Physiological effects of food carbohydrates: A symposium co-sponsored by the Division of Carbohydrate Chemistry and the Division of Agricultural and Food ... 11-12, 1974 (ACS symposium series ; 15)

Physiologica Effects o Food Carbohydrates

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

Physiological Effects of Food Carbohydrates Allene Jeanes and John Hodge, Editors

A symposium co-sponsored by the Division of Carbohydrate Chemistry and the Division of Agricultural and Food Chemistry at the 168th Meeting of the American Chemical Society, Atlantic City, N . J., Sept. 11-12, 1974.

ACS SYMPOSIUM SERIES

AMERICAN CHEMICAL SOCIETY WASHINGTON, D. C.

1975

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

15

Library of Congress

Data

Physiological effects of food carbohydrates (ACS symposium series; 15) Includes bibliographical references and index. 1. Carbohydrates—Physiological effect—Congresses. 2. Carbohydrate metabolism—Congresses. I. Jeanes, Allene Rosalind, 1906-. II. Hodge, John E., 1914. III. American Chemical Society. Division of Carbohydrate Chemistry. IV. American Chemical Society. Division of Agricultural and Food Chemistry. V. Series: American Chemical Society. ACS symposium series; 15. [DNLM: 1. Carbohydrates— Metabolism—Congresses. QU75 A512p 1974] QP701.P48 ISBN 0-8412-0246-X

612'.396 75-14071 ACSMC8 15 1-355 (1975)

Copyright © 1975 American Chemical Society All Rights Reserved PRINTED IN THE UNITED STATES OF AMERICA

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

ACS Symposium Series Robert F. Gould, Series Editor

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

FOREWORD The

ACS

SYMPOSIUM SERIES

a medium for publishing symposia quickly i n book form. The format of the SERIES parallels that of its predecessor, ADVANCES IN CHEMISTRY SERIES, except that in order to save time the papers are not typeset but are reproduced as they are submitted by the authors i n camera-ready form. As a further means of saving time, the papers are not edited or reviewed except by the symposium chairman, who becomes editor of the book. Papers published i n the A C S SYMPOSIUM SERIES are original contributions not published elsewhere in whole or major part and include reports of research as well as reviews since symposia may embrace both types of presentation.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

PREFACE Significant progress has been made in evaluating the physiological ^ effects that have been attributed to food carbohydrates. Because the findings are related to the structure, enzymology, and complexing interactions of carbohydrates, as well as to improved compositions of processed foods, this symposium was organized in the interests of the Division of Carbohydrate Chemistr and the Division of Agricultural and Food Chemistry. Some common disorders definitely associated with dietary carbohydrates are diabetes, stress responses resulting from hypoglycemia, dental caries, eye cataracts, flatulence, and fermentative diarrhea. The postulated role of sugar in the development of atherosclerosis and coronary heart disease has been impugned and debated, but, more importantly, it is being carefully investigated. Investigations center on underlying causes of carbohydrate-induced disorders—e.g., altered enzyme activity in the digestive tract and elevated insulin, cholesterol, and triglyceride levels in serum. Numerous studies show differences according to the type of carbohydrate ingested. Because refined sugars and starches have been referred to as "empty calories," one might wonder whether carbohydrate is needed at all in the diet. Some popular reducing diets contain little carbohydrate. The Food and Nutrition Board of the National Research Council, National Academy of Sciences, has stated in the 1974 edition of "Recommended Dietary Allowances": Carbohydrates can be made in the body from some amino acids and the glycerol moiety of fats; there is therefore no specific requirement for this nutrient i n the diet. However, it is desirable to include some preformed carbohydrate in the diet to avoid ketosis, excessive breakdown of body protein, loss of cations, especially sodium, and involuntary dehydration. Fifty to 100 g of digestible carbohydrate a day will offset the undesirable metabolic responses associated with high fat diets and fasting. The topics of this symposium are related to improved carbohydrate and mineral balance in foods of the future. As food supplies for expanding populations become more critical, the more abundant and economical carbohydrates should be used to maximum advantage to spare the less abundant proteins and fats. However, to establish the optimum ratio of carbohydrates to fats, the physiological effects of their combinations should be delineated more clearly. It now appears that indigestible, as ix In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

well as digestible, carbohydrate should be considered i n formulating processed foods. The postulated benefits of dietary fiber (largely indi­ gestible polysaccharides ) in aiding the elimination of toxins and in reduc­ ing serum cholesterol are discussed. Other advantages that might accrue from ingesting or infusing certain types of carbohydrate in place of other types are cited here. Disadvantages in refining high-carbohydrate cereals and sugar to the point of near depletion of essential mineral content are viewed from a physiological basis. During this century significant changes have taken place in the types of carbohydrate that are made available in our food supplies. U.S. De­ partment of Agriculture statistics show that we are being exposed more to refined sugars and less to starch and dietary fiber as the proportions of fat (mainly) and protei sugars, the proportion of D-glucose, and the maltose and maltooligosaccharides of starch hydrolyzates. The increasing incorporation of starch sirups, including those that contain D-fructose produced by glucose isomerase, is expected to accentu­ ate this trend. The level of lactose, ingested mainly from milk and dairy products rather than from added lactose, has remained about constant. Critics declare that consumers now have less control over their carbohydrate intake than their forebears had because the compositions of prepared convenience foods and beverages vary significantly from those of natural foods. Expanding urban populations dictate an increasing sup­ ply of stable processed foods; therefore, the benefits of adding sugars and modified polysaccharides to improve the stability and acceptance of pre­ pared foods should be weighed against adverse nutritional effects. A l ­ though processing practices have been viewed with alarm in some sectors, we really cannot know whether the changes in carbohydrate composition are innocuous until the physiological effects of the additives have been defined under conditions of normal use. This symposium and others like it attest to the activity of scientists in different disciplines who are supply­ ing answers to the questions raised about the healthfulness of refined sugars and gums. Some of the subjects selected for this symposium were critically reviewed in the International Conference on Sugars in Nutrition held at the Vanderbilt University School of Medicine in 1972 ( "Sugars in Nutri­ tion," H. L. Sipple and K. W. McNutt, Eds., Academic Press, New York, 1974). However, more recent experimental results and additional infor­ mation on the physiology of dietary and infused sugars are presented here, and mostly by different authorities. Parts A and Β of this symposium might therefore be regarded as a supplement to "Sugars i n Nutrition." Part C covers physiological effects of polysaccharides, which were not cov­ ered i n the Vanderbilt conference, including some food additive gums χ In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

and dietary fiber. This information also can be expanded by reference to the Symposium on Fiber in Human Nutrition held by the Nutrition Society at the University of Edinburgh School of Medicine (Proc. Nutr. Soc. (1973) 32, 123-167). Another symposium publication, "Molecular Structure and Function of Food Carbohydrate" (G. G. Birch and L. F. Green, Eds., Wiley, New York, 1973), contains several papers that are related to the topics of this symposium. JOHN E. HODGE

Northern Regional Research Laboratory Agricultural Research Service, U S D A Peoria, Ill. December 1974

xi In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

1 The Physiology of the Intestinal Absorption of Sugars ROBERT K. CRANE College of Medicine and Dentistry of New Jersey, Rutgers Medical School, Department of Physiology, Piscataway, N. J. 08854

This review has t physiology absorption of sugars and should properly begin with a brief discussion of the components of the physiological system which carries out this indispensable task. The small intestine where the absorption of sugars takes place is a tube connecting to the stomach at its upper end and to the large intestine at its lower. In the human adult the tube is about 280 cm (9 feet) in length and an average 4 cm (1-1/2 inches) in internal diameter. The area of the inner surface of the tube is much greater than implied by these two measurements because the inner surface is heavily folded and everywhere on the folds there are to be found numerous projec­ tions called v i l l i (1). Villi are readily seen under a micro­ scope of low power and there are perhaps as many as 25,000,000 v i l l i in all. As indicated in Figure 1, each villus is covered by a sheet of absorptive epithelial cells punctuated at intervals by the so-called goblet cells which supply protective mucous. Between the v i l l i are to be found crypts within which the cells are produced and from which they migrate outward along the sur­ face of a villus during a short 3-4 days of active life before being extruded into the lumen of the gut where they disintegrate and are digested. The v i l l u s i s the working u n i t o f the s m a l l i n t e s t i n e . I t i s on t h i s s t r u c t u r e t h a t the i n n e r ends of the a b s o r p t i v e c e l l s are brought i n t o c l o s e p r o x i m i t y t o the blood and lymph which must p i c k up absorbed n u t r i e n t s and c a r r y them t o the other p a r t s of the body. The outer ends of the a b s o r p t i v e c e l l s are i n contact w i t h the contents of the i n t e s t i n e and are s p e c i a l i z e d to perform t h e i r necessary work. The outer end of each c e l l i s a "brush border" made up o f c l o s e l y packed, p a r a l l e l c y l i n d r i c a l processes c a l l e d m i c r o v i l l i . The l i m i t i n g plasma membrane of t h e c e l l f o l l o w s the contours o f the m i c r o v i l l i . J u s t beneath the brush border along the s i d e s of the c e l l s are t o be found s p e c i a l i z e d j u n c t i o n a l s t r u c t u r e s by means of which the absorpt i v e c e l l s are h e l d together i n t o a more or l e s s continuous sheet. 2 In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

1.

CRÂNE

Intestinal Absorption

of Sugars

3

Expanding our h o r i z o n t o i n c l u d e the whole of the i n t e s t i n a l s u r f a c e w h i l e r e t a i n i n g our view o f i t s microscopic appearance i t i s c l e a r t h a t , so f a r as concerns d i g e s t i o n and a b s o r p t i o n , t h e c o l l e c t i v e brush borders of the e p i t h e l i a l sheet form a j u n c t i o n a l b a r r i e r between the o u t s i d e of the body and the i n s i d e through which n u t r i e n t s must pass i n order t o reach the c i r c u l a t i o n and enter metabolism. The c o l l e c t i v e brush borders separate t h e d i g e s t i v e f u n c t i o n s of t h e i n t e s t i n a l lumen c o n t r i b u t e d by the secreted enzymes of the pancreas from t h e metabolic f u n c t i o n s c o n t r i b u t e d by t h e c e l l s . The brush borders a l s o c o n t r i b u t e d i g e s t i v e f u n c t i o n s o f t h e i r own as w e l l as t h e s e l e c t i v i t y , energy t r a n s d u c t i o n and other p r o p e r t i e s of a b s o r p t i o n a n t i c i pated f o r a c e l l membrane occupying t h i s p a r t i c u l a r l o c a t i o n . The brush border membrane acts as a b i l a y e r l i p o i d a l m a t r i x composed of t h e f a t t y chains of p h o s p h o l i p i d s and glycosphingol i p i d s i n t e r s p e r s e d w i t h c h o l e s t e r o l ( 2 ) and p e r f o r a t e d here and there by aqueous channel may pass by d i f f u s i o n . L i p i d i f f u s e r e a d i l y across the m a t r i x of the membrane. However, t h e membrane i s a s u b s t a n t i a l b a r r i e r t o the r a p i d d i f f u s i o n o f large, h i g h l y water s o l u b l e molecules l i k e the hexoses because these do not enter the m a t r i x and the dimensional p r o p e r t i e s of the aqueous channels are too s m a l l , being e q u i v a l e n t only t o those o f pores o f 4-5 £ i n r a d i u s (3), ( 4 ) . There a r e a l s o aqueous channels between the c e l l s because t h e j u n c t i o n a l complexes o f the i n t e s t i n a l e p i t h e l i u m ' a r e n o t t i g h t {5). However, these channels are a l s o too s m a l l f o r the r a p i d passage of hexoses. Those hexoses which do g e t across the brush border membrane r a p i d l y and i n q u a n t i t y ; and t h i s group n a t u r a l l y i n c l u d e s the major d i e t a r y hexoses, glucose, g a l a c t o s e , and f r u c t o s e , do so because they f i t the s p e c i f i c i t y requirements and are able t o b i n d t o membrane t r a n s p o r t c a r r i e r s ( 6 ) . The a c t u a l mode of o p e r a t i o n o f c a r r i e r s i s c u r r e n t l y unknown. However, t h e i r apparent mode o f o p e r a t i o n , i n s o f a r as we can know i t from k i n e t i c s , i s most e a s i l y described as l i k e t h a t of a f e r r y b o a t , capable of s h u t t l i n g water s o l u b l e molecules across the l i p o i d a l matrix. C a r r i e r f u n c t i o n i s diagrammed i n F i g u r e 2 where t h e upper p a r t i s an o p e r a t i o n a l model and t h e lower p a r t i s a k i n e t i c model of a simple s o - c a l l e d f a c i l i t a t e d d i f f u s i o n type of c a r r i e r t o which constants may be assigned as i n d i c a t e d . The assumptions are few and simple. Substrate i n t e r a c t s w i t h the b i n d i n g s i t e o f a c a r r i e r on e i t h e r s i d e of the membrane and i s t r a n s l o c a t e d i n a s s o c i a t i o n w i t h the c a r r i e r . The b i n d i n g s i t e of the c a r r i e r can t r a n s l o c a t e whether or not i t c a r r i e s substrate. A l l i n t e r a c t i o n s are u s u a l l y assumed t o be symmetrical and e q u i l i b r i u m i s then achieved a t equal transmembrane concentrations or a c t i v i t i e s . F o r the most p a r t , f r u c t o s e crosses the brush border membrane by means of a c a r r i e r w i t h these p r o p e r t i e s ( 7 , 8, 9 ) .

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

4

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

BRUSH BORDER MEMBRANE

CELL CONTENTS

INTESTINAL CONTENTS

+ C ^ c s Figure 2.

^

C+ c s

Schematic of a facilitated diffusion (monofunctional) carrier. Ρ is the permeability coefficient.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

1.

CRÂNE

Intestinal Absorption of Sugars

5

Glucose and g a l a c t o s e , however, use a c a r r i e r which though i t i s somewhat the same i s a l s o somewhat and i m p o r t a n t l y d i f f e r ent. The glucose-galactose c a r r i e r , depicted i n F i g u r e 3 both as an o p e r a t i o n a l model and as a k i n e t i c model, i s an e q u i l i b r a t i n g , symmetrical c a r r i e r l i k e the f r u c t o s e c a r r i e r except t h a t i t has two b i n d i n g s i t e s i n s t e a d o f one. The glucoseg a l a c t o s e c a r r i e r r e q u i r e s N a f o r i t s e f f i c i e n t o p e r a t i o n and cotransports N a i n a t e r n a r y complex along w i t h the sugar ( 6 ) . The p a r t i c u l a r v e r s i o n of the Na -dependent c a r r i e r shown i n F i g u r e 3 i n d i c a t e s t h a t the b i n d i n g s i t e can t r a n s l o c a t e e i t h e r empty o r i n a t e r n a r y complex w i t h both o f i t s s u b s t r a t e s , b u t not w i t h sugar alone. There are other v e r s i o n s w i t h other assumptions about the requirements f o r t r a n s l o c a t i o n but the e s s e n t i a l f e a t u r e s are very s i m i l a r ( 1 0 ) . In an i s o l a t e d system, a c a r r i e r w i t h two b i n d i n g s i t e s i s an e q u i l i b r a t i n g c a r r i e The c a r r i e r i t s e l f can s t a t i o n a r y s t a t e would f i n d equal concentrations o f sugar and equal concentrations of N a on the two s i d e s o f the membranes. In the c e l l u l a r system, however, the'Na -dependent glucoseg a l a c t o s e c a r r i e r i s able t o transduce metabolic energy and t o achieve " u p h i l l " o r a g a i n s t t h e g r a d i e n t t r a n s p o r t by coupling t o the t r a n s c e l l u l a r f l u x of Na . The system i n the i n t e s t i n e seems t o work as suggested by the diagram i n F i g u r e 4. M e t a b o l i c energy i n the form of ATP i s put i n t o a sodium pump i n the basol a t e r a l membranes of the a b s o r p t i v e c e l l s t o d r i v e a t r a n s c e l l u l a r f l u x of N a from the brush border end t o the basol a t e r a l end (12). The glucose-galactose c a r r i e r couples sugar entry to t h i s f l u x by being a route f o r the entry of sodium i o n at the brush border membrane and achieves an " u p h i l l " c e l l u l a r accumulation o f sugar a t the expense o f the " d o w n h i l l " f l u x of Na . I n t a c t d i - and higher saccharides do not get across the brush border membrane i n q u a n t i t y and we thus i n f e r t h a t the needed c a r r i e r s do not e x i s t (13). Tiny amounts o f d i e t a r y d i - and o l i g o s a c c h a r i d e s are sometimes found i n the u r i n e o f i n d i v i d u a l s under study but these t i n y amounts are a t t r i b u t a b l e t o d i f f u s i o n o f these l a r g e compounds through regions o f the i n t e s t i n e where the normal b a r r i e r has been broken down by i n j u r y or by disease. Recently our l a b o r a t o r y has i d e n t i f i e d a route of c e l l u l a r e n t r y of monosaccharides i n a d d i t i o n t o t h a t provided by the N a dependent c a r r i e r s , o f F i g u r e s 3 and 4 (14, 15.)· * ^ t o be b r i e f l y d e s c r i b e d l a t e r i s r e l a t e d t o the a c t i v i t y o f those hydrolases which are an i n t r i n s i c p a r t o f the brush border mem­ brane. As i s discussed a t f u r t h e r l e n g t h by Dr. Gary Cray i n t h i s Symposium and as i s shown i n Table I , there are imbedded i n the outer surface o f the brush border membrane a s u b s t a n t i a l l i s t of b o n d - s p e c i f i c h y d r o l y t i c o r t r a n s f e r a c t i v i t i e s . The +

+

+

+

+

+

+

+

+

T h

s

n

e

w

γ ο ι 1

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

θ

6

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

BRUSH BORDER MEMBRANE

INTESTINAL CONTENTS

Να pump

G

1+

C —

C +| G

k.Jfk-,

k|ik-

kJfk-

kjfik-s

4

4

Να 2

2

C-G-Na^C-G-Να Figure 3.

Schematic of a sodium-dependent bifunctional carrier

American Journal of Clinical Nutrition

Figure 4. Schematic of energy transduction between the baso-hteral sodium pump and brush border Να"-dependent carriers by means of the Na through flux (11) +

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

1.

CRÂNE

7

Intestinal Absorption of Sugars

TABLE I BRUSH BORDER ENZYME ACTIVITIES* Oligopeptidase γ-glutamyl t r a n s p e p t i d a s e Enterokinase Glucoamylase ( o l i g o s a c c h a r i d a s e ) Maltase Sucrase Isomaltase (α-dextrinase) Lactase Trehalase P h l o r i z i n Hydrolase ( g l y c o s y l c e r a m i d a s e ) A l k a l i n e Phosphatase as o f 1974 accordin saccharidases among these enzymes; namely, glucoamylase (which i s h i g h l y a c t i v e a g a i n s t o l i g o s a c c h a r i d e s ) maltase, sucrase, i s o ­ maltase, (which Gray would p r e f e r t o c a l l α-dextrinase a f t e r t h e n a t u r a l s u b s t r a t e found as a product o f p a n c r e a t i c amylase a c t i o n ) l a c t a s e , t r e h a l a s e , and p h l o r i z i n hydrolase share the work o f p o l y s a c c h a r i d e d i g e s t i o n w i t h p a n c r e a t i c amylase as suggested i n F i g u r e 5. Digestive-Sequence Polysaccharides P a n c r e a t i c Amylase

^ Oligosaccharides and D i s a c c h a r i d e s

Brush Border Saccharidases

^ Monosaccharides

Figure 5.

Sequential roles in carbohydrate digestion of pancreatic amylase and brush border saccharidases

In t h e a d u l t , p a n c r e a t i c amylase s p l i t s amylose o n l y as f a r as m a l t o t r i o s e and maltose (17) and amylopectin t o m a l t o t r i o s e , maltose and a - d e x t r i n s (18). The brush border saccharidases then take over t o cleave~Tree glucose from these products. The brush border enzymes a l s o c o n t r i b u t e d i r e c t l y the d i g e s t i v e

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

8

PHYSIOLOGICAL

EFFECTS OF

FOOD

CARBOHYDRATES

c a p a c i t y of t h e i n t e s t i n e f o r d i e t a r y d i s a c c h a r i d e s . A good d e a l o f work has made i t c l e a r t h a t t h e brush border membrane i s a d i g e s t i v e - a b s o r p t i v e surface on which the monosaccharide s u b s t r a t e s f o r t h e c a r r i e r s a r e formed by t h e a c t i o n of d i - and o l i g o s a c c h a r i d a s e s , i f they are not provided i n f r e e form i n t h e d i e t ( 1 9 ) · There i s a c l o s e p r o x i m i t y a t the brush border membrane between the s e q u e n t i a l processes of d i g e s t i o n and absorption and because of t h i s only a r e l a t i v e l y s m a l l amount of monosaccharide accumulates i n t h e lumen during t h e d i g e s t i o n of a d i s a c c h a r i d e . I n Figure 6, taken from Gray and Santiago ( 2 0 ) , i t i s seen t h a t only 10 percent of the glucose formed by brush border h y d r o l y s i s of sucrose over a 30 cm segment o f i n t e s t i n e was found i n the lumen; 90 percent having been absorbed. The experience w i t h l a c t o s e and maltose was s i m i l a r . Fructose was l e s s w e l l absorbed than glucose formed a t t h e same time from sucrose because i t s d i f f e r e n t t r a n s p o r t system i s l e s s e f f i c i e n t at equal concentrations glucose formed a t the compete w i t h t h a t glucose f o r the same t r a n s p o r t system and has a lower a f f i n i t y f o r i t . O v e r a l l , i t i s c l e a r t h a t the absorption of t h e monosaccharide products of d i s a c c h a r i d e d i g e s t i o n i s e f f i c i e n t . I n p a r t , as already mentioned, t h i s may be explained by the c l o s e f u n c t i o n a l p r o x i m i t y of the membrane d i g e s t i v e enzymes t o t h e membrane t r a n s p o r t systems; a p r o x i m i t y that we have l a b e l e d " k i n e t i c advantage" (19). A l s o i n p a r t t h i s may be explained by a f u n c t i o n of theïïTsaccharidasesas a route f o r the d i r e c t t r a n s l o c a t i o n of some of t h e i r products without the i n t e r v e n t i o n of t h e normal c a r r i e r mechanisms, as r e c e n t l y documented i n publ i c a t i o n s from our l a b o r a t o r y (14·, 15) and f u l l y corroborated by D i e d r i c h ( 2 1 ) . However, there i s no r e l i a b l e evidence t o support the i d e a t h a t the a b s o r p t i o n o f t h e monosaccharide products of d i s a c c h a r i d e s can be s u b s t a n t i a l l y more e f f i c i e n t than the a b s o r p t i o n o f the f r e e monosaccharides themselves. For t h e past 15 years, a concept has been f l o a t i n g about t o the e f f e c t t h a t there may be an advantage f o r absorption t o feed sugars i n the form of d i s a c c h a r i d e s r a t h e r than as f r e e monosaccharides. T h i s concept got i t s s t a r t w i t h some i n v i t r o experiments of Chain and h i s colleagues (22). Our s t u d i e s (19) d i d nothing t o d e t r a c t from the i d e a and d i r e c t i n v i v o experimental support f o r a s m a l l e f f e c t seemed t o be provided by human s t u d i e s c a r r i e d out by Ian MacDonald ( 2 3 ) . The most recent work on humans does n o t support the i d e a . I n f a c t , i t i s p o s s i b l e t h a t t h e i d e a has f i n a l l y been l a i d t o r e s t by the c a r e f u l s t u d i e s of Cook (24) who has found a b s o l u t e l y no d i f f e r e n c e i n the p o r t a l blood l e v e l s o f f r u c t o s e and glucose whether i t i s sucrose t h a t i s placed i n the lumen o f the i n t e s t i n e or whether i t i s an equimolar mixture of glucose and f r u c t o s e .

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

CRÂNE

Intestinal Absorption of Sugars

9

Gastroenterology

Figure 6. Role of monosaccharides released by the digestion of disaccharides over a 30-cm infusion-to-collection distance in human intestine (20)

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

10

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

The usefulness t o the organism o f the d i r e c t t r a n s l o c a t i o n of monosaccharides by brush border d i g e s t i v e hydrolases i s c u r ­ r e n t l y a p h y s i o l o g i c a l p u z z l e and, f o r t h i s reason, the data base f o r t h i s new t r a n s p o r t pathway w i l l not be developed here t o any great extent. S u f f i c e i t t o say t h a t i n v i t r o s t u d i e s c a r r i e d out under c o n d i t i o n s where the normal c a r r i e r mechanisms f o r glucose t r a n s p o r t are e i t h e r s a t u r a t e d w i t h s u b s t r a t e and thus operating at a maximal r a t e or completely i n h i b i t e d by the omission of N a have demonstrated an a d d i t i o n a l component of glucose e n t r y i n t o the c e l l s when any d i s a c c h a r i d e substrate of a brush border enzyme i s provided. In the case of sucrose, f r u c t o s e as w e l l as glucose enters and accumulates i n the c e l l s . Moreover, the components of t r a n s l o c a t i o n c o n t r i b u t e d by the i n d i v i d u a l enzymes are a d d i t i v e when more than one d i s a c c h a r i d e i s used. C l e a r l y , these systems f o r d i r e c t t r a n s l o c a t i o n i n c r e a s e the t o t a l c a p a c i t y of the i n t e s t i n e f o r carbohydrate absorption s u b s t a n t i a l l monosaccharide c a r r i e r under which t h i s a d d i t i o n a l c a p a c i t y may f u l f i l l a need are f a r from obvious. The reason f o r t h i s , which i s probably not g e n e r a l l y appre­ c i a t e d , i s t h a t t h e c a p a c i t y of the i n t e s t i n e f o r a b s o r p t i o n of the monosaccharides, glucose, g a l a c t o s e and f r u c t o s e i s already t r u l y enormous. As shown i n Table I I , Holdsworth and Dawson ( 2 5 ) +

TABLE I I THE CAPACITY OF THE GUT TO ABSORB SUGARS Measured:

Glucose =

4

P' ^ min. χ 30 cm

Fructose = 0.9 x glucose C a l c u l a t e d : Glucose =

. °'

4 o n

x

mm. χ 30 cm

i d ^ £ i £ . 2 8 0 cm = 5374 g/day X

day

Fructose = 5374 χ 0.9 = 4#37 g/day THUS T o t a l D a i l y Capacity = 10,211 g > 22 l b . > 50,000 c a l . measured the a b s o r p t i v e c a p a c i t y over a 30 cm segment of i n t e s ­ t i n e i n normal humans. At p e r f u s a t e sugar concentrations of 5 g/100 ml they obtained the measured values of 0.4 g/min/30 cm f o r glucose and 90 percent of t h a t value f o r f r u c t o s e . From these i t may be c a l c u l a t e d t h a t the t o t a l d a i l y c a p a c i t y i s 10,211 g of a mixture of glucose and f r u c t o s e ; an amount

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

1.

CRÂNE

Intestinal Absorption of Sugars

11

e q u i v a l e n t t o over 22 pounds o f sugar and more than 50,000 c a l o r i e s . Although not a l l p a r t s o f the i n t e s t i n e have the same c a p a c i t y as the p a r t s t u d i e d by Holdsworth and Dawson, t h e e x t r a p o l a t e d maximal r a t e was n e a r l y twice t h a t of the value assumed i n Table I I ; namely 0.73 g/min/30 cm and the t o t a l capaci t y c a l c u l a t e d i s probably a reasonable compromise. Galactose, t e s t e d alone, was absorbed even more r a p i d l y than glucose. Such a c a p a c i t y f o r sugar absorption i s ten times more than would be needed t o provide f o r even the most unreasonable i n d i v i d u a l c a l o r i c requirements s i n c e foods i n a d d i t i o n t o sugars are g e n e r a l l y a l s o eaten and i t s great s i z e i n d i c a t e s t h a t cont r o l of sugar absorption i s not exerted a t the l e v e l of the processes of d i g e s t i o n and absorption a t the brush border membrane. C o n t r o l i s exerted by a negative feedback mechanism i n v o l v i n g receptors i n the upper i n t e s t i n e and the m o t i l i t y o f the stomach. The r e l a t i o n s h i p are diagrammed i n Figur stomach and o f the stomach i t s e l f are not i n c l u d e d because i t i s a matter o f f a c t t h a t the r e a l l y indispensable f u n c t i o n of the stomach i s t o serve as a r e s e r v o i r f o r f o o d s t u f f s and t o provide f o r t h e i r r e l e a s e i n s m a l l increments i n t o the s m a l l i n t e s t i n e through the i n t e r m i t t e n t opening o f the p y l o r i c v a l v e . The s m a l l i n t e s t i n e d i g e s t s and absorbs these increments as they are r e c e i v e d but i t s a b i l i t y t o do so depends upon c e r t a i n p h y s i o l o g i c a l l i m i t a t i o n s . Perhaps most important i s the f a c t t h a t the mucosal surface o f the s m a l l i n t e s t i n e i s osmoresponsive. That i s t o say, when the contents of the stomach are r e l e a s e d i n t o the upper s m a l l i n t e s t i n e water s h i f t s between the e x t r a c e l l u l a r f l u i d spaces o f the body and the lumen o f the i n t e s t i n e so as t o balance the osmotic a c t i v i t i e s across the mucosal membrane (26). Normally, the process i s g r o s s l y unremarkable and goes unnoticed. Under abnormal circumstances, however, such as f o l l o w i n g surgery of the stomach so extensive as t o e l i m i n a t e i t s r e s e r v o i r funct i o n , the simple a c t o f e a t i n g may l e a d t o sudden and excessive hyperosmolarity i n the upper i n t e s t i n e w i t h consequent water s h i f t s l a r g e enough t o r e s u l t i n the p h y s i o l o g i c a l response known as the "dumping dyndrome" wherein there can be s e r i o u s vasomotor disturbances i n c l u d i n g sweating, nausea, d i a r r h e a , a f a l l i n blood pressure and weakness (27). S i m i l a r l y , the l a r g e i n t e s t i n e i s a l s o osmoresponsive (28) but i t cannot absorb sugars. Thus when, due t o disease o r surgery, the s m a l l i n t e s t i n a l c a p a c i t y f o r sugar d i g e s t i o n o r a b s o r p t i o n i s so g r e a t l y reduced t h a t a s u b s t a n t i a l amount of unabsorbed sugar enters the large, i n t e s t i n e , d i a r r h e a w i l l ensue owing t o the osmotic p r o p e r t i e s o f the sugar i t s e l f as w e l l as t o any i n c r e a s e i n o s m o t i c a l l y a c t i v e molecules through b a c t e r i a l breakdown o f the sugar t o l a c t i c and other acids (2S). I t takes o n l y 54 grams of glucose t o produce one l i t e r of the osmotic equivalent of the e x t r a c e l l u l a r f l u i d s and, thus, a t l e a s t one

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

12

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

l i t e r o f excess e x c r e t i o n . Under normal circumstances the system i s under c o n t r o l and such untoward e f f e c t s do not happen. Foodstuffs i n general, f a t s , e s p e c i a l l y , but p r o t e i n s a l s o as w e l l as carbohydrates, when they enter the i n t e s t i n e through the p y l o r i c v a l v e e l i c i t responses which slow g a s t r i c emptying. The case of sugars i s shown i n F i g u r e 8 by a summation o f many s t u d i e s c a r r i e d out by J. N. Hunt and h i s colleagues. An i n i t i a l "meal" of 750 ml of a s o l u t i o n of c i t r a t e was placed by tube i n t o the stomach. Most of t h e "meal" was del i v e r e d t o the i n t e s t i n e over the next 20 minutes. The volume d e l i v e r e d i n the same span o f time was reduced by the a d d i t i o n of glucose and the degree t o which the d e l i v e r e d volume was r e duced i n c r e a s e d as the glucose c o n c e n t r a t i o n i n c r e a s e d . C l e a r l y , r e c e p t o r s i n the l i n i n g o f the s m a l l i n t e s t i n e respond t o t h e e n t e r i n g glucose i n p r o p o r t i o n t o i t s c o n c e n t r a t i o n and a c t t o reduce g a s t r i c m o t i l i t r e c e p t o r s f o r glucose ar specific. Fructose g e n e r a l l y d i d not e l i c i t an i n h i b i t o r y response at low c o n c e n t r a t i o n s . C l e a r responses t o f r u c t o s e r e q u i r e d concentrations o f about 300 m i l l i m o l e s / l i t e r and more. Sucrose, or a mixture of glucose and f r u c t o s e , as might be expected, were intermediate i n t h e i r e f f e c t s . Once t h e process of stomach emptying s t a r t s the stomach d e l i v e r s i t s contents a t a r a t e roughly p r o p o r t i o n a l t o t h e i r n u t r i t i v e d e n s i t y ( k c a l / m l ) ( J . N. Hunt, p e r s o n a l communication) and i n a p r e d i c t a b l e and e x p o n e n t i a l f a s h i o n u n t i l the stomach i s very n e a r l y empty. During t h i s process, monosaccharides, i n the d i e t or produced by d i g e s t i o n , are moving i n t o and down t h e i n t e s t i n e and are being absorbed by the c a r r i e r mechanisms e a r l i e r mentioned; f r u c t o s e by means of a f a c i l i t a t e d d i f f u s i o n c a r r i e r , glucose and g a l a c t o s e by means of a Na -dependent cotransport c a r r i e r . I t may be asked, why? What i s the value t o the economy o f the organism t h a t these p a r t i c u l a r mechanisms are used and t h a t d i f f e r e n t mechanisms are used f o r d i f f e r e n t kinds of sugar. An answer may be t h a t the needs are best matched i n t h i s way. The fundamental d i f f e r e n c e between the two c a r r i e r systems i s t h a t the one, the Na -dependent c a r r i e r , can be energized t o produce a c t i v e ( a g a i n s t t h e c o n c e n t r a t i o n g r a d i e n t ) t r a n s p o r t whereas the other, the f a c i l i t a t e d d i f f u s i o n c a r r i e r cannot. This d i f f e r e n c e would seem t o match the energy demands of t h e r e s p e c t i v e a b s o r p t i v e problems. In the case of f r u c t o s e , f r u c t o s e l e v e l s i n the blood during i t s absorption are low, being only one-tenth those of glucose during i t s a b s o r p t i o n , (10-15 mg % as against 150-200 mg %) and f r u c t o s e i s r a p i d l y metabolized reducing the l a t e - or p o s t a b s o r p t i v e blood f r u c t o s e t o very low l e v e l s . Consequently, there i s no l a r g e s t a b l e b l o o d - t o - i n t e s t i n a l lumen gradient of f r u c t o s e c o n c e n t r a t i o n and there may simply be no need f o r +

+

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

1.

CRÂNE

13

Intestinal Absorption of Sugars

SMALL

STOMACH

LARGE INTESTINE

INTESTINE

J ' BACTERIAL

„

DIGESTION and ABSORPTION

Γ—ι

1

1

r

Ί

FERMENT­ ATION

Figure 7. Schematic of intestines

VOLUME PLACED IN STOMACH

100 Ο >

200

MILLIMOLES/LITER

300

400

500

OF MONOSACCHARIDE

Figure 8. Effect of carbohydrate con­ tent on the rate of stomach emptying of a test meal. Drawn from data published in graph­ ic form by Hunt and Knox (29).

