. W., 114, 136 Lipschitz, P. A., 64, 66 Lisser, H., 158, 178 Little, J. M., Jr., 16, 43 Lloyd, C. W., 149, 153, 161, 167 (see Behnken), 1 Y5, 178 Lobotsky, J., 149 (see Lloyd), 153, 161, lY8

Locher, F., 277, 292 Locks, M. S., 63, 65 Lockwood, J. E., 107, 135 Loeb, L., 81, 118, 129, 135, 276, 293 Loeser, A., 102, 135 Logan, V. S., 227 (see Allen), 236, 24? Long, C. N. H., 85, 108 (see Sayers), 118, 135, IS?, 138, 185, 186, 193, 201, 214, 298, 299, 300, 388 Longstreet-Taylor, H., 83 (see Keys), 134 Lombroso, C., 34, 43 Loofbourow, J. R., 149, 178 Loomis, W. F., 302, 328 Loraine, J. A., 146, 150, 163, 170, 171, 178

Lotspeich, W. D., 127, 135 Louis, L. H., 200 (see Conn), 201, 219 Lowe, L., 49, 54, 66 Lowenstein, B. E., 108, 155 Lubitz, J. A., 70, 75, Y6 Ludwig, W., 218, 2-49 Lufkin, N. H., 29, 42 Lukacs, J., 218, 249 Lukens, F. D. W., 185,186,187,188,190, 194, 199, 201, 212, 214, 299, 300 (see Long), 528

384

AUTHOR INDEX

Lumry, R., 261 (see Sweat), 265,274, 299, 294

Lundsgaard, E., 210, $14, 302, 303, 928 Lusk, G., 210 (see Reilly), 211, 215 Lyon, R., 164, 178 Lyons, W. R., 89 (see Nelson), 136

M Mabileau, J., 172, 176 MacArthur, C. G., 298, 327 MacArthur, J. W., 73, 76 McCarrison, R., 5, 43, 98, 107, 115, 125, 136 McCaulay, C., 259 (see Samuels), 271, 294

McCollum, E. V., 89, 100, 136, 138 McConnell, K., 121, 196 McCoord, A., 49 (see Clausen), 50, 54, 66, 66 McCoord, A. B., 121, 136 McCormack, G., 158, 165, 168, 1 7 8 McCreary, J. F., 49 (see May), 50, 54, 66 McCrory, W. W., 128 (see Elkinton), 131 MacDonald, J. J., 298, 327 McDonald, M., 162 (see Mack), 165, 168, 179

McDonald, W. J., 9, 4.4 McDougall, E. J., 302, 680 McGee, L. C., 253, 293 McGrew, R. V., 277 (see Marker), 293 Mach, R. S., 154 (see Watteville), 181 McHenry, E. W., 147, 165, 179, 180 Mack, H. C., 162, 165, 168, 179 McKay, A. F., 277, 292 MacKay, E. M., 299 (see Wick), 307, $86, 364,368 1 McKelvey, J. L., 154, 157, 179 MacKenzie, C. D., 227 (see Allen), 236, 247 Mackenzie, K. R., 128 (see Beck), 129 McKibbin, J. M., 111, 112, 113 (see Schaefer), 114, 130, 138 MacLagan, N. F., 147 (see Henderson), 162, 165, 177 MacLeod, D. H., 163 (see Burrows), 176 Macleod, J. J. R., 207, 208, 21.2, 214 McLetchie, N. G. B., 190 (see Dunn), 219

McNaught, M. L., 235 (see Chanda), 236, 237, 247, 248 Macovski, E., 71, 76 McPherson, M., 73 (see Stuart), 75, 77 McQueen-Williams, M., 92, 1Y5 McQueeney, A. C., 111,112,114,115,135 McQuillan, M. T., 229, 230, 249 McShan, W. H., 332, 333, 334 (see Davis), 339, 344, 360, 361, 362, 368, 369

McWilliams, H. B., 123, 124, I32 Madders, K., 203, 214 Maddock, W. O., 81, 82, 83, 135 Madhava, K. B., 125, 136 Maeder, E. C., 89, 136 Magenta, M. A., 196, 213 Magyar, J., 328 Mahorner, H. R., 98, 135 Mainzer, F., 33, 43 Malcolmson, J. G., 19, 34, 43 Malloizel, L., 298, 526 Manning, M. P., 49 (see Filer), 65 Manning, W. K., 89, 135 Manville, I. A., 91, 135 Marazzi, R., 303, 328 Marchesi, F., 93, 135 Margitay-Becht, E., 126, 155 Marin Bonachera, E., 155, i79 Marine, D., 102, 135, 196, 211 Maritz, A., 339 (see Wattenyl), 370 Mark, J., 91, 136, 258, 291 Markardt, B., 64, 65 Marker, R. E., 277, 293 Marks, H. P., 194, 197, $14 Marmorston-Gottesman, J., 251, $94 Marnay, A., 75, 77 Marois, M., 239 (see Courrier), 248 Marrian, G. F., 81, 82, 87, 126, 235, 143, 150, 151, 163, 170 (see Sommerville), 276,179, 180, 277, 278,280, 283, 286, 291, 292, 29.9, 294 Marseli, 335 Martin, G. J., 128, 135 Martin, J. P., 43 Martinet, M., 107 (see Giroud), 110, 132 Martinez, C., 187 (see Houssay), 188, 191, 194, 195, 218 Martins, T.,91, 136 Marx, W., 199, 201, 212, 214 Masina, M. H., 343 (see Huggins), 344, 369

AUTHOR INDEX

Mason, A. S., 173 (see Kepler), 174 (see Butt), 176 Mason, H. L., 12 (see Williams), 4.6, 49 (see Barnes), 66, 178, 256, 271, 272, 277,285 (see Sprague), 286,287,290, 294

Mason, K. E., 48, 49 (see Filer), 62 (see Wright), 63, 66, 66, 67, 80, 81, 82, 90, 91, 92, 93, 94, 126, 136 Masson, G., 280, 294 Masson, G. M. C., 107 (see Schaffenburg), 138 hlathieson, D. R., 287 (see Spraque), 294 Matthew, G. D., 170, 178 Maun, M. E., 96, 136, 136 Maurer, A. P., 49 (see Adlersberg), 62, 65 Mauzey, A. J., 164,179 Mawson, E. H., 62 (see Henry), 66 May, A. M., 33, 43 May, C. D., 49, 50, 54, 66 Mayer, C., 157, 158, 167, 179 Mayer, J., 91, 126, 136 Mayer, M., 168, 170, 179 Mayer, S., 219 (see Roche), 260 Maszocco, P., 319, 327 Meigs, J. V., 159, 179 Meiklejohn, A. P., 8, 43 Meites, J., 82, 83, 84, 126, 136, 140 Mellanby, E., 91, 133 Mellanby, J., 207, 214 Melnick, D., 49, 66 Melzer, F., 259 (see Koenig), 293 Menten, M. L., (see Bessey), 129 Illentha, J., 303, 305, 328 hlering, J. von, 185, 210, 214 Merivale, W. H. H., 165, 168, 179 M e s h , F., 151 (see Jayle), 1Y8 Messina, A., 55 (see Lewis, Morales), 66

Metivier, V. M., 22, 39, 43 Meulemans, O., 59, 66 Meyer, E. G., 147 (see McHenry), 179 hleyer, K., 198 (see Evans), 207, 213 Meyer, R. K., 92, 93, 94, 95, 117, 120, 129, 139, 332, 333, 334 (see Davis), 339, 341 (see Stafford), 342, 343, 344,360, 361, 362, 368, 369, 370 Meyerhardt, M. H., 168 (see Yanow), 181 Meyerhof, O., 205, 214, 319, 328

385

Meyers, A. W., 84, 136 Meyers, G. B., 160 (see Heller), 177 Michel, R., 219 (see Roche), 239 (see Courrier), 248, 260 Mickelsen, O., 83 (see Keys), 134 Milhorat, A. T., 210 (see Deuel), 212 Millen, T. W., 233, 243, 2.48 Miller, A. M., 286, 294 Miller, C. O., 107, 138 Miller, M., 254 (see Schiller), 286 (see M., A. M.), 294 Mills, R. C., 111, 136 Minkowski, O., 185, 214 Minot, G. R., 29 (see Strauss), 44 Minz, B., 9, 44, 128, 136 Mirick, G. S., 119, 136 Mirsky, I. A., 185, 189, 198, 21-4, 319, 326

Missuro, V., 308, 328 Mitchell, E. R., 111 (see Dalton), 112, 130 Mitchell, H. H., 240, 241, 24.9 Miura, K., 10, 16, 17, 18, 4.2, 44 MPlgaard, H., 223, 249 Mollaret, P., 39 (see Guillain), 43 Monetti, G., 102, 136 Monroe, R. A., 220, 222, 249 Montigel, C., 298 (see VersBr), 299, 300, 309, 310, 311, 325, 328, 330 Moore, C. R., 81, 88, 91, 126, 136 Moore, D. G. F., 39, 44 Moore, L. A., 226 (see Thomas), 228,230, 232, 233, 234, 239, 244, 249, 260 Moore, R. A., 91, 136 Moore, T., 48, 57, 63, 64, 66, 121, 130 Morales, S., 55, 66 Morel, F., 239 (see Courrier), 648 Morgan, A. F., 48, 66, 81, 87, 118, 136, 157, 316, 329 Morgan, B. G. E., 89 (see Coward), 107, 111, I30 Morgulis, S., 84, 186 Morikawa, Y., 107, 136 Morley, E. H., 298 (see Ingle), 327, 329 Morley, M., 149 (see Lloyd), 153, 161, 178

Morris, C. J. 0. R., 145 (see Butt), 174, 176

Morris, H. P., 114, 196 Morris, P., 145 (see Butt), 176

386

AUTHOR INDEX

Morrow, A. G., 145 (see Hertz), 165, 177, 179

Morrow, K., 149 (see Lloyd), 153, 161, 178

Morton, J. H., 155, 156, 179 hloustgaard, J., 225, 240 (see Thorbek), 241, 244, 245, 249, 250 Moya, F., 104, 105, 136 Miihlbock, O., 145, 180 Mukherjee, C. L., 171 (see Govan), 177 Mukherjee, R., 240, 241, 249 Mulay, A. S., 107 (see Nadel), 136 Mulinos, M. G., 81, 84, 85, 86, 126, I36 Muller, C., 92, 136 Muller, J. H., 92, 136 Mullick, D. N., 245, 249 Mune, K., 308, 329 Munson, P. L., 256, 286, 294 Muntz, J. A., 322, 323, 329 Muralt, A. von, 10, 44 Murphy, D. P., 161, 179 Murphy, E. A., 89 (see Evans), 132 Murray, S., 303 (see Deuel), 3.26 Mushett, C. W., 111, 114 (see Gaunt), 132, I36 Rlusso, E., 154 (see Watteville), 181 Mutzenbecher, P. von, 218, 249 Myers, G. B., 159 (see Heller), 177 hlyerson, A., 30 (see Alexander), 42

N Nachmansohn, D., 75, 77 Nadel, E., 107, 136 Nalbandow, A. V., 149, 179 Natelson, S., 51, 66 Nathanson, I. T., 159, 179 Needham, D. N., 307, 326, 366, 368 Nelson, A. A., 111, 136 Nelson, D., 320, 329 Nelson, D. H., 253 (see Gassner), 285, 288, 292 Nelson, M. hl., 89, 136 Nelson, N., 185 (see Mirsky), 189, 214 Nelson, W. O., 92, 93, 136, 203, 211, 217 Nesbitt, F. B., 326 Netter, R., 121, 137 Neubiirger, K., 28, 29, 44 Neufeld, A., 185 (see Collip), 212 Neuschloss, S. M., 319, 330

Newerly, K., 49 (see Adlersberg), 62, 66 Newman, B., 63, 66 Nezamis, J. E., 125, 134, 207, 213, 298, 327

Nichol, C. A., 128, 136 Nichols, J., 125, 136 Nicholson, D. P., 223, 249 Niedermeier, R. P., 236 (see Smith), 250 Nielsen, N., 322, 327 Nitzescu, J. J., 299, 329 Nixon, W. L., 168, 334, 370 Noall, M. W., 277, S94 Noble. R. L., 92 (see Drummond), 93, 131

Nolan, F. W., 210 (see Reilly), 211, 216

0 Oakley, W., 171, 179 Oakwood, T. S., 277 (see Marker), 293 Oastler, E. G., 84, 233, 151, 180 Oberle, E. A., 200 (see Ingle), 201, 214 Ochoa, S., 319, 329 Odell, L D., 346 (see Fishman), 3G8 gstergaard, E., 164, 168, I 79 Oesting, R. B., 147, 179 Ogilvie, R. F., 183, 190, 193, 195, 197, 198, 214, 216 Ohkuma, T., 28, 44 Ohno, S., 115, 136 Okey, R., 89, 136 Olson, R. E., 116, 117, 136, 299, 300, 329 O’hlalley, E. O., 322, 326 Opichal, M., 219, 249 Opsahl, J., 362, 369 Opsahl, J. C., 321, 329 Orent, E. R., 100, 136 Orr, 35 Orr, J. B., 98, 136 Orskov, S. L., 323, 329 Oser, B., 62, 66 Oser, B. L., 49, 66 Ostern, P., 313, 329 Ottaway, J. H., 206, 214 Ovando, P., 112 (see Dumm), 131 Overholser, ill. D., 203 (see Nelson), 214 Overman, R., 128, 131 Owen, E. C., 235 (see Chanda), 236, 237, 238, 241, 242, 245, 247, 248, 249