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

MINUTES Figure 9. Time course of glucose absorption from a loop of rabbit intestine, in vivo. Drawn from data published in graphic form by Barany and Sperber (30).

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

1.

CRÂNE

Intestinal Absorption of Sugars

15

f r u c t o s e t o be absorbed by an energized t r a n s p o r t system. Hence, the f a c i l i t a t e d d i f f u s i o n c a r r i e r s u f f i c e s . The case i s d i f f e r ent f o r glucose. The blood i n h e a l t h always contains a p p r e c i able (80-90 mg %) glucose and one may be c e r t a i n t h a t a q u a n t i t a t i v e l y important p a r t o f the absorption o f a load o f glucose w i l l r e q u i r e the p a r t i c i p a t i o n of an energized c a r r i e r because t h a t p a r t o f a b s o r p t i o n w i l l n e c e s s a r i l y be " u p h i l l " from the lumen t o the blood. What takes p l a c e i n the i n t e s t i n e f o l l o w i n g a l o a d of g l u cose i s w e l l i l l u s t r a t e d by the experiments o f Barany and Sperber (30) w i t h l i v e r a b b i t s as shown i n F i g u r e 9. These workers placed a c e r t a i n volume o f a concentrated glucose s o l u t i o n i n t o a c l o s e d loop of the r a b b i t s i n t e s t i n e and sampled the contents o f the loop a t the i n t e r v a l s t h e r e a f t e r . Initially the c o n c e n t r a t i o n o f glucose i n the i n t e s t i n e was higher than glucose i n the blood. Consequently a b s o r p t i o n during t h i s p e r i o d took place down f e r of sugar from the i n t e s t i n no energy input other than d i f f u s i o n a l . This i s the " d o w n h i l l " component. L a t e r , as a b s o r p t i o n progressed, the c o n c e n t r a t i o n of glucose i n the i n t e s t i n e became lower than i n the blood. Continued absorption consequently took p l a c e " u p h i l l " a g a i n s t t h e c o n c e n t r a t i o n g r a d i e n t and would r e q u i r e the i n p u t o f energy other than d i f f u s i o n a l . At t h e o u t s e t , one might suppose t h a t two d i f f e r e n t c a r r i e r s are used; one f o r the d o w n h i l l component and another f o r the uph i l l . However, t h i s i s not the case. The evidence says t h a t the same c a r r i e r s are used f o r both components. I f these c a r r i e r s were t o have the requirement f o r the consumption o f metabolic energy i n the u p h i l l mode b u i l t i n t o t h e biochemical mechanisms they would be w a s t e f u l when o p e r a t i n g i n the d o w n h i l l mode. In Table I I I are compared four types of membrane t r a n s p o r t which are e i t h e r known or have been proposed t o occur i n animal cells. These a r e : ( l ) F a c i l i t a t e d d i f f u s i o n which f o r present purposes i s viewed as having the c h a r a c t e r i s t i c s o f a symmetrical biochemical r e a c t i o n i n which the s t a t i o n a r y s t a t e achieved w i l l be a 1/1 e q u i l i b r i u m between f r u c t o s e i n s i d e and f r u c t o s e o u t s i d e the c e l L F a c i l i t a t e d d i f f u s i o n can operate only " d o w n h i l l " . (2) V e c t o r i a l biochemical r e a c t i o n s of t h e k i n d envisaged i n the phosphorylation-dephosphorylation hypothesis of the 1930 s - 1950*s ( 31) wherein i t was proposed t h a t the energy f o r accumulation was d e l i v e r e d t o the s u b s t r a t e , glucose, by the t r a n s f e r o f phosphate from ATP w i t h subsequent h y d r o l y s i s t o r e l e a s e f r e e sugar. Roseman and h i s colleagues and Kaback have s t u d i e d systems i n b a c t e r i a l membranes which are of t h i s g e n e r a l type except t h a t phosphorylated sugar and n o t f r e e sugar i s accumulated (32). The asymmetry of the biochemical r e a c t i o n s i n such systems i s obvious. ( 3 ) C o v a l e n t l y energized c a r r i e r s which a r e l i k e the 1

f

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

C + ATP C * Ρ + ADP C * Ρ + Go + HOH C + Gi + Ρ

C + Nao C-Na C-Na + Go C-Na-G C-Na-G «r+ C-Na + G i C'Na C + Nai t o pump

(3) C o v a l e n t l y Energized Reaction

(4) Cotransport Energized Carrier

[Gi] > [Go] [Nai] > [Nao]

no

yes

both

[Gi] > [Go]

both

yes

both

[ F i ] = [Fo]

[Gi] > [Go]

no

Downhill only

Stationary State

C = c a r r i e r , F = f r u c t o s e , G = glucose, Ρ = phosphate, ο = o u t s i d e , i = i n s i d e .

C-G6P + ADP C + Gi + Ρ

C + Go + ATP C-G6P + HOH

( 2 ) V e c t o r i a l Biochemical Reaction

C + Fi

C + Fo

C-F

Reaction Involved

(1) Symmetrical Biochemical Reaction ( F a c i l i t a t e d Diffusion)

D e s c r i p t i v e Name

I s Bond Energy Consumed i n Downhill Mode

Downhill or Uphill Transport Capability

Reactions Involved i n and Energy U t i l i z a t i o n by V a r i o u s H y p o t h e t i c a l Types o f Membrane Transport

TABLE I I I

1.

CRÂNE

Intestinal Absorption of Sugars

17

v e c t o r i a l b i o c h e m i c a l r e a c t i o n s i n t h a t they are fundamentally asymmetrical but which d i f f e r i n t h a t t h e energy f o r accumulation i s d e l i v e r e d t o t h e c a r r i e r r a t h e r than t o the s u b s t r a t e . Perhaps the best example of a c o v a l e n t l y energized c a r r i e r i s the c e l l membrane sodium pump which expresses i t s e l f as an Na^K " a c t i vated ATPase (33). (4) Cotransport energized c a r r i e r s o f the k i n d already d e s c r i b e d above. I n the absence o f a N a f l u x t h e r e a c t i o n s o f these c a r r i e r s a r e symmetrical. I n t h e presence o f a N a f l u x t h e r e a c t i o n s are, as i n d i c a t e d , asymmetrical. As a consequence of t h i s d u a l i t y the cotransport energized system i s the o n l y one o f t h e f o u r which i s not only capable o f both u p h i l l as w e l l as d o w n h i l l t r a n s p o r t but which a l s o does n o t have an absolute requirement t o u t i l i z e bond energy i n the d o w n h i l l mode. The c o t r a n s p o r t energized system i s capable o f a d j u s t i n g energy use t o energy need and i s thus c o n s e r v a t i v e . The a b i l i t y of the i n t e s t i n e t o absor quantitie as c a l c u l a t e d above i s evolved i t s f u n c t i o n s under c o n d i t i o n s o f l i m i t e d food supply where s t r e s s would be expected on developing a system w i t h the a b i l i t y t o capture every l a s t a v a i l a b l e molecule. Under t h e same c o n d i t i o n s there would seem t o be an advantage t o a t r a n s p o r t mechanism which d i d n o t waste t h i s precious food i n prov i d i n g energy merely t o s a t i s f y t h e needs o f the mechanism and not the needs of the work. 4

+

+

Summary The f o l l o w i n g p o i n t s can be r e i t e r a t e d i n summary. 1. Sugar i s absorbed i n t h e form o f monosaccharides by means o f s p e c i f i c brush border membrane c a r r i e r s which are i n c l o s e f u n c t i o n a l p r o x i m i t y t o the brush border d i g e s t i v e h y d r o l ases or by means o f an h y d r o l a s e - r e l a t e d d i r e c t t r a n s l o c a t i o n . 2. There i s normally no advantage f o r a b s o r p t i o n t o provide sugar i n t h e form o f d i s a c c h a r i d e . 3. The t o t a l c a p a c i t y of the s m a l l i n t e s t i n e f o r sugar a b s o r p t i o n i s enormous. 4. The r a t e o f carbohydrate a b s o r p t i o n i s c o n t r o l l e d by negative feed-back t o stomach emptying from s u g a r - s p e c i f i c osmor e c e p t o r s l o c a t e d i n t h e upper i n t e s t i n e . The r e c e p t o r s are l e s s responsive t o f r u c t o s e than t o glucose. 5. The a b s o r p t i o n o f glucose and g a l a c t o s e can take p l a c e both up as w e l l as down a c o n c e n t r a t i o n g r a d i e n t from i n t e s t i n e t o blood. The same c a r r i e r s are used i n both the d o w n h i l l and the u p h i l l modes. 6. The c a r r i e r s f o r glucose and g a l a c t o s e are energized by the cotransport of N a thus p r o v i d i n g a p o s s i b l e advantage f o r energy conservation i n t h a t bond energy need not be consumed i n the d o w n h i l l mode. +

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

18

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

Acknowledgments The work o f the author c i t e d i n t h i s review was supported bygrants from the N a t i o n a l Science Foundation and the N a t i o n a l I n s t i t u t e of A r t h r i t i s , Metabolism and D i g e s t i v e Diseases. Mr. R. M i l t o n prepared the i l l u s t r a t i o n s . Literature Cited

1. Bloom, W. and Fawcett, D. W. "A Textbook of Histology", 9th

Edition, pp. 560-568, W. B. Saunders Co., Philadelphia, 1968. 2. Forstner, G. and Wherrett, J. R. Biochim. Biophys. Acta. (1973) 306, 446-459. 3. Lindemann, B. and Solomon, A. K. J. Gen. Physiol. (1962) 45, 801-810. 4. Fordtran, J. S., Rector and Kinney, J. J. 5. Fromter, E. and Diamond, J. Nature New Biology (1972) 235, 9-13. 6. Crane, R. K. in Code, C. F. Editor "Handbook of Physiology, Section 6. Alimentary Canal, Volume 3. Intestinal Absorption," pp. 1323-1351, American Physiological Society, Washington, 1968. 7. Schultz, S. G. and Strecker, C. K. Biochim. Biophys. Acta. (1970) 211, 586-588. 8. Gracey, Μ., Burke, V. and Oshin, A. Biochim. Biophys. Acta. (1972) 266, 397-406. 9. Honegger, P. and Semenza, G. Biochim. Biophys. Acta. (1973) 318, 390-410. 10. Schultz, S. G. and Curran, P. F. Physiol. Revs. (1970) 50, 637-718. 11. Crane, R. K. Amer. J. Clinical Nutrition (1969) 22, 242-249. 12. Diamond, J. M. Federation Proc. (1971) 30, 6-13. 13. Crane, R. K. in M. Florkin and E. Stotz, Editors, "Compre­ hensive Biochemistry, Vol. 17, Carbohydrate Metabolism", pp. 1-14, Elsevier,Amsterdam, 1969. 14. Malathi, P., Ramaswamy, K., Caspary, W. F. and Crane, R. K. (1973) Biochim. Biophys. Acta. 307, 613-622. 15. Ramaswamy, Κ., Malathi, P., Caspary, W. F. and Crane, R. K. (1974) Biochim. Biophys. Acta. 345, 39-48. 16. Crane, R. K. in T. Z. Csaky, Editor, "Intestinal Absorption and Malabsorption", Raven, Press, New York, in press. 17. Messer, M. and Kerry, K. R. (1967) Biochim. Biophys. Acta. 132, 432-443. 18. Walker, G. J. and Whelan, W. J. (1960) Biochem. J. 76, 257-263. 19. Crane, R. K. in Κ. B. Warren, Editor, Symposia of the International Society for Cell Biology, Vol. 5, Intracellular Transport, pp. 71-102, Academic Press, New York, 1966.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

1.

CRANE

Intestinal

Absorption

of

Sugars

19

20. Gray, G. M. and Santiago, N. A. (1966) Gastroenterology 51, 489-498. 21. Diedrich, D. F. and Hanke, D. W. in T. Z. Csaky, Editor, Intestinal Absorption and Malabsorption", Raven Press, New York, in press. 22. Chain, Ε. B., Mansford, K. R. L. and Pocchiari, F. (1960) J. Physiol. 154, 39-51. 23. MacDonald, I. and Turner, L. J. (1968) The Lancet 1, 841-843. 24. Cook, G. C. (1970) Clinical Sci. 38, 687-697. 25. Holdsworth, C. D. and Dawson, A. M. (1964) Clinical Sci. 27, 371-379. 26. Code, C. F., Bass, P., McClary, G. B., Jr., Newnum, R. L. and Orvis, A. L.(1960)Amer. J. Physiol. 199, 281-288. 27. MacDonald, J. M. Webster M.M. Jr. Tennyson C H and Drapanas, T. (1969 28. Phillips, S. F. (1972) Gastroenterology , 29. Hunt, J. N. and Knox, M. T. in Code, C. F., Editor, Handbook of Physiology, Section 6: Alimentary Canal, Vol. 4, Motility, pp. 1917-1935, American Physiological Society, Washington, 1968. 30.Bárány,E. and Sperber, E. (1939) Skand. Arch. Physiol. 81, 290-299. 31. Crane, R. K.(1960)Physiol. Revs. 40, 789-825. 32. Kaback, H. R. in Tosteson, D. C., Editor, The Molecular Basis of Membrane Function, pp. 421-444, Prentice-Hall, Inc., Englewood Cliffs, 1969. 33. Caldwell, P. C. in Bittar, Ε. E., Editor, Membranes and Ion Transport, Vol. 1, pp. 433-461, Wiley-Interscience, New York, 1970.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

2 Metabolic Effects of Dietary Carbohydrates—A Review SHELDON REISER U.S. Department of Agriculture, ARS, Nutrition Institute, Carbohydrate Nutrition Laboratory, Beltsville, Md. 20705

Many environmental changes have been characteristic of societies described as "Western", "urbanized" or "affluent." Among these changes are a decrease in physical activity, an increase in mental stress and changes in dietary patterns. The changes in dietary patterns of carbohydrate intake that have been developing in the United States are shown in Figure 1 (1). The share of total carbohydrate in the U.S. diet provided by sugars as compared to starch has risen from about 32% around the turn of the century until today sugars, predominantly sucrose, contribute more than 50% of the total carbohydrate. While the average per capita consumption of flours and cereals has dropped from 300 lbs/year in 1909 to 141 lbs/year in 1970, the consumption of refined sugar and other sweeteners has in­ creased from 87 to 126 lbs/year (1,2). These are average figures. Experts estimate that the sugar consumption by the young, that is between 6-20 years of age, probably ranges from 140 to 150 lbs/year (2). The metabolic implications of high sucrose intake in the young will be discussed later. These a f f l u e n t s o c i e t i e s are a l s o c h a r a c t e r i z e d by an i n crease i n the i n c i d e n c e o f heart disease and d i a b e t e s . Diabetes and heart disease represent two o f the most c r i t i c a l h e a l t h problems i n the U.S. today. Diabetes now a f f e c t s about 5 m i l l i o n Americans and r e s u l t s i n over 35,000 deaths a n n u a l l y . By 1980, i t i s estimated t h a t more than 10% o f a l l Americans w i l l have diabetes o r the i n h e r i t e d t r a i t o f diabetes ( 3 ) . One out o f every f o u r a d u l t s i n the U.S. between 18-79 years o f age has been diagnosed as having o r suspected o f having heart disease ( 4 ) . A c l o s e r e l a t i o n s h i p between heart disease and diabetes i s i n d i cated by the f i n d i n g s t h a t d i a b e t i c s have a much higher r i s k o f developing heart disease than the general p o p u l a t i o n (5,6) and t h a t 25% o r more o f p a t i e n t s w i t h v a s c u l a r disease are d i a b e t i c CDOne o f the most i n t r i g u i n g aspects o f t h i s changing p a t t e r n o f carbohydrate consumption i n urbanized c u l t u r e s i s the r e l a t i o n s h i p between the i n c r e a s e d i n t a k e o f r e f i n e d carbohydrates 20 In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

2.

21

Metabolic Effects

REISER

683

1909-13

ι * 4ftf

1972

47.2 52.8

10 STARCH SUGAR

20

30

40

50

60

70

PERCENT

Figure I. Carbohydrate from starch and sugar. Carbohydrate consumption in the United States during the indicated years is based on food disappearing into con­ sumption channels. Carbohydrate in foods such as milk, fruit, and sweeteners was assumed to be present mainly as sugar, and carbohydrate in foods such as grain products and vegetables was assumed to be present mainly as starch. Adapted from Réf. 1.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

22

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

such as sucrose and the e t i o l o g y of v a r i o u s d i s e a s e s . In recent years s e v e r a l c o n t r o v e r s i a l hypotheses have i m p l i c a t e d * t h e i n creased i n g e s t i o n of sucrose, as compared to the more complex carbohydrates, as an important f a c t o r i n the e t i o l o g y o f coronary heart disease Ç8-10) and diabetes (10,11). I t i s recognized t h a t many environmental f a c t o r s , i n c l u d i n g d i e t a r y f a c t o r s , as w e l l as genetic f a c t o r s i n f l u e n c e these diseases and thus the unequivocal r o l e of one of these f a c t o r s independent of the others i s d i f f i c u l t to evaluate. However, i n view o f the controversy surroundi n g d i e t a r y carbohydrate, i t i s the purpose o f t h i s review to d e s c r i b e some of the metabolic e f f e c t s observed a f t e r f e e d i n g sucrose to experimental animals and man and to evaluate the r e l a t i o n s h i p between these metabolic changes and the disease s t a t e s . An area of c o n s i d e r a b l e controversy i s the i n t e r p r e t a t i o n of r e t r o s p e c t i v e or e p i d e m i o l o g i c a l s t u d i e s t h a t attempt t o prove the i n c r e a s e d i n c i d e n c e of a disea$e by c o r r e l a t i o n w i t h the increased i n t a k e o s p e c t i v e s t u d i e s do no as t o the causal s i g n i f i c a n c e o f the c o r r e l a t i o n s detected, they are u s e f u l i n i d e n t i f y i n g trends and i n suggesting s p e c i f i c problem areas worthy o f f u r t h e r study. Many r e t r o s p e c t i v e s t u d i e s have i n d i c a t e d a c o r r e l a t i o n between sucrose i n t a k e and heart disease (8,10,12-15), but Γ would l i k e t o concentrate p r i m a r i l y on the data gathered by an i n t e r n a t i o n a l cooperative study on the r e l a t i o n s h i p between d i e t a r y f a c t o r s and deaths from heart disease i n 37 c o u n t r i e s , p u b l i s h e d by M a s i r o n i i n 1970 (16). Table 1 summarizes these r e s u l t s . M o r t a l i t y data from heart disease ( a r t e r i o s c l e r o t i c and degenerative) were taken from "World Health S t a t i s t i c s Annuals" and v a r i o u s age and sex groups were d e f i n e d . D i e t a r y data were taken from the "Food balance sheets" p u b l i s h e d by the Food and A g r i c u l t u r e O r g a n i z a t i o n . From these c o r r e l a t i o n c o e f f i c i e n t s i t can be seen t h a t w h i l e sucrose i s somewhat l e s s s t r o n g l y c o r r e l a t e d w i t h death r a t e s than i s f a t , there i s nonetheless a s t r o n g p o s i t i v e c o r r e l a t i o n . Since the i n t a k e o f d i e t a r y c a l o r i e s , f a t and sucrose are s i m i l a r l y c o r r e l a t e d , these r e s u l t s are s t i l l open to v a r i o u s i n t e r p r e t a ­ t i o n s . Although on the average d i e t a r y sucrose comprises w e l l over 50% of the simple sugars of the d i e t , i t appears to be much more s t r o n g l y c o r r e l a t e d w i t h heart disease deaths than the t o t a l of a l l the simple d i e t a r y sugars which a l s o i n c l u d e l a c t o s e , f r u c t o s e and glucose. This suggests t h a t sucrose has a s p e c i f i c a c t i o n on heart disease not shared by these other sugars. I t i s a l s o apparent t h a t a s t r o n g i n v e r s e r e l a t i o n s h i p e x i s t s between the amount of complex carbohydrate i n the d i e t and deaths from heart disease. This r e l a t i o n s h i p supports the contention of B u r k i t t (17) and Trowel1 (18) t h a t d i e t a r y f i b e r may exert an important p r e v e n t a t i v e a c t i o n against the i n c i d e n c e of heart disease. The question then a r i s e s as t o what metabolic p r o p e r t i e s o f d i e t a r y sucrose or i t s component monosaccharides can mediate

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975. ?

-0.72 -0.63

0.32 0.24 0.52

0.64 0.56 0.75

0.70 0.84 0.70

Males, 45-54 years

Both sexes, a l l ages

Both sexes, a l l ages (1940 s-1950 s)

Death r a t e s per were taken from and the v a r i o u s balance sheets"

(16)

100,000 p o p u l a t i o n f o r a r t e r i o s c l e r o t i c and degenerative heart disease "World H e a l t h S t a t i s t i c s Annuals" (World H e a l t h O r g a n i z a t i o n , 1958, 1968) groups d e f i n e d by age and sex. D i e t a r y data were taken i n from "Food p u b l i s h e d by the Food and A g r i c u l t u r e O r g a n i z a t i o n (1949, 1966).

Adapted from

-0.71

0.33

0.64

0.55

Females, 55-64 years

,

-0.59

0.31

0.66

0.74

Males, 55-64 years

f

-0.74

Simple sugars

Sucrose

Age-sex

Total fat

Complex carbohydrates

C o r r e l a t i o n c o e f f i c i e n t s between death r a t e s from heart d i s e a s e and d i e t a r y f a c t o r s f o r 37 c o u n t r i e s d u r i n g the 1960 s

Table 1

24

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

an i n c r e a s e d i n c i d e n c e o f v a s c u l a r c o m p l i c a t i o n s , heart d i s e a s e and d i a b e t e s . Since a fundamental d i f f e r e n c e between dietarys t a r c h and sucrose r e s i d e s i n the nature o f the monosaccharide u n i t s comprising t h e r e s p e c t i v e molecules, much o f the i n t e r e s t i n d i e t a r y sucrose has focused on t h e metabolic e f f e c t s o f f r u c t o s e . S t u d i e s w i t h humans and experimental animals have e s t a b l i s h e d t h a t d i e t s c o n t a i n i n g sucrose o r f r u c t o s e produce l a r g e r i n c r e a s e s i n blood l i p i d s (19-23), e s p e c i a l l y the t r i g l y c e r i d e f r a c t i o n (24-28), and i n h e p a t i c l i p o g e n i c enzymes (29-35) than d i e t s c o n t a i n i n g an e q u i v a l e n t amount o f glucose or glucose polymers. The magnitude and d u r a t i o n o f the hyperl i p e m i a i s c o n t r o l l e d by f a c t o r s such as amount o f sugar f e d , age and sex o f t h e s u b j e c t , t h e nature o f the other d i e t a r y i n g r e d i e n t s and g e n e t i c p r e d i s p o s i t i o n . Younger s u b j e c t s and premenopausal females show less i n c r e a s e than o l d e r s u b j e c t s and males (25,26). An important f a c t o r i n t h i s sucrose e f f e c t appears t o be the amoun fat d i e t s , i . e . , l e s s tha s y n t h e s i s from carbohydrate i s a necessary and expected p h y s i o l o g i c a l process. Table 2, adapted from the work o f Macdonald (36), shows t h a t when young men were f e d a d i e t c o n t a i n i n g 60% carbohydrate, 30% f a t and 9% p r o t e i n f o r 5 days, the magnitude of the t r i g l y c e r i d e m i a was dependent on the nature o f both the carbohydrate and the f a t . Sucrose s i g n i f i c a n t l y i n c r e a s e d blood t r i g l y c e r i d e s o n l y when the d i e t a r y f a t was cream and not when i t was sunflower o i l . In c o n t r a s t , glucose d i d not i n c r e a s e the t r i g l y c e r i d e s w i t h e i t h e r f a t . The e f f e c t o f sunflower o i l may be due t o a c c e l e r a t i o n i n the removal o f endogenous t r i g l y c e r i d e s s i n c e N e s t e l and B a r t e r (37) have shown t h a t s u b j e c t s consuming d i e t s r i c h i n polyunsaturated fat have f a s t e r clearance r a t e s than s u b j e c t s f e d s a t u r a t e d fat. These r e s u l t s a l s o might e x p l a i n apparent c o n t r a d i c t i o n s found i n the l i t e r a t u r e as t o the e f f e c t o f d i e t a r y sucrose on serum l i p i d s i n t h a t unsaturated f a t t y a c i d s may mask t h i s e f f e c t . Work from Yudkin's l a b o r a t o r y (Table 3, (38)) shows t h a t sucrose produces a l a r g e r i n c r e a s e i n serum t r i g l y c e r i d e s and c h o l e s t e r o l than does s t a r c h i n r a t s f e d an atherogenic d i e t c o n t a i n i n g 16% hydrogenated coconut o i l and 1% c h o l e s t e r o l for 100 days. The carbohydrate comprised 47% o f the d i e t . By 180 days, t h e l e v e l s o f the blood l i p i d s had f a l l e n , but there was s t i l l the same r e l a t i v e i n c r e a s e i n c h o l e s t e r o l and t r i g l y c e r i d e l e v e l s produced by the sucrose as compared t o s t a r c h . These r e s u l t s again i n d i c a t e t h a t sucrose together w i t h other d i e t a r y f a c t o r s can produce a combination t h a t i s p o t e n t i a l l y more d e t r i m e n t a l t o the h e a l t h o f t h e consumer than e i t h e r o f the f a c t o r s alone. The major e f f e c t o f d i e t a r y sucrose on l i p i d metabolism i n v o l v e s the t r i g l y c e r i d e s . The r i s k f a c t o r i n v o l v e d i n e l e vated l e v e l s o f blood t r i g l y c e r i d e has r e c e n t l y been confirmed by a j o i n t statement on D i e t and Coronary Disease from the

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

Ρ <0.025

Adapted from (56)

75 + 7

71 + 9

69 + 4

95 + 6

78 + 5

mg/100 ml

Triglycerides

The d i e t s contained approximately 9% o f the c a l o r i e s as p r o t e i n (calcium c a s e i n a t e ) , 60% of t h e c a l o r i e s as carbohydrate ( e i t h e r sucrose o r glucose) and 30% o f the c a l o r i e s as f a t ( e i t h e r cream or sunflower o i l ) . Each value represents the mean +_ S.E.M. from 5 s u b j e c t s .

Glucose-sunflower o i l

Glucose-cream

Sucrose-sunflower o i l

Sucrose-cream

Control

Diet

E f f e c t o f d i e t a r y carbohydrate and l i p i d on f a s t i n g blood t r i g l y c e r i d e s i n young men a f t e r 5 days on d i e t

Table 2

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

195

sucrose

Ρ < 0.001

Ρ <0.02

Adapted from (38)

517

392

114

starch P< 0.001

787

262

577

sucrose

P< 0.001

mg/100 ml

Cholesterol

144

mg/100 ml

Triglycerides

starch

Carbohydrate

Adult r a t s were f e d a d i e t c o n s i s t i n g o f 47 p a r t s carbohydrate ( e i t h e r s t a r c h or s u c r o s e ) , 25 p a r t s c a s e i n , 16 p a r t s hydrogenated coconut o i l , 5 p a r t s mineral s a l t s , 5 p a r t s c e l l u l o s e v i t a m i n s , 1 p a r t c h o l e s t e r o l and 1 p a r t c h o l i c a c i d . Each value represents the mean values from at l e a s t 4 r a t s .

180

100

days

Duration o f experiment

E f f e c t o f an atherogenic d i e t c o n t a i n i n g s t a r c h o r sucrose on blood l i p i d s i n r a t s

Table 3

2.

REISER

Metabolic Effects

27

N a t i o n a l Academy o f Science, N a t i o n a l Research C o u n c i l and the American Medical A s s o c i a t i o n C o u n c i l s on Food and N u t r i t i o n (39). In a d d i t i o n a recent p r o s p e c t i v e study of the occurrence of new events of heart disease i n 3000 Scandinavian men showed t h a t a high f a s t i n g plasma t r i g l y c e r i d e l e v e l was as important a r i s k f a c t o r as a h i g h c h o l e s t e r o l l e v e l and was independent of the l e v e l of plasma c h o l e s t e r o l (40). The p h y s i o l o g i c a l s i g n i f i c a n c e of some o f the s t u d i e s r e l a t i n g sucrose i n t a k e to increased t r i g l y c e r i d e s has been questioned because of the comparatively l a r g e q u a n t i t i e s o f sucrose used. Table 4, adapted from work by Mukherjee and coworkers (41), shows t h a t on d i e t s c o n t a i n i n g r e f i n e d sugar at l e v e l s c o n s i s t e n t w i t h those consumed i n many areas o f the w o r l d , i . e . , 12%, but lower than the sucrose l e v e l s i n the U.S. d i e t , sucrose, as compared t o s t a r c h or glucose, i n c r e a s e d the l e v e l s o f blood t r i g l y c e r i d e , blood c h o l e s t e r o l d l i v e triglycerid i rat a f t e r 30 days. Fructos to i n c r e a s e blood c h o l e s t e r o compare w i t h s t a r c h , tended t o decrease t r i g l y c e r i d e l e v e l s and i n crease c h o l e s t e r o l l e v e l s . While e f f o r t s to show s i m i l a r e f f e c t s of low l e v e l s o f d i e t a r y sucrose on t r i g l y c e r i d e l e v e l s i n humans have not been e q u a l l y s u c c e s s f u l (42), i t has been c o n s i s t e n t l y shown t h a t when there i s a g r e a t l y reduced d i e t a r y i n t a k e of sucrose by humans, t r i g l y c e r i d e l e v e l s decrease (4345). The f a l l i n serum t r i g l y c e r i d e i s greater f o r those subjects w i t h h i g h e r i n i t i a l l e v e l s than f o r those w i t h lower i n i t i a l levels. The importance o f the i n c r e a s e i n blood t r i g l y c e r i d e s by d i e t a r y sucrose has been questioned because, i n many cases, the e f f e c t i s only t r a n s i t o r y and, a f t e r a p e r i o d o f a d a p t a t i o n , t r i g l y c e r i d e s r e t u r n t o normal (46). In g e n e r a l , these events can be explained on the b a s i s t h a t d i e t a r y f r u c t o s e or sucrose induces an i n c r e a s e i n l i v e r l i p o g e n e s i s so t h a t the i n f l u x o f t r i g l y c e r i d e s i n t o the plasma exceeds the removal c a p a c i t y . C o n t r i b u t i n g t o t h i s t r i g l y c e r i d e m i a i s the decreased a c t i v i t y o f adipose t i s s u e l i p o p r o t e i n l i p a s e or c l e a r i n g f a c t o r observed a f t e r the feeding of sucrose or f r u c t o s e as compared to glucose (27). This adaptation s t i l l i n v o l v e s an increased throughput o f d i e t a r y sucrose as t r i g l y c e r i d e i n the normal person w i t h the p o t e n t i a l danger t h a t a defect or breakdown i n t h i s adapt a t i o n might produce t r i g l y c e r i d e m i a . More important, there are segments o f the p o p u l a t i o n t h a t appear t o have a genetic p r e d i s p o s i t i o n t h a t r e s u l t s i n an i n a b i l i t y to adapt t o changes i n d i e t a r y carbohydrate and who show a l a r g e and permanent i n crease i n blood t r i g l y c e r i d e s as a r e s u l t of an i n c r e a s e i n the i n t a k e o f sucrose. This type o f h y p e r l i p e m i a has been d e s c r i b e d as carbohydrate-induced or Type IV by F r e d r i c k s o n , Levy and Lees (47). Carbohydrate-induced h y p e r l i p e m i a has been shown to be a s s o c i a t e d w i t h abnormal glucose t o l e r a n c e , diabetes and heart disease. Wood et a l . , (48) have estimated t h a t 8.6% of

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

8.30 + 1.91

20.30 + 0.25

15.80 + 0.50

46.3 + 1.4

43.2 + 5.3

98.7 + 6,9

25.5 +_ 4.3

97.2 +1.7

95.0 + 1.8

12% glucose 52% s t a r c h

12% f r u c t o s e 52% s t a r c h

12% sucrose 52% s t a r c h

Each value represents the average o f 8 experiments c a r r i e d out i n d u p l i c a t e +_ S.E.M.

Adapted from (41)

11.80 + 0.07

37.0 + 1.7

59.4 + 2.5

mg/g

Liver t r i g l y c e r i d e

64% s t a r c h

Serum c h o l e s t e r o l mg/100 ml

Serum t r i g l y c e r i d e mg/100 ml

D i e t a r y carbohydrate

E f f e c t o f d i e t a r y sugars on serum and l i v e r l i p i d s i n r a t s

Table 4

2.

REISER

29

Metabolic Effects

f r e e - l i v i n g v o l u n t e e r s i n C a l i f o r n i a , aged 25-79, showed the Type IV l i p o p r o t e i n e m i a p a t t e r n , 4.8% i n women and 13% i n men. Numerous s t u d i e s have demonstrated t h a t i n p a t i e n t s w i t h carbohydrate-induced l i p e m i a , sucrose produced much l a r g e r i n c r e a s e s i n blood t r i g l y c e r i d e s than s t a r c h (49-56). F i g u r e I I summarizes s t u d i e s from Kuo's l a b o r a t o r y (54) on the e f f e c t o f d i e t a r y sucrose on the l e v e l s o f blood t r i g l y c e r i d e s i n Type IV h y p e r l i p e m i c p a t i e n t s . A s e l f - s e l e c t e d d i e t produced a marked t r i g l y c e r i d e m i a , 120 mg/100 ml being considered about normal. A d i e t c o n t a i n i n g the 60% carbohydrate p r i m a r i l y as sucrose increased the t r i g l y c e r i d e s f u r t h e r w h i l e a 60% s t a r c h d i e t d r a m a t i c a l l y lowered the t r i g l y c e r i d e s . Each d i e t a r y p e r i o d was o f 3 weeks d u r a t i o n . Table 5, adapted from the work o f L i t t l e and Antar (55,56), shows the p a t t e r n o f blood t r i g l y c e r i d e s i n p a t i e n t s w i t h e i t h e r Type I I ( f a t s e n s i t i v e ) or Type I I I , IV and V l i p e m i a s as a f u n c t i o n o f the nature o f the d i e t a r y carbohydrate f a t , s a t u r a t e d or unsaturated e x h i b i t e d higher l e v e l s o f t r i g l y c e r i d e s , c h a r a c t e r i s t i c o f these types o f l i p e m i a s , and showed a much l a r g e r i n c r e a s e i n t r i g l y c e r i d e due t o sucrose than the Type I I p a t i e n t s . In c o n t r a s t t o the sucrose-induced i n c r e a s e o f t r i g l y c e r i d e s i n normal s u b j e c t s , the nature o f the f a t , s a t u r a t e d o r unsaturated, d i d not e f f e c t the magnitude o f the sucrose i n c r e a s e . However, the blood t r i g l y c e r i d e l e v e l s were lower i n d i e t s c o n t a i n i n g unsaturated than i n those c o n t a i n i n g s a t u r a t e d f a t s . From these r e s u l t s i t i s apparent t h a t d i e t a r y sucrose i s an important e n v i ronmental f a c t o r i n the expression o f c a r b o h y d r a t e - s e n s i t i v i t y i n genetically susceptible subjects. In order t o determine the primary biochemical defect chara c t e r i z i n g the i n t e r a c t i o n between d i e t and g e n e t i c e x p r e s s i o n , the c a r b o h y d r a t e - s e n s i t i v e BHE s t r a i n o f r a t has been used e x t e n s i v e l y by s c i e n t i s t s at the N u t r i t i o n I n s t i t u r e o f the U.S. Department o f A g r i c u l t u r e (57-71). BHE r a t s have been shown t o gain more body weight and accumulate more carcass and l i v e r l i p i d (60,61,63,66), have increased a c t i v i t i e s o f h e p a t i c l i p o g e n i c enzymes (64,71) and have h i g h e r l e v e l s o f blood l i p i d s (63,71) than other s t r a i n s o f r a t s f e d the same high carbohydrate d i e t . Studies by Berdanier and coworkers (68) have demonstrated t h a t the BHE r a t e x h i b i t s a marked h y p e r i n s u l i n e m i a e a r l y i n l i f e as compared t o s i m i l a r l y f e d W i s t a r r a t s (Figure I I I ) . F i f t y days o f age corresponds t o about 5-7 years o f age i n a human. By 100 days o f age the i n s u l i n l e v e l s had decreased d r a m a t i c a l l y . The importance o f t h i s e a r l y h y p e r i n s u l i n e m i a was confirmed by t e s t s w i t h other r a t s t r a i n s . Wistar r a t s made h y p e r i n s u l i n e m i c a

a

The BHE s t r a i n r e s u l t s from a cross between the Pennsylvania S t a t e C o l l e g e s t r a i n and the Osbome-Mendel ( a l s o c a l l e d Yale) s t r a i n s . BHE animals are c u r r e n t l y a v a i l a b l e from Flow Laboratories, Dublin, V i r g i n i a .