AUTHOR INDEX

P

387

Perlmutter, M., 307,329 Peter, F., 115, 1.40 Peters, V. B., 111 (see Dalton), 112, IS0 Page, J. A., 19, 20, 25, 27, 44 Petersen, W. E., 219 (see Blaxter), 229, Page, J. E., 248 235, g47, 249 Pallister, R. A., 16, 21, 26, 34, 39, 43 Pettit, G . H. N., 233 (see Yates), 260 P'An, S. Y., 92, 136 Phillips, P. A., 62, 66 Papanicoloau, G. N., 81, 137 Phillips, P. H., 6, 43, 89 (see Boyer), 100, Pappenheimer, A. M., 30, 4.4 101, 111 (see Mills), 130, 196, 322 Paracchi, P., 172, 179 (see Boyer), 326 Parente, N., 271 (see Kochakian), 293 Piaux, G., 165 (see SBguy), 168, 180 Park, C. R., 307, 328 Pigeaud, H., 164, 165, 168, 169, 171, 179 Park, E. A., 56, 66 Parkes, A. S., 81, 82, 87, 126, 136, 137, Pijoan, M., 30 (see Alexander), 42 Pilhorn, H. R., 147, 176, 274, 891 221, 224, 248, 291 Parkhurst, R. T., 70 (see Lubitz), 75, 76 Pincus, G., 111, I S ? , 148, 149, 162, 179 Parks, A. E., 162 (see Mack), 165, 168, Pitt-Rivers, R., 221, 249 Plotz, J., 147, 154, 156, 157, 158, 163, 179 168, 169, 179 Parrot, F. O., Jr., 113, 119, 120, 122, 127, Politou, V. C., 150, 165, 280 132 Polley, H. F., 287 (see Sprague), 894 Parrot, J. L., 102, 132 Polskin, L. J., 48, 59, 66 Parviainen, S., 171, 179 Paschkis, K. E., 84 (see Gomez Mont), Pomerantz, L., 81 (see Mulinos), 84, 85, 86, 126, 136 139, 145 (see Rakoff), 179, 272 (see Pomeroy, B. S., 229 (see Petersen), 249 Cantarow), 283, 289, 291,294 Ponz, F., 238, 249 Patterson, H. R., 151 (see Engel), 176 Popjak, G., 62, 66, 137 Patterson, J. W., 192, 195, 196, 216 Popper, H., 49, 51, 56, 64, 66, 121, 137. Patzelt, K., 121, 137 Porges, O., 298, 329 Paul, H., 234, 8.48 Porter, R. R., 7 (see Swank), 46 Paul, O., 236, 848 Pottinger, S. R., 70, 77 Paul, W. D., 189 (see Clark), 212 Pottner, M. M., 261 (see Samuels), 263, Paulson, M., 114 (see Wintrobe), 140 Pauly, H., 221, 849 894 Potts, F. A., 76 Paye, R. S., 49, 66 Poulsen, E., 223, 249 Payne, F. L., 169, 179 Pearlman, W. H., 146, 162, 179, 286, 294 Poussel, H., 75, 77 Power, M. H., 173 (see Kepler), 178, 285 Pearse, R., 147 (see McHenry), 179 (see Sprague), 287, 894 Pearson, P. B., 95, 137 Powers, J. M., 285 (see Cuyler), 29.3 Pearsons, G., 99 (see Sharpless), 138 Prado, J. L., 104 (see Moya), 105 (see Pedersen, J., 172, 179 Dontigny), 105 (see DeGrandprB), Pedersen-Bjergaard, G., 147, 152, 153, 130,131, 133,136 162, 163, 179 Pedersen-Bjergaard, K., 147, 152, 153, Prados, M., 7, 46 Prato, G. S., 99 (see Sharpless), 138 157, 158, 159, 162, 163, 179 Pratt, H. S., 77 Peel, J., 171, 179 Prelog, V., 252 (see Tagmann), 277, 294 Pekelharing, C. A., 4, 34, 44 Presnall, A. K., 103, 197 Pencharz, R., 89 (see Okey), 136 Prestrud, M. C., 105 (see Ingle), 134, 327 Pentz, E. J., 300 (see Engel), 321, 327 Peraita, M., 19, 22, 26, 27, 34, 38, 39, 44 Preti, L., 192, 216 Price, H., 232, 247 PerjBs, J., 115, 129, 137 Price, R. K., 44 Perla, D., 251, 294

388

AUTHOR INDEX

Price, W. H., 205, 816 Prickett, C. O., 6, 30, 44 Priestley, J. T., 185, 816 Proust, M. A., 41, 4.4 Prunty, F. T. G., 191, 216 Prsylecki, St. J., 307, 329 Putter, A,, 72, 77 Pulver, R., 320, 321, 322, 323, 329 Purr, A., 191, 816 Purves, H. D., 99, 125, 134, 137

Q Quaife, M. L., 48, 63, 66 Quastel, J. H., 358, 359, 360, 367, 369 Querido, A., 147 (see Kassenaar), 178 Quick, A. J., 49, 55, 56, 66, 137 Quimby, F. H., 81, 85, 137

R de Raadt, 0. L. E., 38, 44 Rabinovitch, J., 84, 137 Radcliffe, J., 185 (see Priestley), 215 Raffy, A., 70 (see Fontaine), 74, 76, 89, 130 Rafsky, H. A., 63, 66 Ragan, C., 107 (see Hyman), 108, 126, 134

Ragins, A. B., 64, 66 Ragsdale, A. C., 226 (see Reinecke), 228 (see Ralston), 235, 244, 249 Rak, K., 162, 179 Rakoff, A. E., 145, 179 Ralli, E. P., 111, 112 (see Dumm), 115, 131, 137, 320, 387, 329 Ralston, N. P., 228, 235, 244, 240 Ramos, A. P., 172, 179 Randall, L. M., 146 (see Gastineau), 177 Randall, R. M., 97 (see Albanese), 129 Randall, S. s., 221, 248, 2.49 Randoin, L., 121, 187 Ranson, S. W., 26, 44 Rapala, R. T., 253 (see Gassner), 285,292 Rasch, G., 151, 177 Ratsimamanga, A. R., 107, 137 Ratsimamanga, R., 107, 132 Rauchenstein, E., 233 (see Jensen), 248 Ravid, J. M., 169 (see Siegler), 180 Ray, S. C., 234 (see Smith), 850

Ray, S. N., 107, 133 Raynaud, R., 95, 126, 130 Reade, B., 118, 137 Reece, R. P., 84, 96, 137, 140, 224 (see Hurst), 230, 234, 848, 2.49 Reed, C. I., 103 (see Bartoli, Deutsch), 129,191,137

Reed, J. O., 82, 83, 84, 136 Regan, J. F., 203 (see Barnes), 211 Reich, H., 254 (see West), 256, 277 (see Samuels), 287, 291, 294, 295 Reich, R., 253 (see Gassner), 285, 292 Reichert, F. L., 198 (see Evans), 816 Reichstein, T., 253, 277, 291, 294 Reid, E., 199 (see Cotes), 200, 205, 206, 818, 216

Reilly, F. H., 210, 211, 215 Reinecke, E. P., 219, 220, 221, 222, 226, 235, 239, 245 (see Mullick), 247, 249 Reinecke, R. M., 299, 300, 389 Reinhardt, H. L., 165, 179 Reiss, M., 105, 132 Remington, R. E., 98, 135 Remp, D. G., 48, 66 Reymond, A., 147, 179 Reynoso, A., 209 Rhoads, C. P., 163 (see Dobriner), 176, 253, 277 (see Lieberman), 286, 287, 892, 293

Rice, C., 159 (see Nathanson), 179 Richards, R. L., 23 (see Ungley), 46 Richardson, K. C., 198, 215 Richert, D., 299 (see Olson), 329 Richli, R., 169, 180 Richter, F., 226 (see Reinecke), 234, 260 Richterich, R., 315, 330 Riddell, J. D., 17, 44 Riegel, B., 278, 283, 294 Riesser, O., 311, 329 Rietti, C. T., 196 (see Houssay), 813 Riley, G. M., 161, 180 Rinaldini, L. hl., 81, 82, 83, 91, 137 Robbins, S. L., 164, 180 Roberts, J. Z., 323 (see Cowie), 326 Roberts, R. B., 323 (see Cowie), 326 Roberts, S., 367, 370 Robertson, E., 320, 328, 335, 341, 342, 343, 369 Robertson, J. D., 239, 250 Robey, M., 156, 165 (see SBguy), 168,180

AUTHOR INDEX

Robinson, H. L., 35 (see Adolph), 42 Robinson, M., 222, 223, 260 Rocha, A., 91 (see Martins), 236 Roche, J., 219, 222, 239 (see Courrier), 248, 260, 319 (see Abul-Fadl), 326 Rodriguez, R., 104 (see Moya), 105, 136 Rodriguez, R. R., 188 (see Foglia, Lewis), 195, 203, 213, 214, 216 Rogers, J., 150, 154, 180 Rogerson, A. G., 128 (see Elkinton), 131 Rogoff, J. M., 298, 329 Rohdewald, M., 307, 321, 330 Rohrmann, E., 277, 293 Rohse, W. G., 129 Rokhlina, M. R., 112, 137 Roper, E. A., 103 (see Sherwood), 138 Roseman, E., 6 (see Aring), 7, 9, 42 Rosen, S. H., 102 (see Marine), 136 Rosenbaum M., 6 (see Aring), 7, 9, 42 Rosenberg, A., 49 (see Sobel), 53, 54, 55, 56, 57, 59, 60, 61, 62, 67 Rosenberg, T., 360 (see Adman), 368 Rosenblum, L. A., 33 (see Jolliffe), 40,43 Rosenmund, H., 148,180 Rosentahl, M. C., 128, IS9 Ross, E., 278 (see Dorfman), 292 Ross, R. A., 154 (see Hamblen), 177 Roth, 0. A., 156, 176 Roth, P., 112 (see Dumm), 131 Rothstein, A,, 323, 329 Rowland, S. J., 234 (see Bartlett), 235, 236, 237, 238, 247 Rowlands, I. W., 92, 137 Rubinstein, 1. M., 341 (see Stafford), 342, 343, 344, 369 Runnstrom, J., 329 Rusch, H. P., 81 (see Boutwell), 85, 115, 130 Ruschig, H., 276 (see Slotta), 294 Russel, W. R., 31, 4 S Russell, J. A., 199 (see Wilhelmi), 216, 320, 329 Rust, W., 160, 180 Ruzicka, L., 252 (see Tagmann), 277, S94, 296 Ryan, A. E., 49 (see Jones), 66 S

Sabol, M., 98 (see Sharpless), 138 Sadhu, D. P., 103, 257, 1S9

389

Saffran, J. C., 148, 153, 177 Sailer, E., 303, 314, 315, 330 Saint, J. H., 189 (see Burtness), 212 Salhanick, H. A., 111, 137, 168, 277 (see Noall), 294 Salinger, S., 149 (see Watteville), 181 Salmon, W. D., 6 (see Prickett), 44, 111, 116, 151, 137 Salter, H. P., 90, 91, 140 Samuels, L., 80, 137 Samuels, L. T., 80, 81, 88, 91, 95, 96, 104 (see Sayers), 126, 127, 236, 137, 146 (see West), 154, 157, 169, 179, 181, 251,253 (see Gassner), 254, 255, 256, 259, 261, 263, 265 (see Sweat), 266 (see Levedahl), 269 (see Bryson), 270, 271, 272, 273, 274 (see Lumry), 276, 277, 280, 281, 285, 287, 288 (see Nelson), 291, 291, 292, 293, 294, 296, 303 (see Deuel), 326 Santa, N., 70, 75, 77, 107 (see Giroud), 110, 132 Sarason, E. L., 112, 137 Sass-KortsBk, A., 300, 329 Savage, E. E., 126 (see Deuel), 130 Savard, K., 104 (see Moya), 105, 136 Sawyer, C. H., 339, 368 Sayers, G., 104, 108, 120, 137, 138 Sayers, M. A., 104, 108, 120, 137, 138 Schiifer, A., 102, 138 Schaefer, A. E., 114, 138 Schaffenburg, C., 107, 138 Scharf, A. J., 55, 67 Schenker, V., 110, 118, 122, 138 Scheps, M., 102 (see Elmer), 132 Scheube, B., 4, 10, 16, 18, 44 Schilder, P., 29, 4.2 Schiller, S., 153, 180, 254, 294, 300 (see Engel), 319 (see Hayano), 321, 327, 350, 351, 3669 Schinkel, M., 151 (see Engel), 176 Schneider, H. A., 49 (see Lewis), 51, 53, 55, 62, 66 Schneider, J. J., 271, 272, 286, 290, 294 Schober, M., 102, 138 Schondorff, B., 210, 616 Schoeneck, F. J., 169, 180 Schrader, G. A., 6 (see Prickett), & Schraffenberger, E. J., 89, 140 Schretzenmayr, A., 17, .64

390

AUTHOR INDEX

Schroeder, M. S., 150, 165, 177 Schteingart, M.,127, 131 Schulman, J. H., 53 (see Frazer), 66 Schultz, L. H., 236 (see Smith), 260 Schultze, A. B., 231, 260 Schulze, E.,95, 102, 103, 127, 134, 138 Schulze, W., 186,816 Schuman, H., 305, 321,329 Schwter, N., 188 (see Foglia), 213 Schuurmans, D., 224,226,160 Schuyl, J. W., 89, 138 Schwartz, T.B., 339, 369 Scarlet, H.,70 (see Camien), 76 Scattergood, L. W., 71, 77 Scott, G.I., 16,35, 39, 44 Scott, H.H.,16, 19,21, 34, 39, 44 Scott, W. W., 63, 67, 343 (see Huggins),

Shaver, A,, 121 (see Bennett), 129 Shaw, J. H., 111 (see Mills), 115, 119, 120, 122 (see Deane), 123, 127, 130, 136, 138 Shaw, J. J., 89 (see Boyer), 100, 101, 1 30 Sheehan, H. L., 190 (see Dunn), 213 Shelling, D. H., 50, 67 Sheppard, R., 189 (see Ingle), 200, 201, 813,214

Sherman, M.,51 (see Sobel), 53,54,55,67 Sherwood, C. R.,128,131 Sherwood, T.C., 103,138 Shettles, L. B., 97, 133,138 Shils, M.E., 89, 100,138 Shimazono, J., 10,16, 44 Shimotori, N., 48, 66 Shin, H., 87, 138 369 Shipley, R. A., 197, 216, 278 (see DorfScow, R. O.,334, 370 man), 286 (see Howitt), 292,193 Scowen, E. F., 102, 139 Shippel, S. L., 173, 180 Seath, D. M., 243, 260 Shive, W., 69 (see Williams), 77 Sebrell, W. H., 34, 44, 89, 111, 130 Shock, N. W., 63 Seckel, H.P. G., 307, 389, 366, 369 Shohl, A. T.,208 (see Cannon), 209, 212 Segal, A. D., 63, 67 Shourie, K.L., 41, 44 Segaloff, A., 153,176 Shuman, C. R., 321, 329 SBguy, J., 156, 165, 168, 180 Siderius, P.,147 (see Kassenaar), 178 Seitchik, J., 143, 170, 180 Siegler, S. L., 154, 169, 180 Sellers, D. M.,259 (see Samuels), 271,294 Siehrs, A. E., 107, 138 Sellers, K. C., 49,66 Silberbush, F. F., 239,247 Selman, B. C., 63 (see Scott), 67 Silvette, H.,298 (see Britton), 299,326 Seldenrath, H.C., 147 (see Kassenaar), Simms, H. D., 107, 111, 136 178 Simonnet, H., 154, 157, 158, 174 (see Selye, H., 81, 85, 86, 103, 104 (see B6clBre), 176 Moya), 105 (see Hay, DeGrandprB, Simonsen, J. L., 41 (see Anderson), 4.2 Henriques), 109, 110, 111, 112, 115, Simpson, G. K., 221, 860 118,122, 124,125,130, 131,153, 136, Simpson, J., 27, 4.4 138, 185 (see Collip), 812, 294, 308, Simpson, M.E., 84 (see Lee), 88,90,126, 3.99, 349, 369 132,136, 139, 148,149,152,153,162, Selzer, L.,308, 327 164, 168, 172, 176, 198 (see Evans), Semmons, E. M., 147 (see McHenry), 199 (see Li), 201 (see Becks, Marx), 212, 213, 214, 320 (see Fraenkel165, 179, 180 Conrat), 3.87, 335,337, 369 Senarclens, F. de, 154, 180 Severinghaus, A. E., 90,1% Sinclair, H. M., 80, 139 Sevringhaus, E. L., 286 (see Miller), ,994 Singer, E., 92,94,157, 139 Shah, S. R. A., 41, 4 Singer, T. P.,192,811, 216 Shapiro, B., 302,319 Siperstein, D. M., 81,139 Sharman, I. M.,57, 63, 64,66 Skelton, F. R., 111, 115, 139 Sharples, G. R., 98, 99, 138 Skinner, J. T., 101, 139 Skutta, G., 115,139 Sharples, L. R., 19, 44 Shattuck, G.C., 9,29, .61 Slanetz, C. A., 92 (see P'An), 136

AUTHOR INDEX Slaughter, D., 202, 207, 210, 213 Slein, M. W., 205 (see Colowick), 212, 216, 319, 326 Slocumb, C. H., 287 (see Sprague), 294 Slotta, K. H., 276, 294 Smelzer, J., 81 (see Mulinos), 126, 1% Smidt, W., 102 (see Sturm), 13'9 Smirk, F. H., 26, 42 Smith, B. F., 12 (see Williams), 4.6 Smith, C. H., 17 (see Riddell), 44 Smith, D. A., 19, 21, 27, 35, 44 Smith, G. V. S., 143 (see S., O.), 153, 163, 164, 169, 170, 171, 180 Smith, H., 319, S29 Smith, H. W., 210, 616 Smith, J. A. B., 234, 235, 260 Smith, M. H., 161 (see Riley), 180 Smith, 0. W., 143, 147,153, 163,164, 169, 170, 171, 180 Smith, P. E., 84, 91, 136, 139 Smith, R. H., 205 (see Reid), 206, 216, 233 (see Jensen), 248 Smith, V. R., 236, 260 Smits, G., 311, 329 Smitskamp, H., 21, 23, 25, 44 Sneddon, I. B., 35,39, 42 Snow, S., 51 (see Sobel), 53, 54, 55, 67 Snyder, A. F., 165 (see Weisman), 181 Sobel, A. E., 47, 48 (see Polskin), 49 (see Kramer), 50, 51 (see Natelson), 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 66,66,67 Sobotka, H., 49 (see Adlersberg), 62, 66 Soiva, K., 171 (see Parviainen), 179 Soll, S. N., 272 (see Glass), 292 Somers, G. F., 248 Sommerville, I. F., 143, 150, 170, 180, 253 (see Dobriner), 278, 279, 280, 285, 287, 292, 294 Somogyi, J. C., 321, 329, 3SO Sorba, M., 172, 180 Soulairac, A,, 303, 316, 320, 329 Soule, S. D., 165, 168 (see Yanow), 180, 181

Spector, H., 96, 129 Speirs, R. A., 117, 120, f39 Spence, A. W., 102, 1.99 Spiegelman, A. R., 203, 216 Spielman, A., 229 (see Petersen), 249 Spielmann, F., 167, 172, 177, fS0

39 1

Spies, T., 11, 46 Upies, T. D., 6 (see Aring), 7, 9, 42 Spillane, J. D., 16, 35, 39, 44 Sprague, R. G., 173 (see Kepler), 178, 285,287, 294, 300, S29 Sprawson, C. A., 17, 44 Ssobolew, L. W., 186, 216 Stadie, W. C., 319, 329 Staehelin, D., 302, 311, 312, 313, 329 Stafford,R. O., 341, 342, 343, 344, 369 Stangl, E., 187, 216 Stanier, J. E., 62 (see Henry), 66 Stanletr, C. A., 55, 67 Stannus, H. S., 19, 21, 22, 26, 34, 38, 39,

44 Starr, 193, 213 Starr, P., 80, 100, 139 Stecher, R. M., 145 (see Ungar), 180 Steenbock, H., 101 (see Waddell, Skinner), 159 Stefanini, M., 128, 139 Stefko, W., 84, 139 Steigman, F., 49 (see Popper), 56, 66 Stein, S. I., 92, 139 Steinberg, C. L., 67 Stenram, U., 316, 317, 328, 329 Stephens, D. J., 81, 84, 139 Stern, D., 147, 165, 168, 169, 180 Stetson, R. P., 203 (see Gessler), 213 Stetten, D., Jr., S29 Stevenson, M. F., 150, 180 Stewart, C. A., 81, 139 Stewart, G. N., 298, 329 Stewart, H. C., 53 (see Frazer), 66 Stocknrd, C. R., 81, 137 Stoerk, H. C., 119, 139 Stokem, M. B., 286 (see Lieberman), 893 Stokes, J., Jr., 128 (see Elkinton), 131 Stone, S., 49, 67 Stott, H., 98, 139 Stran, H. M., 150 (see Jones), 154, 163, 178

Strauss, M. B., 9, 29, 44, 299 (see Buell), 326

Stroink, J. A., 145, 180 Struck, H. C., 103 (see Dmtsch), 131 Stuart, C. A., 72, 73, 75, 77 Studer, P. E., 286 (see Lieberman), 993 Sturgis, S. H., 153,154, 160, 162, 185,180 Sturm, A., 102, 1.99

392

AUTHOR INDEX

Siillmann, H., 302, 328, 330 Sulman, F., 165, 166 (see Zondek), 167, 172, 181 Supplee, G. C., 111, 114, 139. Sure, B., 59, 67 Suter, S., 190, 214 Sutherland, 318 Sutherland, E. S., 163, 180 Sutherland, E. W., 197, 205, 212, 216, 318, 329 Sutton, D. C., 83, 139 Sutton, T. S., 62 (see Esh), 66,90, 139 Swank, R. L., 7, 8, 46 Swam, H. G., 112, 139 Swanson, E. W., 226, 234, 250 Swanson, R. G., 226, 231, 260 Sweat, M. L., 261 (see Samuels), 263, 294 Swhgle, W. W., 298, 329 Swyer, G. I. M., 165, 168, 180 Sydenham, A., 83, 139 Sydenstricker, V. P., 33 (see Cleckley), 40, @, 45 Sykes, J. F., 226 (see Thomas), 228, 232, 233,260 Szanto, G., 102, 129 Szego, C., 259 (see Koenig), 293 Szego, C. M., 367, 370 Szent-Gyorgyi, A., 107, 139 Saorengyi, E., 322, 328 Szumowski, P., 225, 260

T Tabor, C. W., 57 (see Klopp), 66 Tagmann, E., 252, 294, 295 Takamatsu, H., 316, 329 Talbot, J., 319 (see Eisenberg), 327 Talbot, N. B., 105, 122 (see Gardner), 132, 139, 151 (see Zygmuntowicz), 181 Tallman, J., 72 (see Stuart), 77 Tanaka, T., 29, 4.6 Tausk, M., 276 (see Adler), 291 Taylor, H. C., 163 (see Dobriner), 176 Telford, I. R., 94, 139 TeLinde, R. W., 280, 293 Terbruggen, A,, 102, 139 Teresa, S. I., 88, 139 Terkildsen, K., 169, 177

Thacker, E. A., 103 (see Reed), 1% Thaddea, S., 329 Thalberg, J., 59, 67 Thatcher, H. S., 59, 67 Thayer, S. A., 148, 149, 180, 299 (see Olson), 329 ThBrien, M., 109, 110, 131, 139 Thomas, E. M., 55 (see Kagan), 66 Thomas, J. W., 226, 228, 230, 232, 233, 234, 244, 260 Thompson, J., 98, 139 Thompson, S. Y., 70, 75, 76, 234 (see Bartlett), 235, 236, 237, 238 (see Houston), 247, 2.48,250 Thomson, S. Y., 62 (see Henry), 65 Thorbek, G., 225,240, 241, 244, 245,249, 260 Thorn, G. W., 118 (see Lewis), 136, 201, 216, 286 (see Engel), 2'92, 300, 303 (see Koepf), 321, 328 Thygessen, J. E., 340, 369 Tillman, A. J. B., 29, 45 Tipton, I. H., 334 (see T., S. R.), 370 Tipton, S. R., 308, 319, 329, 334, 370 Tissiires, A., 317, 330 Tobian, L., 164, 171, 180 Tobin, S. M., 169 (see Siegler), 180 T$nnesen, M., 152, 153, 157, 158, 159, 179 Tolentino, J. C., 56, 67 Tompsett, S. L., 151, 180 Tonhazy, N. E., 347, 368, 370 Toompas, C. A., 109, 129 Torda, C., 348, 362, 370 Trager, W., 70, 77 Traill, D., 221, 250 Treager, H. S., 126, 139 Trikojus, V. M., 229 (see McQuillan), 230, 249 Truscott, B. L., 102, 137, 139 Tsai, C., 207 (see Evans), 213 Tsao, M., 55, 67 Tschopp, E., 146, 180 Tsunoda, T., 5, 46 Tuchmann-Duplessis, H., 101, 139 Tuckich, E. B., 220 (see Frieden), 2@ Tullner, W. W., 145 (see Hertz), 177 Tulsky, A. S., 168, 177 Turn Suden, C., 315,330 Turcatti, E., 319, 330

AUTHOR INDEX Turner, C. W., 84,137,218 (see Herman), 219, 220, 221 (see Reinecke), 222, 226, 227, 228 (see Ralston), 229 (see McQuillan), 231, 235, 239, 244, 248, 249, 250 Turner, J. C., 107, 108, 126 (see Hyman), 134 Twombly, G. H., 173, 180 Tyler, F. B., 255 (see West), 256, 272, 273, 274, 276, 288 (see Nelson), 294, 296 Tyler, F. H., 146 (see West), 181 Tyslowitz, R., 118, 139

U Umbreit, W. W., 339, 347, 367, 368, 370 Underhill, S. W. F., 92, 94, 15’9 Ungar, F., 145, 180 Ungley, C. C., 23, 45 Unna, K., 111,136 Uribe, C., 122 (see Gardner), 132

V Vail, V. N., 314, 315, 328, 3S0, 335, 342, 369 Valdecasas, F. G., 128, 230 Van Der Rijst, M. P. J., 91 (see Arons), 129 Van Donk, E., 101 (see Skinner), 139 Van Dyke, H. B., 92 (see P’An), 136 Van Landingham, A. H., 234, 235, 237, 244, 247, 250 Van Os, P. M., 91, 126, 139 Van Wagenen, G., 92, IS9 Varangot, J., 165, 115 Vartiainen, S., 171 (see Parviainen), 179 Vass, C. C. N., 191, 116 Vavich, M. G., 236 (see Kemmerer), 237, 248 Vedder, E. B., 5, 6, 16, 18, 34, 45 Venning, E. H., 144, 153, 156 (see Browne), 163, 164, 166, 168, 170, 175, 180, 256, 280, 289, 295 Verne, J., 74, 77, 316, 330 Verzdr, F., 115, 140, 297, 298, 299, 300, 301, 303, 305, 307, 308,309,310, 311, 315,320, 321,322,323,324,326, 327, 328,329, 330,364, 365,366,369,370

393

Vestergaard, P., 151, 181 Viale, G., 319, 330 Vidal, E., 19, 4.6 Viehoever, A., 70,73, 74, 77 Vignos, P. J., Jr., 145 (see Ungar), 180 Villee, C. A., 304, 330 Voegtli, W., 303 (see Mentha), 305, 307, 311, 312, 313, 328, 329 Voegtlin, C., 5, 46 Volk, B. W., 49, 51, 66 Vollmer, E. P., 83, 140 Vollmer, H., 48, 67 Von Kolnitz, H., 98, 135 Von Querner, F., 121, 140 Votta, R. A., 147, 181

W Waddell, J., 101, 140 Wade, N. J., 116, 133, 299 (see Olson), 329 Wagener, H. P., 29, 45 Waisbren, B. A., 191, 214 Walkling, A. A., 145 (see Rakoff), 179 Wallach, D. P., 221 (see Reinecke), 249 Waller, L., 89 (see Coward), 230 Wallner, E., 126, 136 Walshe, F. M. R., 6, 8, 46 Wang, F. C., 299,300 (see Sass-KortsBk), 301, 308, 329, 330 Wangerin, D. M., 97 (see Holt), 13s de Wardener, H. E., 31, 32, 46 Ware, L. L., 190 (see Hughes), 613 Waring, E. J., 19, 46 Warkany, J., 61, 67, 89, 1.60 Warren, F. L., 163 (see Burrows), 176 Warren, S., 187, 615 Watson, E. M., 97, 14.0, 313, 315, 330, 341, 342, 5’70 Wattenwyl, H. von, 157, 181 Wattenyl, H., 339, 5’70 Watteville, H. de, 141, 149, 150, 154, 156, 162, 163, 164, 168, 169, 172, 180 Weakley, C. E., 237, 24Y Weakley, C. E., Jr., 235 (see Van Landingham), 237, 244, 250 Weatherby, E. J., 96, 140 Webster, B., 99, 140, 147, I79 Wechsler, I., 29, 45 Weichselbaum, A., 187, 216

394

AUTHOR INDEX

Weick, G., 55, 6Y Weill, J., 83 (see Zimmer), 140 Weir, J. F., 29, 46 Weisman, A. I., 165, 181 Welch, J. W., 103 (see Reed), 13Y Wells, B. B., 210, 816 Welt, I. D., 329 Wendt, H., 128, l 4 O Wenner, V., 303, 305, 307, 308, 330, 364, 365, 3Y0 Werner, S. C., 81, 82, 83, 85, 140 Wernicke, C., 28, 29, 32, 46 Wertheimer, E., 192, 212 West, C. TI., 146, 181,251,254,255,256, 270, 271, 272, 273, 274, 276, 296 West, M. M., 49 (see Sobel), 54, 59, 60, 61, 6Y Westfall, B. B., 145 (see Hertz), lY7 Weetphal, U., 154, 163 (see Kaufmann), 168, 178, 181, 276, 280, 291 Whaley, E. M., 46 Wharton, J. D., 343 (see Huggins), 344, 369 Wheatley, V. R., 147 (see Henderson), 162, 165, l Y ? Wheeler, C. E., 200 (see Conn), 201, 21.2 White, A., 85, 108 (see Sayers), 113, 120, 131, 137, 138, 321, 32Y, 330, 339 (see Holman), 369 White, P., 171, 181 218, 235, 238, 2-48 Whitehead, R., 114, 140 Whitney, J. E., 103, 127, 140 Whittaker, J., 85 (see Selye), 138 Wick, A. M., 364 (see Bartlett), 368 Wick, A. N., 299, 326,330 Wickson, M. E., 118, 140 Wiebelhaus, V. D., 124 (see Betheil), 129 Wiener, H., 190, 616 Wilbrandt, W., 302, 330 Wilder, R. M., 12 (see Williams), 46 Wilhelmi, A. E., 199, 216, 320, 389 Wilkinson, J. If.,147 (see Henderson), 162, 165, 177 Wilkinson, P. B., 35, 46 Willardson, D. G., 288 (see Nelson), 294 Willcox, W. H., 16, 46 Williams, D. C., 145 (see Butt), 176 Williams, H. L., 97, 1.60, 313, 315, 330, 341, 342, 370 Williams, R. D., 12, 46