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

697

700\-

600—

I

50

°i

o

400

300

200

183

100

SELF SELECTED HOME DIET

SUCROSE DIET

1

STARCH DIET

Figure II. Effect of dietary carbohydrate on the blood triglycerides in type TV hyperlipemia patients. Each value represents the mean serum triglyceride level observed in six hyperglyceridemic patients during the consumption of a home diet and when an equivalent amount of carbohydrate was supplied primarily as sucrose or starch. Adapted from Kef. 54.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

saturated

saturated

unsaturated

Type I I

Type I I I , IV and V

Type I I I , IV and V

starch sucrose

starch sucrose

starch sucrose

Carbohydrate

70

51

13

Q

0.

sucrose i n c r e a s e

Adapted from (55, 56)

190 324

251 392

109 123

mg/100 ml

Blood t r i g l y c e r i d e

The h y p e r l i p e m i c p a t i e n t s were c l a s s i f i e d as t o Type according t o F r e d r i c k s o n et a l . (47). Each value represents the mean from a t l e a s t 5 p a t i e n t s .

Fat

Patient

Blood t r i g l y c e r i d e s i n Type I I and Type I I I , IV and V h y p e r l i p e m i a p a t i e n t s as a f u n c t i o n o f the nature o f the d i e t a r y carbohydrate and f a t

Table 5

32

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

1201-

I00h 3

w

3 CO

3
80h

60h

40

ill

20^

50

100 300 BHE

5 0 100 300 WISTAR

AGE ( DAYS) Figure III. Fasting serum immune-reactive insulin levels of 50-, 100-, and 300-day old BHE and Wistar rats. Each value represents the mean and the S.E.M. from at least 10 rats. Adapted from Kef. 68.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

2.

REISER

Metabolic Effects

33

at an e a r l y age by the i n j e c t i o n o f i n s u l i n or by feeding t o l butamide, developed a p a t t e r n o f increased h e p a t i c l i p o g e n i c enzyme a c t i v i t y s i m i l a r t o t h a t observed i n BHE r a t s . I t was a l s o found t h a t feeding sucrose t o the BHE r a t s from weanling to 50 days o f age appeared t o exert l o n g - l a s t i n g metabolic e f f e c t s on parameters such as serum l i p i d s and i n s u l i n even a f t e r the sugar had not been fed f o r 100 days (72). These f i n d i n g s taken together w i t h the p a t t e r n o f very high sucrose i n t a k e by the young suggest t h a t the c o n d i t i o n s f o r the induct i o n o f the metabolism c h a r a c t e r i z i n g c a r b o h y d r a t e - s e n s i t i v i t y i s optimal at an e a r l y age. Figure IV shows t h a t adipose t i s s u e from BHE r a t s e x h i b i t e d a much s m a l l e r s e n s i t i v i t y t o i n s u l i n , as measured by CO2 r e l e a s e from glucose, than d i d adipose from Wistar r a t s (73). With i n c r e a s i n g age, t i s s u e s e n s i t i v i t y of both s t r a i n s was reduced but the s t r a i n d i f f e r e n c e p e r s i s t e d . These r e s u l t s suggest t h a t the h y p e r i n s u l i n e m i a observed i n the BHE r a t occurs as a consequenc to i n s u l i n and t h a t n o r m a l i z a t i o represent a gradual d e p l e t i o n o f p a n c r e a t i c i n s u l i n s t o r e s . This c o n c l u s i o n was supported by f i n d i n g t h a t at 50 days the pancreas from BHE r a t s contained s l i g h t l y more t o t a l i n s u l i n than Wistar r a t s but at 150 days t h i s s i t u a t i o n was reversed (73). In a r e l a t e d study, Vrana and coworkers (74) demonstrated a decreased adipose t i s s u e s e n s i t i v i t y o f sucrose-fed as compared to s t a r c h - f e d Wistar r a t s (Table 6). These r e s u l t s show t h a t w h i l e i n s u l i n d i d not s i g n i f i c a n t l y i n c r e a s e glucose i n c o r p o r a t i o n i n t o l i p i d i n the sucrose-fed r a t s even at 1000 yUnits i n s u l i n / m l , s t a r c h - f e d r a t s i n c o r p o r a t e d 2.7 times as much glucose i n t o l i p i d due t o i n s u l i n . Rats fed equal amounts o f s t a r c h and sucrose gave intermediate i n s u l i n responses. L o n g i t u d i n a l s t u d i e s o f the events r e s u l t i n g from the carbohydrate s e n s i t i v i t y have shown t h a t BHE r a t s developed a glucose i n t o l e r a n c e as a f u n c t i o n of age and at 425 days o f age showed symptoms of a r t e r i o s c l e r o s i s of the coronary v e s s e l s (71). Therefore, the r e l a t i o n s h i p between heart disease and diabetes might be a t t r i b u t e d t o a common metabolic defect t h a t produces a h y p e r i n s u l i n e m i c response t o a sucrose s t r e s s , e s p e c i a l l y i n those i n d i v i d u a l s g e n e t i c a l l y predisposed toward carbohydrate s e n s i t i v i t y . In t h i s connection i t has been r e ported t h a t high sucrose i n t a k e produced h y p e r i n s u l i n i s m i n about 1/3 of the human s u b j e c t s t e s t e d and t h a t the i n c r e a s e was greater i n p a t i e n t s w i t h p e r i p h e r a l v a s c u l a r disease than i n normal subjects (75). Many of the f i n d i n g s r e l a t i n g d i e t a r y sucrose t o diabetes have come from the work of Dr. Aharon Cohen i n I s r a e l (10,33,51 76-90). R e t r o s p e c t i v e s t u d i e s l i n k i n g diabetes t o sucrose have been based on the emergence o f diabetes as a s e r i o u s h e a l t h problem i n s p e c i f i c e t h n i c groups t h a t have r e c e n t l y undergone changes i n t h e i r t r a d i t i o n a l d i e t a r y p a t t e r n s c h a r a c t e r i z e d by an increased i n t a k e of r e f i n e d carbohydrate at the expense o f

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

34

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

3000H

ε

II

2000

ο ο

w

g-

1000

J

M l

l

i

50

150

50

BHE

150

WISTAR AGE (DAYS)

Figure IV. Adipose tissue sensitivity to insulin of BHE and Wistar rats at 50 and 150 days of age. Tissue sensitivity is expressed as the difference in counts per minute be­ tween the basal- and insulin-stimulated C0 release from radioactive glucose by slices of epididyml fat pads. Each value represents the mean ± S.E.M from 24 rats. Adapted from Ref. 73. 2

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

1905 2019

S t a r c h + sucrose

Sucrose

1.1

1.9

2.7

Adapted from (74)

2240

3640

6290

A d u l t r a t s were f e d d i e t s c o n t a i n i n g 20% c a l o r i e s as p r o t e i n , 10% c a l o r i e s as f a t and 70% c a l o r i e s as carbohydrate, e i t h e r s t a r c h and/or sucrose as i n d i c a t e d . Each value represents the mean values from 6 r a t s .

2371

i n s u l i n increase

I n s u l i n c o n c e n t r a t i o n (yUnits/ml) 0 1000 cpm/mg p r o t e i n

Starch

D i e t a r y Carbohydrate

E f f e c t o f d i e t a r y sucrose on the i n s u l i n s e n s i t i v i t y o f r a t adipose t i s s u e as measured by i n c o r p o r a t i o n o f glucose i n t o l i p i d

Table 6

36

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

crude, u n r e f i n e d carbohydrate (11). These r e t r o s p e c t i v e s t u d i e s are t y p i f i e d by r e s u l t s obtained w i t h the Yemenite Jews. In a survey of the prevalence o f diabetes i n I s r a e l d u r i n g 1958-1959 i t was found t h a t o f the 5000 Yemenites examined who had been l i v i n g i n I s r a e l f o r l e s s than 10 y e a r s , only 3 cases o f d i a betes were found (76). In c o n t r a s t , among Yemenites who had l i v e d i n I s r a e l f o r more than 25 years the i n c i d e n c e o f diabetes was 2.9%, or even more than t h a t o f Jews coming from Western c o u n t r i e s . On a n a l y z i n g the d i f f e r e n t environmental f a c t o r s t h a t might have caused t h i s i n c r e a s e , the d a i l y d i e t a r y p a t t e r n s of the Yemenites i n Yemen and i n I s r a e l were compared (Table 7) (10). The t o t a l c a l o r i c i n t a k e was only s l i g h t l y l e s s i n the Yemen than i n I s r a e l . Two outstanding d i f f e r e n c e s i n the type of food consumed were apparent. F i r s t , i n the Yemen the f a t s consumed were mainly o f animal o r i g i n . Vegetable o i l was r a r e l y used. I I s r a e l th s e t t l e d Yemenite d s i m i l a r amounts of t o t a and, i n a d d i t i o n , abou gram vegetabl , i n the Yemen the carbohydrate consumed was mainly s t a r c h w i t h almost no sucrose used. In I s r a e l there was a t e n f o l d i n c r e a s e i n d i e t a r y sucrose. I t was concluded, t h e r e f o r e , t h a t i f d i e t were an environmental f a c t o r i n the e t i o l o g y o f diabetes i n t h i s e t h n i c group, the most l i k e l y d i e t a r y suspect was sucrose. The hypothesis t h a t sucrose i s an e t i o l o g i c a l f a c t o r i n diabetes has been t e s t e d i n g e n e t i c a l l y s e l e c t e d r a t s (84). These r a t s were bred on the b a s i s o f glucose t o l e r a n c e t e s t s . Animals showing the highest r i s e i n blood sugar, c a l l e d the "Upward s e l e c t i o n , " were mated and r a t s w i t h the lowest r i s e i n blood glucose v a l u e s , or "Downward s e l e c t i o n , " were mated. The blood glucose values of these animals 60 minutes a f t e r an i n t r a g a s t r i c glucose load i n both the Upward and Downward s e l e c t e d l i n e s , as a f u n c t i o n o f generation and d i e t a r y carbhydrate, are shown i n F i g u r e V (89). In succeeding generations of the Upward l i n e fed sucrose the l e v e l of blood glucose rose to high values and a c o n s i d e r a b l e number o f the animals d e v e l oped a d i a b e t i c - l i k e syndrome w i t h h i g h f a s t i n g blood glucose and spontaneous g l u c o s u r i a . This e f f e c t could be shown w i t h as l i t t l e as 25% o f the d i e t a r y carbohydrate as sucrose. In c o n t r a s t , the s i b l i n g Upward s t r a i n fed s t a r c h d i d not show a r i s e i n blood glucose. In the o f f s p r i n g of the Downward sel e c t e d l i n e , blood glucose d i d not r i s e i n r a t s fed e i t h e r the sucrose or the s t a r c h d i e t . The diabetes appearing i n the sucrose-fed Upward s e l e c t e d l i n e showed other symptoms o f human adult-onset diabetes such as i n i t i a l increased f a s t i n g plasma i n s u l i n l e v e l s , p e r i p h e r a l i n s u l i n r e s i s t a n c e and r e t i n a l and r e n a l v a s c u l a r c o m p l i c a t i o n s (89). These r e s u l t s show the need f o r i n t e r a c t i o n between g e n e t i c and d i e t a r y f a c t o r s f o r the expression o f the d i a b e t i c syndrome t o become evident. A prominent e x p l a n a t i o n f o r the increased diabetogenic and l i p o g e n i c c a p a c i t y of d i e t a r y sucrose as opposed t o d i e t a r y

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

2559

Yemenites; o l d s e t t l e r s

86

80

g

Protein

30

14

g

Oil

372

351

g

Total

63

6

g

Sucrose

Carbohydrates

Adapted from (10) families.

51

43

g

Animal + Margarine

Each v a l u e r e p r e s e n t s the average d a i l y food i n t a k e from 20

2237

Calories

Yemenites; new immigrants

Groups

Fat

D a i l y food i n t a k e p a t t e r n o f Yemenites l i v i n g i n Yemen as compared t o Yemenites l i v i n g i n I s r a e l

Table 7

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

• — • SUCROSE-FED RATS • " · STARCH —FED RATS

Figure V. Blood glucose values at 60 minutes after a gastric glucose load in genetically selected sucrose- and starch-fed rats. The Upward selection represents the progeny resulting from the mating of males and females with highest blood glucose values following a glucose tolerance test. The Downward selection represents the progeny resulting from the mating of males and females with the lowest blood glucose values following a glucose tolerance test. Each rat was given an intragastric glucose load of 350 mg/100 g body weight. Each point represents the mean from at least 12 rats. Adapted from Ref. 89.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

2.

REISER

39

Metabolic Effects

s t a r c h i s based on d i f f e r e n c e s i n the r a t e s of i n t e s t i n a l d i g e s t i o n and absorption o f the component monosaccharides (78,81,91,92). The feeding o f d i e t s high i n sucrose has been shown t o produce an adaptive i n c r e a s e i n the a c t i v i t y o f i n t e s t i n a l sucrase (93-96) w h i l e s t a r c h feeding does not appear t o i n f l u e n c e the a c t i v i t y or r a t e o f r e l e a s e o f p a n c r e a t i c amylase. A more r a p i d and e f f i c i e n t absorption of glucose from sucrosefed than from s t a r c h - f e d s u b j e c t s , as already suggested by work from Crane's l a b o r a t o r y (97,98), would produce increases i n p o s t p r a n d i a l blood glucose w i t h a r e s u l t i n g s t r o n g s t i m u l a t i o n of the i n s u l i n response. Such s t i m u l a t i o n , when repeated, might produce a c h r o n i c h y p e r i n s u l i n e m i a which e v e n t u a l l y could impair the i n s u l i n producing system and produce d i a b e t i c l i k e symptoms. The i n i t i a l p e r i o d o f c h r o n i c h y p e r i n s u l i n e m i a could s i g n a l a p a t t e r n of enzyme i n d u c t i o n s which could d i r e c t the pathways o f carbohydrat metabolis toward increased l i p o genesis, thus e x p l a i n i n enzymes found a f t e r sucros feeding f a c t o r s , f r u c t o s e metabolism i n the l i v e r may a l s o c o n t r i b u t e to a reduced glucose t o l e r a n c e i n sucrose-fed animals by i n c r e a s i n g the l e v e l s of glucose 6-phosphatase a c t i v i t y (33). This enzyme i s r e q u i r e d t o form blood glucose. Fructose-fed r a t s have a l s o shown d e f i c i e n t h e p a t i c u t i l i z a t i o n o f glucose (99). I f d i e t a r y sucrose i s an e t i o l o g i c a l f a c t o r i n any d i s e a s e , an understanding of the s p e c i f i c metabolic c h a r a c t e r i s t i c s of f r u c t o s e may provide i n f o r m a t i o n as to the mechanisms t h a t may be i n v o l v e d . There are marked d i f f e r e n c e s i n the metabolism of absorbed f r u c t o s e i n the s m a l l i n t e s t i n e s of v a r i o u s s p e c i e s . These d i f f e r e n c e s appear t o be o f fundamental importance i n determining the subsequent metabolic f a t e o f f r u c t o s e . In the r a t (100) and i n man (101) f r u c t o s e i s p o o r l y metabolized and appears i n the p o r t a l blood p r i m a r i l y as f r u c t o s e together w i t h a s m a l l amount o f l a c t a t e . In c o n t r a s t , i n the small i n t e s t i n e of the guinea p i g f r u c t o s e i s mainly converted t o glucose (102). The p h y s i o l o g i c a l importance of t h i s d i f f e r e n c e i n i n t e s t i n a l metabolism can be seen i n the f a i l u r e of f r u c t o s e feeding to induce h y p e r t r i g l y c e r i d e m i a i n guinea p i g s as i t does i n r a t s and man (27). I t i s , t h e r e f o r e , p o s s i b l e to c o r r e l a t e the h y p e r t r i g l y c e r i d e m i c e f f e c t o f f r u c t o s e feeding w i t h the appearance o f unchanged f r u c t o s e i n the c i r c u l a t i o n . Studies u s i n g both humans (103) and r a t s (104) have shown t h a t f r u c t o s e i n f u s i o n r e s u l t s i n a dramatic decrease o f h e p a t i c adenine n u c l e o t i d e s , e s p e c i a l l y ATP. Glucose i n f u s i o n d i d not produce comparable decreases i n the n u c l e o t i d e l e v e l s (103). These r e s u l t s suggest t h a t f r u c t o s e may act as an ATP s i n k " i n the l i v e r . Since the c l e a r i n g o f blood t r i g l y c e r i d e s by the l i v e r has been reported t o be an a c t i v e process (46,105), a decreased l e v e l o f h e p a t i c ATP may prevent normal c l e a r i n g (106) thereby c o n t r i b u t i n g t o increased l e v e l s of blood t r i g l y c e r i d e s . The M

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

40

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

f a t t y l i v e r s found i n sucrose-fed r a t s could a l s o be e x p l a i n e d on the b a s i s of a decreased ATP-dependent s y n t h e s i s of the phospholipids r e q u i r e d t o m o b i l i z e l i v e r l i p i d . In Conclusion: During t h i s review, the metabolic e f f e c t s o f d i e t a r y sucrose reported by numerous i n v e s t i g a t o r s and the i m p l i c a t i o n s of these r e s u l t s as they p e r t a i n t o the h e a l t h and w e l l - b e i n g o f humans and experimental animals have been des c r i b e d . In some s t u d i e s , p a r t i c u l a r l y w i t h humans, these e f f e c t s have not been c o n s i s t e n t l y observed or when observed have been i n t e r p r e t e d d i f f e r e n t l y . However, the r e s u l t s o f the s t u d i e s described i n t h i s review are c o n s i s t e n t w i t h the f o l l o w ing: CI) Although sucrose alone has not as yet been shown to be an important r i s k f a c t o r i n the e t i o l o g y o f heart disease and diabetes i n the m a j o r i t y o f the p o p u l a t i o n , sucrose together w i t h other environmental f a c t o r s may produce a combination t h a t i s more l i p o g e n i c or diabetogeni tha f th f a c t o r alone (2) Sucrose alone may b i n heart disease and diabete segmen populatio described as "carbohydrate s e n s i t i v e . " I t i s apparent t h a t much more work on t h i s problem i s r e q u i r e d , e s p e c i a l l y i n e a r l y i d e n t i f i c a t i o n o f c a r b o h y d r a t e - s e n s i t i v e i n d i v i d u a l s . These f i n d i n g s , however, i n d i c a t e t h a t a r e v e r s a l of the present trend of increased consumption of r e f i n e d sugar to an increased consumption o f the more complex and n a t u r a l forms o f carbohydrates should be encouraged. Questions: Q. Did the Yemenites have d i f f e r e n t d i e t a r y f i b e r i n t a k e i n Yemen and i n I s r a e l ? A. Since the d a i l y carbohydrate i n t a k e o f the Yemenites was very s i m i l a r i n the Yemen and i n I s r a e l and s i n c e the sucrose i n take had increased so d r a m a t i c a l l y i n I s r a e l , i t i s safe t o assume t h a t the i n t a k e of d i e t a r y f i b e r was less i n I s r a e l than i n the Yemen. However, a c t u a l f i b e r contents i n these d i e t s were not reported. Q. Are there s t u d i e s c u r r e n t l y underway t o i d e n t i f y carbohydratesensitive individuals? A. We are c u r r e n t l y t r y i n g t o fund a cooperative study w i t h Dr. Cohen i n I s r a e l t h a t we b e l i e v e can e s t a b l i s h parameters f o r the i d e n t i f i c a t i o n o f c a r b o h y d r a t e - s e n s i t i v i t y . These s t u d i e s are a c o n t i n u a t i o n o f the survey o f the Yemenites who migrated to I s r a e l p r i o r to 1958 and who e x h i b i t e d p r a c t i c a l l y no diabetes at t h a t time. We p l a n t o measure v a r i o u s blood hormones and blood l i p i d s i n a sample of t h i s p o p u l a t i o n and t o c o r r e l a t e the l e v e l s of these parameters w i t h the i n c i d e n c e of symptoms of diabetes and heart disease, the l e v e l o f d i e t a r y sugar and the f a m i l i a l c l u s t e r i n g of the s u b j e c t s . We hope t o determine whether the a f f e c t e d i n d i v i d u a l s w i l l have had a high i n t a k e o f sugar, whether they were g e n e t i c a l l y r e l a t e d and whether they showed s p e c i f i c i n c r e a s e s i n the blood parameters t e s t e d .

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

2.

REISER

Metabolic Effects

41

Literature Cited:

1. Page, L. & Friend, B. "Sugars in Nutrition," edited by Sipple, H. & McNutt, J., p. 93. Nutrition Foundation Monograph Series, Academic Press, New York, 1974. 2. "Hearings before the Select Committee on Nutrition and Human Needs of the United States Senate, Part 2 - Sugar in diet, diabetes and heart diseases," p. 147, U.S. Government Printing Office, Washington, D.C., 1973. 3. "Hearings before the Select Committee on Nutrition and Human Needs of the United States Senate, Part 2 - Sugar in diet, diabetes and heart diseases," p. 146, U.S. Government Printing Office, Washington, D.C., 1973. 4. Weir, C. E . , "An Evaluation of Research in the United States on Human Nutrition, Report No. 2, Benefits from Nutrition Research," p. 16, U.S. Department of Agriculture, Washington, D.C. 1971 5. Ostrander, L. D. Dávalos, R. & Cole, , p.365, , , 1970. 6. Keen, H . , "Early Diabetes," edited by Camerini-Dávalos, R. & Cole, H. S., p. 437, Academic Press, New York, 1970. 7. Wahlberg, F. & Thomasson, B., "Carbohydrate Metabolism and its Disorders," Vol. 2, edited by Dickens, Ε., Whelan, J . J . & Randle, P. J., p. 188, Academic Press, New York, 1968. 8. Yudkin, J., Lancet, (1957), ii, 155. 9. Michaels, L . , Brit. Heart J., (1966), 28, 258. 10. Cohen, A. M., Bavly, S. & Poznanski, R., Lancet, (1966), ii, 1399. 11. Cleave, T. L. & Campbell, G. D., "Diabetes, Coronary Thrombosis, and the Saccharine Disease," p. 15, John Wright & Sons, Ltd., Bristol, 1969. 12. Antar, Μ. Α., Ohlson, M. A. & Hodges, R. E . , Am. J. Clin. Nutr., (1964), 14, 169. 13. Osancova, K., Hejda, S. & Zvolankova, Κ., Lancet, (1965), i, 494. 14. Lopez, Α., Krehl, W. Α., Hodges, R. E. & Good, Ε. I., Am. J. Clin. Nutr., (1966), 19, 361. 15. Yudkin, J. & Morland, J., Am. J. Clin. Nutr., (1967), 20, 503. 16. Masironi, R., Bull. Wld. Hlth. Org., (1970), 42, 103. 17. Burkitt, D. P., Walker, A. R. P. & Painter, N. S., JAMA, (1974), 229,1068. 18. Trowell, Η., Am. J. Clin. Nutr., (1973), 25, 926. 19. Winitz, Μ., Graff, J. & Sudman, D. Α., Arch. Biochem. Biophys., (1964), 108, 576. 20. Hodges, R. E. & Krehl, W. Α., Am. J. Clin. Nutr., (1965), 17, 334. 21. Macdonald, I. & Roberts, J. B., Metabolism, (1965), 14, 991.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

42

PHYSIOLOGICAL EFFECTS OF FOOD CARBOHYDRATES

22. Dalderup, L. Μ., Nesser, W. & Keller, Α. Η. Μ., Voeding, (1968), 29, 245. 23. Naismith, D. J., "Sugar. Chemical, Biological and Nutritional Aspects of Sucrose," edited by Yudkin, J., Edelman, J. & Haugh, L., p. 123, Butterworth's, London, 1971. 24. Macdonald, I. & Braithwaite, D. M., Clin. Sci., (1964), 27, 23. 25. Macdonald, I., Clin. Sci., (1965), 29, 193. 26. Macdonald, I., Am. J. Clin. Nutr., (1966), 18, 369. 27. Bar-On, H.&Stein, Y., J. Nutr., (1968), 94, 95. 28. Szanto, S. & Yudkin, J., Postgrad. Med. J., (1969), 45, 602. 29. Nikkila, E. A. & Ojala, K., Life Sci., (1965), 4, 937. 30. Fábry, P., Poledrie, R., Kazdová, L.&Braun, T., Nutr. Diet., (1968), 10, 81. 31. Naismith, D. J., Proc. Nutr. Soc., (1971), 30, 259. 32. Pereira, J. N.& Jangaard 392. 33. Cohen, Α., Briller, S.& Shafrir, E., Biochim. Biophys. Acts, (1972), 279, 129. 34. Chevalier, M. M., Wiley, J. H.& Leveille, G. Α., J. Nutr. (1972), 102, 337. 35. Michaelis, O. Ε., IV&Szepesi, B., J. Nutr., (1973), 103, 697. 36. Macdonald, I., Am. J. Clin. Nutr., (1967), 20, 345. 37. Nestel, P. J. & Barter, P. J., Am. J. Clin. Nutr., (1973), 241. 38. Qureshi, R. U., Akinyanju, P. A.& Yudkin, J. Nutr. Metabol., (1970), 12, 347. 39. Nutr. Reviews, (1972), 30, 223. 40. Carlson, L. Α. & Böttiger, L. E., Lancet, (1972), i, 865. 41. Mukherjee, S., Basu, M.& Trivedi, Κ., J. Atheroscler Res. (1969), 10, 261. 42. Truswell, A. S., "Sugar and Human Health," edited by Stewart, S. C., p. 50, The International Sugar Research Foundation, Bethesda, Md., 1972. 43. Rifkind, B. M., Lawson, D. H.& Gale, M., Lancet, (1966), ii, 1379. 44. Mann, J. I., Truswell, A. S., Hendricks, D. A.&Manning, E., Lancet, (1970), i, 870. 45. Roberts, A. M., Proc. Nutr. Soc., (1971), 30, 71A. 46. Ahrens, R. Α., Am. J. Clin. Nutr., (1974), 27, 403. 47. Fredrickson, D. S., Levy, R. I.& Lees, R. S., New Eng. J. Med., (1967), 276, 34, 94, 148, 215, 273. 48. Wood, P. D. S., Stern, M. P., Silver, Α., Reaven, G. M. & von der Groeben, J., Circulation, (1972), XLV, 114. 49. Kaufmann, Ν. Α., Gutman, Α., Barzilai, D., Eshchar, J., Blondheim, S. H.& Stein, Y., Israel J. Med. Sci., (1965), 1, 389.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

2.

REISER

Metabolic

Effects

43

50. Kuo, P. T. & Bassett, D. R., Ann. Intern. Med., (1965), 62, 1199. 51. Cohen, A. M., Kaufmann, N. A. & Poznanski, R., Brit. Med. J., (1966), 1, 339. 52. Kaufmann, Ν. Α., Poznanski, R., Blondheim, S. Η. & Stein, Y., Am. J. Clin. Nutr., (1966), 18, 261. 53. Kaufmann, Ν. Α., Poznanski, R., Blondheim, S. H. & Stein, Y., Israel J. Med. Sci., (1966), 2, 715. 54. Kuo, P. T., Feng, L., Cohen, N. N., Fitts, W. T., Jr. & Miller, L. D., Am. J. Clin. Nutr., (1967), 20, 116. 55. Little, J. Α., Birchwood, B. L., Simmons, D. Α., Antar, Μ. Α., Kallos, Α., Buckley, G. C. & Csima, A. Atherosclerosis, (1970), 11, 173. 56. Antar, Μ. Α., Little, J. Α., Lucas, C., Buckley, G. C. & Csima, Α., Atherosclerosis, (1970), 11 191. 57. Marshall, M. W. & Womack M. J Nutr. (1954) 52 51 58. Womack, M. & Marshall 59. Marshall, M. W., Hildebrand, , Dupont, , M., J. Nutr., (1959), 69, 371. 60. Marshall, M. W. & Hildebrand, Η. Ε., J. Nutr., (1963), 79, 227. 61. Adams, Μ., Home Econ. Res. Rept., No. 24, USDA, (1964). 62. Durand, Α. Μ. Α., Fisher, M. & Adams, M., Arch. Path., (1964), 77, 268. 63. Taylor, D. C., Conway, E. S., Schuster, Ε. M. & Adams, Μ., J. Nutr., (1967), 91, 275. 64. Chang, M. L. W., Schuster, Ε. Μ., Lee, J. Α., Snodgrass, C. & Benton, D. Α., J. Nutr., (1968), 96, 368. 65. Durand, Α. Μ. Α., Fisher, M. & Adams, Μ., Arch. Path. (1968), 85, 318. 66. Marshall, M. W., Womack, M., Hildebrand, H. & Munson, A. W., Proc. Soc. Exp. Biol. Med., (1969), 132, 227. 67. Berdanier, C. D., Szepesi, Β., Moser, P. & Diachenko, S., Proc. Soc. Exp. Biol. Med., (1971), 137, 668. 68. Berdanier, C. D., Marshall, M. W. & Moser, P., Life Sci., (1971), 10, 105. 69. Marshall, M. W., Durand, A. M. A. & Adams, Μ., Fourth International Symposium of ICLA., p. 383. Nat. Acad. Sci., Washington, D.C.,1971. 70. Chang, M. L. W., Lee, J. A. S Simmons, Ν., Proc. Soc. Exp. Biol. Med., (1971), 138, 742. 71. Berdanier, C. D., J. Nutr., (1974), 104, 1246. 72. Moser, P. B. & Berdanier, C. D., J. Nutr., (1974), 104, 687. 73. Berdanier, C. D., Diabetologia, (1974), 10, 1. 74. Vrána, Α., Slabochová, Ζ., Kazdova, L. & Fábry, P., Nutr. Rep. Int., (1971), 3, 31. 75. Yudkin, J., Szanto, S. & Kakkar, V. V., Postgrad. Med. J . , (1969), 45, 608. 76. Cohen, Α. Μ., Metabolism, (1961), 10, 50. 77. Cohen, Α. Μ., Am. Heart J . , (1963), 65, 291.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

44

PHYSIOLOGICAL EFFECTS OF FOOD CARBOHYDRATES

78. Cohen, A. M.& Teitelbaum, Α., Am. J. Physiol., (1964), 206, 105. 79. Cohen, Α. Μ., Teitelbaum, Α., Balogh, M.& Groen, J. J., Am. J. Clin. Nutr., (1966), 19, 59. 80. Cohen, A. M., Am. J. Clin. Nutr., (1967), 20, 126. 81. Cohen, Α. Μ., Life Sci., (1968), 7, 23. 82. Cohen, A. M.& Rosenmann, E., Diabetologia, (1971), 7, 25. 83. Rosenmann, E., Teitelbaum, A.& Cohen, A. M., Diabetes, (1971), 20, 803. 84. Cohen, Α. Μ., Teitelbaum, A.& Saliternik, R., Metabolism, (1972), 21, 235. 85. Yanko, L., Michaelson, I. C.& Cohen, A. M., Israel J. Med. Sci., (1972), 8, 1633. 86. Cohen, A. M., Acta Med. Scand. Suppl., (1972), 542, 173. 87. Cohen, Α. Μ., Michaelson, I. C.& Yanko, L., Am. J. Ophth., (1972), 73, 863. 88. Shafrir, E., Teitelbaum Sci., (1972), 8, 990. 89. Cohen, Α. Μ., "Hearings before the Select Committee on Nutrition and Human Needs of the United States Senate, Part 2 - Sugar in diet, diabetes and heart diseases," p. 167, U.S. Government Printing Office, Washington, D.C. 1973. 90. Rosenmann, E., Palti, Z., Teitelbaum, A.& Cohen, A. M., Metabolism, (1974), 23, 343. 91. Crossley, J. N.& Macdonald, I., Nutr. Metabol., (1970), 12, 171. 92. Jourdan, Μ. Η., Nutr. Metabol., (1972), 14, 28. 93. Blair, D. G., Yakimets, W.& Tuba, J., Can. J. Biochem. Physiol., (1963), 41, 917. 94. Deren, J. & Zamcheck, Ν., Fed. Proc, (1965), 24, 203. 95. Deren, J. J., Broitman, S. A.& Zamcheck, N., J. Clin. Invest., (1967), 46, 186. 96. Reddy, B. S., Pleasants, J. R.&Wostmann, B. S., J. Nutr., (1968), 95, 413. 97. Malathi, P., Ramaswamy, K., Caspary, W. F.& Crane, R. K., Biochim. Biophys. Acta, (1973), 307, 613. 98. Ramaswamy, K., Malathi, P., Caspary, W. F & Crane, R. Κ., Biochim. Biophys. Acta, (1974), 345, 39. 99. Hill, R., Baker, N.& Chaikoff, I. L., J. Biol. Chem., (1954), 209, 705. 100. Mavrias, D. A.& Mayer, R. J., Biochim. Biophys. Acta, (1973), 291, 531. 101. Cook, G. C., Clin. Sci., (1969), 37, 675. 102. Mavrias, D. A.& Mayer, R. J., Biochim. Biophys. Acta, (1973), 291, 538. 103. Bode, C., Schumacher, Η., Goebell, Η., Zelder,O.& Pelzel, Η., Hormone Metab. Res., (1971), 3, 289. 104. Woods, H. F., Eggleston, L. V.&Krebs, Η. Α., Biochem. J.,(1970), 119, 501.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

2.

REISER

Metabolic

Effects

105. Havel, R. J., Proceedings of the Second International Symposium on Atherosclerosis, p. 210, edited by Jones, R. J., New York, Springer Verlag, 1970. 106. Schotz, M. C., Arnesjö, B.& Olivecrona, T., Biochim. Biophys. Acta, (1966), 125, 485.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

3 Factors Influencing Adipose Tissue Response to Food Carbohydrates 1

DALE R. ROMSOS and GILBERT A. L E V E I L L E Food Science and Human Nutrition, Michigan State University, East Lansing, Mich. 48824

Interest has bee y bohydrates in human nutrition. Not only is the quantity of carbohydrate in the diet changing but the types of carbohydrate consumed by the Western World has also changed during the past 50 years. These changes, in some cases, have been implicated as a contributing factor to numerous diseases. For example, sucrose has been implicated as a major factor in the development of cardiovascular disease (1). However, it should be pointed out that not all researchers are in agreement on this point (2, 3). In this review we will discuss our efforts to understand the role of dietary carbohydrate in the control of adipose tis­ sue fatty acid synthesis. Since the metabolic response of one organ, in some cases, influences metabolism in another organ, our studies have involved lipid metabolism in liver as well as in adipose tissue. Dietary Factors Influencing Adipose Tissue Response to Carbo­ hydrates Studies concerning the influence of the quantity of car­ bohydrate in the diet on adipose tissue fatty acid synthesis are complex since modification of one dietary variable imposes a change on another d i e t a r y v a r i a b l e . F r e q u e n t l y d i e t a r y c a r bohydrate and f a t are interchanged i n s t u d i e s on the i n f l u e n c e of d i e t on l i p i d metabolism. I f the carbohydrate content of the d i e t i s m o d i f i e d without a concomitant change i n another n u t r i e n t , the n u t r i e n t :energy r a t i o of the d i e t i s changed. G e n e r a l l y a r e d u c t i o n i n the carbohydrate content of the d i e t w i l l depress f a t t y a c i d s y n t h e s i s i n adipose t i s s u e prepar a t i o n s of both r a t s and p i g s ; however, there are e x c e p t i o n s .

Supported by Grant HL-14677 from the N a t i o n a l I n s t i t u t e of H e a l t h , U.S. P u b l i c H e a l t h S e r v i c e . J o u r n a l A r t i c l e No. 7023 Michigan A g r i c u l t u r e Experiment S t a t i o n . 46 In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

Library EmerTcsn Chemical Society 3.

ROMSOS

LEVEiLLE

AND

Adipose Tissue Response

47

These exceptions may a i d i n our understanding of the c o n t r o l o f l i p i d metabolism i n adipose t i s s u e . S u b s t i t u t i o n of mediumc h a i n - t r i g l y c e r i d e s (MCT) f o r d i e t a r y glucose d i d not i n f l u e n c e adipose t i s s u e l i p o g e n e s i s i n the r a t even though the carbohydrate content of the d i e t was decreased (4) (Table 1). L i p o genesis i n p i g adipose t i s s u e was depressed l e s s by the a d d i t i o n of 10% MCT to the d i e t than by the a d d i t i o n of l a r d o r corn o i l (5) (Table 1). S i m i l a r l y , replacement o f d i e t a r y g l u cose with 1,3-butanediol (BD) d i d not depress adipose t i s s u e l i p o g e n e s i s i n the r a t o r p i g (6, 7) (Table 2). Thus, i t i s apparent that the r o l e o f carbohydrate content of the d i e t i n the r e g u l a t i o n of adipose t i s s u e metabolism i s complex and i n v o l v e s i t s r e l a t i o n s h i p with other d i e t a r y i n g r e d i e n t s . Both MCT and BD have marked i n f l u e n c e s on h e p a t i c metabolism (4,6, 8). T h e i r metabolism e l e v a t e s c i r c u l a t i n g ketone body l e v e l s . P r e f e r e n t i a l o x i d a t i o n of ketone bodies by muscle would spare glucose f o r use i n adipos TABLE 1. IN VITRO FATTY ACID SYNTHESIS IN ADIPOSE TISSUE FROM RATS AND PIGS FED MEDIUM-CHAIN TRIGLYCERIDES Diet Species

Control

MCT

Rat

1143 + 63

1170 + 72

Pig

659 + 46

526 + 28

From (4) and (5). D i e t s fed f o r 3 weeks. Rat - 12% MCT. P i g - 10% IjjIgT. F a t t y a c i d s y n t h e s i s expressed as nmoles glucose-U- C i n c o r p o r a t e d i n t o f a t t y acids/100 mg t i s s u e / 2 hr. TABLE 2.

IN VITRO FATTY ACID SYNTHESIS IN ADIPOSE TISSUE FROM RATS AND PIGS FED BUTANEDIOL Diet 17% BD energy

Species

Control

Rat

844 + 77

700

Pig

184 + 28

250 + 29

7 2

±

From (6) and (7). D i e t s ^ f e d f o r 3 weeks. R e s u l t s expressed as nanomoles glucose-U- C i n c o r p o r a t e d i n t o f a t t y acids/100 mg t i s s u e / 2 hr.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

48

PHYSIOLOGICAL

EFFECTS O F FOOD

CARBOHYDRATES

• 4 0 0 \- Fructose Fed Substrate Glucose-U- C I4

300r-

Fructose-U- C ,4

2 00 Ο

l o o

50

100

150

200

Substrate concentration, m M

Biochimica et Biophysica Acta

Figure 1.

14

14

In vitro conversion of glucose-U- C and fructose-U- C to fatty acids in liver slices from glucose- or fructose-fed rats (19)

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

3.

ROMSOS

AND

49

Adipose Tissue Response

LEVEiLLE

P a t t e r n of food intake a l s o a l t e r s adipose t i s s u e l i p i d metabolism. The c a p a c i t y of adipose t i s s u e preparations from meal-fed r a t s o r pigs to convert carbohydrate t o f a t t y a c i d s i s increased d r a m a t i c a l l y (9,10). Thus, i t i s apparent that f a c t o r s which a l t e r the p a t t e r n of carbohydrate intake may a l s o i n f l u e n c e l i p i d metabolism i n adipose t i s s u e . The remainder of our d i s c u s s i o n w i l l center on the i n f l u e n c e of d i e t a r y f r u c t o s e on adipose t i s s u e metabolism. I n t e s t i n a l Transport

and P e r i p h e r a l

Fructose

Levels

V i r t u a l l y a l l species studied can absorb and u t i l i z e f r u c tose. However, i t i s important to recognize that the i n t e s t i n e has s p e c i e s - s p e c i f i c i n f l u e n c e s on f r u c t o s e metabolism. In the guinea p i g and hamster f r u c t o s e i s l a r g e l y converted to glucose during t r a n s f e r across the i n t e s t i n a l w a l l whereas i n other s p e c i e s , i n c l u d i n g man tose i s absorbed l a r g e l approximately 85% of a f r u c t o s e - C dose unchanged (11). In the r a t at l e a s t , f r u c t o s e absorption appears to occur v i a an a c t i v e carrier-mediated mechanism (12,13). Thus, i t i s apparent that i n a c o n s i d e r a t i o n of the i n f l u e n c e of d i e t a r y f r u c t o s e on adipose t i s s u e metabolism one must be aware of the s p e c i e s s p e c i f i c response of the i n t e s t i n e to f r u c t o s e . Feeding d i e t s c o n t a i n i n g f r u c t o s e to species that absorb f r u c t o s e i n t a c t increases the p o r t a l v e i n concentration of f r u c tose markedly; however, p e r i p h e r a l c i r c u l a t i n g l e v e l s of f r u c tose are elevated to a much l e s s e r extent (14). This has been taken to i n d i c a t e that hepatic metabolism of f r u c t o s e i s extensive. D i e t a r y Fructose

and Hepatic and Adipose Tissue

Lipogenesis

I t i s c l e a r that s u b s t i t u t i n g f r u c t o s e f o r glucose i n the d i e t of r a t s leads to changes i n l i p i d metabolism. To increase our understanding of the i n f l u e n c e of d i e t a r y carbohydrates on metabolism we have examined h e p a t i c and adipose t i s s u e responses to d i e t a r y f r u c t o s e . I t i s g e n e r a l l y accepted that feeding f r u c t o s e to r a t s increases the r a t e of f a t t y a c i d synthesis i n the l i v e r ; however, r e s u l t s from i n v i t r o estimates of hepatic f a t t y a c i d synthesis are not a l l i n agreement (15,16,17,18). We examined the i n f l u e n c e of s u b s t r a t e source and concent r a t i o n on h e p a t i c l i p o g e n e s i s i n r a t s ^ f e d glucose or f r u c t o s e c o n t a i n i n g d i e t (Figure 1). Glucose- C conversion to f a t t y a c i d s increased as the concentration of substrate increased, probably r e l a t e d to the a b i l i t y of high l e v e l s of glucose to saturate ç^ucokinase and thus increase a c e t y l CoA formation. Fructose-^ C conversion to hepatic f a t t y acids was higher than glucoseC conversion to f a t t y acids when the substrate con-

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

50

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

c e n t r a t i o n was 10 mM; however as s u b s t r a t e concentration was i n c r e a s e d , h e p a t i c conversion of f r u c t o s e to f a t t y a c i d s was markedly decreased. T h i s decrease i n f r u c t o s e conversion to f a t t y acids when the f r u c t o s e l e v e l i n the i n c u b a t i o n media exceeded 50 mM was probably r e l a t e d to the r a p i d phosphorylation of f r u c t o s e and the subsequent decrease i n ATP l e v e l s . We measured ATP l e v e l s i n l i v e r s l i c e s and observed that ATP l e v e l s were depressed by more than 50% when the media contained 100 mM f r u c t o s e r a t h e r than 100 mM glucose (19). We then examined i n v i v o adenine n u c l e o t i d e l e v e l s i n l i v e r from r a t s fed glucose or f r u c t o s e . ATP l e v e l s were elevated i n l i v e r s of r a t s fed f r u c t o s e (Table 3). Whether t h i s i n c r e a s e i n h e p a t i c ATP l e v e l would allow r a t s consuming a l a r g e dose of f r u c t o s e , such as might occur i n meal-fed animals, to more e f f e c t i v e l y dispose of t h i s dose under i n v i v o c o n d i t i o n s remains to be e s t a b l i s h e d . TABLE 3.

EFFECT OF DIETAR ADENINE NUCLEOTIDE LEVELS IN VIVO D i e t a r y Carbohydrate Glucose

Fructose

ATP

25

39

ADP

9

11

AMP

6

5

ym/liver

From (19).

D i e t s were fed f o r 3 weeks.

Various techniques have been employed to o b t a i n i n v i v o estimates of f a t t y a c i d s y n t h e s i s i n r a t s fed v a r i o u s carbohydrates. O r a l , i n t r a p e r i t o n e a l or intravenous a d m i n i s t r a t i o n of a t r a c e r dose of l a b e l e d substrate i s complicated by p o s s i b l e d i f f e r e n c e s i n r a t e s of substrate absorption, by i n t e s t i n a l metabolism of the substrate or by p o s s i b l e i s o t o p e ^ d i l u t i o n e f f e c t s at the t i s s u e l e v e l . We e l e c t e d to u t i l i z e H-labeled water as a t o o l to obtain t o t a l r a t e s of f a t t y a c i d s y n t h e s i s , l a r g e l y independent of the source of a c e t y l groups which are incorporated i n t o f a t t y a c i d s . I t i s c l e a r that the i n v i v o r a t e of f a t t y a c i d synthesis i n l i v e r of f r u c t o s e fed r a t s was higher than observed i n glucose fed animals (Table 4).

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

3.

ROMSOS

AND

TABLE 4.

LEVEiLLE

Adipose Tissue Response

51

EFFECT OF DIETARY CARBOHYDRATE ON IN VIVO FATTY ACID SYNTHESIS D i e t a r y Carbohydrate Tissue

Fructose

Glucose

Liver

47 + 6

80 + 13

E x t r a Hepatic

14 + 1

7+1

From (19).

Results expressed as dpm χ 10

per gram.

Recognizing that d i e t a r y f r u c t o s e elevated the r a t e of h e p a t i c l i p o g e n e s i s , we examined l i p i d metabolism i n adipose t i s s u e , the predominan fructose. In çgididyma from f r u c t o s e - C was lower than observed from glucoseC regardless of d i e t a r y carbohydrate f e d (Table 5 ) . Further, d i e t a r y f r u c t o s e depressed l i p o g e n e s i s from both s u b s t r a t e s . These r e s u l t s suggested to us that the r e l a t i v e importance of the adipose t i s s u e to f a t t y a c i d s y n t h e s i s may be decreased i n f r u c t o s e - f e d r a t s . Estimates of the i n v i v o r a t e of f a t t y a c i d synthesis obtained with t r i t i a t e d water a l s o i n d i c a t e d that the e x t r a h e p a t i c r a t e of f a t t y a c i d s y n t h e s i s was decreased i n r a t s fed f r u c t o s e (Table 4 ) . Although the t o t a l r a t e of f a t t y a c i d s y n t h e s i s i n r a t s f e d f r u c t o s e was unchanged, the r e l a t i v e importance of the l i v e r was increased and that of the e x t r a hep a t i c t i s s u e s decreased when f r u c t o s e , r a t h e r than glucose, was fed to r a t s . TABLE 5.

INFLUENCE OF DIETARY CARBOHYDRATE SOURCE ON IN VITRO FATTY ACID SYNTHESIS IN RAT ADIPOSE TISSUE D i e t a r y Carbohydrate

Substrate 14 C 14 Fructose-U- C Glucose-U-

Glucose

Fructose

540 + 61

271 + 57

191 + 19

87 + 26

From (19). Results expressed as nmoles substrate converted to f a t t y a c i d s per 100 mg t i s s u e per 2 h r s .

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

52

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

Several f a c t o r s are probably i n v o l v e d i n t h i s s h i f t i n metabolism i n the presence of d i e t a r y f r u c t o s e . Froesch (20) has r e c e n t l y reviewed f r u c t o s e metabolism i n adipose t i s s u e . He noted that f r u c t o s e t r a n s p o r t i n t o the adipocyte appears to be mediated by a c a r r i e r w i t h a r e l a t i v e l y high apparent Κ f o r f r u c t o s e ; thus s i g n i f i c a n t t r a n s p o r t of f r u c t o s e occurs onTy when blood f r u c t o s e l e v e l s are h i g h . F u r t h e r , i n s u l i n does not s t i m u l a t e f r u c t o s e uptake by r a t adipose t i s s u e . Since adipose t i s s u e l a c k s f r u c t o k i n a s e , hexokinase i s probably i n v o l v e d i n the phosphorylation o f f r u c t o s e i n t h i s t i s s u e . P h y s i o l o g i c a l l y , f r u c t o s e - C conversion t o f a t t y a c i d s i n r a t adipose t i s s u e probably occurs^only a f t e r p r i o r h e p a t i c conversion of f r u c t o s e C t o glucose- C. One of the key c o n t r o l p o i n t s i n the conversion of d i e t a r y carbohydrate t o f a t t y a c i d s i n r a t adipose t i s s u e i s at the l e v e l of glucose entry i n t o the adipocyte, an i n s u l i n dependent process. Thus, v a r i o u f a t t y acid synthesis i c u l a t i n g i n s u l i n l e v e l s . Bruckdorfer e t a l (21) have demon­ s t r a t e d that r a t s f e d f r u c t o s e have lower c i r c u l a t i n g i n s u l i n l e v e l s than do r a t s f e d glucose. A l s o , an o r a l dose of f r u c t o s e does not i n c r e a s e plasma i n s u l i n l e v e l s i n f a s t e d r a t s whereas a comparable glucose load doubled plasma i n s u l i n l e v e l s . Results of these s t u d i e s suggest that the r a p i d metabolism of f r u c t o s e by the l i v e r coupled w i t h lower c i r c u l a t i n g i n s u l i n l e v e l s i n f r u c t o s e f e d r a t s c o n t r i b u t e to the decreased r a t e of f a t t y a c i d s y n t h e s i s observed i n these r a t s . Conclusions

and Speculation

D i e t a r y carbohydrates do i n f l u e n c e adipose t i s s u e f a t t y a c i d s y n t h e s i s i n r a t s and p i g s . One of the key c o n t r o l p o i n t s i n the conversion of d i e t a r y carbohydrates t o f a t t y a c i d s i s a t the l e v e l of glucose entry i n t o the adipocyte, an i n s u l i n de­ pendent process. A r e d u c t i o n i n carbohydrate i n t a k e g e n e r a l l y would be expected t o depress i n s u l i n s e c r e t i o n ; however i f com­ pounds r e a d i l y converted t o ketones, such as MCT o r BD, are sub­ s t i t u t e d f o r the carbohydrate p o r t i o n of the d i e t i n s u l i n , s e ­ c r e t i o n might not decrease s i n c e ketones a l s o s t i m u l a t e i n s u l i n r e l e a s e . Meal-eating a l s o increases i n s u l i n r e l e a s e and glucose s t i m u l a t e s a greater i n s u l i n r e l e a s e than does f r u c t o s e . I f the adipocyte maintains i t s s e n s i t i v i t y t o i n s u l i n , these d i e t a r y c o n d i t i o n s would be expected to increase glucose entry i n t o the c e l l thereby s t i m u l a t i n g f a t t y a c i d s y n t h e s i s . The r o l e of d i e t a r y f a c t o r s i n m a i n t a i n i n g i n s u l i n s e n s i t i v i t y of the a d i ­ pocyte are not c l e a r .

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

3.

ROMSOS

AND

LEVEILLE

Adipose Tissue Response

53

Literature Cited

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

Yudkin, J. Proc. Nutr. Soc. (1972) 31, 331-337. Walker, A. R. P. Atherosclerosis (1971) 14, 137-152. Keys, A. Atherosclerosis (1971) 14, 193-202. Wiley, J. Η., and Leveille, G. A. J. Nutr. (1973) 103, 829-835. Allee, G. L., Romsos, D. R., Leveille, G. A. and Baker, D. H. Proc. Soc. Exp. Biol. Med. (1972) 139, 422-427. Romsos, D. R., Sasse, C. and Leveille, G. A. J. Nutr. (1974) 104, 202-209. Romsos, D. R., Belo, P. S., Miller, E. R. and Leveille, G. A. J. Nutr. (1974) Submitted. Romsos, D. R., Belo, P. S. and Leveille, G. A. J. Nutr. (1974) 104 In press Leveille, G. A. Allee, G. L., Romsos H. J. Nutr. (1972) 102, 1115-1122. Leveille, G. Α., Akinbami, T. K. and Ikediobi, C. O. Proc. Soc. Exp. Biol. Med. (1970) 135, 483-486. Macrae, A. R. and Neudoerffer, T. S. Biochim. Biophys. Acta (1972) 288, 137-144. Gracey, M., Burke, V. and Oshin, A. Biochim. Biophys. Acta (1972) 266, 397-406. Topping, D. L. and Mayes, P. A. Nutr. Metabol. (1971) 13, 331-338. Chevalier, M. M., Wiley, J. H. and Leveille, G. A. J. Nutr. (1972) 102, 337-342. Zakim, D., Pardini, R. S., Herman, R. H. and Sauberlich, H. E. Biochim. Biophys. Acta (1967) 144, 242-251. Kritchevsky, D. and Tepper, S. A. Med. Exp. (1969) 19, 329-341. Bender, A. E. and Thadoni, P. V. Nutr. Metabol. (1970) 12, 22-39. Romsos, D. R., and Leveille, G. A. Biochim. Biophys. Acta (1974) 360, 1-11. Froesch, E. R. Acta Med. Scand. (1972) Suppl. 542, 37-46. Bruckdorfer, K. R., Khan, I. H., and Yudkin, J. Biochem. J. (1972) 129,439-445.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

4 Benefits of Dietary Fructose in Alleviating the Human Stress Response J. DANIEL PALM Department of Biology, St. Olaf College, Northfield, Minn.

The structural characteristic from glucose result in differential physiological properties. For some uses glucose is the sugar of choice. For other situa­ tions fructose has distinct advantages. It has recently been found that when pure fructose is exchanged for most other carbohy­ drates in a good high-protein low-carbohydrate diet, it deserves a reputation of being "nature's tranquilizer", or the ideal "cravecontrol" food, and the answer to most of the problems associated with hypoglycemia. Hypoglycemia is not a distinct disease. The name of the dis­ order indicates a deficiency of sugar in the blood sufficient to maintain the nervous system control. It is a physiological dis­ order that is commonly found in persons with diagnoses of schizo­ phrenia, alcoholism, migraine headaches, hyperactivity, severe overweight and many other disorders. Hypoglycemia is a stress which normally results in a response which raises the blood sugar concentration. Everyone experiences some hypoglycemia each day. It is hypoglycemia which triggers the morning and afternoon treks to the coffee and sweet-roll counter. It causes students, and sometimes others, to fall asleep during classes and conferences. It is hypoglycemia that causes the tremor of the hands and the recognition of hunger when meals are delayed. Hypoglycemia ap­ pears to be the most repetitive stress for all members of society. For some unfortunate persons hypoglycemia is not a temporary con­ dition which is rapidly corrected by normal homeostatic responses. In some it persists because insufficient adrenalin is released to convert the liver glycogen to glucose. Such persons remain phys­ iologically and emotionally depressed. In others hypoglycemia persists despite the mobilization of massive amounts of adrenalin which normally increases the blood sugar concentration. The per­ sons with this variety of hypoglycemia develop various symptoms of hypomania. The association of hypoglycemia with both physical and emotional disorders deserves attention. Ever s i n c e hypoglycemia was recognized as a consequence o f the i n j e c t i o n o f e x c e s s i v e amounts o f i n s u l i n i n d i a b e t i c patients, 54 In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

4.

PALM

Fructose and Human Stress

55

i t has been known that most cases o f hypoglycemia do not r e s u l t from h y p e r i n s u l i n i s m . Nor i s hypoglycemia due t o a d e f i c i e n c y o f carbohydrates i n the d i e t . (1) Most cases a r e f u n c t i o n a l hypoglycemias which are t r i g g e r e d by d i e t a r y carbohydrates without any evidence of o v e r - m o b i l i z a t i o n of i n s u l i n . For t h i s reason, Conn, a quarter of a century ago, introduced a h i g h - p r o t e i n low-carbohydrate d i e t f o r hypoglycemics t o provide f o r gluconeogenesis from the p r o t e i n s and f o r a l e s s e n i n g o f the chances f o r i n s u l i n r e lease by d i e t a r y carbohydrates. (2) U n t i l now t h i s has been the d i e t a r y regimen o f preference f o r hypoglycemics although i t d i d not r e s o l v e a l l o f the problems f o r these persons. A d i e t that r e l i e s p r i m a r i l y on p r o t e i n s f o r the energy and s u b s t r a t e r e q u i r e ments o f the b r a i n i s n o t only expensive and somewhat u n p a l a t a b l e but i t runs the r i s k o f gout and other problems of e x c r e t i o n o f the m e t a b o l i c end-products o f p r o t e i n u t i l i z a t i o n , (3) U n f o r t u n a t e l y , the b r a i n cannot u t i l i z e f a t s f o r a l l o f i t s requirements The a l t e r n a t i v e i s t o p r o v i d l i n r e l e a s e but y e t provide system. Glucose causes i n s u l i n r e l e a s e . I n s u l i n causes the s t o r age o f excess sugar as glycogen i n the l i v e r and so lowers the blood sugar c o n c e n t r a t i o n . Pure f r u c t o s e does not cause i n s u l i n r e l e a s e . (4) Although f r u c t o s e has long been used by d i a b e t i c s i n Europe i t s use has not been promoted i n the U n i t e d States where i n s u l i n therapy and d i e t a r y balance programs are predominant. (5, 6) Although an e x t e n s i v e l i t e r a t u r e on f r u c t o s e metabolism i n the human as w e l l as i n experimental animals has accumulated, most s t u d i e s have been conducted u s i n g c h a l l e n g e doses by d i e t a r y or i n j e c t i o n routes without regard f o r the abnormalacy o f these cond i t i o n s . (7, 8) U n f o r t u n a t e l y , s e v e r a l s t u d i e s have been r e p o r t ed as s t u d i e s o f f r u c t o s e which were a c t u a l l y s t u d i e s o f sucrose i n comparison t o glucose, l i q u i d glucose, and s t a r c h . (9, 10) Yet the d i f f e r e n c e s found as a r e s u l t o f such d i e t s were a t t r i b uted t o the f r u c t o s e component o f the sucrose w i t h o u t any attempt to study the e f f e c t s o f pure f r u c t o s e . Such conclusions seem w h o l l y u n j u s t i f i e d . S e v e r a l s t u d i e s have demonstrated t h a t i n s u l i n can promote the storage o f f r u c t o s e as glycogen. (11, 1 2 ) . I f the d i e t i n c l u d e s i n s u l i n - i n d u c i n g carbohydrates, the m e t a b o l i c advantages o f f r u c t o s e may be expected t o be d i m i n i s h e d . Glucose i s a c t i v e l y transported from the gut so that i t s conc e n t r a t i o n r i s e s r a p i d l y a f t e r d i e t a r y i n t a k e . Fructose i s pass i v e l y moved through the c e l l s by d i f f u s i o n . (13) There i s i n s u f f i c i e n t evidence t o support the c o n t e n t i o n that any s i g n i f i c a n t amount o f f r u c t o s e i s r e g u l a r l y converted t o glucose e i t h e r i n the gut or i n h e p a t i c c e l l s . (14) Since sugars are p r i m a r i l y absorbed by c e l l s i n the upper d i g e s t i v e t r a c t , the i n g e s t i o n o f l a r g e amounts o f f r u c t o s e a t any one time can l e a d , i n some persons, to increased water r e t e n t i o n i n the l a r g e bowel and the onset o f d i a r r h e a . (15) A simple p r e c a u t i o n against such an event i s t o provide the d i e t a r y f r u c t o s e i n smaller amounts t o decrease the

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

56

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

l i k e l i h o o d of f r u c t o s e passing i n t o the lower d i g e s t i v e t r a c t . I t i s w e l l known that excess carbohydrates, those which exceed the immediate demand f o r c e l l u l a r energy, are stored e i t h e r as glycogen or f a t . When f r u c t o s e i s provided i n a d i e t i n exchange f o r i n s u l i n - i n d u c i n g carbohydrates, i t would be expected that a l l excess f r u c t o s e must be converted to f a t . There are some s t u d i e s w i t h experimental animals which support t h i s general r e c o g n i t i o n . (16, 17) This has l e d to a concern that an exchange of f r u c t o s e f o r glucose would r e s u l t i n a r i s e i n plasma t r i g l y c e r ides and c h o l e s t e r o l . S t r e s s c o n d i t i o n s cause the p h y s i o l o g i c a l s t r e s s responses. Hypoglycemia i s a s t r e s s . Normally i t i s c o r r e c t e d by the changes which r e s u l t from the s t r e s s response. The pioneer work by Hans Selye on the General S t r e s s Response i s w e l l known and the i n t e r r e l a t i o n s h i p s to many d i s o r d e r s have been e s t a b l i s h e d . (18, 19) A l l s t r e s s c o n d i t i o n s , whether they come from the emotions, physi c a l trauma, t o x i c i t y , o by c e l l s of the nervou mus of the b r a i n . These c e l l s d i r e c t l y s t i m u l a t e sympathetic nerves to secrete n o r a d r e n a l i n . The hypothalamic c e l l s a l s o secrete r e l e a s e f a c t o r s i n t o the blood of the p o r t a l v e s s e l s which supply the p i t u i t a r y . When the p i t u i t a r y i s s t i m u l a t e d by these r e l e a s e f a c t o r s i t secretes ACTH ( a d r e n o c o r t i c o t r o p h i c hormone) to cause the adrenal c o r t e x to elaborate s p e c i f i c hormones. The s t e r o i d hormones of the adrenal cortex have s e v e r a l e f f e c t s . They cause the m o b i l i z a t i o n of both f a t and p r o t e i n s f o r c e l l u l a r use and cause the r e l e a s e of a d r e n a l i n from the adrenal medulla. I t i s t h i s a d r e n a l i n , i n a d d i t i o n to i t s other a c t i o n s , which i n i t i a t e s the changes r e q u i r e d to convert l i v e r glycogen to glucose which can be s u p p l i e d to the b r a i n . I t i s p r i m a r i l y t h i s p o r t i o n of the s t r e s s response that i s r e l a t e d to carbohydrate metabolism. Only r e c e n t l y has i t been p o s s i b l e to study i n d e t a i l the r e l a t i o n s h i p between s t r e s s and the sympathetic nervous system hormones. I t has only been i n the past few years that a l l of the metabolic products of the sympathetic hormones have been known. (20) I n j u s t over one decade, von E u l e r r e c e i v e d a Nobel P r i z e f o r h i s discovery of n o r a d r e n a l i n as the primary t r a n s m i t t e r of the p e r i p h e r a l sympathetic nerve c e l l s . A x e l r o d was awarded a Nobel P r i z e f o r h i s d i s c o v e r y of the enzyme COMT (catechol-0methyl t r a n s f e r a s e ) which c a t a l y z e s the m e t h y l a t i o n of the sympat h e t i c hormones and thus d e a c t i v a t e s these molecules. Sutherland was then the r e c i p i e n t of a Nobel P r i z e f o r h i s discovery of c y c l i c AMP ( 3 5 ' AMP) and i t s r e l a t i o n s h i p to the conversion of glycogen to glucose under the i n f l u e n c e of a d r e n a l i n . Many other i n v e s t i g a t o r s have provided other i n s i g h t s and methodologies f o r the a n a l y s i s of the metabolism of these hormones. (21, 22) A l l of t h i s i n f o r m a t i o n was necessary before other ideas about s t r e s s and the s t r e s s response could be t e s t e d . I t i s a p a r t of our general dogma that s t r e s s c o n t r i b u t e s to such v a r i e d d i s o r d e r s as coronary thrombosis, hypertension, p e p t i c !

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

4.

PALM

Fructose and

Human Stress

57

u l c e r s and g a l l stones i n a d d i t i o n to a v a r i e t y of nervous d i s orders. A s i m p l i s t i c , layman's d e s c r i p t i o n concludes that s c h i z ophrenia i s a d i s o r d e r o f o r i e n t a t i o n and behavior which develops when a person i s exposed to s t r e s s which exceeds h i s a b i l i t y to cope. The common depressants which are used to decrease the symptomatology of s c h i z o p h r e n i a suppress the a c t i v i t y of the symp a t h e t i c system. (23) Conversely, a d r e n a l i n and the drugs which mimic the sympathetic hormones are known to exacerbate the symptoms o f s c h i z o p h r e n i a . (24) Many p s y c h i a t r i s t s b e l i e v e that the o r i g i n s of s c h i z o p h r e n i a r e l a t e to an i n a p p r o p r i a t e p e r c e p t i o n of s t r e s s so that these persons over-respond to s t r e s s e s which would not d i s r u p t the l i v e s of most persons. (25) Yet these p r a c t i t i o n e r s a l s o recognize the p h y s i o l o g i c a l r e g u l a t i o n s and i m p l i c a t i o n s of the s t r e s s responses. The r e c o g n i t i o n of the involvement of s t r e s s i n the e t i o l o g y of s c h i z o p h r e n i a adds another o r i e n t a t i o n to the study o f s t r e s s r e l a t e d d i s o r d e r s . The t i o n o f b e h a v i o r a l change of an i n h e r i t a b l e component of s c h i z o p h r e n i a has l e d to a search f o r metabolic d e f e c t s i n the r e g u l a t i o n o f the hormones which are a s s o c i a t e d w i t h the sympathetic system. Many of the concepts about the general s t r e s s response can now be extended to behaviora l d i s o r d e r s and s t u d i e d as b i o c h e m i c a l problems. The complete pathways and products of the enzymatic d e a c t i v a t i o n of the catecholamine hormones, n o r a d r e n a l i n and a d r e n a l i n , a r e known. A f t e r the m o b i l i z a t i o n of these hormones o f the s t r e s s r e sponse the m e t a b o l i c products accumulate i n the u r i n e . The u r i n e thus serves as a r e p o s i t o r y of evidence of the s t r e s s response. I t i s apparent t h a t a d e f i c i e n c y i n any one of the enzymes i n v o l v ed i n the d e a c t i v a t i o n of the hormones would r e s u l t i n the absence of p a r t i c u l a r end-products i n the c o l l e c t e d u r i n e . (26) When e a r l y morning u r i n e samples from a group of i n s t i t u t i o n a l i z e d but non-medicated s c h i z o p h r e n i c s were t e s t e d by gas chromatographic s e p a r a t i o n of the components and flame i o n i z a t i o n d e t e c t i o n of each molecular v a r i e t y , a l l of the normal end-products of noradr e n a l i n and a d r e n a l i n were found i n each of the u r i n e s . (27) This demonstrated that s c h i z o p h r e n i c s as a group are not charact e r i s t i c a l l y d e f i c i e n t i n any of the enzymes which are normally i n v o l v e d i n the d e a c t i v a t i o n of the sympathetic system hormones. The data d i d p r o v i d e one enigma. The n i g h t - t i m e accumulation of these products i n the u r i n e appeared to be much g r e a t e r than had been found i n the samples obtained from a c o n t r o l p o p u l a t i o n . The magnitude of the accumulation of these catecholamine products provided the b a s i s f o r a hypothesis that these s c h i z o p h r e n i c p a t i e n t s were responding to s t r e s s c o n d i t i o n s even d u r i n g t h e i r p e r i o d s of sleep which probably i n v o l v e p h y s i o l o g i c a l s t r e s s f a c t o r s . I t was then d i s c o v e r e d that the records of many of these p a t i e n t s cont a i n e d i n f o r m a t i o n about glucose t o l e r a n c e t e s t s which had a l l been i n d i c a t i v e of hypoglycemia. The simultaneous f i n d i n g s of hypoglycemia and the exceedingly

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

58

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

h i g h values of the sympathetic system products appear i n i t i a l l y to be l o g i c a l l y i n c o n s i s t e n t . Normally a d r e n a l i n i n i t i a t e s the conv e r s i o n of glycogen to glucose. The presence of l a r g e amounts of catecholamine products i n the u r i n e i n d i c a t e s h i g h sympathetic system a c t i v i t y which would be expected to be a s s o c i a t e d w i t h an abundance of blood sugar. This d i s c o v e r y of the hypoglycemia ass o c i a t e d w i t h evidences of h y p e r a c t i v i t y of the sympathetic system l e d to another hypothesis. I t i s proposed that among schizophreni c s an i n i t i a t i n g s t r e s s c o n d i t i o n i s a d e f i c i e n c y of blood sugar which r e q u i r e s the m o b i l i z a t i o n of a d r e n a l i n which f o r some reason i s unable to e f f e c t i v e l y r a i s e the blood sugar l e v e l to the r e q u i r e d l e v e l and the s t r e s s response i s continued. The focus of t h i s hypothesis i s t h a t s i n c e a l l s t r e s s e s are a d d i t i v e the s t r e s s of hypoglycemia should be seen as a s u f f i c i e n t , i f not r e q u i r e d , s t r e s s to p r e c i p i t a t e s c h i z o p h r e n i c behavior. Some of the symptom f schizophrenic t b related d i r e c t l y to the d e f i c i e n c w h i l e other symptoms appea hig t r a t i o n of a c t i v e sympathetic system hormones. Such a hypothesis r e c o n c i l e s the hypoglycemia w i t h the presence of h i g h c a t e c h o l a mine products i n the u r i n e and suggests a b a s i s f o r the b i o l o g i c component of s c h i z o p h r e n i a . U n f o r t u n a t e l y the d i e t of most i n s t i t u t i o n a l i z e d schizophreni c s i s preponderantly carbohydrate and contains a p a u c i t y of prot e i n s . Such a d i e t i s d i s a s t e r o u s f o r a person w i t h tendencies to f u n c t i o n a l hypoglycemia. The depressive drugs which are commonly used to c o n t r o l the behavior of the s c h i z o p h r e n i c s i n t e r f e r e w i t h the normal a b i l i t i e s f o r r a i s i n g the blood sugar c o n c e n t r a t i o n . Therefore, a t e s t program among i n s t i t u t i o n a l i z e d s c h i z o p h r e n i c s to evaluate the e f f e c t i v e n e s s of a program to e x t e r n a l l y r e g u l a t e the blood sugar l e v e l by exchanging f r u c t o s e f o r the normal i n s u l i n - i n d u c i n g carbohydrates i s v i r t u a l l y i m p o s s i b l e . I t i s the b e h a v i o r a l c h a r a c t e r i s t i c s which d i s t i n g u i s h the s c h i z o p h r e n i c from the a l c o h o l i c , the h y p e r a c t i v e or hypomanic, and the crave-eater who becomes obese. These are a l s o s t r e s s induced d i s o r d e r s i n which the behavior o f t e n appears to accomodate or compensate f o r the s t r e s s f a c t o r s . The a l c o h o l i c has learned that a l c o h o l depresses the p e r c e p t i o n of s t r e s s even i f he does not recognize that a l c o h o l does not supply the b r a i n w i t h the sugar i t needs. The h y p e r a c t i v e f e e l s b e t t e r adjusted i f he can i n c r e a s e h i s a c t i v i t y and by t h i s means accomplish some i n c r e a s e i n blood sugar. He might j u s t take a b r i s k walk to get more adr e n a l i n and more sugar r a t h e r than a c t u a l l y be "burning o f f " energy. Too much blood sugar makes a person s l u g g i s h , not a c t i v e . The s t r e s s - e a t e r consumes candies, p a s t r i e s , and f r u i t s and thus keeps the blood sugar h i g h enough to t e m p o r a r i l y avoid the s t r e s s response. I n these persons the e f f e c t i v e n e s s of the i n s u l i n cont r o l of the sugar storage system p r e d i c a t e s a r e i n i t i a t i o n of the f u n c t i o n a l hypoglycemia and a r e t u r n of the s t r e s s . I f the same reasoning i s extended to s c h i z o p h r e n i a i t can be

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

4.