Williams, R. J., 69, Y Y , 99, 140 Williams, R. R., 11, 46 Williams, T. L., 272 (see Cantarow), $991 Williamson, M. B., 62, 67 Willstatter, R., 307, 321, 330 Wilson, 46 Wilson, H., 151 (see Engel), 176, 181, 253 (see Dobriner), 286, 287, 292 Wilson, H. E. C., 210 (see Deuel), 212 Wilson, S. A. K., 6, 46 Winkler, C., 4, 34, .64 Winkler, H., 101, 107, 140 Winkler-Julesz, E., 101, 134 Winter, B., 147 (see Bachman), 176 Winternitz, W., 49 (see Adlersberg), 62, 66 Wintersteiner, O., 276, 291 Wintrobe, M. M., 18, 46, 114, 128 (see Hamilton), 233, 140 Winzler R. J., 220 (see Frieden), 248 Wirs, H., 321, 330 Wise, G. H., 236 (see Allen), 24Y Wiswell, J., 281, 296 Wittle, E. L., 277 (see Marker), 293 Wohl, M. G., 128, 140 Wolbach, S. B., 50, 57, 66, 90, 91, 140 Wolberg, H., 343, 369 Wolcott, M. W., 187 (see Lukens), 199,

.@I4

Wolfe, J. K., 286, 2996 Wolfe, J. M., 82, 90, 91, 92, 136, 140 Wolff, H. G., 348, 362, 370 Wolff, M., 72, Y? Wolfson, W. Q., 285, 296 Wollaeger, E. E., 49 (see Barnes), 66 Wollman, S. H., 334, 370 Wolterink, L. F., 84, 136, 221 (see Reinecke), 249 Woltman, H. W., 29, 42 Wood, M., 151 (see Zgymuntowicz), 181 Wood, M. S., 105 (see Talbot), 139 Wood, W. A., 241, 24Y Woodbury, L. A., 108 (see Sayers), 138 Woodward, T. E., 233 (see Jensen), 248 Woollard, H. H., 5, 46 Woollett, E. A., 248 Work, E., 302, 330 Work, Th. S., 302, 330 Wortis, H., 9 (see Jolliffe), 30, 31, 43 Wrenn, T. R., 233 (see Sykes), 260

395

AUTHOR INDEX

Wright, H., 5, 45 Wright, M. D., 92 (see Drummond), 93, 131 Wright, S. W., 49 (see Filer), 62, 66, 67 Wright, W. S., 208 (see Cannon), 209,212 Wiist, H., 314, 328 Wyman, L. C., 315, 330

Y Yadu, K. B., 126, 140 Yamada, E. Y., 305 (see Hayano), 319, 327,352, 353,354, 355,358,362,367, 369 Yamagiwa, K., 5,445 Yanow, M., 165, 168, 180, 181 Yates, F., 233, 260 Yeomans, A., 7 (see Swank), 46 Yiengst, M. J., 63, 67 Young, F. C., 105, 1.60, 190 (see Hughes), 194, 197, 198, 199, 200 (see Cotes), 203, 205 (see Reid), 206, 207 (see Evans), 212, 218, .%'l,$,216, 319, 329 Young, W. C., 277 (see Fish), 291

Z

Zabin, I., 113, 140 Zaffaroni, A., 286, 296 Zamcheck, N., 126 (see Treager), 139 Zander, J., 163 (see Kaufmann), 168, 178 Zanelli, C. F., 41, 4s Zarrow, I. G., 111 (see Salhanick), 137 Zarrow, M. X., 111 (see Salhanick), 137, 162, 277 (see NoaII), 294 Zeller, E. A., 339 (see Wattenyl), 370 Zepplin, M., 99 (see Halverson), 133 Zetterstrorn, R., 63 (see Ermster), 66 Ziegler, J. A., 323, 328 Zimmer, R., 83, 140 Zimmerman, H. M., 5, 18, 46 Zondek, B., 91, 140, 145, 163, 165, 166, 167, 172, 181, 256, 280, 296 Zorn, W., 226,234, 260 Zucker, T. F., 119, 183 Zuntz, N., 210, 116 Zwemer, R. L., 108, 135 Zygmuntowicz, A. S., 105 (see Talbot), 139, 151, 181

Subject Index A

uterine, 348 ovariectomy and, 348 ACTH, see under Adrenocorticotropic estrogen and, 348 Adenosinetriphosphate, hormone effect on enzyme substrate oxidation in ATP, see under Adenosinetriphosphate kidney, following adrenafectomy, Abortion, chorionic gonadotropin blood level in, 348 166 Adenylic acid, estrogen blood level in, 167 effect on enzyme substrate oxidation in hormonal disturbances in, 166 kidney following adrenalectomy, pregnanediol excretion and, 167, 168 348 Absorption, Adrenal cortex, of fat-soluble vitamins, 47, 48-56 acetylcholine synthesis in brain and, selective, intestinal of sugar, 302 348 adrenal cortex and, 302, 303, 316 ascorbic acid in, 107 alkaline phosphatase and, 302, 313, and function of, 107 316 cell metabolism and, 298 21-Acetoxypregnenolone, cholesterol and, 125 effect on glycogen metabolism in rat choline and, 116, 117 diaphragm, 365 dehydroisoandrosterone and, 289 Acetylcholine, effect on activity of flavines, 320 synthesis, in brain, 348 on diabetogcnic activity of estrogens, adrenal cortex and, 348 202 on pantothenie acid activity, 320 hypophysectomy and, 348 ACTH and, 348 independent function of different layers thiamine and, 9 of, 112 transmission of nerve impulses and, enzymic oxidation of A6-3(p)-ols in, 287 9-10 3-Acetylpyridine1 pantothenic acid deficiency and, 111toxic effect on Daphnia, 75 115 Acne vulgaris, phosphorylating enzymes and, 301ff., vitamin A and, 59 303, 309 Acrodynia, 19 urinary steroids deriving probably Actomyosin, 348 from, 285-287 in uterus, 348 vitamin D and, 103 effect of castration on, 348 Adrenal glands, androgenic ketosteroids and, 252, 253 of estrogen on, 348 of ovariectomy on, 348 carotene in, 121 Addison's disease, dehydroisoandrosterone and, 289 effects of inanition on, 85-87 blood sugar and, 298 Adenosinetriphosphatase, species differences in, 86-87 effect of desoxycorticosterone on, 363 of pyridoxine deficiency on, 119ff. steroid hormones and tissue, 367 phosphorylation and, 303 396

SUBJECT INDEX

397

progestins from, 276 effect on amino acid oxidase, 339 vitamin A in, 121 of stress on requirement for, 109 stress and, 121 Adrenocortical steroids, see also under Adrenal tumors, Adrenocortical hormones and under effect on urinary dehydroisoandrosnames of individual steroids diabetogenic activity, 201, 299, 300 terone, 255 Adrenalectomy, effect on alanine deaminase, 320, 321 alloxan diabetes and, 193, 194 on alkaline phosphatase, 314, 315, effect on aminooxidase of kidney and 317, 318, 341 liver, 338, 339 on arginase, 320 on arginase of liver, 320, 335, on enzymes of carbohydrate metabolism, 297-330 337 on blood sugar, 298 on gluconeogenesis from proteins, on diabetes following pancreatec300 on glutamic deaminase, 320, 321 tomy, 186, 201 on glycogen metabolism, 300, 305on enzyme substrate oxidation in kidney, 348 309, 319, 325 ATP and, 348 on hexokinaee activity, 319 adenylic acid and, 348 on hyaluronidase, 321, 362 cortisone and, 348 on oxidase systems, 319-320 on glycogen formation, 298,299,301, on protein enzymes, 320, 321 on serum peptidase, 321 303-305, 364 insulin and, 305 on lactacidogen of muscle, 319 on liver enzymes, 347 metabolism, 285 by tissues, 287-289 on kidney proline oxidase, 347 cortisone and, 347 respiration and, 308 on oxidation of tissue enzyme sub- Adrenocorticotropic hormone, activity, KCl and, 127 strates, 368 cortisone and, 368 ascorbic acid deficiency and, 128 carbohydrate metabolism and, 193 on phosphatase activity, 315, 316, choline deficiency and secretion of, 117, 317,318 on phosphocreatine of muscle, 319 118 on phosphorylase activity, 309-313 diabetogenic action, 200-201, 205 mechanism of, 200 on selective intestinal sugar absorpeffect on acetylcholine synthesis in tion, 302, 316 on succinoxidase of liver, 334 brain following adrenalectomy, electrolyte metabolism and, 321 348 Adrenaline, on androgens in adrenal vein blood, diabetogenic action, 201-202, 208, 209 253 on arginase activity, 320 Adrenocortical extract, on brain cell respiration following effect on glycogen metabolism in liver, castration, 356 366 of dietary protein on secretion and on kidney and liver arginase, 335 synthesis of, under stress, 104-107 synergism with anterior pituitary exon glycogen metabolism, 200 tract, 205 on hexokinase activity, 319 Adrenocortical hormones, see also under on serum peptidase, 321 Adrenocortical steroids and under function of, 105 names of individual hormones growth-inhibiting activity of, 201 anoxic anoxia and secretion of, 119 hypoferremia and, 128 ascorbic acid and, 107, 108-111

398

SUBJECT INDEX

inanition and, 85,86 number of, 105 nutrition and response to, 116 purine metabolism and, 193 riboflavin deficiency and secretion of, 119 urinary steroids following administration of, 285, 287 vitamin K deficiency and, 128 Adrenocorticotropin, see under Adrenocorticotropic hormone Age, effect on absorption and transportation of vitamin A, 63-64 DL-Alanine, effect on inhibition of Dmmino acid oxidase by desoxycorticosterone, 353,354,355 Alanine deaminase, effect of adrenalectomy on, 320 Alarm reaction, 116 choline deficiency and, 116 inanition and, 117 pantothenic acid deficiency and, 111 Alcoholism, peripheral neuritis and, 9,29 Wernicke’s encephalopathy and chronic, 30 3(a),20(a)-Allopregnandiol, 277 as metabolite of progesterone, 278 3,20-AllopregnanedioneI 277 as metabolite of progesterone, 278 Allopregnan-3(a)-ol-20-onel277 as metabolite of progesterone, 278 Allopregnan-3(8)-ol-20sne, 277 as metabolite of progesterone, 278 3(o), 11 (B), 17(a),2O12l-AUopregnanepentol, effect on glycogen metabolism, 365 3(~),16(a),20(~)-Allopregnanetriol,277 as metabolite of progesterone, 278 Allopregnane-3(a),17(a),2l;triol-2O-one effect on D-amino acid oxidaae, 356 Allopregnene3(a),6(a)-diol-20-one, 277 as metabolite of progesterone, 278 Alloxan, ascorbic acid and, 190, 191, 196 dehydroascorbic acid and, 195 derivatives, diabetogenic activity, 192 diabetogenic action, 190ff.

adrenalectomy and, 193, 194 ascorbic acid and, 190, 191, 196 factors influencing, 191 hypophysectomy and, 193 mechanism of, 191-192 thiouracil and, 193 effect on pancreas, 191, 192,194 Amenorrhea, excretion of FSH in, 159 of gonadotropin in, 158 of pregnanediol in, 158 Amino acid deficiency, effect on gonads, 96-98 Amino acid oxidase, effect on kidney, 337 steroid hormones and tissue, 367 D-Amino acid oxidase, effect of adrenalectomy on, 320 effect of steroid hormones on, 319,320, 352-358 inhibitory effect of desoxycorticostePone on, 352-355, 356, 358, 362363, 367 Dmlanine and, 353,354, 355 enzyme concentration and, 353 incubation time and, 353 isoalloxazine adenine nucleotide and, 354,355 possible mechanism of, 353, 354 L-Amino acid oxidase, effectof desoxycorticosterone on, 363 Amylase, effect of desoxycorticosterone on, 363 Androgens, adrenal, testicular androgens and, 258 in blood, 146 diabetogenic activity, 203 effect on D-amino acid oxidase, 356,357 on arginase of kidney and liver, 336338 on carboxylase, 362 on enolase, 362 on hyaluronidase, 363 on malic oxidase, 362 on phosphatase in tissues, 341, 343, 345 on succinoxidase of liver, 361 metabolism, 252-276 in kidney, 269-271 in liver, 258-269ff., 272-274,275,276

SUBJECT INDEX

possible pathways, 257 origin, 252 testicular, adrenal androgens and, 258 liver and, 258 urinary excretion, 147 during menstrual cycle, 154 in pregnancy, 103 and tumors, 172 3(a),17(p)-Androstanediol, effect on *amino acid oxidase, 356

3(8),17(?)-Androstanediol, effect on glycogen metabolism in rat diaphragm, 365 3(p),17 (8)-Androstanediol, effect on kidney arginase, 337, 338 effect of 17-methyl derivatives on, 338 3(a),17(p)-Androstanediol, as metabolite of testosterone, 254, 255 of androsterone, 272 Androstane-3 (a),l1(p)-diol-17-one, see under 1I-Kydroxyandrosterone 3,17-Androstanedione, effect on kidney arginase, 338 on oxidation of a-glycerophoaphate, 359 as metabolite of androsterone, 272 of testosterone, 254, 255 urinary excretion following ACTH administration, 287 Androstan-3(a)-ol-11,17-dione,see under 11-Ketoandrosterone A&-Androstene-3( B ) , 17(j3)-diol, effect on o-amino acid oxidase, 357 on oxidation of a-glycerophoaphate, 359, 360 on glycogen metabolism, 365 As-Androstene-3 ( 8 ) )17(a)-diol, as metabolite of dehydroisoandrosterone, 256, 271 As-Androstene-3(p), 17(/3)-diol, adrenocortical origin of urinary, 286 A4-Androstene-3,17-dione, effect on D-amino acid oxidase, 356 on choline acetylase, 362 on oxidation of a-glycerophosphate, 359, 360 on tissue respiration, 350 as metabolite of dehydroisoandrosterone, 256

399

of testosterone, 255, 258, 261, 264 metabolites of, 255 As-Androstene-3, 17-dione, as metabolite of testosterone, 290 Ag-Androsten-3(a)-ol-17-one, adrenocortical origin of urinary, 286 A~-Androsten-3(~)-ol-l’l-one,see under Dehydroisoandrosterone Ax-Androsten-I T-one, from androsterone, 254 AS-Androstene-3(8),16(a),17(p)-triol, adrenocortical origin of urinary, 286 As-Androstene-3(@),16(@),17(@)-triol, as metabolite of dehydroisoandrosterone, 271 Androsterone, 252 adrenocortical origin of urinary, 286 effect on D-amino acid oxidase, 356 on kidney arginase, 338 metabolism of, 255 in liver, 272 as metabolite of A4-androstene-3,17dione, 255 of dehydroisoandrosterone, 256 of testosterone, 252, 253-254 sodium sulfate, effect on D-amino acid oxidase, 356 on liver succinoxidase, 361 on malic oxidase, 362 urinary excretion, 148 foIlowing ACTH administration, 287 as glucuronide, 256 in pregnancy, 163 Anesthetics, glycosuria due to, 209 Anorexia, thiamine deficiency and, 6 Anoxia, anoxic, secretion of adrenocortical hormones and, 119 Antuitrin G, calcium retention and, 127 Arginase, I action, 334 activity, adrenalectomy and, 320 adrenocortical steroids and, 320 effect of desoxycorticosterone on liver, 363 of steroid hormones on liver and kidney, 334-338