PALM

Fructose and Human Stress

59

suggested that t h i s behavior compensates f o r a d e f i c i e n c y o f blood sugar r e g u l a t i o n even i f i t i s o l a t e s the person from e f f e c tive social interactions. Since hypoglycemia i s a common c h a r a c t e r i s t i c o f a l l o f these d i s o r d e r s i n which behaviors may compensate f o r the d e f i c i e n c y i n r e g u l a t i o n o f the blood sugar l e v e l s , the i n v e s t i g a t i o n s o f the e f f e c t i v e n e s s o f f r u c t o s e i n p r o v i d i n g sugar f o r the b r a i n without p r e c i p i t a t i n g i n s u l i n r e l e a s e and the r e t u r n o f the hypoglycemia can be done on persons who have no known problems i n blood sugar r e g u l a t i o n . S e v e r a l questions remain concerning the a p p l i c a b i l i t y of f r u c t o s e exchange d i e t s . I f f r u c t o s e i s given i n s m a l l amounts i n h o u r l y i n t e r v a l s does an a p p r e c i a b l e amount get converted t o glucose which could i n i t i a t e the i n s u l i n release? I f f r u c t o s e i s exchanged f o r most o f the other carbohydrates i n a d i e t does i t promote the accumulation o f plasma t r i g l y c e r i d e s and plasma chol e s t e r o l ? I f f r u c t o s e i s used i n a d i e t a r y program t o prevent hypoglycemia i s there a demonstrabl the enzymatic products o i s l o g i c a l t o assume t h a t the magnitude o f the s t r e s s c o n d i t i o n w i l l be m i r r o r e d i n the magnitude o f the measured s t r e s s response? Three d i f f e r e n t i n v e s t i g a t i v e programs have been conducted t o answer these q u e s t i o n s . Experiments Blood a b s o r p t i o n curves o f sugars were determined from blood samples obtained from experimental s u b j e c t s every f i v e minutes a f t e r i n g e s t i o n o f the sugars. The sugar c o n c e n t r a t i o n s were measured u s i n g a Beckman Glucose A n a l y z e r . Determinations were made on three separate days f o r each o f the s u b j e c t s . One day 50 grams of sucrose syrup was i n g e s t e d a f t e r four consecutive samples showed the blood glucose c o n c e n t r a t i o n t o be n e a r l y s t a b l e a t 90 mg./ 100 ml. o f blood. Another day 25 grams o f glucose was i n g e s t e d . The t h i r d t e s t u t i l i z e d 25 grams o f f r u c t o s e . Least-squares f i t ted l i n e s were computed from the data obtained from the blood g l u cose c o n c e n t r a t i o n s . F i g u r e 1 i l l u s t r a t e s a t y p i c a l set o f g l u cose c o n c e n t r a t i o n curves from these experiments. Pure glucose entered the blood stream more r a p i d l y than glucose obtained from sucrose (slope = 1.54 + S.D. 0.0656 versus a slope o f 0.983 + S.D. 0.555) although the p r a c t i c a l i t y o f t h i s d i f f e r e n c e i s n e g l i g i b l e . Although i t i s necessary t o d i g e s t the sucrose t o glucose and f r u c t o s e before i t s a b s o r p t i o n , the delay imposed on the absorpt i o n o f glucose from sucrose i s e s s e n t i a l l y i n c o n s e q u e n t i a l . When the blood glucose determinations were made f o l l o w i n g the i n g e s t i o n of 25 grams o f f r u c t o s e , the slope o f the l i n e was not found t o be s i g n i f i c a n t l y d i f f e r e n t from the normal v a r i a t i o n o f the blood glucose as a f u n c t i o n of the s t r e s s response t o the hypoglycemia together w i t h the s t r e s s o f the sample c o l l e c t i o n . The only b i t of value o f t h i s data i s t o show t h a t f r u c t o s e i s n o t converted t o glucose i n any s i g n i f i c a n t amounts i n reasonable d i e t a r y exchange

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

60

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

programs. In order t o determine i f an exchange of 100 grams o f pure f r u c t o s e f o r most o f the other carbohydrates o f a normal d i e t would a f f e c t the c o n c e n t r a t i o n o f plasma t r i g l y c e r i d e s and c h o l e s t e r o l these substances were measured from plasma obtained from 17 h e a l t h y persons who were not f o l l o w i n g any d i e t a r y regimens. These persons were then asked t o continue t h e i r r e g u l a r e a t i n g h a b i t s but t o exchange 100 grams o f f r u c t o s e f o r most of the i n s u l i n - i n d u c i n g carbohydrates f o r a p e r i o d o f three days. The plasma c h o l e s t e r o l and plasma t r i g l y c e r i d e s were measured a t the end o f t h i s three day d i e t program. Table 1 and F i g u r e s 2, 3 i l l u s t r a t e the l e v e l s of these l i p i d substances. There i s no e v i dence o f c o n s i s t e n t change i n these substances as the r e s u l t o f the f r u c t o s e provided i n 8-10 grams each hour. A p r o j e c t to t e s t the e f f e c t i v e n e s s o f d i e t a r y f r u c t o s e t o r e g u l a t e the blood sugar c o n c e n t r a t i o n u t i l i z e d the u r i n a r y ac cumulation of catecholamin response i n i t i a t e d by hypoglycemia were c o l l e c t e d from 20 normal, h e a l t h y a d u l t s who f o l l o w e d the h i g h - p r o t e i n low-carbohydrate d i e t w i t h f r u c t o s e one day and w i t h other carbohydrates another day. I n i t i a l l y i t was intended t o u t i l i z e a h i g h pressure l i q u i d chromatographic automated system to determine a l l of the catecholamine m e t a b o l i t e s i n the u r i n e . T e c h n i c a l d i f f i c u l t i e s encountered i n s c r e e n i n g some contaminants which obscured the d e f i n i t i v e s e p a r a t i o n o f the catecholamine m e t a b o l i t e s prevents the i n c l u s i o n o f t h i s data a t t h i s time. The t o t a l catecholamines ( n o r a d r e n a l i n and a d r e n a l i n ) and the methylated i n t e r m e d i a t e s (normetadrenalin and metadrenalin) were determined u s i n g the Bio-Rad Catecholamine Test K i t s . F i g u r e 4 demonstrates the r e l a t i o n s h i p s of these compounds. VMA was d e t e r mined from the same samples a t two independent commercial medical l a b o r a t o r i e s . One o f the l a b o r a t o r i e s used the P i s a n o , Crout, and Abraham method (28) w h i l e the other used the method o f G i t l o w , et a l (29). Both the c o n t r o l and the d i e t t e s t day samples of u r i n e were determined by the same l a b o r a t o r y a t the same time. The u r i n e s were c o l l e c t e d i n g l a s s jugs c o n t a i n i n g 15 m l . cone. HCl. Care was taken to i n s u r e a l l o f the b o t t l e s were f i t ted w i t h t e f l o n cover i n s e r t s t o e l i m i n a t e contamination o f the samples by cork or paper products. A l l o f the catecholamine u r i n e samples were h y d r o l y z e d a t pH 0.5 f o r 20 minutes i n capped tubes p l a c e d i n b o i l i n g water. The Bio-Rad system o f a n a l y s i s was m o d i f i e d s l i g h t l y t o provide an i n t e r n a l standard f o r each sample and thus reduce the problems o f f l u o r e s c e n c e quenching and t o i n crease the r e l i a b i l i t y of d e t e r m i n a t i o n o f a l l o f the products. Catecholamines were e l u t e d from the d i s p o s a b l e columns w i t h 10 m l . of 4.0 N. b o r i c a c i d . The e l u a t e was d i v i d e d i n t o four equal port i o n s . P o r t i o n 1 remained as the c o n t r o l . P o r t i o n 2 was the unknown sample. P o r t i o n 3 was s p i k e d w i t h 1 ug o f n o r a d r e n a l i n standard. P o r t i o n 4 was s p i k e d w i t h 2 ug of n o r a d r e n a l i n standard. A l l p o r t i o n s were brought to the same volume w i t h g l a s s d i s t i l l e d

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

4.

61

Fructose and Human Stress

PALM

Figure 1.

Plasma glucose concentration following

0

80

160

0

ingestion of three different sugars

80

MG. TRIGLYCERIDES/100 ML. PLASMA Figure 2.

Distributions of plasma triglyceride levels

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

160

62

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

Table 1. Effect of Diet on Plasma Cholesterol and Plasma Triglycerides. Diet Program Exchanged 100 grams Fructose for 100 grams of Other Carbohydrates

SUBJECT CODE

CB LJ IJ MJ GJ JD KK OM JK IN DJ GB GK IM AC SK VO

CONTROL DIET CHOLESTEROL mg./100 m l . plasma 217 197 256 214 158 193 197 170 186 222 170 190 232 138 127 173 301 X= 196.529 S.E.= 10.3174 S.D.=42.5399

FRUCTOSE DIET CHOLESTEROL mg./100 m l . plasma

CONTROL DIET TRIGLYCERIDES mg./100 m l . plasma

FRUCTOSE DIET TRIGLYCERIDES mg./100 m l . plasma

181 205 243 216 183 214 222

137 91 114 61 53 68 151

178 129 61 57 38 78 106

246 187 235 232 163 148 170 363

46 76 50 79 35 27 61 84

59 41 55 70 67 43 73 103

X= 211.176 S.E.=11.7584 S.D.=48.481

X= 73.0588 S.E.=8.2629 S.D.=34.0688

X= 73.5294 S.E.=8.94432 S.D.=36.8784

F=.876673 (1,32 D.F.)

F=.150008E-2 (1,32 D.F.)

Probability=.99999

Probability=.99999

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

4.

63

Fructose and Human Stress

PALM

FRUCTOSE EXCHANGE DIET

CONTROL DIET X=196.529 S.D.=42.54

to -» ζ ο to

ANALYSIS OF VARIANCE OF TEST POPULATIONS

o£ 3 LU

Û-

F=.877 (1,32 D.F.) PROBABILITY= 0.999

LU β

X=211.118 S.D.=48.48

1

100

200

300 MG. CHOLESTEROL/100 ML. PLASMA

Figure 3.

Distributions of plasma cholesterol levels as a function of dietary program

DOPAMINE —> —> —>NORADRENALIN —> —> — » ADRENALIN

^ ^ ^ Dihydroxy Mandelio Aldehyde Dihydroxy Mandelio Aoid

Figure 4.

Dihydroxy Phenyl Glycol

NORMETADRENALIN METADRENALIN Si Τ" Ν */ Methoxy-Hydroxy Mandelio Aldehyde ^Methoxy-Hydroxy Phenyl Glycol

Pathways of metabolic conversions of catecholamine excretion products

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

64

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

water. The f l u o r e s c e n c e of P o r t i o n s 2,3, and 4 were measured i n comparison to t h a t of P o r t i o n 1 w i t h a Turner Fluorometer w i t h a h i g h s e n s i t i v i t y adapter. The three f l u o r e s c e n c e readings f o r each u r i n e sample were fed i n t o a computer to determine a l e a s t squares l i n e of the three d e t e r m i n a t i o n s , to p r i n t out the slope of the l i n e and the p o i n t of i n t e r c e p t as w e l l as determine the standard d e v i a t i o n of the i n t e r c e p t . The computer program then m u l t i p l i e d the determined values by the amount of u r i n e i n the i n i t i a l 24 hour sample to g i v e the amount of t o t a l catecholamines excreted i n the 24 hour p e r i o d . The O-methyl d e r i v a t i v e s , normetadrenalin and metadrenalin were e l u t e d from the columns w i t h 10 ml. 4.0. N. ammonium hydroxi d e a f t e r the columns had been washed w i t h d i s t i l l e d water. The 10 ml. e l u a t e was d i v i d e d i n t o 4 p o r t i o n s ; c o n t r o l , unknown, unknown + 3 ug of normetadrenalin standard, and unknown + 9 ug of normetadrenalin standard. The samples were d i l u t e d to equal v o l umes and read a t 360 nm absorbance a t 350 nm wa n a t i o n which may i n f l u e n c e the determinations a t 360 nm. The same computer program which was used f o r the computations of c a t e cholamine c o n c e n t r a t i o n s was a l s o used to determine the values of the methylated d e r i v a t i v e s of the catecholamines. I n a l l 20 cases the measured c o n c e n t r a t i o n of the t o t a l c a t e cholamines excreted was higjher on the day of the c o n t r o l d i e t than on the day of the f r u c t o s e exchange d i e t . The data obtained from the two t e s t s e t s (Table 2) shows t h a t the decrease i n c a t e c h o l a mine e x c r e t i o n on the day of the f r u c t o s e d i e t i s s i g n i f i c a n t l y d i f f e r e n t at the 99% confidence i n t e r v a l . The e x c r e t i o n of the O-methylated dérivâtes (metadrenalin) showed no s i g n i f i c a n t changes (Table 3). F i g u r e s 5, 6 i l l u s t r a t e the p o p u l a t i o n s h i f t as a f u n c t i o n of the d i e t a r y change. The VMA c o n c e n t r a t i o n s (Table 4) w h i l e not h i g h e r i n every case on the day of the c o n t r o l d i e t , d i f f e r s i g n i f i c a n t l y between the c o n t r o l and t e s t day to demonstrate a decrease i n VMA accumulation on the day of the f r u c tose exchange d i e t a t the 95% confidence i n t e r v a l . I n the cases where the VMA c o n c e n t r a t i o n s were g r e a t e r on the day of the f r u c tose d i e t these samples were obtained from female s u b j e c t s who were menstruating on the day t h a t t h i s sample was c o l l e c t e d . Conclusions and I m p l i c a t i o n s The exchange of f r u c t o s e f o r other d i e t a r y carbohydrates (100 grams exchanged f o r most of the i n s u l i n - i n d u c i n g carbohydrates of the c o n t r o l d i e t ) maintains a s u f f i c i e n c y of blood suga r to prevent the s t r e s s of hypoglycemia. This c o n c l u s i o n i s j u s t i f i e d by the s i g n i f i c a n t decrease i n the u r i n a r y accumulation of t o t a l catecholamines and VMA on the day of the f r u c t o s e d i e t . The i m p l i c a t i o n s of these f i n d i n g s have wide a p p l i c a t i o n and s i g n i f i c a n c e . General experience among the hundreds of persons who have r e g u l a r l y used f r u c t o s e i n exchange f o r glucose i n q u a s i -

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

4.

PALM

Table 2.

SUBJECT

RC YJ QL RJ RS QB QR FL AE UJ AW CB SB OB GB HB IB QG QV uc

65

Fructose and Human Stress Daily Excretion of Urinary Catecholamines

CONTROL FRUCTOSE DIET DIET VOLUME VOLUME ml/24 h. ml/24 h.

1700 1725 1610 1820 1280 1445 1900 2460 1230 2005 815 1420 1550 1610 1020 710 1800 1710 1910 850

CONTROL DIET CATECHOL. ug/24 h.

S.D.

FRUCTOSE DIET CATECHOL. ug/24 h.

S.D.

1800 1600 1400 2365 900 600

159.4 87.0 152.5 282.0 175.5 125.5

1.04 0.59 0.78 1.68 1.46 0.89

132.0 80.7 104.7 75.7 68.9 69.8

0.52 0.53 0.78 0.45 1.16 1.16

178 1360 1310 1550 1700 1810 1965 1230 690 1810 1030 2320 1080

86.4 103.9 210.6 116.9 236.6 94.2 65.3 70.5 136.4 195.4 217.8 100.8

0.62 0.59 1.34 0.57 1.55 0.67 1.14 1.27 0.38 1.20 0.68 0.64

73.4 72.0 75.1 40.9 158.9 74.9 61.5 45.3 105.7 96.9 125.3 73.4

0.42 0.82 0.33 0.47 0.55 0.54 0.60 0.76 0.33 1.61 0.27 1.17

X= 146.41 S.E.=13.26 S.D.=59.32

X= 87.4 S.E.=6.77 S.D.=30.2786

F=15.6986 (1,38 D.F.) Probability=0.00055

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

66

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

Table 3. Daily Excretion of Urinary Catecholamine Metholated Products

JBJECT

RC YJ QL RJ RS QB QR FL AE UJ AW CB SB OB GB HB IB QG QV UC

CONTROL DIET METADR. ug/24 h.

S.D.

1526.5 1351.0 2192.0 1571.2 1369.8 991.4 1012.0 2190.1 2098.8 1247.6 2005.8 1564.3 2722.9 1459.1 814.4 581.0 2367.0 1747.2 1648.9 1917.0

FRUCTOSE DIET METADR. ug/24 h.

S.D.

9.29 8.46 7.74 8.96 10.75 6.95 5.46

1731.5 1365.0 1491.5 2165.8 1847.6 973.0 945.1

5.09 9.07 10.87 9.65 20.80 17.58 6.61

6.27 12.87 5.60 17.61 9.38 8.34 8.45 7.18 10.80 4.64 12.06

1242.0 1894.8 1683.4 2456.0 1266.0 712.5 432.8 1627.4 1232.4 993.5 1502.9

9.75 6.16 5.04 6.87 6.49 6.10 6.68 4.63 12.18 2.41 14.36

X= 1618.9 S.E.=122.66 S.D.=548.537

X= 1456.7 S.E.=112.21 S.D.=501.809

F=0.951672 (1,38 D.F.) Probability=.99999

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

4.

Fructose and Human Stress

PALM

^0

80

120

160 200

ug T o t a l Figure 5.

67

Urinary

240

280

40

80

120

Catecholamînes/24 Hours

Frequency distributions of total urinary catecholamines excreted during day of dietary exchange program

1

3

5

7

9

11

0

2

4

6

8

10

MG. VMA EXCRETED/24 HOURS Figure 6.

Frequency distribution of VMA excretion during day of dietary exchange

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

68

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

Table 4. Daily Excretion of Urinary VMA

SUBJECT

RC YJ QL RJ RS QB QR FL AE UJ AW CB SB OB GB HB IB QG QV UC

CONTROL DIET VMA mg/ 24 h.

FRUCTOSE DIET VMA mg/ 24 h.

10.2 9.7 6.0 7.6 4.6 5.6 1. 9. 6.4 7.9 4.3 5.1 9.1 8.0 3.1 2.3 3.8 6.0 4.1 6.4

X= 6.118

7.3 9.1 5.5 9.6 4.1 2.9

4.4 6.2 3.8 3.6 5.7 4.6 1.8 1.3 3.3 4.3 2.6 7.6

X= 4.47

F=4.01786 (1,38 D.F.) Probability=0.0494

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

4.

PALM

Fructose and

Human Stress

69

c l i n i c a l t e s t programs d u r i n g the past two years has i n d i c a t e d that 100 grams of f r u c t o s e per day together w i t h the e l i m i n a t i o n of i n s u l i n - i n d u c i n g carbohydrates i n the d i e t completely e l i m i n ates the hunger, f a t i g u e and a n x i e t y that accompany most weight r e d u c t i o n d i e t s . The same amount of f r u c t o s e can completely e l i m i n a t e the i n s a t i a b l e demand f o r a l c o h o l among a l c o h o l i c s . Such an exchange of sugars can reduce or e l i m i n a t e migraine headaches f o r persons who are prone to such problems. The p r o v i s i o n of f r u c t o s e does not make angels out of a l l h y p e r a c t i v e c h i l d r e n but the general experience has shown them to be much more manageable. I t i s necessary to remember that f r u c t o s e i s not a drug. I t i s only a food t h a t i n i t s e l f i s d e f i c i e n t i n v i t a m i n s , amino a c i d s , and m i n e r a l s . Fructose does not change behavior. It allows a person to change the behavior i f the s t r e s s problem d i s o r d er i n v o l v e s the r e g u l a t i o n of blood sugar The l o g i c f o r the use of f r u c t o s e i n t h i s manne the known p r o p e r t i e s o t e n s i o n of the ideas i n t o c l i n i c a l a p p l i c a t i o n s and t e s t s w i l l be l e f t to the p h y s i c i a n s and p s y c h o l o g i s t s to determine the extent of the advantages of such a program. The immediate i n c l u s i o n of t h i s d i e t a r y program can be expected to be b e n e f i c i a l f o r persons w i t h c e r e b r a l hemorrhage or coronary damage which depress the r h y t h m i c i t y of adrenal c o r t i c a l s t e r o i d hormones and who t h e r e f o r e are kept i n p h y s i o l o g i c a l as w e l l as p s y c h o l o g i c a l depression and are unable to manage t h e i r blood glucose r e g u l a t i o n s u f f i c i e n t l y . F o r t u n a t e l y f r u c t o s e can be purchased i n many good food s t o r e s . The advantages of i t s use can be recognized by anyone who f o l l o w s the simple exchange of f r u c t o s e f o r the m a j o r i t y of the glucose of a good d i e t . To i n s u r e some glycogen i s kept a v a i l a b l e the i n c l u s i o n of glucose i n the form of t o a s t , c e r e a l , and f r u i t s f o r the f i r s t meal of the day i s a d v i s a b l e f o r most persons. I t i s apparent that the exchange of f r u c t o s e f o r other c a r bohydrates i s advantageous to many who have no recognized p a t h o l ogy. Fructose provides d i s t i n c t advantage to the a t h l e t e s i n c e i t w i l l not induce i n s u l i n r e l e a s e . I f other sugars are consumed before strenuous a c t i v i t y the presence of the i n s u l i n and the r e lease of a d r e n a l i n prevents e i t h e r of these hormones from accomp l i s h i n g t h e i r normal f u n c t i o n of blood sugar r e g u l a t i o n . Among those persons who d r i n k a l c o h o l i c beverages the p r o v i s i o n of f r u c tose w i l l prevent hypoglycemia as w e l l as i n o t h e r s , but i t has the added advantage of preventing the n e c e s s i t y of the s t r e s s r e sponse s t e r o i d r e l e a s e of f a t s i n t o the blood. When f a t s are r e leased they compete f o r the same enzymes as those used to metabol i z e the a l c o h o l . I f the f a t s and the a l c o h o l are not completely m e t a b o l i z e d they are converted to ketones which can cause nausea and headaches. Fructose has been used to prevent c o l i c i n s m a l l babies. The l i s t can go on and on. Although f r u c t o s e does not promote d e n t a l plaque as e f f e c -

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

70

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

t i v e l y as glucose the h o u r l y i n t a k e of f r u c t o s e i n candies, hot and c o l d beverages, s a l a d d r e s s i n g s , and j e l l o - l i k e deserts w i l l r e s u l t i n c a v i t i e s unless there i s an i n c r e a s e i n d e n t a l care. A s m a l l p o r t i o n of the p o p u l a t i o n i s d e f i c i e n t i n the enzymes necessary to u t i l i z e f r u c t o s e . This h e r e d i t a r y c o n d i t i o n of f r u c t o s e i n t o l e r a n c e prevents these persons from e l i m i n a t i n g hypoglycemia by exchanging f r u c t o s e f o r other carbohydrates. Such persons have a d i s l i k e f o r candies and sweetened foods although they can u s u a l l y e a t f r u i t s without d i s c o m f o r t . Acknowledgements The author wishes t o g i v e p a r t i c u l a r r e c o g n i t i o n to Mr. John B. Hawley, J r . , a Minneapolis i n d u s t r i a l i s t , f o r h i s f i n a n c i a l support o f t h i s r e s e a r c h . Thanks are a l s o extended t o my a s s i s t ant, Ms. M a r c i a D i e t z , f o r her v a l u a b l e help and to my c o l l e a g u e s Duane Olson and Stewart The help provided t o th Hanson and John Swanson i s a l s o a p p r e c i a t e d .

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

4.

PALM

Fructose

and

Human

Stress

71

Literature Cited

1. Hearn, W.R., E. J. Webel, R.W. Randolph, and N.E. Parks, Proc. Soc. Exp. Biol. (1961) 107, 515. 2. Conn, J.W. and H.S. Seltzer, Amer. J. Med., (1955) 19, 460. 3. Talbott, J.H., "Gout", Grune & Stratton, New York (1964). 4. Aitken, J.M. and M.G. Dunningan, Brit. Med. J. (1969) 3, 276. 5. Craig, J.W., W.R. Drucker, M.Owens et al., Proc. Soc. Exp. Biol. (1951) 78, 698. 6. Roch-Norlund, A.E., E. Hultman, and L.H. Nilsson, in "Symposi­ um on Clinical and Metabolic Aspects of Fructose", E.A. Nikkila and J.K. Huttunen eds., Acta Medica Scandinavica. Suppl. (1972) 542. 7. Woods, H.F., L.V. Eggleston and H.A. Krebs, Biochem J. (1970) 119. 8. Yu, D.T. and M.S. Phillips J. Ultrastructure Res (1971) 36 222. 9. Yudkin, J. Edelman an Houg (eds.) "Sugar", , London (1971). 10. Yudkin, J. and J. Roddy, Lancet (1964) 2, 6. 11. Weinstein, J.J. and J. H. Roe, J. Lab. Clin. Med. (1952) 40 39. 12. Pozza, G., G. Galarsino, H. Hoffeld, and P.D. Foa, Amer. J. Physiol. (1958) 192, 497. 13. Adelman, R.C., in "Symposium on Clinical and Metabolic Aspects of Fructose", E.A. Nikkila and J.K. Huttunen eds., Acta Medica Scandinavica, Suppl. (1972) 542. 14. Crane, R.K., Physiol. Rev. (1960) 40, 789. 15. Papadopoulos, N.M. and J.H. Roe, Amer. J. Physiol. (1957) 189 301. 16. Bar-On, Η., and Y. Stein, J. Nutr. (1968) 94, 95. 17. Nikkila, E.A., Scand. J. Clin. Lab. Invest., 18, Suppl. (1966) 92, 76. 18. Selye, H., "Stress of Life", McGraw Hill, New York (1956). 19. Selye, Η., "Hormones and Resistance", Springer-Verlag, Heidelberg, (1971). 20. Axelrod, J., Recent Prog. Horm. Res., (1965) 21, 597. 21. von Euler, U.S., and F. Lishajko, Acta Physiol. Scand. (1961) 51, 348. 22. Weil-Malherbe, H., and E.R.B. Smith, Pharmacol. Rev. (1966) 18, 331. 23. Cooper, J.R., F.E. Bloom and R.H. Roth, "The Biochemical Basis of Neuropharmacology", p. 80, Oxford Univ. Press, London (1970) 24. Blum, J.J., ed., "Biogenic Amines as Physiological Regulators", Prentice-Hall Inc., Englewood Cliffs, N.J. (1970). 25. Szara, J. Axelrod, and S. Perlin, Amer. J. Psychiat. (1958) 15, 162. 26. Palm, J.D., Schizophrenia (1969) 1, 161. 27. Palm, J.D., Schizophrenia (1970) 2, 198.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

72

PHYSIOLOGICAL EFFECTS OF FOOD CARBOHYDRATES

Literature Cited continued 28. Pisano, J.J., J.R. Crout, and D. Abraham, Clin. Chim. A c t a . (1962) 7, 285. 29. G i t l o w , S.E., L.M. B e r t a n i , A. Rausen, D. G r i b e t z , and S.W. D z i e d z i c , Cancer (1970) 25, 1377.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

5 The Metabolism of Infused Maltose and Other Sugars ELEANOR A. YOUNG and ELLIOT WESER Division of Gastroenterology, Department of Medicine, The University of Texas Health Science Center, San Antonio, Tex. 78284

Introduction

Provision of adequate nutrition when oral feeding or tubefeeding is difficult or impossible, can be achieved if essential nutrients and calories are introduced directly into the vein. For over three hundred years, man has searched for ways to provide life-dependent nutrients intravenously. As early as 1656 Sir Christopher Wren had already utilized a goose quill attached to a pig's bladder to introduce ale, wine and opium into dog veins (1). Richard Lower of Oxford, in 1662 reported intravenous injections into animals, and Jean B. Davis, a French physician in Paris, transfused lamb blood into man in 1667 (1). In 1843, almost two centuries later, Claude Bernard first infused sugars into animal veins (2), and in 1896, Biedl and Kraus reported the intravenous infusion of dextrose solutions in man (3). Perhaps one of the most exciting and challenging developments in modern medicine, has been the achievement of providing total parenteral nutrition to man. This has been possible largely as a result of advances in the knowledge of the nutritional require­ ments of man, availability of essential nutrients, and means of parenteral delivery over extended periods of time. Several re­ views (4,5,6) and national and international symposia (7,8,9,10, 11,12) attest to the distinguished achievements made within the past decade, largely as a result of numerous clinical trials in which calories and all essential nutrients...amino acids, carbo­ hydrates, fats, vitamins and minerals...have been effectively de­ livered intravenously to man. Numerous c l i n i c a l s i t u a t i o n s a r i s e i n which p a r e n t e r a l nut r i t i o n may not only be t h e r a p e u t i c , but e s s e n t i a l t o l i f e . Some of these are summarized i n Table 1. Notwithstanding the recent progress made i n p a r e n t e r a l a l i m e n t a t i o n , current problems focus on s u p e r i o r vena cava c a t h e r i z a t i o n v i a the s u b c l a v i a n v e i n ; i n f e c t i o n and s e p s i s i n v o l v i n g catheter e n t r y s i t e , contamination of the catheter and s o l u t i o n ; and hyperglycemic, hyperosmolar dehyd r a t i o n (13). 73 In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

74

PHYSIOLOGICAL

EFFECTS

OF FOOD

CARBOHYDRATES

TABLE I INDICATIONS FOR INTRAVENOUS FEEDING Malnutrition Chronic diarrhea Chronic vomiting G.I. obstruction Bowel resection Inflammatory bowel disease G.I. fistulas and anomalies Malignant disease Anorexia nervosa Coma Preoperative preparation

The Search For C a l o r i e Sources From a n u t r i t i o n a l p o i n t of view, the search f o r s u i t a b l e c a l o r i e sources has been of fundamental importance. As seen i n Table I I , a v a r i e t y of compounds have been e x p l o r e d . T A B L E II CALORIE SOURCES FOR INTRAVENOUS FEEDING Glucose Fructose Xylitol Sorbitol Ethanol Maltose Fat emulsions Amino Acids

I t i s the purpose o f t h i s paper to review b r i e f l y the carbohydrate c a l o r i e sources s t u d i e d t o date, and f i n a l l y t o summarize the p o s s i b i l i t i e s o f the d i s a c c h a r i d e , maltose, as a carbohydrate subs t r a t e i n intravenous f e e d i n g . Glucose. Since glucose i s the carbohydrate normally found i n the b l o o d , i t would seem t o be the obvious and i d e a l choice of c a l o r i e s . Glucose i s an economic and r e a d i l y a v a i l a b l e carbohyd r a t e , i s e f f i c i e n t l y u t i l i z e d p h y s i o l o g i c a l l y and a l s o has the h i g h e s t u t i l i z a t i o n r a t e i n normal man. Notwithstanding the advantages o f glucose as an intravenous source of c a l o r i e s , i t

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

5.