400

SUBJECT INDEX

Arginine, deficiency, effect on testes, 97 Ascorbic acid, adjustment to stress and, 107-110 in adrenal cortex, 107 alloxan and, 190, 191, 196 antithyrotropic effect of vitamin P and, 102 cortical hormones and, 107, 108 in crustaceans, 75 deficiency, ACTH and, 128 effect on pituitary-adrenal system, 107 on response to thyrotropin, 127 effect on anterior pituitary, 101 gonadotropin and, 127 in milk, effect of iodinated proteins on, 236-237, 239 of inorganic iodide on, 237 in pituitary, 110-111 requirements, infection and, 110 toxic doses of drugs and, 110 resistance to cold and, 109 thyrotropic hormone and, 102 Ascorbic acid oxidase, effect of desoxycorticosterone on, 362, 363 Atabrine, antitoxic liver factor and, 123, 124 Ataxia, in dietary deficiencies, 39 Atrophy, muscular, vitamin E and, 4 Avitaminosis, due to impaired absorption, 50, 56 to impaired transportation, 56, 57, 63-64

B Beriberi, “alcoholic” peripheral neuritis and, 9 clinical symptoms, 10-16 cranial nerve lesions in, 38 etiology, 5 deficiency of thiamine and other factors, 6ff., 12 high alcohol consumption, 12 incidence among prisoners of war, I 1 neurological findings in, 14-18

pathology, 4-15 polyneuritis gravidarum and, 9 predisposing factors, 11-14 retrobulbar neuropathy and, 37 Bernard puncture, diabetes due to, 187, 207 Bile acids, vitamin A metabolism and, 49 Biotin, deficiency, effect on gonads, 89 Bisdehydrodoisynolic acid, effect on glycogen metabolism in rat diaphragm, 365 Blood, androgens in, 146, 253 effect of ACTH on, 253 17-hydroxycorticoids in, 288 effect of cortisone on, 288 Blood sugar, Addison’s disease and, 298 adrenalectomy and, 298 Brachyura, meIanin in, 74 riboflavin in, 74 Brain, effect of steroid hormones on cell respiration in, 349-352 C

CG, see under Gonadotropin, chorionic Calcium, antuitrin G and retention of, 127 deficiency, effect on gonads, 89 effect on thyroid, 98 metabolism, effect of iodinated casein on, 242 of thyroxine on, 242 Cancer, AD-etiocholenolone excretion and, 173 Carbohydrate metabolism, ACTH and, 193 activities of different corticoids in, 300-301 , 308 effect of corticoids on enzymes of, 297330 pituitary and, 196 potassium metabolism and, 321-325 Carbon monoxide, glycosuria due to, 209

SUBJECT INDEX

Carhoxylase, effect of steroid hormones on, 362, 363, 364 Carotene, in adrenal glands, 121 effect on estrous cycle of rat, 103 in milk, effect of iodinated proteins and thyroxine on, 236 Casein, iodinated, see also under Proteins, iodinated, as galactopoietic agent in dairy animals, activity of, 218, 223, 224 effect on calcium metabolism, 242 on composition of milk, 234-240 on disease resistance, 243 on milk yield, 224, 226-229 on mortality of young, 244 on niacin content of milk, 237 on phosphorus metabolism, 242 on reproduction, 244 on thyroid, 231 homeostatic effects, 229-234 endocrine effects, 230-232 nutritional effects, 232-233 practical aspects of, 245-246 as source of thyroxine for dairy cows, 219 Castration, effect on brain cell respiration, 349 on kidney and liver arginase, 335, 336 on kidney phosphatases, 340, 343 on serum cholinesterase, 340 on succinic dehydrogenase in tissues, 334 Celiac disease, effect on vitamin A metabolism, 49 Cell metabolism, adrenal cortex and, 298 effect of sex hormones on, 276, 325 A-cells, of pancreas, alloxan and, 190 B-cells, of pancreas, alloxan and, 190, 191, 192 diabetes and, 188-189, 197, 204 effect of dithiaone on, 196 of oxine on, 196 glutathione content and activity of, 194 hydropic generation, 187, 194

401

synthesis of glutathione in, 192 of insulin in, 189, 192 Chastek paralysis, thiamine deficiency and, 30 Chloroform, glycosuria and, 209 21-Chloroprogesterone, effect on D-amino acid oxidase, 357 A4-ChoIesten-3-one, effect on oxidation of a-glycerophosphate, 359 Cholesterol, adrenal cortex and, 125 effect on D-amino acid oxidase, 357, 358 on choline acetylase, 362 on oxidation of a-glycerophosphate, 359, 360 sodium sulfate of, 357, 358 Cholic acid, effect on oxidation of a-glycerophosphate, 359 Choline, adrenal cortex and, 116, 117 deficiency, effect on ACTH secretion, 117, 118 on kidneys, 116, 117 role in nutrition of crustaceans, 75 Choline acetylase, adrenal cortex and, 348-349 effect of steroid hormones on, 362, 367 Cholinesterase, serum, action, 339 effect of castration on, 340 of testosterone on, 339, 340 steroid hormones and, 367 Chorioepithelioma, chorionic gonadotropin secretion and, 169 pregnanediol excretion and, 169 Chromobacter, pigment of, 75 Cladocera, culture media for, 72-73 nutritiona1 requirements of, 75-76 Cold, resistance to, ascorbic acid and, 109 riboflavin and, 118 Compound E, Kendall’s, see under Cortisone

402

SUBJECT INDEX

Compound F, Kendall’s, see under 17Hydroxycorticosterone Compound S, Reichstein’s, see under 17-Hydroxy-1 I-desoxycorticosterone Corticoids, see also under Adrenocortical hormones, Adrenocortical steroids and under names of individual compounds chemical estimation of formaldehydogenic, 151 of reducing, 151 effect on carbohydrate metabolism, 297-330 Corticosterone, diabetogenic action, 201, 299 effect on D-amino acid oxidase, 356 on arginase of liver, 335, 337 on glycogen metabolism in rat diaphragm, 365 on hyaluronidase, 321 on tissue phosphatases, 341 Corticosterone-like hormones, gluconeogenesis and, 112 pantothenic acid deficiency and formation of, 112-113 Cortin, riboflavin-deficiency, and, 118 Corpus luteum, isolation of progesterone from, 276 Cortisone, anti-insulin effect, 121 ascorbic acid and, 107, 108 compounds, effect of Cushing’s disease on circulating, 346 of stress on, 346 desoxycorticosterone and, 107 diabetogenic action, 201, 299 effect on alkaline phosphatase, 341 on D-amino acid oxidase, 356 on arginase, 335, 337, 338 on enzyme substrate oxidation in kidney following adrenalectomy, 348, 368 on 8-glucuronidase, 346 on glycogen formation and, 301, 307, 364, 365, 366 on proline oxidase, 349 on scurvy, 107 isolation from adrenals, ‘185 metabolism, 151, 285, 288-289

steroid excretion following administration of, 287 urinary excretion following ACTH administration, 287 cow, metabolism, effect of iodinated proteins and thyroxine on, 240-243 milk yield, effect of iodinated proteins and thyroxine on, 218, 224-226 of nutrition on, 232 sources of thyroxine for dairy, 219-220 Crayfish, xanthine oxidase in liver of, 74 Creatinine, urinary excretion, 147 Crustaceans, see also under names of individual members amino acids in, 70 ascorbic acid in, 75 mineral requirements, 73 nutrition of, 69-77 role of choline in, 75 of pantothenic acid in, 75 of thiamine in, 75 riboflavin in, 64-75 vitamin A in, 75 vitamin content of, 70, 75 Cushing’s disease, effect on blood serum p-glycuronidase, 346 on circulating cortisone compounds, 346 Cysteine, diabetogenic action of alloxan and, 191 effect on phosphorylase, 312, 313 Cystine, blood glutathione and, 191, 192 Cytochrome c, effect of diethylstilbestrol on, 361 ffavine enzymes and, 320 Cytochrome oxidase, effect of desoxycorticosterone on, 363 of steroid compounds on, 339, 362, 363 of testosterone on, 339

D DPN, see under nucleotide

Diphosphopyridine

BUBJECT INDEX

Daphnia, culture media for, 72-73 requirements for inositol, 75 for minerals, 73 for vitamin B, 74 for vitamin E, 70, 73-74, 76 Deafness, due to dietary deficiencies, 38 Dehydroascorbic acid, alloxan and, 195 diabetogenic action, 195 mechanism of, 195-196 Dehydrocholic acid, effect on oxidation of a-glycerophosphate, 359 11-Dehydrocorticosterone effect on D-amino acid oxidase, 356 on glycogen metabolism in rat diaphragm, 365 in liver, 366 on hyaluronidase, 321 on liver and kidney arginase, 336, 337 on tissue alkaline phosphatase, 34 urinary excretion of pregnane-3 (a),20diol-11-one following administration of, 287 11-Dehydro-17-hydroxycorticosterone, see under Cortisone Deh ydroisoandrosterone, adrenal cortex and, 256, 289 effect on D-amino oxidase, 356 on oxidation of a-glycerophosphate, 359, 360 on succinoxidase, 361 on tissue respiration, 350, 351 metabolism of, 256 in liver, 271-272, 275 sodium sulfate of, 356, 358 urinary, adrenal tumors and, 255 adrenocortical origin, 286 I3esoxycorticosterone, acetate, effect on oxidation of a-glycerophosphate, 359 ascorbic acid and, 107 cortisone and, 107 diabetogenic activity, 299 effect on alanine deaminase, 320, 321 on alkaline phosphatase, 314, 341 on arginase of liver and kidney, 335, 337

403

on blood sugar following adrenalectomy, 298 on carbohydrate metabolism, 299ff ., 305-309, 366 on enzyme systems, 319, 320, 352ff., 362-364 on flavine enzymes, 320 on glutamic deaminase, 320, 321 on glycogen metabolism, 305, 306, 307,366 on phosphorylase, 305, 309, 310, 311 on protein enzymes, 320 on tissue respiration, 349, 350-351 mechanism of, 351-352 site of, 352 glucoside, effect on glycogen metabolism, 365 inactivation, 288 inhibition of o-amino acid oxidase by, 352-355, 356, 358, 362-363, 367 DGalanine and, 353, 354, 355 as compared with other steroids, 356-357 FAD and, 354, 355 incubation time and, 353 possible mechanism of, 353, 354, 367 insulin and, 364 Desoxycorticosterone-like hormones, electrolyte balance and, 112 formation, pantothenic acid deficiency and, 114 site of, 112 Diabetes, see also under Glycosuria B-cells of pancreas and, 188, 189, 204 estrogens and, after menopause, 203 hormonal, 196-206 idiohypophyseal, 197, 198 mechanism of, 205 metahypophyseal, 197, 198, 199 metathyroid, 204 thyroid, 204 incidence, in the United Kingdom, 184 in the USA, 184 mortal t y from, 184 produced by Bernard puncture, 187 by high caloric diet, 186-187 effect of insulin on, 189 by subtotal pancreatectomy, 187188

404

SUBJECT INDEX

estrogens and, 188 insulin and, 189 sex differences in, 188 testosterone and, 188 Dialuric acid, diabetogenic action of, and methyl derivative, 192 Dianisalacetone, effect on liver succinoxidase, 361 Diaphragm, of rat, glycogen metabolism in, 303ff. adrenalectomy and, 303,304 effect of steroid hormones on, 305307,364,366 work and, 304 Dienestrol, effect on liver succinoxidase, 361 on malic oxidase, 362 Diet, high caloric, diabetes and, 186-187, 189 Diethylstilbestrol, diabetogenic activity, 299 effect on cytochrome c, 361 on malic oxidase, 362 on oxidation of a-glycerophosphate, 359,360 on succinoxidase of liver, 361 quinone, effect on oxidation of a-glycerophosphate, 359 Digitalis, glycosuria and, 209 Dimercaptopropanol (BAL), diabetogenic action of alloxan and, 191 3,4-Diphenylhexane-phydroxy-p’-oxyacetate, effect of mono- and disodium, on liver succinoxidase, 361 Diphenylthiocarbazone, diabetogenic action, 196 mechanism of, 196 Diphosphopyridine nuc!cotide, enzymic destruction of testosterone and, 260,261 Dispersing agents, for fat-soluble vitamins, 53, 55 Dithizone, sec under Diphenylthiocarbazone Dlws, effect on requirement for unknown nutrients, 123

of toxic doses on vitamin C requirements, 110 Dysmenorrhea, progesterone and, 155

E Edema, in beriberi, 12, 13, 18 thiamine deficiency and, 18 Electrolyte metabolism, adrenalectomy and, 321 Encephalitis haemorrhagica superior, 28 Encephalopathy, niacin deficiency and, 32-33 Wernicke’s, 28-32 alcoholism and, 30 clinical symptoms, 30ff. pathology, 28-29, 32 thiamine deficiency and, 28, 29-30 treatment, 32 Enolase, effect of steroid hormones on, 362 Enzymes, destruction of testosterone by, of liver, 258, 259-269 of kidney, 269-271 of other tissues, 271 effect of corticoids on, of carbohydrate metabolism, 297-330 phosphorylating, see also under names of individual enzymes adrenal cortex and, 301ff ., 303, 309 adrenalectomy and, 318 Enzyme systems, effect of steroids on, 352-364, 366-368 mechanism of, 366-368 tissue specificity in, 367 Epithelioma, effect of testosterone on respiration of primary Brown-Pearce, 352 20,21-Epoxy-A‘-pregnenediol-3-one, effect on D-amino acid oxidase, 357, 358 Equilin, diabetogenic action, 202 effect of, and sodium sulfate of, on D-amino acid oxidase, 357 Esterme, effect of ovariectomy on uterine and ovarian, 346