YOUNG

AND WESER

Infused Maltose

75

nevertheless has s e v e r a l s e r i o u s l i m i t a t i o n s . I f a 5% glucose s o l u t i o n i s used as the s o l e source of c a l o r i e s , 10 l i t e r s o f s o l u t i o n would be r e q u i r e d t o provide the c a l o r i c needs o f the average a d u l t male. I n f u s i o n o f hypertonic s o l u t i o n s o f glucose i n t o p e r i p h e r a l v e i n s may lead to complications such as thrombop h l e b i t i s , and h i g h u r i n a r y l o s s e s o f the sugar w i t h a consequent osmotic d i u r e s i s (14,15,16). Furthermore, glucose, being dependent on i n s u l i n f o r i t s u t i l i z a t i o n , may be c o n t r a i n d i c a t e d i n c o n d i t i o n s i n which i n s u l i n a v a i l a b i l i t y and/or a c t i v i t y may be diminished o r absent, such as s u r g i c a l s t r e s s a s s o c i a t e d w i t h e l e vated catecholamines and g l u c o c o r t i c o i d s (17,18,19), as w e l l as i n diabetes (20,21,22,23), hypothermia (17), p a n c r e a t i t i s (20,22), and sometimes i n uremia (20,24). The problems of s i g n i f i c a n t l y impaired glucose t o l e r a n c e i n p a t i e n t s w i t h l a t e n t o r overt diabetes m e l l i t u s , p a n c r e a t i t i s , s t r e s s , s e p s i s o r shock, can o f t e n be c o n t r o l l e d i f the p a r e n t e r a l a d m i n i s t r a t i o n of h y p e r t o n i c glucose solutions i s i n i t i a t e d a nous i n s u l i n response, o r 26). S t i l l another l i m i t a t i o n o f glucose as an intravenous source of c a l o r i e s i s the M a i l l a r d r e a c t i o n that takes place during s t e r i l i z a t i o n and storage of combined glucose-amino a c i d s o l u t i o n s , i n a c t i v a t i n g e s s e n t i a l amino a c i d s , e s p e c i a l l y l y s i n e (27). For these reasons, other carbohydrates that might overcome some o f the l i m i t a t i o n s of glucose, have been s t u d i e d . Fructose. Comparative s t u d i e s of glucose and f r u c t o s e suggest that f r u c t o s e may be more r a p i d l y metabolized than glucose (28,29, 30), i n f u s e d v e i n s may have a higher t o l e r a n c e f o r f r u c t o s e ( 3 1 ) , and the u r i n a r y e x c r e t i o n o f f r u c t o s e i s l e s s than that o f glucose (32,33,34). The i n i t i a l uptake of f r u c t o s e and i t s subsequent phosphorylation to fructose-l-phosphate c a t a l y z e d by f r u c t o k i n a s e i n the l i v e r and adipose t i s s u e i s independent o f i n s u l i n (16,20, 31,34). Some s t u d i e s have shown t h a t f r u c t o s e i n f u s i o n s t i m u l a t e s i n s u l i n r e l e a s e (36,37), w h i l e other s t u d i e s do not confirm t h i s (20). Nevertheless, some i n f u s e d f r u c t o s e i s converted t o glucose (38,39) and consequently r e q u i r e s i n s u l i n f o r i t s f u r t h e r metabol i s m (17,32). Thus, w h i l e o v e r a l l u t i l i z a t i o n o f glucose and f r u c t o s e are s i m i l a r i n normal man, f r u c t o s e may have c e r t a i n advantages over glucose, e s p e c i a l l y f o r p a t i e n t s i n the immediate p o s t o p e r a t i v e s t a t e when there i s a known i n s u l i n antagonism (20, 30,40,41), diabetes m e l l i t u s (22,23,29), c e r t a i n l i v e r diseases (20) and i n pancreatectomy (20,22). I n s p i t e o f the advantages of f r u c t o s e as a c a l o r i e source f o r intravenous i n f u s i o n , a number of s e r i o u s l i m i t a t i o n s have been r e p o r t e d , i n c l u d i n g an increase i n l a c t i c a c i d l e v e l s i n the l i v e r (42,43), increased blood l a c t a t e l e v e l s f o l l o w i n g r a p i d i n f u s i o n (44,47), d e p l e t i o n o f high-energyphosphate as w e l l as i n o r g a n i c phosphate i n the l i v e r (48,49), r e duction o f i n o r g a n i c phosphate i n the serum (23), and h y p e r u r i c e mia (44,50). The use of intravenous f r u c t o s e can thus have s e r i ous and profound metabolic e f f e c t s under c e r t a i n circumstances,

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

76

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

r a i s i n g s e r i o u s questions and c a u t i o n concerning i t s use as a c a l o r i e s u b s t r a t e i n p a r e n t e r a l a l i m e n t a t i o n (51,53). S o r b i t o l . S o r b i t o l , a hexahydric a l c o h o l with the same c a l o r i c value as glucose and f r u c t o s e (4.1 k c a l / g ) , i s not d i r e c t l y o x i d i z e d f o r energy production, but i s converted to f r u c t o s e v i a s o r b i t o l dehydrogenase, and metabolized as i s t h i s sugar (54). A second and q u a n t i t a t i v e l y l e s s important metabolic pathway i s the conversion of s o r b i t o l to glucose v i a aldose reductase (55). One advantage of s o r b i t o l i s that i t does not i n t e r a c t with amino acids i n the M a i l l a r d r e a c t i o n (56). S o r b i t o l i s not reabsorbed by the r e n a l t u b u l e s , and i f administered r a p i d l y , can provoke a d i u r e s i s (57,58). Thus, from a n u t r i t i o n a l p o i n t of view, i f the r a t e of u t i l i z a t i o n and t o l e r a n c e i s l e s s than that of glucose or f r u c t o s e , s o r b i t o l does not o f f e r any important advantage over these sugars, and shares the l i m i t a t i o n s of these two sugars as a c a l o r i e source i n intravenou Xylitol. The p e n t i o l , x y l i t o l , i s a n a t u r a l intermediate i n carbohydrate metabolism, and i s r a p i d l y o x i d i z e d to L - x y l u l o s e by s o r b i t o l dehydrogenase, and i s then shunted i n t o the pentose phosphate c y c l e . X y l i t o l s t i m u l a t e s i n s u l i n r e l e a s e only when i n f u s e d at high dosage (20,59), and shares with f r u c t o s e and s o r b i t o l , i t s p a r t i a l insulin-independence c h a r a c t e r i s t i c s (20,59), X y l i t o l can then be r e a d i l y converted to glucose (27). I n f u s i o n rates are l i m i t e d (36), and i n a d d i t i o n , x y l i t o l r a i s e s serum u r i c a c i d and b i l i r u b i n (60,62), and has adverse e f f e c t s with regard to a wide spectrum of metabolic a b n o r m a l i t i e s , i n c l u d i n g metabolic a c i d o s i s , r e n a l t u b u l a r e p i t h e l i a l - c e l l damage, i n t r a l u m i n a l dep o s i t s of calcium oxalate c r y s t a l s i n the r e n a l tubules, a l t e r e d c e r e b r a l f u n c t i o n and h e p a t o c e l l u l a r i n j u r y (60,62). Nevertheless, S p i t z et a l (59) have reported s u c c e s s f u l u t i l i z a t i o n of x y l i t o l i n healthy subjects and p a t i e n t s with r e n a l d i s e a s e , and suggest the use of t h i s c a l o r i e source i n uremia and i n other c o n d i t i o n s c h a r a c t e r i z e d by carbohydrate i n t o l e r a n c e and i n s u l i n r e s i s t a n c e . Ethanol. Ethanol contains 7.1 k c a l / g , and thus p o t e n t i a l l y i s a higher c a l o r i e source than other intravenous compounds with the exception of l i p i d s . A l c o h o l i s o x i d i z e d by a l c o h o l dehydrogenase to acetaldehyde, which i s then converted to a c e t y l CoA and shunted i n t o the t r i c a r b o x y l i c a c i d c y c l e f o r complete o x i d a t i o n . I t i s thought that i n s u l i n i s not e s s e n t i a l f o r o x i d a t i o n of a l c o h o l (63). Ethanol has some v a s o d i l a t o r a c t i o n at the s i t e of f l u i d entry and may thus reduce the i n c i d e n c e of thrombophlebitis when i t i s used i n hyperosmotic carbohydrate s o l u t i o n s (64). In a d d i t i o n ethanol provides c a l o r i e s without an accompanying osmotic l o a d , and has minimal u r i n a r y e x c r e t i o n r a t e s (64). However, the c o n c e n t r a t i o n of ethanol i n the body f l u i d s must be kept w i t h i n t o l e r a b l e l i m i t s to avoid pronounced pharmacological e f f e c t s (64). D i r e c t t o x i c e f f e c t s of ethanol on the l i v e r (65) makes the use

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

5.

YOUNG

AND

of ethanol

WESER

Infused Maltose

77

questionable.

Combination of C a l o r i e Sources. Because no s i n g l e c a l o r i e source seems to be i d e a l , s e v e r a l i n v e s t i g a t o r s are c u r r e n t l y searching f o r appropriate combinations of carbohydrate and p o l y a l c o h o l that can be used i n t r a v e n o u s l y . I t i s p o s t u l a t e d that c a p i t a l i z i n g on the most d e s i r a b l e c h a r a c t e r i s t i c s of each i n d i v i d u a l source, some r a t i o of combined sources may prove to be a more s u i t a b l e means of p r o v i d i n g energy s u b s t r a t e (66). I t i s a l s o of importance to note that d i f f e r e n t carbohydrates have varying i n s u l i n o g e n i c p o t e n t i a l . The s t i m u l a t i o n of i n s u l i n i s a maj o r key to anabolism and may thus i n f l u e n c e the choice of carbohydrate s u b s t r a t e i n p a r e n t e r a l feeding. U t i l i z a t i o n of Intravenous Disaccharides as a Source of C a l o r i e s After oral ingestio s m a l l amounts of thes under normal circumstances c i r c u l a t i n g l e v e l s as w e l l as u r i n a r y e x c r e t i o n of these sugars i s minimal. For these reasons, the i n f u s i o n of the double sugars has r e c e i v e d l i m i t e d c o n s i d e r a t i o n as a p o s s i b l e source of c a l o r i e s i n intravenous n u t r i t i o n . W i t h i n the past decade a v a r i e t y of animal and human s t u d i e s suggest that i n fused maltose, u n l i k e the other d i s a c c h a r i d e s , may be s u i t a b l e as a carbohydrate s u b s t r a t e i n intravenous n u t r i t i o n . Animal S t u d i e s . When r a d i o l a b e l e d l a c t o s e or sucrose are adm i n i s t e r e d i n t r a v e n o u s l y i n the r a t , o n l y s m a l l amounts of isotope appear i n the e x p i r e d CO2 over a 24-hr p e r i o d , w h i l e 62-68% i s exc r e t e d i n the u r i n e (Table I I I ) (67). This confirms previous s t u d i e s which showed that when these two d i s a c c h a r i d e s are admini s t e r e d p a r e n t e r a l l y i n the r a t , they are r a p i d l y and almost quant i t a t i v e l y excreted i n the u r i n e (68,69). In c o n t r a s t , 55-59% of i n t r a v e n o u s l y administered l a b e l e d maltose appears as 14c02> w i t h minimal u r i n a r y e x c r e t i o n of l ^ C . The recovery of l a b e l e d from maltose was comparable to that from i n j e c t i o n of l a b e l e d g l u cose or other monosaccharide mixtures (67). The extensive metabolism of i n j e c t e d maltose to ^C02 suggested a p o s s i b l e r e c i r c u l a t i o n through the i n t e s t i n a l mucosa w i t h subsequent o x i d a t i o n of maltose by i n t e s t i n a l maltases, o r , the p o s s i b i l i t y t h a t t i s s u e s other than s m a l l bowel mucosa might possess maltase a c t i v i t y . As seen i n Table IV, the h y d r o l y s i s and subsequent metabolism of i n t r a v e n o u s l y administered maltose was not s i g n i f i c a n t l y i n f l u e n c e d by s e l e c t i v e removal of the s m a l l bowel, kidneys, or 70% of the l i v e r (67). A n a l y s i s of maltase act i v i t y i n s e l e c t e d r a t organs (Table V) i n d i c a t e s the presence of maltase i n a v a r i e t y of t i s s u e s (67). Other estimates of r a t t i s sue maltase a c t i v i t y are comparable (70,71,72). I t t h e r e f o r e seems u n l i k e l y that c i r c u l a t i o n of i n j e c t e d maltose to s m a l l bowel mucosa p l a y s a s i g n i f i c a n t r o l e i n i t s o v e r - a l l metabolism, or

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

78

PHYSIOLOGICAL

TABLE 1

EFFECTS

4

No. animals

Glucose-1- C Glucose-U- C Glucose-1- C + galactose** Glucose-U- C + fructose-U- Ct Maltose-1- C Maltose-U- C Lactose-1- C l4

14

CARBOHYDRATES

III

Metabolism of C-labeled disaccharides iv administration in the rat*

Sugar

O F FOOD

1 4

C0

after

Urine

2

1

4

C

% dose/24 hours 5.3 ± 4.7 14.8 ± 10.3

5 5

62.0 ± 11.6 64.0 ± 12.0

4

52.0 ± 9.7

5 5 5 6 5

50.7 54.6 58.6 6.2 7.6

l4

9.8 ± 6.6

14

14

l4

14

14

Sucrose-U- C 14

± ± ± ± ±

19.3 4.8 3.2 62.1 68.4

7.9 7.0 5.8 2.7 2.4

± ± ± ± ±

4.6 3.9 3.0 13.5 10.8

* Animals received 5 mg of suger in 0.5 ml (1 μο per ml). * * M i x t u r e contained 2.5 mg of each sugar and 0.5 μο g l u c o s e - 1 - C . tMixture contained 2.5 mg and 0.25 μο of each sugar. 14

Journal of Clinical Investigation (67)

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

5.

YOUNG

79

Infused Maltose

AND WESER

T A B L E IV Oxidation

of

1

4

l 4

C-labeled sugars t o C 0 after iv injection in partially eviscerated rats 2

1 4

Organ removed

l4

l4

48.3 46.2 50.1 45.0

2

Glucose-1- C

Maltose-1- C

Sham Kidneys Liver (70%) Small bowel

C0

% dose/24 hours 55.3 ± 19.5 (3) 45.5 ± 17.6 (3) 43.9 ± 4.7 (3)

± 7.7(4)* ± 11.3 (4) ± 9.2 (5) ±

Lactose-1- C 14

2.9 ± 0.9 (3) 16.9 ± 5 . 9 (3) 2.4 (1)

* Number of rats is given in parenthesis. Journal of Clinical Investigation (67)

TABLE V Maltase activity in homogenates of rat organs

Maltase Organ

Intestinal mucosa Kidney Brain Liver Pancreas Spleen Muscle Serum Human serum

1

2

3

U*

U 390 61

205 73

485 17 14 2

U

1.6 4

1 0.1 9.1 0.3

0.1 0.3 12.5 0.1

4.0 1.7 5.6 0.2 0.3 8.9 0.2

*One U equals 1 μιτιοΐβ maltose hydrolyzed per minute per g protein. Journal of Clinical Investigation (67)

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

80

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

that a s i n g l e t i s s u e maltase was r e s p o n s i b l e f o r maltose o x i d a ­ t i o n . I t i s more probable that c i r c u l a t i n g maltose, u n l i k e l a c ­ tose o r sucrose, may be hydrolyzed by e x t r a i n t e s t i n a l maltases i n s e v e r a l t i s s u e s and subsequently metabolized. To explore whether m a l t o s y l o l i g o s a c c h a r i d e s were a l s o metab­ o l i z e d i n v i v o , the o x i d a t i o n of a t r a c e r dose of u n i f o r m l y l a ­ beled - ^ C - m a l t o t r i o s e to -^CC^ a f t e r intravenous i n j e c t i o n i n the r a t was measured (73). As seen i n F i g u r e 1, t r a c e r doses of u n i ­ formly l a b e l e d ^ C - m a l t o t r i o s e , as w e l l as u n i f o r m l y l a b e l e d 1*0maltose may be o x i d i z e d to xC>2 a f t e r intravenous i n j e c t i o n i n the r a t as e f f i c i e n t l y as U - C - g l u c o s e , w i t h 64.2 + 4.2%, 65.5 + 8.3% and 60.5 + 4.8% of the i n f u s e d dose recovered as 1*0)2, r e s p e c t i v e l y (73). When i n s u l i n i s s i m u l t a n e o u s l y administered w i t h g l u c o s e 1-14 maltose-l--^C to r a t s , the percentage of i n j e c t e d -^C ex­ p i r e d as ^CC>2 was the same as the recovery of 14C02 from adminis­ t r a t i o n of the sugar withou j e c t i o n of glucose-l-l^C. the e x p i r e d CO2 over a 6-hr p e r i o d . When i n s u l i n was added to the i n j e c t i o n s o l u t i o n , 47.5 + 6.6% of the l a b e l e d glucose was e x p i r e d as The f r a c t i o n of i n j e c t e d l^C recovered as 14(χ>2 f o l l o w ­ i n g m a l t o s e - l - l ^ C i n j e c t i o n , w i t h and without i n s u l i n was a l s o the same, these values being 59.7 + 4.9 and 59.7 + 5.0%, r e s p e c t i v e l y . L i k e w i s e , when the two sugars are compared, there was no s i g n i f i ­ cant d i f f e r e n c e i n the amount of glucose or maltose o x i d i z e d to CO2 w i t h or without i n s u l i n . As seen i n F i g u r e s 2 and 3, i n s u l i n d i d cause a more r a p i d o x i d a t i o n of both sugars to 1^C02« S p e c i f i c a c t i v i t y curves showed s i g n i f i c a n t l y e a r l i e r peaks when i n s u l i n was given w i t h e i t h e r glucose or maltose, than when these sugars were administered alone. When the peak e x c r e t i o n curves a f t e r maltose and glucose a d m i n i s t r a t i o n are compared, i t i s c o n s i s t e n t ­ l y observed t h a t the peak o x i d a t i o n time a f t e r maltose i s delayed, suggesting a "precursor-product" r e l a t i o n s h i p which r e q u i r e s time f o r maltose to be hydrolyzed to glucose. I n s u l i n a l s o enhances the i n c o r p o r a t i o n of i n t r a v e n o u s l y i n ­ j e c t e d glucose and maltose i n t o r a t epididymal l i p i d s (Figure 4 ) . The s p e c i f i c a c t i v i t y of e x t r a c t e d l i p i d s of r a t epididymal l i p i d s f o l l o w i n g intravenous i n f u s i o n of glucose p l u s i n s u l i n was 37% g r e a t e r than when glucose was i n f u s e d alone. Of p a r t i c u l a r i n t e r ­ e s t i s the s i m i l a r response noted when i n s u l i n was i n j e c t e d w i t h maltose, r e p r e s e n t i n g a 29% i n c r e a s e over i n c o r p o r a t i o n observed when maltose was given alone. Rat epididymal t i s s u e was e q u a l l y i n s u l i n - s e n s i t i v e when e i t h e r glucose or maltose was the sugar donating i t s l a b e l e d carbon to the s y n t h e s i s of l i p i d s (74). These s t u d i e s i n d i c a t e t h a t glucose and maltose respond s i m i l a r l y to i n s u l i n stimulation. The o x i d a t i o n of i n t r a v e n o u s l y administered t r e h a l o s e has a l s o been s t u d i e d (75). This double sugar was s e l e c t e d f o r study because i t c l o s e l y resembles maltose, c o n s i s t i n g of two glucose molecules j o i n e d i n a n / - 1 , 1 l i n k a g e . Figure 5 p l o t s the 14

o r

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

YOUNG

AND WESER

Infused Maltose

ro

ο X

Minutes Biochimica et Biophysica Acta

Figure 1. Oxidation of uniformly labeled [ C] maltotriose, [ C] malt­ ose, and [ C] glucose to C0 after intravenous injection in the rat. Each point represents the mean value of five animate (73). 14

14

14

14

2

Time (Hours) Endocrinology 14

Figure 2. Effect of insulin on the oxidation of circulating maltose-l- C to C0 . Specific activity curve following maltose (50 mg) iv (O); maltose (50 mg) plus insulin (0.2 U)iv(m) (74).

14

2

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

Time (Hours) Endocrinology 14

14

Figure 3. Effect of insulin on the oxidation of circulating glucose-l- C to C0 L Specific activity curve following glucose (50 mg) iv (O); glucose (50 mg) plus insulin(0.2)iv{%) (74). 2

GLUCOSE

GLUCOSE INSULIN

MALTOSE

MALTOSE INSULIN Endocrinology

Figure 4. Effect of insulin on the incorporation of r e labeled glucose, glucose plus insulin, maltose and maltose plus insulin into rat epididymal tissue. Bars represent the means and S.D. for 6, 7, 6 and 6 animals, respectively (74).

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

5.

YOUNG

AND WESER

Infused Maltose

83

o x i d a t i o n o f u n i f o r m l y l a b e l e d glucose, maltose, sucrose and t r e halose f o l l o w i n g intravenous a d m i n i s t r a t i o n i n the r a t . A f t e r the i n j e c t i o n o f trehalose-U-l^C and sucrose-U-l^C only 5 . 2 6 + 0 . 8 8 % and 8.11 + 1.25% o f the i s o t o p e appeared i n the expired C02, r e s p e c t i v e l y . I n c o n t r a s t , a f t e r maltose o r glucose i n j e c t i o n , 59.60 + 2.30% and 49.30 + 3.50% r e s p e c t i v e l y , was excreted as 1*C02. Only 3 t o 5% o f the i n f u s e d maltose and glucose was excreted i n the u r i n e , w h i l e 40 to 47% o f the t r e h a l o s e and sucrose was excreted (75). The r e s u l t s o f t h i s study i n d i c a t e that w h i l e t r e halose and maltose both c o n s i s t o f two glucose molecules, t h e i r metabolic f a t e i s q u i t e d i f f e r e n t . U n l i k e maltose, t r e h a l o s e i s m i n i m a l l y o x i d i z e d t o CO2 and l a r g e l y excreted i n the u r i n e . I t has been suggested that the absence o f t r e h a l a s e and sucrase i n r a t serum and kidney (71,72,76,77) probably accounts f o r the m i n i mal o x i d a t i o n o f these sugars. I n c o n t r a s t , maltase i s r e l a t i v e l y high i n r a t kidney (67,77,78) and serum (67,77) which would exp l a i n the d i f f e r e n c e i given p a r e n t e r a l l y . I a c t i v i t y i n mammalian kidney may p l a y a r o l e i n t u b u l a r reabsorpt i o n o f t r e h a l o s e and maltose, as w e l l as glucose (79). Rat k i d ney s l i c e s have been shown to o x i d i z e l^C-maltose and l^C-maltot r i o s e t o ^C02 more than other t i s s u e s , w i t h the exception of s m a l l bowel mucosa (78). A f t e r the intravenous a d m i n i s t r a t i o n o f u n i f o r m l y l a b e l e d t r e h a l o s e and sucrose, the kidney t i s s u e accumulates the h i g h e s t cpm/g t i s s u e when compared to recovery o f the l a b e l i n other t i s s u e s (75). These s t u d i e s lend f u r t h e r support to the suggestion t h a t the presence of the d i s a c c h a r i d a s e i n the r e n a l t u b u l a r t i s s u e may be a major determinant o f the e f f i c i e n c y of d i s a c c h a r i d e metabolism subsequent t o intravenous i n f u s i o n . I n an animal such as the r a b b i t w i t h h i g h t r e h a l a s e a c t i v i t y i n the kidney (72,78), 64% o f i n t r a v e n o u s l y administered trehalose-U-l^C was o x i d i z e d t o ^*C02> w h i l e l e s s than 2% was excreted i n the u r i n e (75). The recovery o f 1*C as ^C02 a f t e r i n f u s i o n o f t r e halose i n the r a b b i t i s seen i n Figure 6. The e f f i c i e n t o x i d a t i o n o f i n t r a v e n o u s l y administered maltose poses a question concerning the entry o f t h i s sugar i n t o the c e l l p r i o r to i t s metabolism. A comparative study of the uptake of maltose and other sugars i n t o r a t diaphragm c e l l s i n d i c a t e d that at e q u i l i b r i u m a l l sugars entered c e l l s by d i f f u s i o n (80). A t a l l equimolar c o n c e n t r a t i o n s , d i s a c c h a r i d e t r a n s p o r t i n t o i n t r a c e l l u l a r water was e q u a l , but o n l y 50% that o f monosaccharides. When i n t r a c e l l u l a r sugar was c a l c u l a t e d on a weight b a s i s (mg/ml) t r a n s p o r t of a l l d i s a c c h a r i d e s and monosaccharides was equal (80). In c e l l s that possess i n t r a c e l l u l a r d i s a c c h a r i d a s e a c t i v i t y , h y d r o l y s i s o f d i s a c c h a r i d e s and monosaccharides would account f o r t h e i r subsequent metabolism. The slow 24-hr i n f u s i o n s o f n u t r i e n t s o l u t i o n c o n t a i n i n g 25% maltose hydrate, amino a c i d s , v i t a m i n s and e l e c t r o l y t e s i n r a t s over a 14 day p e r i o d r e s u l t e d i n severe weight l o s s e s and up t o 52% u r i n a r y e x c r e t i o n of the i n f u s e d maltose. S i m i l a r i n f u s i o n s

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

14

14

2

14

14

14

Comparative Biochemistry and Physiology

Figure 5. Oxidation of intravenously administered glucose-U- C, maltose-U- C, sucrose-U- C, and trehalose-U- C to C0 in rats over a 6-hour period. Each point represents the mean value for 6, 6, 4 and 7 animals respectively (75).

HOURS

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

Figure 6. 2

Comparative Biochemistry and Physiology

Oxidation of circulating trehalose to C0 in the rabbit. Points represent mean ± S.D. for one animal (75).

14

HOURS

86

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

of glucose s o l u t i o n s r e s u l t e d i n weight maintenance and l i t t l e o r no u r i n a r y e x c r e t i o n (81). While t h i s study concludes that l o n g term p a r e n t e r a l maltose cannot serve as a t o t a l c a l o r i c s u b s t i t u t e f o r glucose i n complete p a r e n t e r a l n u t r i t i o n , r e s u l t s are l i m i t e d to observations i n only two animals. A d d i t i o n a l long-term parent e r a l s t u d i e s are needed. Human S t u d i e s . A f t e r l a c t o s e o r sucrose i n f u s i o n i n man, these d i s a c c h a r i d e s are l a r g e l y excreted i n the u r i n e (67,82). As seen i n Table V I , only s m a l l q u a n t i t i e s o f i n f u s e d maltose appear i n the u r i n e , suggesting that t h i s d i s a c c h a r i d e i s metabolized (67). O x i d a t i o n curves o f i n t r a v e n o u s l y administered maltose to h e a l t h y , normal s u b j e c t s are seen i n F i g u r e 7 (83). When 10 g maltose c o n t a i n i n g 5 u C i maltose-U-l^C given i n t r a v e n o u s l y , t h i s d i s a c c h a r i d e was r e a d i l y metabolized to CO2 w i t h 60% of the administered r a d i o a c t i v i t hr p e r i o d . Less than 8 i n the u r i n e e i t h e r as maltose or as glucose (83). The metabolic response o f intravenous i n f u s i o n of maltose and glucose t o normal s u b j e c t s i s compared i n F i g u r e 8. Blood glucose concentrations d i d not i n c r e a s e s i g n i f i c a n t l y a f t e r maltose i n f u s i o n , although a s i g n i f i c a n t r i s e i n t o t a l reducing substances was noted, i n d i c a t i n g the presence of t h i s d i s a c c h a r i d e i n the blood. This suggests t h a t e x t r a c e l l u l a r h y d r o l y s i s of maltose to glucose i s minimal. Since human serum c o n t a i n s almost no maltase a c t i v i t y (67,72), i t i s probable t h a t maltose enters t i s s u e c e l l s i n t a c t and i s subsequently metabolized. I n i t i a l l y , there was a f o u r f o l d i n c r e a s e i n serum i n s u l i n c o n c e n t r a t i o n a f t e r glucose and a t h r e e f o l d i n c r e a s e a f t e r maltose i n f u s i o n . T h e r e a f t e r , serum i n s u l i n c o n c e n t r a t i o n s g r a d u a l l y d e c l i n e d i n a s i m i l a r manner f o r both sugars. Data from t h i s study (83) demonstrates t h a t maltose i s r e a d i l y a v a i l a b l e as a metabolic s u b s t r a t e , and may provide the r e q u i r e d m e t a b o l i t e ( s ) necessary t o i n i t i a t e i n s u l i n s e c r e t i o n . The plasma f r e e f a t t y a c i d s a t 15 min decreased 371 u E q / l i t e r a f t e r glucose and 338 u E q / l i t e r a f t e r maltose i n f u s i o n . The r e s u l t s of t h i s study i n d i c a t e t h a t the u t i l i z a t i o n o f i n f u s e d maltose e l i c i t s s i m i l a r metabolic e f f e c t s as glucose i n the normal subject. Table V I I shows the s p e c i f i c a c t i v i t y (counts/minute per m i l l i g r a m ) of serum glucose and maltose a f t e r intravenous admini s t r a t i o n of 55 u C i maltose-U-^C i one s u b j e c t . Although there was no change i n serum glucose c o n c e n t r a t i o n , the s p e c i f i c a c t i v i t o f glucose s l o w l y i n c r e a s e d d u r i n g the 60 min p e r i o d , l i k e l y r e p r e s e n t i n g r e e n t r y o f l a b e l e d glucose from t i s s u e sources. On the other hand, the s p e c i f i c a c t i v i t y o f the i n j e c t e d maltose r e mained r e l a t i v e l y constant (83). The metabolism of maltose and glucose a f t e r intravenous i n j e c t i o n was compared i n normal and m i l d l y d i a b e t i c s u b j e c t s (84). The recovery o f as ^ 0 0 2 i normal s u b j e c t s was s i m i l a r a f t e r w

a

s

n

n

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

YOUNG

A N D WESER

Infused Maltose

T A B L E VI Disaccharide recovered in 24-hour urine sample, after iv administration of 10 g in adult humans

Disaccharide infused Subject

Lactose

Sucrose

Maltose

g

9

9

J.S. I.R. B.B. B.G.

10.5

7.2

0.09

4.8

0.12

6.8

0.15

7.1

0.08

8.6

Mean±SD

8.

Journal of Clinical Investigation ( 6 7 )

T A B L E VII Specific Activity of Serum Glucose and Maltose after Intravenous Administration of 10g Maltose-U- C* 14

Specific activity

Minutes

Serum glucose

Estimated maltose

mg/100 ml

mg/100 ml

Maltose

Glucose

cpm/mg

0

87

0

0

0

15

92

61

209

5773

30

90

50

487

6153

45

90

34

878

5933

60

94

31

1000

6975

* 5 5 MCI Maltose-U- C. 14

Journal of Clinical Investigation ( 8 3 )

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

PHYSIOLOGICAL

EFFECTS

OF FOOD

CARBOHYDRATES

180'

MINUTES Journal of Clinical Investigation 14

Figure 7. Fraction of injected C recovered as expired C0 per millimole C0 over a 6-hr period after the intravenous administration of 10 g C-hbeled maltose. Points are mean values for five subjects (83). 14

2

2

14

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

5.

YOUNG

89

Infused Maltose

A N D WESER

300 GLUCOSE INFUSION

250-

MALTOSE INFUSION 255200175·

GLUCOSE mg/100 ml

150 I25-| 100' 75

255 H 200-| TOTAL REDUCING |75SUBSTANCES mq/IOOml | 5 Q

125· 100-I

75-1 0

50-1 40-| SERUM INSULIN pU/ml

3020· I0H

900 700

PLASMA FREE * FATTY ACIDS μ E./liter 4

~i—ι—ι 0

20

40

1

60

1

80

1

100

Γ 120 140

Figure 8. Blood glucose, total reduc­ ing substances, insulin and free fatty acids for six subjects following the in­ travenous administration of 25 g malt­ ose or glucose. Points represent the means ± S.E.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

90

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

the a d m i n i s t r a t i o n of l a b e l e d glucose and maltose, with 33.3 + 1.6%, 37.7 + 3.4% and 3 6 . 5 + 1 . 9 % recovered a f t e r i n f u s i o n of maltose-U-l^C., glucose-U-l^C and glucose-l-^C., r e s p e c t i v e l y (Table V I I I ) . D i a b e t i c subjects excreted 25.4 + 1.3% of administered maltose-U- C as C 0 , while a s i m i l a r amount, 28.3 + 0.7% was excreted a f t e r g l u c o s e - l - ^ C i n f u s i o n . Both normal and d i a b e t i c subjects showed a delayed peak e x c r e t i o n of approximately 100 min a f t e r maltose i n f u s i o n as compared to glucose i n f u s i o n . The l^co2 e x c r e t i o n curves f o r the d i a b e t i c subjects are shown i n Figure 9. Our s t u d i e s i n d i c a t e that d i a b e t i c subjects have a decreased c a p a c i t y to shunt a loading dose of both glucose and maltose i n t o C02 pathways. The u r i n a r y e x c r e t i o n of was somewhat greater a f t e r maltose i n f u s i o n than a f t e r glucose i n f u s i o n but t h i s d i f f e r e n c e was not s i g n i f i c a n t except f o r lower u r i n a r y exc r e t i o n a f t e r g l u c o s e - l - l ^ C . The r e n a l threshold and clearance r a t e s f o r maltose i n man have not been determined and the a c t u a l amount of glucose and maltos upon the concentration an i s no maltase a c t i v i t y i n human serum, human kidney t i s s u e does have maltase a c t i v i t y (85). Chromatographic separation of the urinary i n d i c a t e s that some 50-55% of the excreted r a d i o a c t i v i t y f o l l o w i n g maltose i n f u s i o n i s excreted as l ^ C glucose (84). The metabolic response to the intravenous i n f u s i o n of maltose and glucose to normal and d i a b e t i c subjects i s shown i n F i g ures 10 and 11 and the s t a t i s t i c a l a n a l y s i s i s summarized i n Table IX. Serum glucose concentration a f t e r maltose i n f u s i o n r e mained l e s s than 95 mg/100 ml over the e n t i r e 2-hr p e r i o d , i n cont r a s t to the e l e v a t i o n of serum glucose f o l l o w i n g glucose administ r a t i o n . The increase i n t o t a l serum reducing substances was simi l a r a f t e r maltose and glucose a d m i n i s t r a t i o n . In normal and d i a b e t i c s u b j e c t s , there was a s i g n i f i c a n t r i s e i n serum i n s u l i n a f t e r maltose i n j e c t i o n , however t h i s increase was greater a f t e r glucose. D i a b e t i c subjects showed an abnormal i . v . glucose t o l erance t e s t and higher serum i n s u l i n l e v e l s at 60, 90, and 120 min a f t e r glucose i n f u s i o n as compared to normal s u b j e c t s . I n s u l i n disappearance curves i n both normal and d i a b e t i c subjects f o l l o w ing maltose i n f u s i o n i n d i c a t e a slow r a t e of removal of i n s u l i n from the serum. The r e l a t i o n s h i p between the delayed disappearance of serum i n s u l i n and the serum glucose concentrations (which remain below 95 mg/100 ml) i s not known. High l e v e l s of c i r c u l a t i n g maltose (as r e f l e c t e d i n the concentrations of t o t a l serum sugars) at t h i s same time i n t e r v a l may d i r e c t l y stimulate i n s u l i n r e l e a s e . I t i s not known i f i n s u l i n i s required f o r maltose entry i n t o mammalian c e l l s . Intravenously administered maltose appears to be as e f f i c i e n t l y u t i l i z e d as glucose i n m i l d l y d i a b e t i c and normal s u b j e c t s . Further s t u d i e s i n severely d i a b e t i c p a t i e n t s are needed to determine whether maltose may be e f f i c i e n t l y metabo l i z e d despite a reduced i n s u l i n response (84). Several studies i n Japan (86,87,88) have reported the use of maltose s o l u t i o n s i n a v a r i e t y of s u r g i c a l p a t i e n t s . In a study 14

1 4

2

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

IV. M a l t o s e - U - C (5) V . G l u c o s e - l - C (5)

Diabetic

1 4

11+ III III V IV V

14

14

14

11.0 ± 2 . 4 7.3 ± 2.0 NS ρ < 0.05 NS NS NS NS

240 ± 10 138 ± 15 ρ < 0.001 ρ < 0.001 NS ρ < 0.001 NS NS

NS NS NS NS ρ < 0.05 ρ < 0.05

Journal of Clinical Endocrinology and Metabolism (84)

t Mean ± S E . + Statistical comparisons are between groups as indicated.

1 4

25.4 ± 1.3 28.3 ± 0.7

Dose ± 1.6t ± 3.4 ± 1.9

2

% Dose 11.8 ± 1.9 6.9 ± 1.2 5.1 ± 0 . 6

% 33.3 37.7 36.5

C0

1 4

C-labeled maltose or glucose to

Minutes 235 ± 10 110 ± 13 120 ± 6

4

Urinary

1

Peak C

1 4

* Number in parenthesis indicates number of subjects,

1 vs I vs II vs IV vs 1 vs II vs

1. M a l t o s e - U - C (5)* II. G l u c o s e - U - C (5) III. G l u c o s e - l - C (4)

Normal

14

Carbohydrate

after the intravenous administration of 25 g normal and diabetic subjects

Subjects

l4

Recovery of C

T A B L E VIII

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

16.0-r

14.0-

"o

I2.0H

50

100

150

200

250

300

350

400

MINUTES Journal of Clinical Endocrinology and Metabolism 14

14

Figure 9. Fraction of injected C recovered as expired C0 over a 6-hr period after the intravenous administration of 25 g glucose-l- C or maltose-U- C to five mildly diabetic subjects (84) 2

14

14

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

YOUNG

AND WESER

Infused Maltose 240-

MINUTES

Figure 10. Metabolic response of 14 control and five mildly diabetic subjects follounng the intravenous administration of 25 g glucose-l- C. Each point represents mean value ± S.E. 14

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

94

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

MINUTES

Figure 11. Metabolic response of 14 control and five mildly diabetic subjects to the intravenous administration of 25 g maltose-U- C. Each point represents mean value ±S.E. 14

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975. Journal of Clinical Endocrinology and Metabolism (84)

loading. III. Diabetic maltose loading. IV. Diabetic glucose loading.

I. Normal maltose loading. II. Normal glucose

NS NS NS NS ρ < 0.05 NS NS ρ < 0.01 NS ρ < 0.01 NS NS NS ρ < 0.01 NS NS

ρ < 0.05 ρ < 0.01 ρ < 0.05 NS

NS NS NS NS

I vs II III vs IV I vs IM II vs IV

"Statistical comparisons are between subject groups indicated:

Serum Insulin, μΙΙ/ml

NS NS NS

NS

NS

ρ < 0.01 NS NS ρ < 0.05 ρ < 0.01 NS NS ρ < 0.05

ρ < 0.01 ρ < 0.05 NS NS

NS NS NS NS

NS NS NS NS

NS NS ρ < 0.01 NS

NS

ρ < 0.05 NS NS ρ < 0.05

NS NS NS ρ < 0.05

NS NS NS ρ < 0.05

ρ < 0.05 ρ < 0.001 NS NS

120

90

ρ < 0.001 ρ < 0.001 NS NS

Minutes after infusion 60 30

NS* NS NS NS

0

15

of glucose and

I vs III

II IV III IV

I vs III vs I vs II vs

Total Serum Sugars. mg/100 ml

"Apparent Maltose/' mg/100 ml

II IV III IV

I vs III vs I vs II vs

Serum Glucose mg/100 ml

Subject groups

response to the intravenous administration

maltose to normal and diabetic subjects

Statistical comparisons of metabolic

T A B L E IX

96

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

of 67 p a t i e n t s , no secondary e f f e c t s , c l i n i c a l a b n o r m a l i t i e s , or hyperosmolar problems were recognized (84), Conclusions To date, there i s no i d e a l carbohydrate source of c a l o r i e s for parenteral alimentation. Studies of i n t r a v e n o u s l y administer­ ed d i s a c c h a r i d e s i n d i c a t e that maltose and glucose are e f f i c i e n t ­ l y and s i m i l a r l y metabolized i n man and i n the r a t . Maltose, be­ cause of i t s c a l o r i s d e n s i t y and a b i l i t y to be metabolized, may be of importance i n p a r e n t e r a l n u t r i t i o n , and warrants f u r t h e r study. Literature Cited

1. Annan, G. L., Bull. Ν. Y. Acad. Med. (1939) 15:622. 2. Foster, M. "Claude Bernard". Longmans Green Company, New York. 1899. 3. Biedl, A. and Kraus 4. Geyer, Robert P., Physiol. Rev. (1960) 40:150. 5. Law, David Η., Adv. Intern. Med. (1972) 18:389. 6. Shils, Maurice Ε., J. Amer. Med. Assoc. (1972) 220:14. 7. Wretlind, A. (Editor). "Colloquim on intravenous feeding". Colloquim held at The Royal Society of Medicine. London, May 1-2, 1962. Nutr dieta (1963) 5:295. 8. "Parenteral Nutrition Symposium". Symposium held at Kungalv, Sweden, Nov. 9-10, 1962. Acta Chir. Scand. (1964) Suppl. 325: 1. 9. Meng, H.C. and Law, David H. (Editors). "Parenteral Nutri­ tion". International Symposium on Parenteral Nutrition. Vanderbilt University School of Medicine, 1968. Charles C. Thomas, Springfield, 1970. 10. Cowan, G. S. M. and Scheetz, W. L. (Editors). "Intravenous Hyperalimentation". U. S. Army Institute of Surgical Research, San Antonio, Texas. 1970. Lea and Febiger, Philadelphia, 1972. 11. Wilkison, A. W. (Editor). "Parenteral Nutrition".. An Inter­ national Symposium in London, April 20, May 1, 1971. Williams and Wilkins Company, Baltimore, 1972. 12. American Medical Association. "Symposium on Total Parenteral Nutrition". Symposium held at Nashville, Tennessee, Jan. 1719, 1972. American Medical Association, Council on Food and Nutrition, Chicago, 1974. 13. Dudrick, S. J., Steiger, Ε., Long, J. M., Ruberg, R. L., Allen, T. R., Vars, H. M. and Rhoads, J. E. In "Intravenous Hyperalimentation", George Cowan, Jr. and Walter Scheetz, (Editors), p. 3. Lea and Febiger, Philadelphia, 1972. 14. Wyrick, W. J., Rea, W. S. and McClelland, R. Ν.,J.Amer. Med. Assoc. (1970) 211:1967. 15. Winters, R. W., Scaglione, P. R., Nahas, G. G. and Verosky, M. J. Clin. Invest. (1964) 43:647.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

5.

YOUNG

AND

WESER

Infused

Maltose

97

16. Thoren, L., Nutr. Dieta, (1963) 5:305. 17. Froesch, E. R. and Keller, U. In "Parenteral Nutrition". A. W. Wilkinson, (Editor). An International Symposium, Lon­ don, April 30, May 1, 1971, Williams and Wilkins Company, Baltimore, 1972, p. 105. 18. Hayes, M. A. and Brandt, R. L., Surgery (1952) 32:819. 19. Elman, R. and Weichsclbaum, Τ. Ε., Arch. Surg. (1951) 62:683. 20. Mehnert, Η., Forster, Η., Geser, C., Α., Haslbeck, Μ., Dehmel, Κ. H., In "Parenteral Nutrition". H. C. Meng and D. H. Law (Editors). International Symposium on Parenteral Nutrition. Vanderbilt University School of Medicine, 1968. Charles C. Thomas, Springfield, 1970, p. 112. 21. Schaefer, H. F., Med. Welt. (1960) 32:1632. 22. Daughaday, W. H. and Weichselbaum. Τ. Ε., Metabolism (1953) 2:459. 23. Miller, M., Drucker W R. Owens J. E. Craig J. W and Woodward, Jr., Η. 24. Luke, R. G., Dinwoodie, J., , Kennedy, C., J. Lab. Clin. Med. (1964) 64:731. 25. Hinton, P., Littlejohn, S., Allison, S. P. and Lloyd, J., Lancet (1971) 1:767. 26. Dudrick, S. J., MacFadyen, Β. V., VanBuren, C. T., Ruberg, R. L., Ann. Surg. (1972) 176:259. 27. Bassler, Κ. H. In "Parenteral Nutrition". H. C. Meng and D. H. Law (Editors). Proceedings of an International Sympo­ sium, Vanderbilt University School of Medicine, Nashville, Tennessee, 1968. Charles C. Thomas, Springfield, 1970, p.96. 28. Weichselbaum, T. E., Elman, R., and Lune, R. H., Proc. Soc. Explt. Biol. (1950) 75:816. 29. Albanese, Α. Α., Felch, W. C., Higgons, R. Α., Vestal, B. L. and Stephanson, L., Metabolism (1952) 1:20. 30. Elman, R., Amer. J. Clin. Nutr. (1953) 1:287. 31. Thoren, L., Acta. Chir. Scand., (1964). Suppl. 325, p.75. 32. Wretlind, Α., Nutr. and Metabol. (1972) 14:Suppl. p.l. 33. Moncrief, J. Α., Coldwater, Κ. B. and Elman, R., Arch. Surg. (1953) 67:57. 34. Miller, M. J., Craig, W., Drucker, W. R. and Woodward, Η., Yale J. Biol. and Med. (1956) 29:335. 35. Froesch, E. R., Zapf, J., Keller, U. and Oelz, O., Europ. J. Clin. Invest. (1971) 2:8. 36. Heuckenkamp, P. U. and Zollner, Ν., Nutr. Metabol. (1972) 14:Suppl. p.58. 37. Aitkin, J. M. and Dunnigan, M. G., Brit. Med. J. (1969) 3:276. 38. Weinstein, J. J. and Roe, J. Η., J. Lab. Clin. Med. (1952) 40:47. 39. Pletscher, Α., Helv. Med. Acta. (1953) 20:100. 40. Mehnert, Η., Mahrhofer, Ε., and Forster, Η., Munchen Med. Wschr. (1964) 106:193. 41. Sunzel, Η., Acta Chir. Scand. (1958) 115:235. 42. Berry, Μ. Ν., Proc. Roy. Soc. Med. (1967) 60:1260.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

98

PHYSIOLOGICAL EFFECTS OF FOOD CARBOHYDRATES

43. Woods, H. F., Oliva, P. Α., Amer. J. Med. (1970) 48:209. 44. Bergstrom, J., Hultman, E., and Roch-Norlund, A. E., Acta. Med. Scand. (1968) 184:359. 45. Craig, G. M. and Crane, C. W., Brit. Med. J. (1971) 4:211. 46. Sahebjami, H. and Scalettar, R., Lancet (1971) 1:366. 47. Cook, G. C. and Jacobson. J., Brit. J. Nutr. (1971) 26:187. 48. Woods, H. F., Eggleston, L. U. and Krebs, Η. Α., Biochem. J. (1970) 119:501. 49. Bode, C., Schumacher, Η., Goebell, Η., Zelder, D., and Pelzel, Η., Hormone Metab. Res. (1971) 3:289. 50. Fox, I. H. and Kelley, W. N., Metabolism (1972) 20:713. 51. Pearson, J. F. and Shuttleworth, R., Amer. J. Obstet. Gynec. (1971) 111:259. 52. Woods, H. F. and Alberti, K. G. Μ. Μ., Lancet (1972) 2:1354. 53. Harries, J. T. In "Parenteral Nutrition" A W Wilkinson (Editor). An Internationa May 1, 1971. William 54. Blackley, R. L., Biochem. J. (1951) 49:257. 55. Hers, H. G., J. Biol. Chem. (1955) 214:373. 56. Lee, Η. Α., Morgan, A. G., Waldrom, R., and Bennet, J. In "Parenteral Nutrition". An International Symposium in Lon don, April 30, May 1, 1971. A. W. Wilkinson (Editor). Williams and Wilkins, Baltimore, 1972, p.121. 57. Bye, P. Α., Brit. J. Surg. (1969) 56:653. 58. Seeberg, V. P., McQuarrie, Ε. B., and Secor, C. C ., Proc. Soc. Exper. Biol. (1955) 89:303. 59. Spitz, I. Μ., Rubenstein, Α. Η., Bersohn, I., and Bassler, Κ. H., Metabolism (1970) 19:24. 60. Thomas, D. W., Edwards, J. Β., and Edwards, R. G., New Eng. J. Med. (1970) 283:437. 61. Schumer, W., Metabolism (1971) 20:345. 62. Forster, Η., Meyer, Ε., and Ziege, Μ., Klin. Wschr. (1970) 48:878. 63. Mirsky, I. A. and Nelson, Ν., Amer. J. Physiol. (1939) 127:308. 64. Coats, D. A. In "Parenteral Nutrition". A. W. Wilkinson (Editor). An International Symposium in London, April 30, May 1, 1971. Williams and Wilkins, Baltimore, 1972, p.152. 65. Lieber, C. S., Gastroenterology (1973) 65:821. 66. Bassler, Κ. H. and Bickel, H. In "Parenteral Nutrition". A. W. Wilkinson (Editor). An International Symposium in London, April 30, May 1, 1971. Williams and Wilkins, Baltimore, 1972, p.99. 67. Weser, Elliot and Sleisenger, Marvin Η., J. Clin. Invest. (1967) 46:499. 68. Dahlqvist, A. and Thompson, D. L., Acta. Physiol. Scand. (1964) 61:20. 69. Dahlqvist, A. and Thompson, D. L., J. Physiol. (1963) 162:193.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

5.

YOUNG

AND

WESER

Infused

Maltose

99

70. Semenza, G. In "Alimentary Canal". C. F. Code (Editor). Williams and Wilkins, Baltimore, 1968, Vol. 5, Section 6, p.2543. 71. Bittencourt, Η., Sleisenger, Μ. Η., and Weser, E. Gastro­ enterology (1969) 57:410. 72. Van Handel, F., Comp. Biochem. Physiol. (1968) 26:561. 73. Weser, Ε., Friedman, Μ., and Sleisenger, Μ. Η., Biochim et Biophys. Acta. (1967) 136:170. 74. Young, J. M. and Weser, E., Endocrinology (1970) 86:426. 75. Flores, M., Weser, Ε., and Young, Ε. Α., Comp. Biochem. Physiol. (1974) 50B: (In press) 76. Dahlqvist, A. and Brun, Α., J. Histochem. Cytochem. (1962) 10:294. 77. Courtois, J. E. and Demelier, J. E., Bull. Soc. Chim. Biol. (1966) 48:277. 78. Sacktor, Β., Proc Nat Acad Sci (1968) 60:1007 79. Silverman, Melvin. 80. Young, E. A. and Weser, , (1974) 81. Yoshimura, N. N., Ehrlich, H., Westman, T. L., and Deindoerfer, F. H., J. Nutr. (1973) 103:1256. 82. Deane, N., Schreiner, G. E. and Robertson, J. S., J. Clin. Invest. (1951) 30:1463. 83. Young, J. M. and Weser, E., J. Clin Invest. (1971) 50:986. 84. Young, E. A. and Weser, E., J. Clin. Endocrin. and Metab. (1974) 38:181. 85. Drayfus, J. C. and Alexandre, Υ., Biochim. Biophys. Res. Commun. (1972) 48:914. 86. Sunada, Tenitake, et al, Diag. and Treat. (1971) 59:2386. (Japanese) 87. Hayasaka, Α., et al, J. New Remedies and Clinics (1972) 21:3 (Japanese) 88. Tanaka, Takaya, et al, J. New Remedies and Clinics (1971) 20:125. (Japanese)

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

6 The Metabolism of Lactose and Galactose R. GAURTH

HANSEN

Utah State University, Logan, Utah 84322 RICHARD GITZELMANN Laboratory for Metabolic Research, Children's Hospital, Zurich, Switzerland

Lactose, which galactos glucose, primary carbohydrate source for developing mammals, and in humans it constitutes 40 percent of the energy consumed during the nursing period. Why lactose evolved as the unique carbohydrate of milk is un­ clear, especially since most individuals can meet their galactose need by biosynthesis from glucose. Whatever the rationale for lactose in milk, the occurrence of galactose in glyco-proteins, complex poly­ saccharides, and lipids, particularly in nervous tissue, has suggested specific functions. The organoleptic and physical properties of galac­ tose and, more specifically, the simultaneous occurrence of calcium and lactose in milk, may be significant evolutionary determinants. Lactose, in contrast to other saccharides, appears to enhance the absorption of calcium, as does vitamin D (1) (2). In man, calcium absorption is associated with the hydrolysis of lactose (3). The extensive literature concerned with galactose occurrence will not be reviewed, only some relevant examples will be cited. Col­ lagen contains glycosylated hydroxylysine, either as galactose or as glucosyl-galactose (4). Bone collagen mainly contains galactose mono­ saccharides, which could be important calcium binding centers, since many carbohydrates bind calcium in aqueous solution. In humans an excretion of three oligosaccharides containing galactose is enhanced by lactose ingestion (5). The three oligosaccharides appear to represent nonreducing terminals of the bloods antigenic determinants. I. Lactose Catabolism A. Microorganisms. The utilization of lactose f o r energy o r structural purposes i s preceded by hydrolysis to the hexoses, which can be absorbed. There i s considerable potential f o r improving the properties and acceptability of lactose i n foods by hydrolyzing i t to the monosaccharides. Toward that end, substantial progress has been 100 In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

6.

HANSEN

AND

GiTZELMANN

Lactose and

Galactose

101

made toward isolating and characterizing the relevant enzyme f r o m l a c tose-fermenting microorganisms (6) (7) (8). In microorganisms, galactosidases are induced by galactose and by other sugars that have configurations related to galactose. B. Mammalian Digestion. In mammals, the r a t e - l i m i t i n g step in lactose digestion appears to be the hydrolysis of the disaccharide into absorbable monosaccharides. Most mammalian disaccharidase activity is localized i n the brush-border fraction of the mucosal c e l l s of the s m a l l intestine (9)· 1. Lactase deficiencies. Whether lactase i s induced or constitutive i n man has not been c l e a r l y established. We do know, however, that i n populations that traditionally depend on m i l k as a significant source of energy, throughou lyze lactose. But when lactos adult diet, the capacity to hydrolyze lactose seems to decline over time (10). B y contrast, lactose intolerance i s common i n many non-milk consuming adult populations. Among these people, the ability to metabolize lactose, obviously present during infancy, disappears between the ages of one and four. Evidence that lactase levels respond to an altered lactose intake i s questionable (11). Neither exposure to extra lactose (12) (13) nor r e moval of lactose f r o m the diet for periods of 40 to 50 days altered l a c tase levels i n the intestinal tissues of adults. Perhaps of more s i g n i f i cance, nine i n a group of ten galactosemic children, ages 7 to 17 years, who had carefully avoided lactose-containing materials since e a r l y infancy, had normal blood glucose responses to o r a l lactose loads, suggesting that their lactase levels had not decreased during their long periods of lactose abstinence. The one exception was a 15-year old Negro who was determined by biopsy to be lactase deficient (14). Any potential for an adaptive response to lactose i n humans i s insignificant during the life span. A genetic basis f o r adult lactase deficiency i s indicated by the 70 percent of black A m e r i c a n s who are r e ported to be lactose intolerant, which duplicates the adult intolerance level of black A f r i c a n s , whose exposure to lactose i s probably much l e s s . Although the question has not been conclusively resolved i n humans, adult lactase deficiency may be p r i m a r i l y under genetic control. Intestinal lactase may have occurred i n i t i a l l y i n adult humans as a consequence of the domestication of milk-producing livestock. This concept i s substantiated by the lifelong presence of lactose i n most western European adults and in those ethnically derived f r o m Europe, These people can effectively digest the lactose i n d a i r y foods. A c c o r d -

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

102

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

ing to this hypothesis, adaptation to the presence of lactose in the diet required many generations and a period of several thousand years. Adult blacks in the United States have not developed intestinal lactase after exposure to lactose f o r 300 years. When milk-producing animals were domesticated it i s postulated that some adults with lactase deficiency became lactase producers through gene mutation (15). The specific selective advantage of the lactose-tolerant adult was associated with the lactose-induced enhancement of calcium absorption in an environment that provided a low dietary supply of vitamin D. M i l k consumption thus may be advantageous for lactose-tolerant individuals because m i l k supplies both calcium and a factor promoting its absorption. The r a r e l y o c c u r r i n g congenital lactose intolerance that i s due to a deficiency of lactase d b H o l z e l (16) "Alactasia " A limited number of infant the mode of inheritance i s not clear. Another congenital lactose intoler ance i s associated with the inability of infants to hydrolyze lactose and the subsequent appearance of lactose i n the urine. These phenomena generally seem to be secondary to mucosal damage associated with acute infectious diarrhea, and probably constitute a different and more complex syndrome than alactasia. A voluminous and important literature i s developing describing deficiencies of lysosomal beta- and alpha-galactosidases (17) (18). In F a b r y s Disease, there i s a deficiency of the ^-galactosidase which normally hydrolyzes ceramide trihexoside, resulting i n the accumulation of trihexosylceramide in various organs, p r i m a r i l y i n kidneys. Three other sphingolipidoses are attributed to the deficiency of an enzyme h y d r o l y z i n g ^ - g a l a c t o s i d i c bonds: Krabbe s Disease, Ceramidelactoside Lipidosis, and Generalized Gangliosidosis. Galactosidases obviously play an important role in the regulation of glycolipid levels in tissues. 1

T

II. Lactose Biosynthesis It was e a r l y concluded (19) that UDP-galactose was the donor and glucose-1-phosphate the acceptor during the synthesis of lactose. The product of the reaction, the phosphate ester of lactose, was postulated to have a role i n its excretion by the glandular tissue. By using C isotopes in the lactating cow and isolating the postulated i n t e r mediates and products, investigators concluded that a major pathway for lactose synthesis had UDP-galactose as the donor, and free glucose (not glucose-l-phosphate) as the acceptor (20). Tissue extracts subsequently were found to catalyze the synthesis of lactose f r o m UDPgalactose and glucose but, contrary to expectations, the yields were 1 4

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

6.

HANSEN

AND

GiTZELMANN

103

Lactose and Galactose

very low (21). This work confirmed the isotope studies in the whole animal. A. Lactose Synthetase. Our understanding of lactose synthesis phenomena was substantially advanced when the lactose synthetase was resolved into two components: a mixture of "A" protein fractionated from mammary glands, and of ^ f - l a c t a l b u m i n , a protein normally found i n m i l k (22) (23). In the absence of Pf-lactalbumin, the "A" protein w i l l catalyze galactosyl transfer to N-acetylglucosamine (24). Glucose i s not a good acceptor, however, having a high apparent M i c h a e l i s constant of 1 M. In the presence of c^f-lactalbumin, the A protein effectively catalyzes the synthesis of lactose, decreasing the K about 1,000 fold (25). Thus c^-lactalbumin i s a specifier protein for the synthesis of lactose The net effect of of-lactalbumin i s to con vert a glycosyl transferas plex polysaccharides to an efficient system f o r lactose synthesis (Figure 1). The mammary gland i s unique i n that it can produce ôf-lactalbumin, and in so doing it makes glucose an effective substrate f o r the galactosyl transferase enzyme. The p r i n c i p a l function of the galactosyl transferases that originate in tissues other than lactating mammary glands i s to transfer galactose to an appropriate carbohydrate side chain of glycoproteins and lipids. On this basis it i s expected that these transferases are widely distributed. Significantly, bovine 4) glycosidic linkage, and such a linkage i s hydrolyzed in the lysozyme reaction. It has been speculated, therefore, that the two proteins have related evolutionary origins. A s determined by disc gel electrophoresis, eC-lactalbumins isolated f r o m the m i l k of pigs, sheep, and goats have two electrophoretically distinct f o r m s , both of which are active i n the lactose synthetase reaction and appear to be charged i s o m e r s resulting f r o m slight variations in amino acid composition (27). Genetic variance i n bovine of-lactalbumin has been observed i n electrophoretic separation, hence the two f o r m s observed i n the pig, goat, and sheep may also represent genetic variance. The "A" protein i n bovine skim m i l k has been purified of contaminating proteins. It i s a single-chain glycoprotein of molecular weight of 44, 000 (28) (29). In the presence of one of the substrates, the "A" protein and c ^ l a c t a l b u m i n f o r m a stable complex that contains one molecule of each protein. M

m

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

T T

104

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

ΙΠ· Galactose Biosynthesis i n the M a m m a r y Gland It i s generally assumed that galactose i s formed from UDPglucose i n the mammary gland, catalyzed by the C-4 epimerase. In addition to defining a p r i m a r y pathway of lactose formation, with UDPgalactose and free glucose being the immediate p r e c u r s o r s , the p r e ­ viously mentioned isotope studies revealed the interesting possibility of an unusual mode of galactose synthesis (20). When 1, 3 C - l a b e l e d glycerol was injected into lactating mammary glands, the patterns of labeling of glucose and galactose differed. The galactose portion of lactose contained more C than did glucose. Further, Carbons 4 and 6 of the galactose particularly reflected the injected substrate. UDP-gal­ actose generally contained more label than did UDP-glucose, even though the latter was thought to be the normal biosynthetic p r e c u r s o r Alternative explanation discrete centers of metaboli activity, g l y c e r o (o glyceraldehyde) i s intercepted differentially at these centers. 2) UDP-hexose i s labeled by direct incorporation of triose. The e a r l i e r but unconfirmed report of UDP-dihydroxyacetone offered one possible intermediate (30). M o r e recently, i n the conversion of galactopyranose to galactofuranose at the nucleotide level, an open chain hexose derivative has been postulated that could incorporate triose by a transaldolase-like exchange (31) (Figure 2). 14

1 4

IV.

Galactose Metabolism.

Galactose that i s consumed i n excess of developmental needs i s metabolized for energy. In humans the l i v e r appears to be the p r i m a r y site f o r a l l meta­ b o l i s m of galactose; however, other tissues including the red c e l l have this capacity and hence can be conveniently used to evaluate the meta­ bolic capability of the individual. Fundamental studies using m i c r o ­ organisms and animals, together with analyses of the metabolic prob­ lems i n the human genetic disorders, have c l a r i f i e d the pathways of galactose metabolism i n man. The p r i m a r y metabolic reactions of galactose are illustrated i n Figure 3. A. Reduction of Galactose. A non-specific reduction of the aldehyde at carbon-1 leads to the product galactitol (32) (33). This process i s especially significant since the lens of the eye forms galactitol f r o m excess galactose, and, i n some genetically defective humans, who have no further capacity to metabolize galactitol, it accu­ mulates and gives r i s e to cataracts (34).

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

6.

HANSEN

AND

GLUCOSE

GLC-6-P

ATP

U TP

PPi

CH 0H

CH 0H

CH 0H

2

2

2

2

UDP-GALACTOSE

UDP-GLC

GLC - 1 - Ρ

ADP

CH 0H

105

Lactose and Gahctose

GiTZELMANN

Synthetase OH

HO • UDP

OH

a-Lactalbumin "A" Protein

OH OH LACTOSE

Figure 1.

GLUC0SE^GLUC0SE-6-P-»GLUC0SE-l-P-*UDP-GLUC0SE

CH 0H 2

R = URIDINE- DIPHOSPHATE

Figure 2. Galactose furanose formation

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

ο

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

6.

HANSEN

AND

GiTZELMANN

Lactose and Galactose

107

B. Dehydrogenation of Galactose. Dehydrogenation at carbon-1, which precedes the formation of galactonic acid, occurs i n man and other animals (35). In man, whether galactonic acid a r i s e s by the action of a specific enzyme or a general aldehyde dehydrogenase o r some phosphorylated intermediates has not been solved, but galactonic acid i s excreted i n cases of galactosemia and of galactokinase deficiency when galactose i s consumed (36) (37). It does serve as a base f o r quantitative estimation of galactose (38). C. Oxydation of Galactose. Microorganisms contain an enzyme that catalyzes an oxidation of galactose at carbon-6 to galactose hemialdehyde (39). While this reaction i s of no known significance i n humans it does provide a basis for quantitative estimates of their levels of galactose and some of its derivatives D. Tagatose Pathway phosphorylatio galactos carbon-6 i s of questionable significance i n man. Some evidence for the occurrence of gal-6-P has been presented (40), but as an alternate to direct phosphorylation, the gal-6-P could a r i s e f r o m g a l - l - P with a mutase type reaction (41). Indications are that when g a l - l - P i s present, gal-6-P i s not formed (42). In microorganisms, gal-6-P can be metabolized i n a manner analogous to the way glc-6-P i n glycolysis in a series of reactions (catalyzed by inducible enzymes) that are distinct f r o m those of the glucose pathway (43). The sequential products are gal-6-P, tagatose-6-P, tagatose-1, 6-diP, then glyceraldehyde-3-P and dihydroxyacetone-P (two trioses common to the glucose pathway) ( F i g ure 4). E. L e l o i r Pathway. Phosphorylation catalyzed by a kinase initiates the L e l o i r pathway of galactose metabolism i n man. The r e sulting product (gal-l-P) was identified i n 1943 i n rabbit l i v e r following ingestion of galactose (44). Human l i v e r tissue normally metabolizes most of the ingested galactose through the L e l o i r pathway, which r e quires two more enzymes i n addition to the kinase, namely, a t r a n s f e r ase and an epimerase (45); a)

b)

c)

kinase galactose + A T P transferase g a l - l - P + UDP-glc epimerase UDP-gal ^

> gal-l-P + ADP

^

i UDP-gal + g l c - l - P

^

UDP-glc

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

Tagatose pathway Figure 4. In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

6.

HANSEN

AND

GiTZELMANN

Lactose and Galactose

Sum of b + c: gal-l-P

s

109

glc-l-P

1. Biosynthesis of uridine diphosphate glucose and uridine diphosphate galactose. In addition to the function of UDP-glc as a cofactor i n the L e l o i r pathway f o r galactose utilization, it serves broader needs of the c e l l (46) (47). The synthesis of glycogen requires UDP-glc. Many chemical compounds needed f o r growth and f o r differentiation con­ tain a variety of saccharides. UDP-glc i s the p r i m a r y source of glycosyl residues f o r the biosynthesis of many of these saccharides. It i s not surprising, therefore, that the enzyme f o r catalyzing the synthesis of UDP-glc i s ubiquitous and abundant: d)

pyrophosphorylase glc-l-P + UT

In aqueous extracts of l i v e r , more than 0.5% of the protein i s this syn­ thetase f o r UDP-glc (48) (49). It i s relevant here to note that the r e ­ action i s reversible; the equilibrium i n vitro favors reactants as written, which fact gives r i s e to the enzyme being commonly designated as a pyrophosphorylase. In addition to being abundant, the pyrophosphorylase i s not highly specific. It w i l l catalyze the reaction with UDP-gal at about 5 % of the rate with UDP-glc (50). d') g a l - l - P + U T P

v

UDP-gal + P P i

F. Pyrophosphorylase Pathway of Galactose Metabolism. Isselbacher (51) has proposed a pyrophosphorylase pathway of galactose utilization which i s significant i n humans: T

d ) pyrophosphorylase gal-l-P + UTP c)

d)

epimerase UDP-gal pyrophosphorylase UDP-glc + P P i

>• UDP-gal + P P i

*

UDP-glc

* UTP + glc-l-P

!

Sum d , c and d: gal-l-P —

±> g l c - l - P

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

110

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

Due to its low specificity, the UDP-glc pyrophosphorylase w i l l catalyze both reactions d and d (although claims have been made that a separate enzyme (52) (53) exists i n human l i v e r for each of the two r e actions). f

G. Uridine Diphosphate Galactose C-4-epimerase. The epimerase, which i s required together with the pyrophosphorylase, seems to be ubiquitous and occurs e a r l y i n human development (54). The epimerase-catalyzed reaction i s the p r i m a r y site of glucose formation f r o m galactose when this sugar i s being metabolized for energy. Conversely, when galactose i s needed f o r structural purposes, it i s derived f r o m glucose at the nucleoside diphosphate hexose level, v i a the same r e action. The UDP-gal-C-4 epimerase reaction requires D P N as a cofactor. When the enzym D P N usually dissociate however, retain D P N more tenaciously. Reduced pyridine nucleotide strongly inhibits the animal enzymes. In hemolysates f r o m red cells f r o m newborn infants, two d i s tinct bands of epimerase are distinguishable on electrophoresis, while f r o m adult red cells only one distinctly different band i s found (55). When N A D i s added to the electrophoresis gel containing epimerase, the two-banded pattern characteristic of infant hemolysate i s seen. A n N A D dependent structural interchange must be responsible f o r this observation. The specific hydrogen atoms involved i n the hexose interconversion are removed f r o m one hexose substrate and replaced on the other i n the opposite steric mode at Carbon 4. This requires a substantial change i n the conformation of the hexoses during the reaction, with the carbon-bound hydrogens being retained. In the epimerase reaction, a keto intermediate at Carbon 4 was established by Nelsestuen (56) and confirmed by an isotope effect on the reaction rate (57) (56) (58) (59) (60). Ethanol oxidation generates an increase i n N A D H 2 , which apparently inhibits the UDP-galactose C-4-epimerase (61). This i n turn inhibits the clearance of galactose f r o m the blood (62). Adding N A D to a l i v e r preparation has partially prevented the enhancement effect of ethanol on N A D H 2 . In pregnant women the capacity of the l i v e r to remove galactose i s accelerated and the inhibitory effect of ethanol i s greatly reduced (63). +

+

+

+

+

+

H. Genetic Defects. Galactosemia i s an inborn e r r o r of metab o l i s m (64) caused by an inherited deficiency of g a l - l - P u r i d y t r a n s f e r ase, which normally catalyzes the second step i n the conversion of

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

6.

HANSEN

AND

GiTZELMANN

Lactose and Galactose

111

galactose to glucose. Disease manifestations are cataracts, l i v e r , and kidney dysfunction and disturbed mental development due to organic brain damage. Symptoms are apparent when galactose i s ingested and g a l - l - P (i.e., the product of the kinase reaction) accumulates. Defects in catalysis during the other two steps in the L e l o i r pathway have now been described in humans. Following the discovery of the transferase defect, a number of kinaseless f a m i l i e s were reported. In patients with a defect i n the kinase (65), galactose and galactitol, but not galactose-l-phosphate, accumulate i n the tissues and cataracts develop early i n infancy. The other toxic manifestations of transferaseless galactosemia do not occur in persons lacking the kinase. Since cataracts develop in patients with either defect, galactitol must be the causative agent for cataract formation in both the kinase and transferase defects. Galactokinase deficienc who had eye cataracts (66) develope g their f i r s t year of life. In humans, an epimerase deficit i n blood cells has been reported (67) (54). If the l i v e r and other organs are also epimerase deficient, the function of the epimerase reaction in metabolism would dictate a carefully controlled galactose intake. The dietary problem would be to provide enough galactose to meet the requirements for this sugar and its derivatives, but not so much as to produce toxic quantities of i n t e r mediates. In the absence of epimerase, presumably the kinase and pyrophosphorylase reactions would provide a biosynthetic route f o r UDP-gal. 1. Genetics of transferase deficiency. Since the absence of g a l a c t o s e - l - P - u r i d y l transferase activity i s the basis for diagnosing galactosemia, the development of quantitative methods f o r measuring the transferase in erythrocytes allowed precise definition of i n t e r r e l a tionships in families of patients with the disease (68) (69) (70). Direct measurements of the enzyme i n erythrocytes confirmed little o r no transferase activity i n a l l tested galactosemics. Further, the parents, some siblings, and some other relatives of the tested patients had, on the average, only half the normal level of enzyme. Both parents, as well as a paternal and a maternal grandparent, must therefore be c a r r i e r s , o r genetic hétérozygotes, before the disorder w i l l be expressed in offspring. This finding c l e a r l y establishes galactosemia as an autosomal-recessive disease. In tested galactosemic families, the offspring have demonstrated the expected mendelian ratios of one galactosemic:two hétérozygotes: one normal. At least one of the maternal and one of the paternal grandparents of the patient and other relatives have been hétérozygotes i n the

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

112

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

frequency predicted f r o m the hypothesized mode of inheritance. 2. Genetic variants of transferase deficiency. A so-called Duarte variant of g a l - l - P - u r i d y l transferase has been defined by a r e finement of the chemical assay and by electrophoretic separation of blood c e l l proteins having transferase activity. F a m i l y relationships have been defined for the Duarte mutant. The hétérozygote has about three-fourths the normal transferase activity. The homozygote has one-half the normal value of the transferase. The g a l - l - P u r i d y l transferase i n the Duarte variant appears to be a structural mutation since starch gel electrophoresis reveals two faster moving proteins with catalytic activity (71) (72). The importance of having a detailed structural analysis of the transferase i s apparent. On the basis of electrophoresis and the u r i d y l transferase r e d c e l l value, individuals hav and the Duarte structura cal galactosemics i s revealed by immulogical procedures, but i s i n active according to chemical assays (74) (75). Structural alteration i s assumed to prevent the less active Duarte variant and the inactive galactosemia protein f r o m performing its normal catalytic function (76). Other transferase variants have been identified. It has been shown (77) that a so-called Negro variant could have a 10 percent norm a l transferase activity based on assays of uridyl transferase in l i v e r and intestinal tissues, even though the r e d cells showed no activity. Since this i s difficult to explain morphologically, a more rigorous characterization of this mutant must precede a tenable interpretation. The enzyme i n the Rennes mutant that was found in two galactosemic siblings migrates more slowly than normal transferase on electrophoresis (78). A further variation, which was revealed by an unstable transferase (Indiana variant), has been found i n a galactosemic f a m i l y (Z9).