SUBJECT INDEX

of estrogens on, 346 of simultaneous administration of progesterone and estrone on liver and kidney, 346 8-glucuronidase and, 345, 346 lipide content of tissues and, 346 steroid hormones and tissue, 346, 367 Estradiol, assay in blood, 145 in urine, 145, 148 diabetogenic activity of, and its derivatives, 202, 290 effect on brain cell respiration following castration, 350 on phosphorylase activity, 310 of simultaneous treatment with insulin and, on alloxan diabetes, 193, 194 on tissue respiration, 350 pancreotropic action, 195 urinary excretion, 148 during menstrual cycle, 153 8-Estradiol, effect on kidney arginase, 337, 338 Estradiol benzoate, assay in pregnancy, 145 effect on diabetes after menopause, 203 Estriol, diabetogenic action, 203 urinary excretion, 148 during menstrual cycle, 153 in pregnancy, 163 Estrogens, see also under names of individual compounds activity, nutrition and, 128 antidiabetogenic: effects, 203 assay in blood, 145 in urine, 145, 150 blood levels of, abortion and, 167 diabetogenic action, 188, 202, 203, 299 adrenal cortex and, 202 effect on alanine deaminase, 320 on u-amino oxidase, 357 on ATP-ase, 348 on esterase, 346 on 8-glucuronidase, 345, 346 on glutamic deaminase, 320 on liver succinoxidase, 361 possible mechanism of, 361-362

405

on malic oxidase, 362 on oxidation of a-glycerophosphate, 359 of progesterone on metabolism of, 153 on tissue alkaline phosphatase, 341, 342 inactivation by liver, 258 phosphate esters, inhibitory effect on kidney alkaline phosphatase, 360 sodium sulfate, effect on D-amino acid oxidase, 357, 358 on glycogen metabolism, 365 urinary excretion, 147 in amenorrhea, 158 hydatid mole and, 169 in menopause, 159 during menstrual cycle, 153 in ovarian disorders, 157 in pregnancy, 162, 164, 165 in toxemia of pregnancy, 170 tumors and, 172 Estrone, diabetogenic action, 203 effect on D-amino acid oxidase, 357 on esterase, 346 on oxidation of a-glycerophosphate, 359 on tissue respiration, 350 glucoside, effect on kidney alkaline phosphatase, 360 progesterone and, 346 sodium sulfate, effect on u-amino acid oxidase, 357, 358 on liver succinoxidase, 361 urinary excretion, 148 during menstrua1 cycle, 153 17-Ethyny1testosterone1 effect on kidney arginase, 338 3(a), 17(a)-Etiocholanediol, as metabolite of 3~a)-etiocholanol-l7one, 272 3(a),l7(~)-Etiocholanediol, as metabolite of dehydroisoandrosterone, 256 of 3(cu)-etiocholanol-l7-one, 272 Etiocholane-3(a),ll (D)-diol-17-one (11hydroxyetiocholanolone), adrenocortical origin of urinary, 286

406

SUBJECT INDEX

urinary excretion following ACTH adminstration, 287 following cortisone, 287 Etiocholane-3(a),l7(j3)-diol-ll-one (1 1ketoetiocholanediol) , adrenocortical origin of urinary, 286 Etiocholane-3,17-dione, urinary excretion following ACTH administration, 287 3,17-Etiocholanedione, as metabolite of 3(a)-etiocholanol-17one, 272 of testosterone, 254 Etiocholan-3(a)-ol-ll,l7-dione (ll-ketoetiocholanolone), adrenocortical origin of urinary, 286 urinary excretion following ACTH administration, 287 following cortisone administration, 287 Etiocholanolone, adrenocortical origin of urinary, 286 urinary excretion, 148 as glucuronide, 256 in pregnancy, 163, 164 3(a)-Etiocholanol-l7-one, metabolism in liver, 272 as metabolite of A4-androstene-3,17dione, 255 of dehydroisoandrosterone, 256 of testosterone, 253, 254 AO-Etioc holenolone, urinary excretion in cancer, 173 AQ-Etiocholen-3(a)-ol-17-one, adrenocortical origin of urinary, 286

F FAD, see under Isoalloxazine adenine dinucleotide FSH, see Hormone, follicle-stimulating Factors, unknown, in liver, 123 Fat, effect of thyroxine or iodionated casein on milk, 234 Fatty acids, essential, deficiency, effect on gonads, 89 growth hormone activity and, 126 Fertility, vitamin E deficiency and, 93

‘ I Fesselungsdiabetes,” 208 Flay ne enzymes, desoxycorticosterone and, 320 Flavines, activity, ltdrenal cortex and, 320 Folic acid, requirement, hyperthyroidism and, 128

G General adaptation syndrome, 86, 115 nutrition and, 103-125 Genetotrophic factors, 99 Glands, endocrine, see also under name of individual glands nutrition and, 80 Gluconeogenesis, adrenocortical hormones and, 112, 118, 300 blood sugar and, 208 inanition and, 85 in phlorizinized animals, 21 1 adrenals and, 21 1 thyroid and, 211 Glucose, intraperitoneal effect on pancreas, 188-189 phosphorylation, 205 hexokinase and, 205 8-Glucuronidase, action, 34 of blood serum, 346 pregnancy and, 346 steroid hormones and, 346 esterase and, 345, 346 of tissues, 345 estrogen and, 346 ovariectorny and, 346 steroid hormones and, 367 uterine, estrogens and, 345 Glutamic deaminase, effect of adrenalectomy on, 320 of desoxycorticosterone on, 320 of estrogens on, 320 of testosterone on, 320 Glutaminase, effect of desoxycorticosterone on, 362, 364 Glutathione, alloxan diabetes and, 191

407

SUBJECT INDEX

of blood, 191 cystine and, 191, 192 dehydroascorbic acid and, 196 methionine and, 196 effect of anterior pituitary preparations on, in tissues, 195 on phosphorylase activity, 313 synthesis by B-cells of pancreas, 192 a-Gly cerophosphate, oxidation, 358, 359, 360 sources, 358, 359 a-Glycerophosphate dehydrogenase, effect of 3-ketosteroids on, 367 of steroid hormones on, 358-360 brain homogenates and, 358 Glycogen, effect of corticoids on, 300, 301, 305309 formation, adrenalectomy and, 298300, 303-305 metabolism, effect of corticoids on, 305308, 325 possible mechanism of, 308-309 of steroid hormones on, 364-366 in liver, 366 effect of adrenocortical extract on, 366 of corticoids on, 307, 366 in rat diaphragm, 303ff. adrenalectomy and, 303, 304 corticoids and, 305-307 work and, 304 potassium and, 321-323 Glycogenolysis, 207-210 due to nervous damage, 207-208 Glycolysis, potassium and, 325 Glycosuria, see also under Diabetes alimentary, 206-207 asphyxial, 209 emotional, 208-209 mechanism of, 209 etiology, 189 experimental, 183-215 elicited by administration of hormones, 185, 196-206 of phlorizin, 210-211 by anesthetics, 209 by reduction of insulin supply, 185, 196-206

Goats, effect of iodinated casein on milk yield, 218 Goitrogenic agents, effect on iodine metabolism, 99 on anterior pituitary, 100 in vegetables, 98-99 Gonadotropin, chorionic, abortion and, 166 bioassay, 150 blood level in pregnancy, 146 chorioepithelioma and, 169 level in hyperemesis gravidarum, 169 secretion, hydatidiform mole and, 168 urinary excretion, 147 in normal and ectopic pregnancy, 146, 162, 164, 172 pituitary, ascorbic acid and, 127 vitamin B12deficiency and response to, 126 Gonadotropins, see also under names of individual hormones assay, 141-181 effect on ovarian development in hyperthyroid rats, 124 of vitamin deficiencies on, 87-95 secretion, effect of inanition on, 81-87 of protein and amino acid deficiencies, 95-98 urinary excretion, during menstrual cycle, 152 in menopause, 159 in ovarian disorders, 157 in pregnancy, 158 tumors and, 172 Growth hormone, of anterior pituitary, composition, 199 diabetogenic action, 199-200 mechanism, 199 inanition and, 83, 84 nutrition and response to, 126 requirement for pantothenic acid and, 127 vitamin A deficiency and, 127

H Hexestrol, effect on liver succinoxidase, 361

408

SUBJECT INDEX

on malic oxidase, 362 Hexokinase, adrenocortical extracts and, 319 effect of steroid hormones on, 362 inhibition by anterior pituitary extracts, 205, 206 319 insulin and, 319 role in glucose phosphorylation, 205, 206 Hormone(s), see also under names of individual compounds action mechanism of, 331 adrenocorticotropic, see under Adrenocorticotropic hormone assays in obstetrics, 141-181 cell-stimulating, bioassay, 149 urinary excretion during menstrual cycle, 153 diabetogenic action, 185, 196-206 follicle-stimulating, bioassay, 149 urinary excretion in amenorrhea, 159 in menopause, 159 ovulation and, 160 gonadotropic, see under Gonadotropin and under names of individual members lactogenic, see under Lactogenic hormone pituitary, see also under names of individual hormones effect of malnutriture on, 81-101 of nutritive state on response to, 126-127 sex, see under Sex hormones steroid, see under Steroid hormones vitamin A transportation and, 64 Hyaluronidase, effect of adrenocortical steroids on, 321, 362 11(8)-Hydroxyandrosterone, adrenocortical origin of urinary, 286 urinary excretion following ACTH administration, 287 following cortisone administration, 284 17-Hydroxycorticoids, see also under names of individual compounds in blood, 288 following cortisone administration, 288

17-Hydroxycorticosterone, diabetogenic action, 201 effect on D-amino acid oxidase, 356, 358 on glycogen metabolism in rat diaphragm, 364, 365 isolation from adrenals, 285 metabolism, 285 urinary, adrenocortical origin of, 285 excretion following ACTH administration, 287 following cortisone administration, 287 17-Hydroxy-1 I -dehydrocorticosterone, 8ee under Cortisone 17-Hydroxy-ll-desoxycorticosterone, adrenocortical origin of urinary, 286 effect of D-amino acid oxidase, 356 metabolism, 285 3(8)-Hydroxyetiocholenic acid, effect on glycogen metabolism in rat diaphragm, 365 3-Hydroxy-17-ketosteroids, as metabolites of testosterone, 264 1(4’-Hydroxyphenyl)-2-phenylethane, effect on succinoxidase system, 362 17-Hydroxypregnanediol (Pregnanetriol), adrenocortical origin of urinary, 286 17-Hydroxypregnanolone, adrenocortical origin of urinary, 286 17-Hydroxy-A6-pregnenol-3(8)-2O-one, adrenocortical origin of urinary, 286 17(a)-Hydroxyprogesterone, effect on n-amino acidoxidase, 357 on glycogen metabolism in rat diaphragm, 365 metabolism, 285 &Hydroxyquinoline, see under Oxine Hyperemesis gravidarum, chorionic gonadotropin level in, 169 urinary excretion of estrogens during, 170 of pregnanediol during, 170, 171 Hypergl ycemia, effect of pancreatic islets, 199 emotional, 208-209 mechanism of, 209 muscular exertion and, 209 Hyperkeratosis,

409

SUBJECT INDEX

therapeutic effect of vitamin A in, 56, 57 Hypertension, in painful feet syndrome, 25-26, 28 Hyperthyroidism, vitamin requirements and, 110, 128 Hypoferremia, ACTH and, 128 Hy pophysectomy , effect on acetylcholine synthesis in brain, 348 ACTH and, 348 on acid phosphatase in liver, 343 on amino acid oxidase in kidney and liver, 338, 339 on arginase in liver, 336, 337 on diabetes following alloxan administration, 193, 194 following pancreatectomy, 186, 201 on succinoxidase in liver, 334 nutrition and response to, 127 pseudohypophysectomy and, 85

I ICSH, see Hormone, cell-stimulating Inanition, adrenocorticotropin and, 85, 86 effect on gluconeogenesis, 85 on lymphocytes, 85 on pituitary hormones, 81-87 on sex organs, 81, 83 on thymus gland, 85 on thyroid, 84 Inositol requirement, of Daphnia, 75 Insulin, action mechanism, 206 adrenocortica steroids and, 121, 305, 364 B-cells of pancreas and, 189, 192 effect on diabetes following forcefeeding, 189 following pancreatectomy, 189 on diabetogenic action of anterior pituitary extract, 199 on hexokinase, 205, 206, 319 of simultaneous administration of, and estradiol on alloxan diabetes, 193, 194

glycosuria and reduction of, supply, 185, 196-206 vitamin A deficiency and sensitivity to, 121 Intestine, alkaline phosphatase in, 302 histochemical demonstration of, 316317 selective sugar absorption by, 302, 303 alkaline phosphatnse and, 313 iodoacetate and, 302,303 phlorizin and, 302 phosphorylation theory of, 302 Iodide, inorganic, effect on ascorbic acid content of milk, 237 Iodine, deficiency, effect on anterior pituitary, 98, 100 excretion, by mammary giand, 239 renal function and, 99 sulfanilamide and, 99 metabolism, effect of goitrogens on, 99 Isoalloxazine adenine nucleotide, effect on inhibition of D-amino acid oxidase by desoxycorticosterone, sterone, 354, 355 Ieoandrosterone, effect on arginase of liver and kidney, 337, 338 as metabolite of androsterone, 272 of testosterone, 254 17-Isopregnan-3(a)-oI-20-one, 277 a8 metabolite of progesterone, 278

K Kendall’s compound E, see under Cortisone 11-Ketoandrosterone, adrenocortical origin of urinary, 286 7-Ketocho1esterol effect on oxidation of a-glycerophosphate, 359 Ketosteroids, see also under names of individual compounds urinary excretion in pregnancy, 163

410

SUBJECT INDEX

3-Ketosteroids, effect on a-glycerophosphate dehydrogenase, 367 17-Ketosteroids, chemical estimation, 151 concentration in pregnancy, sex of fetus and, 163 conjugation with glycuronic and sulfuric acids, 256, 272 liver disease and, 272 formation from testosterone, 261, 266, 267 urinary, adrenals and androgenic, 252 excretion of, 146, 147 endocrine disorders and, 174 during menstrual cycle, 154 ovariectomy and, 253 tumors and, 173 origin of, 252, 285 I-Keto-l,2,3,4-tetrahydrophenanthrene, effect on liver succinoxidase, 361 Kidney, alkaline phosphatase of, 302,314,317318 adrenalectomy and, 317 localization, 317 selective sugar absorption and, 313 testosterone and, 314 arginase in, steroid hormones and, 334, 335,336 effect of adrenalectomy on proline oxidase in, 347 cortisone and, 347 of castration on u-amino acid oxidase in, 337 of choline deficiency on, 116, 117 of phlorizin on, 210 of vitamin B deficiencies on, 347 inactivation of testosterone by, 258, 269-271 iodine excretion and functon of, 99 metabolism of steroids by, 290 Kidney stones, vitamin A deficiency and, 63 L

Labor, premature, hormonal disturbances and, 166

Lactacidogen, of muscle, adrenalectomy and, 319 Lactation, ascorbic acid requirements and, 110 Lactogenic hormone, diabetogenic action, 204-205 inanition and, 83, 84-85 Lactose, in milk, effect of iodinated casein or thyroxine on, 234-235 Lathyrism, 41 Leukoplakia vulvae, therapeutic effect of vitamin A on, 64 Lipase, effect of desoxycorticosterone on, 362, 363 Lipide extracts, androgenic activity, 251 Lipides, tissue esterase and, 346 Liver , antitoxic factor of, 123-124 adrenocortical hormones, 125 growth activity of, 125 requirement for, 123 drugs and, 123, 124 stress and, 123 arginase in, nutrition and, 337 steroid hormones and, 334, 335, 336, 337 damage, glycosuria and, 209-210 disease, effect on androgen metabolism, 272-274 on conversion of progesterone to pregnanediol, 283-284 enzymes, adrenalectomy and, 347 glycogen metabolism by, steroid hormones and, 307, 366 metabolism of steroid hormones by, 258-276, 280ff., 290 of androgens, 258-269 272-274 of cortisone, 288-289 of desoxycorticosterone, 288 of progesterone, 280ff. storage of vitamin A in, 48, 52, 53 succinoxidase in, effect of adrenalectomy on, 334 of hypophysectomy on, 334 of steroid hormones on, 360-362 unknown factors in, 123

41 1

SUBJECT INDEX Lobster, xanthine oxidase in liver of, 74 Luteotropin, see underlactogenic hormone Lymphocytes, inanit:on and, 85

M Malk oxidaee, effect of androgens on, 362 of estrogens on, 362 mechanism of, 362 Malnutriture, definition, 80 effect on pituitary-adrenal system, 104 on pituitary hormones, 81-101 Mammary gland, effect of thyroxine on, 229 excretion of iodine by, 239 Manganese, anterior pituitary and, 100-101 deficiency, effect on gonads, 89 hlelanin, in Brachyura, 74 Menopause, urinary excretion of estrogen in, 159 of FSH in, 159 of gonadotropin in, 159 Menstrual cycle, hormone excretion during, 152 Metabolism, intermediary, of androgens, 252-276 of non-benzenoid steroid hormones, 251-295 oxidative, steroid hormones and, 349352 Methionine, blood serum glutathione and, 191, 192 7-Methylbisdehydrodoisynolic acid, concentration in organs, 146 2-Methyl-l,4-naphthoquinone, effect on oxidation of a-glycerophosphate, 359 Methyltestosterone, diabetogenic action, 203 effect on D-amino acid oxidase, 338, 356, 357, 358 on arginase in kidney, 337 on diabetes following pancreatectomy, 188 on testosterone destruction b y liver, 267

on tissue respiration, 349-351 metabolism by liver, 265, 266, 267, 268 citrate and, 265, 266 D P N and, 265, 266 kinetics, 266 species differences in, 266 Milk, of dairy animals, composition, effect of iodinated casein and thyroxine on, 234-240 suitability for human consumption of, following administration of iodinated proteins, 239 of thyroxine, 239 thyroxine in, 239 vitamins, fat-soluble in, 57ff. effect of iodinated proteins and thyroxine on, 236 transfer of, to, 57, 59-61 yield, effect of iodinated proteins on, 218, 224-229 of thyroxine on, 218, 224-229 mother’s, vitamin E content of, 62 Mineral deficiencies, effect on anterior pituitary, 98 Mole, hydatidiform, chorionic gonadotropin secretion and, 168 estrogen excretion and, 169 Morphine, glycosuria and, 209 Muscle(s), beriberi and, of limbs, 14-15 potassium in, 325 Myasthenic bulbar paralysis, 39

N Nerve(& cranial, lesions due to dietary deficiencies, 346 ., 38-39 impulses, acetylcholine and transmission of, 9-10 thiamine and, 10 peripheral, in beriberi, 5-10 effect of starvation on, 7 of thiamine deficiency on, 5ff. potassium in, 325 Neuritis, “alcoholic,” peripheral, beriberi and, 9, 29 vitamin B and, 9, 29

412

SUBJECT INDEX

Neuropathies, dietary, 1-45 beriberi, 4-18 cord syndromes in, 39-42 cranial nerve lesions in, 38-39 niacin deficiency encephalopathy, 32 painful feet syndrome, 18-28 retrobulbar, 33-37 beriberi and, 33, 34, 37 clinical symptoms, 35-37 etiology, 37 spinal ataxia and, 39-40 Wernicke’s encephalopathy, 28-32 Niacin, deficiency, encephalopathy and, 32-33 effect on anterior pituitary, 101 on delirium tremens, 33 on enzymic testosterone destruction by liver, 269 of iodinated casein on milk, 237 on toxic psychoses, 33 painful feet syndrome and, 22, 27, 28 Nutrients, factors increasing requirements for essential, 80 unknown, liver as source of, 123 requirements for, 123 Nutrition, anterior pituitary and, 79-140 of crustaceans, 69-77 effect on anterior pituitary hormones, 127 on endocrine glands, 80 on enzymic testosterone destruction by liver, 267-269 on estrogen activity, 128 on milk yield in cows, 232 on response to ACTH, 115 to hypophysectomy, 127 to pituitary hormones, 115, 126127 Nystagmus, thiamine deficiency and, 31

P Pain, glycosuria and, 208 Painful feet syndrome, 18-28 etiology and pathology, 22-27 hypertension and, 25-26, 28

mechanism of pain production in, 23-24 tendon reflexes in, 24-25 treatment of, 21-22 Pancreas, A-cells of, 187, 188, 189 B-cells of, 187, 188, 189 dystrophy, vitamin A metabolism and, 49

effect of intraperitoneal glucose on, 188-189 Pancreatectomy, diabetes following, 185ff., 187ff. andrenalectomy and, 186 estrogens and, 188, 203 hypophysectomy and, 186 insulin and, 189 species differences in, 185 thiouracil and, 188 thyroid and, 187-188 uracil and, 187 Pantothenic acid, adrenal cortex and, 320 deficiency, adrenal cortex and, 111-115 adrenal insufficiency and, 347 alarm reaction and, 111 effect on gonadotropin, 88 on gonads, 88, 89 painful feet syndrome and, 22, 27, 28 requirement, growth hormone and, 127 hyperthyroidism and, 128 response to stress and, 113, 115 role in nutrition of crustacesns, 75 Pellagra, etiology, 34 optic nerve damage in, 34 spinal cord damage in, 39 Pepsin, effect of desoxycorticosterone on, 363 Peptidase, serum, ACTH and, 321 adrenocortical steroids and, 321, 339 Phenocycline, effect on diabetea following subtotal pancreatectomy, 188 Phenols, effect on liver, succinoxidase, 361 Phloririn, action on kidney, 210 adrenalectomy and, 211

SUBJECT INDEX

413

hypophysectomy and, 21 1 effect of iodinated proteins on, 242 thyroidectomy and, 711 Phosphorylase, desoxycorticosterone and, 305, 309, effect on diabetogenic action of anterior pituitary extract, 199 310 of liver, adrenalectomy and, 313 on selective intestinal sugar absorpof muscle, activity, 309, 310 tion, 302, 303 adrenalectomy and, 309ff. glycosuria, 210-21 1 cysteine and, 312, 313 Phosphatase, estradiol and, 310 effect of thyroxine on milk, 238 glutathione and, 313 Phosphatase, acid, progesterone and, 310 in accessory male sex organs, 343testosterone and, 310 344 Phosphorylation theory, effect of castration on, 343, 344 of selective intestinal sugar absorption, of testosterone on, 343, 344 302 activity, 315 Physostigmine, thiamine and, 9 adrenalectomy and, 315 Pituitary-adrenal system, see also under desoxycorticosterone and, 363 General adaptation syndrome steroid hormones and, 315 effect of ascorbic acid deficiency on, in kidney, castration and, 340, 343 107, 108 steroid hormones and, 343 of inanition on, 115 testosterone and, 342, 343 of malnutriture, 104 serum, cancer of prostate and, 344 of overfeeding, 125 Phosphatase, alkaline, of pantothenic acid, 112, 113, 116 activity, 313, 314 of potassium deficiency, 122-123 adrenalectomy and, 314-315 of pyridoxine deficiency, 119-121 adrenocortical steroids and, 314, 315, of rape seed oil, 125 316, 341 of riboflavin, 118 androgens and, 342 of sodium deficiency, 122 castration and, 340, 341 of stress, 103, 109 estrogens and, 341, 360 of thiamine, 115-116 progesterone and, 342 of turnip seed oil, 125 testosterone and, 340, 342 of vitamin A, 121-122 histochemical demonstration in intesPituitary, tine, 316-3 17 anterior, in kidney, 317 effect on glutathione content of in intestinal epithelium, 302 tissues, 195 in kidney tubules, 302 of iodine deficiency on, 98, 100 riboflavin and, 316 of mineral deficiencies on, 98 selective intestinal sugar absorption of vitamin A deficiency on, 89-91, and, 302, 313,316 95 Phosphatases, of vitamin E deficiency, 92 action, 340 sex differences in, 92 effect of steroid hormones on, 340-345, extract of, diabetogenie action, 196367 200, 205, 206 Phosphocreatine, of muscle, insulin and, 199 adrenalectomy and, 319 lactation and, 198 Phosphoglucomutase, 318-319 mechanism of, 195, 206 activity, Bdrenalectomy and, 318 phlorizin and, 199 Phosphorus metabolism, pregnancy and, 198

414

SUBJECT INDEX

species differences in, 197 effect on alloxan diabetes, 193, 195 galactopoietic activity, 218 inhibition of hexokinase, 205 probable identity of diabetogenic and growth principle in, 200 protein storage and, 198 gonadotropin content, circulating gonadotropins and, 83 inanition and, 82 hormones, effect on nutritive state, 127 inanition and, 85 niacin and, 101 nutrition and, 79-140 vitamin A and, 101, 102 vitamin C and, 101 ascorbic acid in, 110-111 carbohydrate metabolism and, 196 cytology of starved, 86 Placenta, progestins from, 276, 277 Polyneuritis, alcoholic, 18 of pregnancy, 18, 29 beriberi and, 9 vitamin B deficiency and, 8ff. Potassium, chloride, effect on ACTH, 127 effect on pituitary-adrenal system, 122-123 glycogen metabolism and, 321-325 glycolysis and, 324, 325 iodide, effect on thyrotropin activity, 127 metabolism, carbohydrate metabolism and, 321-325 in muscle, 325 in nerve, 325 Pregnancy, ascorbic acid requirements in, I10 diagnostic tests for, 164 ectopic, urinary excretion of gonadotropin in, 172 of pregnanediol in, 172 8-glucuronidase and, 346 17-ketosteroid concentration and sex of fetus, 163 trophoblast activity in, 162, 164, 166, 167, 168

urinary excretion of androgen in, 163 of androsterone, 163 of chorionic gonadotropin, 162, 164 of estriol, 163 of estrogen, 162, 164, 165 of etiocholanolone, 163 of pregnanediol, 163, 164, 165 of pregnanolone, 163, 164 of steroids, 162 Pregnanediol, 277 assay in blood, 146 chemical estimation, 150, 151 metabolism by liver, 283 as metabolite of progesterone, 252,278, 279, 280 sterility and, 162 urinary excretion, 147 abortion and, 167, 168 in amenorrhea, 158 chorioepithelioma and, 169 in ectopic pregnancy, 172 in endocrine disorders, 174 hydatid mole and, 169 during menstrual cycle, 153, 156 in ovarian disorders, 156, 157 in pregnancy, 163, 164, 165 in toxemia of pregnancy, 170, 171 tumors and, 173 3(a),20(a)-Pregnanediol, 277 adrenocortical origin of urinary, 286 effect on D-amino acid oxidase, 357, 358 as metabolite of progesterone, 278 Pregnane3(a), 17(a)-diol-llJ2O-dione (21-Desoxytetrahydro E), adrenocortical origin of urinary, 286 urinary excretion following cortisone administration, 287 Pregnane-3 (a),20(a)-diol-ll-one (11ketopregnanediol) , adrenocortical origin of urinary, 286 Pregnane-3(a)-ol-ll,2O-dione (1l-ketopregnanolone) , adrenocortical origin of urinary, 286 Pregnane-3(a) ,2O-diol-l l-one, effect on glycogen metabolism in rat diaphragm, 365 urinary excretion following administration of 1l-dehydrocorticosterone, 287

BUBJECT INDEX

Pregnane-3(a),6(a)-diol-2O-one,277 as metabolite of progesterone, 278 3,2O-Pregnanedione, 277 as metabolite of progesterone, 278 Pregnane-3(a), 11(@),17(a),21-tetrol-20one (tetrahydro F), adrenocortical origin of urinary, 286 urinary excretion following ACTH administration, 287 following cortisone administration, 287 Pregnane-3 (a)-17 (a)2 1-triol-I 1,2O-dione (tetrahydro E), adrenocortical origin of urinary, 286 urinary excretion following ACTH administration, 287 following cortisone administration, 287 3(a)-Pregnanol, 277 as metabolite of progesterone, 278 Pregnanolone, urinary excretion in pregnancy, 163 Pregnan-3 (a)-ol-2O-one (Epipregnanolone), 277 as metabolite of progesterone, 278 urinary excretion following ACTH administration, 287 As-Pregnene-3 (@),20(a)-diol, adrenocortical origin of urinary, 286 A4-Pregnene-17,20-diol-3-one, effect on D-aminO acid oxidase, 357,358 A6-Pregnene-3(@),21-diol-3-one, effect on glycogen production in r at diaphragm, 364, 365 As-Pregnene-3 (@),21-diol-20-one (2l-hydroxypregnenolone) , adrenocortical origin of urinary, 286 urinary excretion following ACTH administration, 287 Pregnenolone, effect on oxidation of a-glycerophosphate, 359, 360 As-Pregnene-3 (p)-o1-20-onel 277 isolation from testes, 277 A6-Pregnene-3(p),17(a),20 (a)-triol, adrenocortical origin of urinary, 286 A6-Pregnene-3(@),17(a)2O(@)-triol, adrenocortical origin of urinary, 286 Premarin, effect on liver succinoxidase, 361

415

Premenstrual tension, therapeutic effect of vitamin A on, 64 Progestins, 276 intermediary metabolism, 276-277 urinary excretion, 351 Progesterone, administration in dysmenorrhea, 155 in threatened abortion, 168 anesthetic activity, 280 hepatectomy and, 280 antifibromatogenic activity, 280 assay, 145, 148 blood levels in pregnancy, 145 conversion to pregnanediol, 278, 279, 283 liver disease and, 283-284 effect on alkaline phosphatase, 342 on D-amino oxidase, 356 on arginase, 338 on estrogen metabolism, 153 on @-glucuronidase, 345, 346 on glycogen metabolism by diaphragm, 364, 365 on oxidation of a-glycerophosphate 359 on phosphorylase activity, 310, 311 of simultaneous administration of estrone and, on esterase of liver and spleen, 346 on tissue respiration, 350 estrone and, 346 isolation from adrenals, 277 from corpus luteum, 276 from placenta, 162, 277 metabolism, 151, 278-285 isotope studies, 278 by liver, 258, 280-282, 283 accelerators, 281, 282 mechanism, 281, 284, 291 species differences in, 285 pathways, 284 rate of, 279-280 by tissues, 280-285 in toxemia of pregnancy, 170 methyl, see under Methyltestosterone pregnanediol as metabolite of, 252 urinary excretion, 148 in pregnancy, 145 of steroids following administration of, 277

416

SUBJECT INDEX

Prolactin, see under Lactogenic hormone Proline oxidase, in tissues, adrenalectomy and, 347 steroid hormones and, 367 vitamin B deficiency and, 347 Promin, antitoxic liver factor and, 123, 124 Prostate, acid phosphatase in, 343, 344 castration and, 344 testosterone and, 344 cancer, effect on serum acid phosphatase, 344 effect of steroid hormones on enzyme systems in, 332, 339, 343-345 Protease, effect of desoxycorticosterone on, 363 Protein enzymes, adrenocortical steroids and, 320 Protein(s), deficiency, gonadotropin secretion and, 95-96 dietary, effect on response to growth hormone, 126 on secretion and synthesis of ACTH under stress, 104, 107 in milk, effect of iodinated casein on, 235 iodinated, as galactopoietic agents for dairy animals, 217-250 activity and thyroxine content, 222, 223, 224 determination of thyroxine in, 221 effect on ascorbic acid in milk, 236-237 on fat soluble vitamins in milk, 236 on metabolism of cow, 240-243 on milk yield, 224-229 as source of thyroxine for dairy COWS, 2 18-222 Pseudohypophysectomy, 85 hypophysectomy and, 85 Psychoses, effect of niacin on “toxic,” 33 vitamin B complex and, 4 Purine metabolism, ACTH and, 193 Pyrexia, ascorbic acid requirements and, 110

Pyridoxine, adjustment to stress and, 120, 121 deficiency, effect on adrenals, 119-120 on gonads, 89 on pihitary-adrenal system, 119.121 protein synthesis and, 121 requirement, hyperthyroidism and, 128

R Rape seed oil, effect on pituitary-adrenal system, 125 Respiration, adrenocortical steroids and, 308 Riboflavin, alkaline phosphatase and, 316 in Brachyura, 74 deficiency, adrenal insufficiency and, 347 ACTH and, 119 pituitary-adrenal system and, 118ff. resistance t o cold and, 118 effect on gonads, 87 in milk, effect of hyperthyroidism on, 237 of thyroxine on, 238 painful feet syndrome and, 22, 23, 26, 27, 28 Ribonuclease, effect of desoxycorticosterone on, 363 S

Scurvy, effect of cortisone on, 107 Scours, vitamin A deficiency in, 62 Serum, vitamin A level of, effect on transfer of vitamin A, 57, 59 on vitamin A in milk, 57, 59 Sex hormones, bioassay in urine, 148 cell metabolism and, 325 determination in blood, 146 Sex organs, acid phosphatase in male accessory, 344 effect of vitamin deficiencies on, 87 inanition and, 81, 83 Shrimps, thiaminase in, 75

SUBJECT INDEX Skin diseases, transportation of fat-sohble vitamins and, 56 Sodium deficiency, effect on pituitary-adrenal system, 122 Somatotropin, see under Growth hormone Soybeans, goitrogens in, 98 Spinal cord syndromes, in dietary deficiencies, 39-42 effect of niacin in, 40 latrhyrism and, 41 Starvation, effect on peripheral nerves, 7 Sterility, female, pregnanediol and, 162 Steroid hormones, see also under Steroids and under names of individual compounds assay, 141-181 blood serum 8-glucuronidase and, 346 conjugation of metabolites, 290 effect on acid phosphatase activity, 315 on arginase of liver and kidney, 334337 on brain cell respiration, 349-350 on choline acetylase, 362 on enzyme systems, 352-364,366-368 on glycogen metabolism in liver, 366 on oxidative metabolism, 349-352 castration and, 349 structure and, 351 on succinoxidase of liver, 360-362 on tissue esterase, 346 enzymes and, 331ff. metabolism, intermediary, of non-benzenoid, 251295 by liver, 258ff. tissue oxidation and, 331-370 urinary, 256 adrenocortical origin of,285-287 conjugation, 256 derived from progesterone, 277 excretion, 253 in pregnancy, 277, 278 Steroids, see also under Steroid hormones and under names of individual compounds assay, 141-181 urinary excretion, in pregnancy, 162

417

tumors and, 173 Stilbestrol, diabetogenic action of, and derivatives thereof, 202,203 effect on blood serum 8-glucuronidase, 346 on glycogen metabolism in rat diaphragm, 364,365 treatment of toxemia of pregnancy with, 170 Stress, activation of pituitary-adrenal system under, 103 adjustment to, ascorbic acid and, 107110 pyridoxine and, 120, 121 effect on blood serum 8-glucuronidase, 346 of dietary protein on ACTH secretion and synthesis under, 104-107 manifestations of non-specific, 115 requirement for ascorbic acid and, 110 for cortical hormones and, 109, 118 for growth hormone and, 125 for nutrients, 103, 123 response to, pantothenic acid and, 113, 115 vitamin A content of adrenals and, 121 Succinic dehydrogenase, action, 333 concentration in tissues, androgens and, 333, 334 castration and, 334 effect of desoxycorticosterone on, 363 Succinoxidase system, 333 adrenalertomy and, 333 effect of 1 (4’-hydroxyphenyl)-2-phenylethane on, 362 of steroid hormones on, 334,360-362 Sugar, selective absorption by small intestine, 302 iodoacetate and, 302,303 phlorizin and, 302, 303 Sulfanilamide, effect on iodine excretion, 99

T Tendon reflexes, in beriberi, 16, 17

418

SUBJECT INDEX

in painful feet syndrome, 24-25, 28 Testes, effect of vitamin E deficiency on, 93 isolation of As-pregene-3,9-ol-20-one from, 277 of testosterone from, 252 progestational activity, 276, 277 Testosterone, 252 assay, 146, 148 blood levels after injections, 146 diabetogenic activity, 203, 299 effect on D-amino acid oxidase, 338, 356, 357, 358 on arginase of kidney, 335, 336, 337, 338 on blood serum ,9-glucuronidase, 346 on cell metabolism, 276 on choline acetylase, 362 on cholinesterase of serum, 339, 340 on deaminases, 320 on diabetes following pancreatectomy, 188 on glycogen metabolism in rat diaphragm, 364, 365 on oxidation of cu-glycerophosphate, 359,360 on phosphatases in tissues, 314,343345 on phosphorylase activity, 310 on respiration of primary BrownPearce epithelioma, 352 of tissues, 349, 350, 351 metabolism, 151 intermediary, 253, 254 possible pathways, 254, 255 by kidney, 258, 269-271, 276 citrate and, 270, 271 DPN and, 270,271 species differences in, 270, 271 by liver, 258, 269-271, 276 citrate and, 259, 260, 2G7, 268 DPN and, 259, 260, 2G1, 267, 268 formation of 17-ketosteroids in, 261, 264 inhibitors of, 259 kinetics of, 264-265 liver disease and, 272-274 methyltestosterone and, 267 niacin and. 269 nicotinamide and, 261

nutrition and, 267-269 species differences in, 261,262,263 tryptophan and, 269 metabolites, 252, 254, 255, 290 conjugation, 280 occurrence, 252 structure, 252 urinary excretion, 148 &-Testosterone, effect on kidney arginase, 338 trans-Testosterone, as metabolite of testosterone, 258 Tetraethylammonium bromide, effect in hypertensive toxemia, 171 Thiaminase, in shrimps, 75 Thiamine, acetylcholine and, 9 activation of pituitary-adrenal system and, 115-116 deficiency, anorexia and, 6 beriberi and, Gff ., 1Off. Chastek paralysis and, 30 effect on gonadotropin secretion, 87, 88 on peripheral nerves, 5-10 on testosterone metabolism by liver, 269 nystagmus and, 31, 33 ophthalmoplegia and, 30, 32 optic nerve damage and, 34 response to growth hormone and, 126 symptoms of, 31 Wernicke’s encephalopathy and, 28, 29-30, 31, 32 in milk, hyperthyroidism and, 237, 238 peripheral neuritis and, 9 physostigmine and, 9 requirement, hyperthyroidism and, 9 role in nutrition of crustaceans, 75 transmission of nerve impulses and, 10 Thioglycolic acid, diabetogenic action of alloxan and, 191 Thiouracil, effect on diabetes following alloxan administration, 193, 194 following pancreatectomy, 188 on mineral constituents of milk, on vitamin A and carotene in milk. 236

SUBJECT INDEX

419

Thymus gIand, nutritional, 232-234 inanition and, 85 practical effects of, 245-246 pyridoxine deficiency and, 119 secretion into milk, 239 Thyroid, isolation of crystalline, 218 antitoxic liver factor and, 123 pantothenic acid metabolism and, 128 effect of calcium on, 98 requirement, factors influencing, 99 on diabetes following pancreatecsources of, for dairy cows, 219-220 tomy, 187, 188 DbThyroxhe, uracil and, 187 sodium salt, galactopoietic activity in of iodinated proteins on, 230-231 ruminants, 220 on milk yield in cows, 218 mode of administration and, 220 of thyroxine on, 231 L-Thyroxine, synthetic, 218 extract, diahetogenic action, 204 as galactopoietic agent in dairy anifactors influencing acceptance of iodine mals, 219, 220 by, 99 Tissues, hormone, see under Thyroxine damage to nervous, in dietary neuroinanition and, 83-84 pathies, 3 vitamin D and, 102 metabolism of androgens by, 256, 258vitamin E deficiency and, 94-95 276 Thyroidectomy, of progesterone by, 28Ck285 diabetogenic action of alloxan and, 191 oxidations, steroids and, 331-370 effect on liver amino acid oxidase, 339 relationship between lipide and esterThyrotropic hormone, see under Thyroase content of, 346 tropin respiration, steroids and, 349-352 Thyrotropin, Toxemia, of pregnancy, activity, potassium iodide and, 127 endocrine dysfunction in, 171 inanition and, 83, 84 progesterone metabolism and, 170 vitamin A and secretion of, 102 steroid excretion and, 170 Thyroxine, theory of, 170 carotene metabolism and, 128 determination in iodinated proteins, Transaminase, effect of desoxycorticosterone on, 362, 221 363 effect on mammary gland, 229 Trauma, on thyroid, 231 glycosuria following, 210 5 s galactopoietic agent in dairy aniTrophoblast, mals, 217-250 pregnancy and, 162, 164, 166, 167, 168 effect on calcium metabolism, 242 Trypsin, on health, 244-245 effect of desoxycorticosterone on, 362, on heat regulation, 242-243 363, 364 on metabolism of cow, 240-243 Tryptophan, on milk yield, 224-229 effect on enzymic testosterone destrucon mineral constituents of milk, tion by liver, 269 235 Tumors, on mortality, 245 on phosphatase titer of milk, 238 excretion of androgens in, 172 on phosphorus metabolism, 242 of estrogens in, 172 on reproduction, 243-244 of gonadotropins in, 172 on vitamins in milk, 236-238 of 17-ketosteroids in, 173 homeostatic effects, 229-234 of pregnanediol in, 172 endocrine, 230-232 of steroids in, 172, 173

SUBJECT INDEX

420

Tyrosinase, inhibitory effect of desoxycorticosterone on, 319, 320, 362, 363

U Uracil, effect on diabetes due to subtotal pancreatectomy, 187 Uric acid, diabetogenic action, 192 Urine, of pregnancy, steroids isolated from, 277, 278 Uterus, actomyosin in, 348 castration and, 348 estrogens and, 348 ovariectomy and, 348 adenosinetriphosphatase in, 348 estrogens and, 348 ovariectomy and, 348 6-glucuronidase in, 345 estrogens and, 345 esterase in, 346 estrogen and, 346

V Vaginal cornification, vitamin A and, 64 Veronal, glycosuria and, 209 Vesicles, seminal, acid phosphatase in, 344 effect of steroid hormones on cytochrome oxidase in, 339 on phosphatases in, 343, 344 on succinic dehydrogenase in, 333 Violacein, 75 Vitamin A, absorption, 47 of aqueous dispersions, 50, 51, 52, 53, 54, 56 mode of administration and, 50 of oily dispersions, 50, 51, 52, 53, 54, 56 in old age, 63 particle size of carrier and, 53, 54 symptoms of impaired, 50

in adrenals, 121 effect of stress on, 121 in crustaceans, 75 deficiency, causcs, 57, 59 effect on anterior pituitary, 89, 9091, 05 growth hormone and, 127 insulin sensitivity and, 121 kidney stones and, 63 effect on adrenal weight, 103 on anterior pituitary, 101 on estrous cycle of rat, 103 metabolism, 49 in milk, effect of administration of iodinated proteins on, 236 of thyroxine on, 236 serum levels, hormonos and, 64 in the newborn, 62 storage in liver, 48 in newborns, 62 therapeutic effect on hyperkeratosis, 57 on leukoplakia vulvae, 64 on premenstrual tension, 64 thyrotropie hormone and, 102 transportation, 47 hormones and, 64 in old age, 64 serum levels and, 57, 59, 60 skin diseases and, 56, 57 ultra-filtrability and, 57 Vitamin BI2, deficiency, effect on gonads, 89, 126 requirement, hyperthyroidism and, 128 Vitamin B complex, painful feet syndrome and, 27 psychoses and, 4 Vitamin D, absorption, of aqueous dispersions, 55 impaired, 50 particle size of carrier and, 50, 51 adrenal cortex and, 103 calorigenic effect, 103 effect on anterior pituitary, 103 storage in body, 48 thyroid and, 102 transportation, infectious diseases and, 50 Vitamin E, absorption, of aqueous dispersions, 55

SUBJECT INDEX deficiency, effect on anterior pituitary, 89, 92-93 sex difference in, 92 on fertility, 93-94 on testes, 93 on thyroid gland, 94-95 muscular atrophy and, 4 requirement, of Daphnia, 70, 73-74, 76 serum levels in the newborn, 62 effect on mother’s milk, 63 storage in body, 48 Vitamin K, absorption, of aqueous dispersions, 55 of water-soluble derivatives, 56 deficiency, ACTH and, 128 storage in body, 48 Vitamin P, antithyrotropic effect, 102 ascorbic acid and, 102 Vitamin deficiencies, effect on gonadotropic hormones, 87-95 Vitamins, fat-soluble, adsorption, 47, 48-56 of aqueous dispersions, 56 mechanism of, 48 deficiency, on adequate intake, 48,49 due to impaired transportation, 63-64 dispersing agents for, 53, 55

421

in milk, effect of administration of iodinated proteins on, 236 transfer to milk, 57, 59-61 transportation, 47, 48, 56-64 avitaminosis due to impaired, 56, 63-64 from mother to offspring, 61-63 skin diseases and, 56, 57 water-soluble, effect of adminstration of iodinated proteins on, in milk, 236-237 Vitamins B, activity, adrenal cortex and, 320 deficiency, effect on gonadotropin secretion, 87 on kidney proline oxidase, 347 polyneuritis and, 8ff. in milk, hyperthyroidism and, 237 requirement, of Daphnia, 74

X Santhine oxidase, effect of desoxycorticosterone, on, 364

Z Zinc, reaction with dithizone, 196 with oxine, 196

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