A Los Angeles variant of galactosemia displays three t r a n s f e r ase bands during electrophoresis of erythrocyte hemolysates (80). In contrast to the Duarte variant, the total amount of transferase activity i n the hemolysate i s normal o r greater. I. Galactose-l-phosphate Toxicity. Galactose-1-phosphate i s the toxic agent causing most of the pathology i n c l a s s i c a l galactosemia. This can be i n f e r r e d f r o m the comparison of disease manifestations i n transferase deficiency with those i n galactokinase deficiency. Cataracts are the only common sign; they are caused by osmotic swelling and disruption of lens fibers due to the accumulation of galactitol (34). No b r a i n , l i v e r , and kidney pathology i s observed in kinase

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

6.

HANSEN

AND

GiTZELMANN

Lactose and Galactose

113

deficiency and, since g a l - l - P cannot be formed i n this disorder, one can conclude that it must play the deciding pathogenetic role in t r a n s f e r ase deficiency. Symptoms other than eye cataracts, which are observed i n patients with transferase deficiency, may be due to a high concentration of g a l - l - P within the c e l l . The blood glucose levels of some acutely i l l infants lacking transferase are subnormal, indicating a disturbed c a r bohydrate metabolism. Three key reactions involving glucose have been reported to be affected by galactose-l-phosphate: the mutase, dehydrogenase, and pyrophosphorylase reactions shown in Figure 5 (81) (82). In each case the evidence was based on kinetic studies of isolated enzymes, not f r o m human tissues; it i s therefore somewhat presumptive. About a 50-fold excess of galactose-l-phosphate over glucose phosphates i s required for significant inhibition of the isolated reactions. G l u c o s e - l - P , uridin side diphosphate-glucose inhibitor constants and calculated intracellular levels of these compounds it has been deduced that the uridine derivatives and glucose-1phosphate could have a physiologically regulatory function. J . Biosynthesis of Galactose-l-phosphate f r o m UDP-galactose. The idea that the pyrophosphorylase pathway i s a means of galactose metabolism i n the human erythrocyte stems f r o m the following observations: Epimerase and UDP-glc are normal red c e l l constituents. Hence UDP-gal i s also available as a substrate f o r other reactions. Incubation of hemolysates lacking transferase (84) with UDP-gal produced g a l - l - P in a reaction that was absolutely dependent on inorganic pyrophosphate and was stimulated by magnesium; the production of g a l - l - P was i n hibited by UDP-glc, by UDP and P i . Under identical conditions, the crystalline enzyme f r o m human l i v e r catalyzes the formation of g a l - l - P f r o m UDP-gal (85) (50). UDP-glucose pyrophosphorylase of both calf and rabbit have also been purified and c r y s t a l l i z e d (48) (85) (50). The biochemical evidence that one protein catalyzes reactions with both glucose and galactose derivatives i s convincing. Throughout purification and c r y s tallization, the ratio of activity of the enzyme towards the various substrates remains constant (86) (85) (50). UDP-gal i s bound to the p u r i fied enzyme as a function of the number of protomer subunits of pyrophosphorylase (49) (87). This bound UDP-gal may then be stoichiom e t r i c a l l y replaced by UDP-glc (87). UDP-glc also l i m i t s the synthesis of g a l - l - P f r o m UDP-gal by hemolysates (84). It i s concluded, therefore, that UDP-gal competes with UDP-glc (although l e s s effectively) for the same site on the enzyme. Immunological evidence supporting this conclusion has also been obtained (88).

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

PHYSIOLOGICAL EFFECTS OF FOOD CARBOHYDRATES

.Glycogen,

Glucose

UTP Glc-6-P^

1

•Glc-l-P

r

UDP-GIc

^ PPi

NHIBITION by Gal-1-P 6-P-Gluconic

acid

Figure 5.

Toxicity of

galactose-l-phosphate

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

6.

HANSEN

AND

GiTZELMANN

Lactose and Galactose

115

The production of g a l - l - P f r o m g l c - l - P by the pyrophosphorylase pathway requires the enzymes pyrophosphorylase and epimerase and their substrates, the UDP-hexoses, U T P and P P i . A substantial concentration of U T P i s maintained i n t r a c e l l u l a r l y f o r nucleic acid synthesis and f o r UDP-glc formation. Besides being a p r e c u r s o r of UDPgal, UDP-glc i s a source of glycosyl donors f o r pentoses, uronic acids, and hexosamines; hence, there i s a constant and vital metabolic flux through UDP-glc. Metabolic interconversions of sugars and their d e r i vatives require epimerases, which appear to be generally distributed i n nature. Hence, the epimerase-catalyzed formation of UDP-gal f r o m UDP-glc i s a common intracellular reaction. Under most circumstances, when nucleotides are extracted f r o m the c e l l , UDP-gal and UDPglc are present i n a ratio of approximately 1/3 to 2/3, providing ample UDP-gal substrate f r o m which g a l - l - P may be enzymatically formed. Inorganic pyrophosphat g a l - l - P i s to be produce UDP-gal Pyrophosphat produc of most biosynthetic processes, including the formation of polysaccharides, proteins, and lipids (89). To permit the biosynthesis of g a l - l - P f r o m UDP-gal f r o m an equilibrium that favors reactants, P P i i s theor e t i c a l l y hydrolyzed by the intracellular pyrophosphatases. Quite aside f r o m the futile energy wastage, if this hydrolysis took place, substantial evidence indicates that not only do the c e l l s r e a l i z e the potential for productive use of the P P i , but P P i may have a regulatory function as well (90) (91). Furthermore, P P i accumulates i n man i n sufficient quantity to c r y s t a l l i z e with calcium and cause some joint disorders (92). The availability of P P i f o r g a l - l - P synthesis i s therefore most probable. The galactosemic has a special problem since the transferase block promotes an accumulation of g a l - l - P f r o m dietary galactose while, at the same time, a substantial need f o r UDP-gal obviously exists. In galactosemia, the pyrophosphorylase pathway appears to have adequate capacity to f o r m UDP-gal f r o m g l c - l - P but cannot readily convert g a l - l - P to UDP-gal. Hence, g a l - l - P accumulates when galactose i s ingested. The following observation indicates that the pyrophosphorylase pathway i s largely responsible f o r galactose metabolism i n red cells f r o m galactosemics. A n antibody to crystalline UDP-glucose pyrophosphorylase f r o m human l i v e r has been prepared (88). This antibody, when incubated with extracts of galactosemic red c e l l s , specifically precipitates the enzyme responsible f o r metabolism of galactose. Hence the galactosemic who lacks the L e l o i r pathway can convert some galactose to glucose in erythrocytes (and probably other tissues), v i a the pyrophosphorylase pathway.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

116

PHYSIOLOGICAL

EFFECTS

OF FOOD

CARBOHYDRATES

K. Uncontrolled Biosynthesis of Galactose-l-phosphate f r o m Glucose. The usual procedure for maintaining patients with galactosemia i s to exclude galactose sources f r o m their diets. When this i s carefully done, levels of g a l - l - P lower than 4 mg/100 m l are maintained in most patients. Any galactose ingestion i s followed by an immediate r i s e i n red c e l l g a l - l - P . Unfortunately, even when their intake of galactose sources i s carefully regulated, uncontrolled biosynthesis of g a l - l - P f r o m UDP-gal has been observed i n some galactosemic s (93). Much evidence, both c l i n i c a l and experimental, currently documents biosynthesis of galactose f r o m glucose i n man. Mayes and M i l l e r (94) have grown transferase-deficient skin fibroblasts i n a medium devoid of galactose and observed the formation of g a l - l - P f r o m glucose. The final concentrations (approx 2 mg/g protein) were s i m i l a r to those attained by re protein) (84). Cultured fibroblasts f r o m galactosemics without transferase activity have converted ( 1 - C ) galactose to C 0 2 (95), with an accumulation of g a l - l - P . Both C O 2 and g a l - l - P formation were inhibited by glucose, suggesting a common intermediate. Galactokinasedeficient cells s i m i l a r l y treated did not produce C O 2 f r o m ( l - l ^ C ) galactose, suggesting the pyrophosphorylase pathway as the alternate route of galactose metabolism i n galactosemics. Galactokinase-deficient children grow and develop normally when maintained on a diet free of galactose. Pregnant women who are hétérozygotes f o r transferase deficiency and have previously had t r a n s ferase-deficient offspring are routinely subjected to a galactose exclusion diet; despite galactose deprivation their fetuses develop normally. One completely transferase-deficient woman who followed a lactosefree regimen throughout pregnancy delivered a healthy infant and, presumably s t i l l on the diet, produced lactose (2.8 g/100 ml) i n her colost r u m on the second post partum day (96). In galactokinase-deficient twin infants on a suitable diet, s m a l l amounts of galactose remained detectable in plasma and urine (97). Both galactokinase- and t r a n s f e r ase-deficient infants, though on galactose exclusion diets, excreted some galactitol i n the urine (97) (98). High g a l - l - P i s commonly r e corded i n the cord blood of transferase-deficient newborns born of mothers on galactose-restricted diets (99). The need for galactose-containing polymers to assure functional and structural integrity of c e l l s and tissues i s satisfied by the biosynthetic reactions that have been detailed. Hence there i s a substantial capacity to synthesize galactose and its derivatives at a l l stages of human development. The g a l - l - P of a transferase-deficient newborn (at age 5 hrs) was 17 mg/100 m l of R B C . At the end of his f i r s t day, 14

1

4

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

6.

HANSEN

AND

GiTZELMANN

Lactose and Galactose

117

the g a l - l - P had actually r i s e n to 26 mg/100 m l , although he had been given intravenous glucose only and had taken no food (88). C l e a r l y his red blood cells had synthesized g a l - l - P , either f r o m endogenous galactose stores o r (more likely) f r o m glucose. We have observed what appears to be uncontrolled biosynthesis of g a l - l - P i n two galactosemic infants. A f t e r an initial exposure to m i l k and following diagnosis, both received a galactose-free formula. V e r y surprisingly, upon initiation of the diet at age 7 and 6 days, respectively, red c e l l g a l - l - P levels dropped rather slowly, and potentially toxic levels were maintained for months. At age 27 days, one infant was fed i s o c a l o r i c amounts of dextrimaltose i n water f o r 36 h r s ; however, the g a l - l - P did not drop but rose slightly. The female infant had breast swellings until her 25th day of age. A t age 19 days, her breast s e c r e tions, collected and compared to those of three other newborns contained what appeared to b synthesized f r o m glucose (93) Such biogenesis of galactose could take place i n tissues other than the red cells and the breast glands, e.g., the l i v e r and the central nervous system. It i s remarkable i n this connection that i n the few r e ported long-term follow-up studies (100) (101), some of the galactosemia patients who had been diagnosed at b i r t h and treated since, showed signs of organic b r a i n damage at school age, evidenced by difficulties in visual perception. Self-intoxication with g a l - l - P through a prolonged maintenance of high levels during infant life has been hypothesized as the cause of the damage. Evidently such biosynthesis of g a l - l - P has constituted a mechanism of potential se If-intoxication in well-treated galactosemic infants (102). Convincing evidence implicates the pyrophosphorylase pathway i n such cases (88). The regulation of this process to l i m i t the amount of UDP-gal formed to that required f o r biosynthetic reactions and thereby prevent the formation of toxic quantities of g a l - l - P i s the central problem of some transferase deficient children. T,

Abbreviations :

glc-l-P : gal-l-P: gal-6-P: UDP-glc: UDP-gal: UTP: ATP: ADP: Pi: PPi:

TT

glucose-l-phosphate galacto se -1 -pho sphate galactose-6-phosphate uridine diphosphate glucose uridine diphosphate galactose uridine triphosphate adenosine triphosphate adenosine diphosphate inorganic phosphate inorganic pyrophosphate

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

118

PHYSIOLOGICAL EFFECTS OF FOOD CARBOHYDRATES

Literature Cited

1. Lengemann, F. W., R. H. Wasserman and C. L. Comar, J. Nutr. (1959) 68:443. 2. Fournier, P., Y. Dupins and A. Fournier, Israel J. Med. Sci. (1971) 7:389. 3. Condon, J. R., J. R. Nassim, F.I.C. Millard, A. Hilbe and Ε. M. Stainthorpe, Lancet (1970) i:1027. 4. Segrest, J. P. and L. W. Cunningham, J. Clin. Invest. (1970) 49:1497. 5. Lundblad, Α., P. Hallgren, A. Rudmark and S. Svensson, Biochem. (1973) 12:3341. 6. Wendorff, W. L., and C. H. Amundson, J. Milk Food Technol. (1971) 34(6):300-306 7. Strom, Roberto, Domenic Turini, Franco Celada and Eraldo Antonini, Eur.J.Biochem. (1971) 23:118-124. 8. Hill, J. Α., and R. E. Huber, Biochim. Biophys. Acta. (1971) 250:530-537. 9. Miller, D., and R. K. Crane, Biochim. Biophys. Acta. (1961) 52:293. 10. Simoons, F. J., Amer. J. Dig. Dis. (1970) 15:695-710. 11. Gilat, T., Gastroenterology (1971) 60:346-347. 12. Cuatrecasas, P., D. H. Lockwood and J. R. Caldwell, Lancet (1965) 1(7375):14-18. 13. Keusch, G. T., F. J. Troncale, L. H. Miller, V. Promadhat and P. R. Anderson, Pediatrics (1969) 43:540-545. 14. Kogut, M. D., G. N. Donnell and Κ. N. Shaw, J. Pediat. (1967) 71:75-81. 15. Flatz, G. and H. W. Rotthauwe, The Lancet (1973) 2:76. 16. Holzel, Α., Proc. Roy. Soc. Med. (1968) 61:1095. 17. Brady, R. O., Advances in Enzymology (1973) 38:293-315. 18. Brady, R. O., Angew. Chem. (1973) 85:28-38. 19. Gander, J. E., W. E. Petersen, P. D. Boyer, Arch. Biochem. Biophys. (1957) 69:85-99. 20. Hansen, R. G., Harland G. Wood, Georges J. Peeters, Birgit Jacobson and JoAnne Wilken, J. Biol. Chem. (1962) 237:10341039. 21. Watkins, W. M. and W. Z. Hassid, J. Biol. Chem. (1962) 237: 1432-1440. 22. Brodbeck, U. and Κ. E. Ebner, J. Biol. Chem. (1966) 241:762. 23. Brodbeck, U., W. L. Denton, N. Tanahashi and Κ. E. Ebner, J. Biol. Chem. (1967) 242:1391-1397.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

6.

HANSEN

AND

GITZELMANN

Lactose

and

Galactose

119

24. Brew, K., T. C. Vanaman and R. L. Hill, Proc. Nat. Acad. Sci., U. S. (1968) 59:491. 25. Morrison, J. F. and Κ. E. Ebner, J. Biol. Chem. (1971) 246: 3992-3998. 26. Brew, K., T. C. Vanaman and R. L. Hill, J. Biol. Chem. (1967) 242:3747-3749. 27. Schmidt, D. V. and Κ. E. Ebner, Biochim. Biophys. Acta (1972) 263:714-720. 28. Clarke, J. T. R., L. S. Wolfe and A. S. Perlin, J. Biol. Chem. (1971) 246(18):5563-5569. 29. Klee, Werner Α., and Claude Β. Klee, J. Biol. Chem. (1972) 247(8):2336-2344. 30. Smith, Ε. Ε. Β., B. Galloway and G. T. Mills, Biochem. Biophys. Res. Comm (1961) 5:148-151 31. Trejo, A. Garcia, J. Baddiley, Biochem. J. (1971) 122:49-57. 32. Hers, H. G., Biochim. Biophys. Acta (1960) 37:120-126. 33. Hayman, S. and J. H. Kinoshita, J. Biol. Chem. (1965) 240:877882. 34. Van Heyningen, R., Exp. Eye Res. (1971) 11:415-428. 35. Cuatrecasas, P. and S. Segal, Science (1966) 153:549-551. 36. Bergren, W. R., W. G. Ng, G. N. Donnell and S. P. Markey, Science (1972) 176:683-684. 37. Gitzelmann, R., H. J. Wells and S. Segal, Europ J. Clin. Invest. (1974) 4:79-84. 38. Wallenfels, K. and G. Kurz, "Methods in Enzymology" Vol. IX. p. 112 (W. A. Wood, ed.) Academic Press, New York, 1966. 39. Avigad, G., D. Amaral, C. Asensio and B. L. Horecker, J. Biol. Chem. (1962) 237:2736-2743. 40. Inouye, T., M. Tannenbaum and D. Y.Y. Hsia, Nature (1962) 193:67-68. 41. Posternak, T. and J. P. Rosselet, Helv. Chim. Acta (1954) 37:246-250. 42. Dahlqvist, A. J. Lab. Clin. Med. (1971) 78(6):931-938. 43. Bissett, D. L. and R. L. Anderson, Biochem. Biophys. Res. Comm. (1973) 52:641. 44. Kosterlitz, H. W., Biochem. J. (1943) 37:318-321. 45. Leloir, L. F., Arch. Biochem. Biophys. (1951) 33:186-190. 46. Caputto, R., H. S. Barra and F. A. Cumar, Ann. Rev. Biochem. (1967) 36:211-246. 47. Ginsburg, V. and E. F. Neufeld, Ann. Rev. Biochem. (1969) 38:371-388. 48. Albrecht, G. J., S. T. Bass, L. L. Seifert and R. G. Hansen, J. Biol. Chem. (1966) 241:2968.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

120

PHYSIOLOGICAL EFFECTS OF FOOD CARBOHYDRATES

49. Levine, S., T. A. Gillett, E. Hageman and R. G. Hansen, J. Biol. Chem. (1969) 244:5729-5734. 50. Turnquist, R. L., T. A. Gillett and R. G. Hansen, J. Biol. Chem. (1974) in press. 51. Isselbacher, K. J., J. Biol. Chem. (1958) 232:429-444. 52. Abraham, H. D. and R. R. Howell, J. Biol. Chem. (1969) 244:545-550. 53. Chacko, C. Μ., L. McCrone and H. L. Nadler, Biochim. Biophys. Acta (1972) 268:113-120. 54. Gitzelmann, R. and B. Steinmann, Helv. paed. Acta (1973) 28:497-510. 55. Bergren, W. R., W. G. Ng and G. Donnell, Biochem. Biophys. Acta (1973) 315:464. 56. Nelsestuen, Gary L. and Samuel Kirkwood, J. Biol. Chem. (1971) 246(24):7533-7543 57. Davis, L. and L. Glaser (1971) 43:1429-1435. 58. Wee, T. G., J. Davis and P. A. Frey, J. Biol. Chem. (1972) 247:1339-1342. 59. Wee, T. G. and P. A. Frey, J. Biol. Chem. (1973) 248:33-40. 60. Maitra, U. S., M. A. Gaunt and H. Ankel, J. Biol. Chem. (1974) 249:3075-3078. 61. Forsander, Olof A., Biochim. Biophys. Acta (1972) 268:253-256. 62. Isselbacher, K. J. and S. M. Krane, J. Biol. Chem. (1961) 236:2394. 63. Kesaniemi, Υ. Α., Κ. Ο. Kurppa and K. R. H. Husman, J. Obs. and Gyn. (1973) 80:344. 64. Segal, S., "The Metabolic Basis of Inherited Disease" (J. B. Stanbury, J. B. Wyngaarden and D. S. Fredrickson, eds), pp. 174-195, McGraw Hill, New York, (1972). 65. Gitzelmann, R. Ped. Res. (1967) 1:14. 66. Beutler, E., F. Matsumoto, W. Kuhl, A. Krill, N. Levy, R. Sparks and M. Degnan, N. Eng. J. Med. (1973) 288:1203. 67. Gitzelmann, R., Helv. Paediat. Acta (1972) 27:125-130. 68. Donnell, G. N., W. R. Bergren, R. K. Bretthauer and R. G. Hansen, Pediatrics (1960) 25:572-581. 69. Schwarz, V., A. R. Wells, A. Holzel, G. M. Komrower, Ann. Hum. Gen. (1961) 25:179. 70. Kirkman, H. N. and E. Bynum, Ann. Hum. Gen. (1958-59) 23:117-126. 71. Mathai, C. K. and E. Beutler, Science (1966) 154:1179-1180. 72. Ng, W. G., W. R. Bergren, M. Fields and G. N. Donnell, Biochem. Biophys. Res. Comm. (1969) 37:451.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

6.

HANSEN

AND

GITZELMANN

Lactose

and

Galactose

121

73. Gitzelmann, R., J. R. Poley and A. Prader, Helv. Paed. Acta (1967) 22:252-257. 74. Hansen, R. G., "Galactosemia" (D. Y. Hsia, ed) p. 55, Charles C. Thomas, Springfield, Ill., (1969). 75. Tedesco, T. A. and W. J. Mellman, Science (1971) 172:727-728. 76. Tedesco, Τ. A., J. Biol. Chem. (1972) 247:6631-6636. 77. Segal, S., A. Blair and H. Roth, Am. J. Med. (1965) 38:62. 78. Schapira, F. and J. C. Kaplan, Biochem. Biophys. Res. Comm. (1969) 35:451. 79. Chacko, C. Μ., J. C. Christian, and H. L. Nadler, J. Ped. (1971) 78:454. 80. Ng, W. G., W. R. Bergren and G. N. Donnell. Ann. Hum. Genet. (1973) 37:1. 81. Sidbury, J. B. Jr. "Galactosemia" (D Y Hsia ed) p 13 Charles C. Thomas 82. Oliver, I. Τ., Biochim. Biophys. Acta (1961) 52:75-81. 83. Segal, S. and S. Rogers, Biochim. Biophys. Acta (1971) 250: 351-360. 84. Gitzelmann, R., Pediat. Res. (1969) 3:279-286. 85. Knop, J. K. and R. G. Hansen, J. Biol. Chem. (1970) 245:24992504. 86. Ting, W. K. and R. G. Hansen, Proc. Soc. Exp. Biol. Med. (1968) 127:960-962. 87. Turnquist, R. L., M. M. Turnquist, R. C. Bachmann and R. G. Hansen, Biochim. Biophys. Acta (1974) 364:59-67. 88. Gitzelmann, R. and R. G. Hansen, Biochim. Biophys. Acta (1974) 372:374-378. 89. Lehninger, A. L., "Bioenergetics, " p. 183, Benjamin, New York (1965). 90. Schwenn, J. D., R. M. Lilley and D. A. Walker, Biochim. Biophys. Acta (1973) 325:586-595. 91. Levine, G. and J. A. Bassham, Biochim. Biophys. Acta (1974) 333:136-140. 92. Russell, R. G. G., S. Bisaz, H. Fleisch, H. L. F. Currey, H. M. Rubinstein, A. A. Dietz, J. Boussina, A. Micheli and G. Fallet, Lancet (1970) II:899-902. 93. Gitzelmann, R., B. Steinmann, R. G. Hansen, in press (1974). 94. Mayes, J. S. and L. R. Miller, Biochim. Biophys. Acta (1973) 313:9-16. 95. Petricciani, J. C., K. Binder, C. R. Merril and M. R. Geier, Science (1972) 175:1368. 96. Roe, T. F., J. G. Hallatt, G. N. Donnell and W. G. Ng, J. Pediat. (1971) 78:1026-1030.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

122

PHYSIOLOGICAL EFFECTS OF FOOD CARBOHYDRATES

97. Olambiwonnu, N. O., R. McVie, W. G. Ng, S. D. Frasier and G. N. Donnell, Pediatrics (1974) 53:314-318. 98. Roe, T. F., W. G. Ng, W. R. Bergren and G. N. Donnell, Biochem. Med. (1973) 7:266-273. 99. Donnell, G. Ν., R. Koch and W. R. Bergren, "Galactosemia, " (D. Y. Y. Hsia, ed) p. 247, Charles C. Thomas, Springfield, Ill. (1969). 100. Komrower, G. M. and D. H. Lee, Arch. Dis. Child. (1970) 45:367-373. 101. Fishier, K., G. N. Donnell, W. R. Bergren and R. Koch, Pediatrics (1972) 50:412-419. 102. Gitzelmann, R. and R. G. Hansen, Abstr. Europ. Soc. Ped. Res., Ann. Meeting, Sevilla (1973); Pediat. Res. (1974) 8:137.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

7 Metabolism and Physiological Effects of the Polyols (Alditols) OSCAR TOUSTER Department of Molecular Biology, Vanderbilt University, Nashville, Tenn. 37235

I. Introduction Polyols, or alditols, are of course not strictly carbohydrates, but since they are produced chemically and biologically from sugars, we can consider them honorary carbohydrates. They are now established in mammalian metabolism and are of some importance in nutrition and pathology. In this paper I survey the roles of polyols and emphasize some of the most pertinent current uses and problems. II. Survey of the Occurrence and Enzymology of the Polyols The occurrence of polyols in animals is summarized in Table I. TABLE I. POLYOLS IN ANIMAL METABOLISM Occurrence in tissues Sorbitol—fetal blood, seminal vesicles and plasma, nerve, lens of alloxan-diabetic rats or rats given cataractogenic dose of D-xylose Xylitol—lens of rats given cataractogenic dose of D-xylose Dulcitol—various tissues after cataractogenic dose of D-galactose Occurrence in urine Erythritol D-arabitol, L-arabitol Sorbitol D-mannitol Utilization by mammals in vivo High--xylitol, ribitol, sorbitol Variable--D-mannitol (oral dose moderately utilized; parenteral--poorly) Poor--arabitol (D andL),dulcitol 123 In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

124

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

S o r b i t o l i s most widely d i s t r i b u t e d , o c c u r r i n g in fetal blood and in seminal v e s i c l e s and plasma, where the p o l y o l is un­ doubtedly a normal metabolic intermediate. S o r b i t o l accumulates i n some t i s s u e s of d i a b e t i c animals, a matter which is discussed i n greater d e t a i l below. X y l i t o l and, to a smaller extent, sor­ bitol accumulate in the lens of r a t s given c a t a r a c t o g e n i c doses of Πτ-xylose (1). D u l c i t o l , or galactitol, accumulates a f t e r c a t ­ aractogenic doses of D-galactose, and it is a l s o found i n the human genetic d i s e a s e , galactosemia. Several p o l y o l s have been i s o l a t e d from human u r i n e : e r y t h r i t o l (2), a r a b i t o l (3), mannitol and s o r b i t o l (4,5). The utilization of p o l y o l s by mammals is a l s o i n d i c a t e d i n Table I, with the wide range of utilization i n d i c a t e d . The most a p p r e c i a b l y used p o l y o l s are xylitol, ribitol, and s o r b i t o l . The main biochemical r e a c t i o n s of p o l y o l s a r e shown below:

Enzymati General

Polyol

Mammals a

L-Xylulose

D-Glucose ^

b

s

s

xylitol

sorbitol

a

a

s

D-xylulose

*D-fructose

P o l y o l s undergo o x i d a t i o n to the corresponding ketose (a) or to the corresponding aldose ( b ) , or phosphorylation to the p o l y o l 1-phosphate ( c ) . Many examples of r e a c t i o n s (a) and (b) a r e found i n mammals. The L - x y l u l o s e - x y l i t o l - D ^ x y l u l o s e i n t e r c o n v e r ­ s i o n occurs i n the g l u c u r o n a t e - x y l u l o s e c y c l e . The glucoses o r b i t o l - f r u c t o s e i n t e r c o n v e r s i o n occurs i n male accessory organs and undoubtedly i n other t i s s u e s as w e l l . However, the phos­ p h o r y l a t i o n of p o l y o l s occurs i n microorganisms but not i n mam­ mals. A review on p o l y o l s w i l l supply the reader with a d d i t i o n a l information i n t h i s biochemical area ( 6 ) .

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

7.

III.

125

Polyols (Alditols)

TOUSTER

X y l i t o l i n Metabolism and N u t r i t i o n

Many years ago, when we were i n v e s t i g a t i n g the metabolism of L r x y l u l o s e , the sugar excreted i n gram q u a n t i t i e s by humans w i t h the genetic metabolic abnormality known as e s s e n t i a l p e n t o s u r i a , we discovered that ^ - x y l u l o s e i s normally u t i l i z e d as shown i n the f o l l o w i n g equations:

CH.OH \

CH.OH I

2

0 0

1

HCOH I HOCH » CH OH

NADPH

ΝADP

1

HOCH I HCOH ο I HOCH CH OH

L-Xylulose

CH 0H I HCOH I HOCH I

NAD

NADH

HCOH I CH OH

D-Xylulose

Xylitol CH 0H I HCOH o 2

I

CH 0H ! C=0 I HOCH I HCOH I CH OH o 2

o 1

CH 0H I HCOH o 2

\

HCOH I

HOCH I

CH OH

HOCH I HOCH I CH OH

L-Arabitol

The J±-xylulose i s reduced t o x y l i t o l by an u n u s u a l l y s p e c i f i c NADPH-linked p o l y o l dehydrogenase (7,8) . The x y l i t o l i s then r e o x i d i z e d t o D-xylulose by an NAD-linked enzyme of r a t h e r broad but d e f i n i t e s p e c i f i c i t y . This enzyme i s most commonly known as s o r ­ b i t o l dehydrogenase. P e n t o s u r i c i n d i v i d u a l s excrete smaller amounts o f L - a r a b i t o l , probably as a " d e t o x i c a t i o n " product of the accumulated L - x y l u l o s e . The s u b s t r a t e s p e c i f i c i t y of s o r b i t o l dehydrogenase, which i s b e t t e r expressed by the name, J v - i d i t o l dehydrogenase, i s shown below: S o r b i t o l dehydrogenase i s s p e c i f i c f o r erythro-1,2,4-polyol c o n f i g u r a t i o n .

CH OH 2

hOH hOH

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

126

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

As can be seen from the formula on the r i g h t , s o r b i t o l , x y l i t o l , and r i b i t o l are good s u b s t r a t e s . The low l e v e l of o x i d a t i o n of L - a r a b i t o l by t h i s enzyme even when h i g h l y p u r i f i e d has not been explained. The work on L - x y l u l o s e metabolism i n our l a b o r a t o r y and of King, Burns and others on L-ascorbic a c i d b i o s y n t h e s i s l e d to the formulation of the glucuronate-xylulose c y c l e (9,10), which i s shown i n F i g u r e 1. This c y c l e occurs i n a l l mammals i n which i t has been studied, the r e a c t i o n s g e n e r a l l y o c c u r r i n g i n the l i v e r and i n the kidney. The f u n c t i o n of the c y c l e i s not completely understood. Since an i n a b i l i t y to c a r r y out the r e d u c t i o n of J±x y l u l o s e to x y l i t o l i s apparently not d e l e t e r i o u s , pentosuria i s an i n h e r i t e d metabolic d i s o r d e r (11,12), not a metabolic d i s e a s e . The e a r l y r e a c t i o n s i n the c y c l e are obviously important. I t appears that most higher animals b i o s y n t h e s i z e L r a s c o r b i c a c i d , although primates, the guinea p i g f l y i n g mammals and i n s e c t s (13) do not. I t has bee the guinea p i g l a c k th L-gulonolactone to the keto d e r i v a t i v e . I would a l s o point out the numerous o x i d a t i o n - r e d u c t i o n s using p y r i d i n e n u c l e o t i d e l i n k e d coenzymes which e f f e c t i v e l y move hydrogen from NADPH to NAD, thereby p o s s i b l y serving a transhydrogenase f u n c t i o n . I should a l s o mention that although i t i s accepted that UDP-glucose and UDP-glucuronate are e a r l y members of the c y c l e , and obviously serve as precursors of sugar moieties i n glyco-polymers, there i s some u n c e r t a i n t y as to how the f r e e glucuronate i s produced. Since, i n the conversion of glucuronate to I±-xylulose v i a L-gulonate, there occurs a decarboxylation of the C5 carbon of glucuronate, t h i s carbon being the same as of glucose, t h i s c y c l e can be considered a o x i d a t i o n pathway f o r glucose. It may then be asked whether much glucose i s o x i d i z e d normally through t h i s pathway. S p e c i f i c studies i n d i c a t e that only a small amount of glucose i s handled v i a t h i s route. From the f a c t that pentosuric i n d i v i d u a l s normally excrete s e v e r a l grams of Lr x y l u l o s e each day and from experiments on the extent of augmenta­ t i o n of t h i s e x c r e t i o n on feeding the precursor Ιλ-glucuronolactone, i t can be estimated that the carbohydrate f l u x through the c y c l e i s between 5 and 15 grams per day (14). I t was t h e r e f o r e of some i n t e r e s t that Winegrad and h i s a s s o c i a t e s reported s e v e r a l years ago that the o x i d a t i o n of glucose i s enhanced i n a l l o x a n - d i a b e t i c r a t s (15) and that L r x y l u l o s e l e v e l s i n the serum of d i a b e t i c humans are s e v e r a l times higher than normal (16). These f i n d i n g s are not w e l l understood. I t does appear, however, that i n diabetes the s o - c a l l e d i n s u l i n dependent pathways are more prominently employed and that more glucose appears to be o x i d i z e d v i a glucuronate and v i a s o r b i t o l , as w i l l be discussed i n more d e t a i l below. The u t i l i z a t i o n of p o l y o l s became of s p e c i a l i n t e r e s t when the metabolic importance of s o r b i t o l and x y l i t o l was discovered. The high conversion of an administered p o l y o l to l i v e r glycogen

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

7.

TOUSTER

127

Poly oh (Alditols)

L- Ascorbic Acid - Keto-L-gulonoloctone L- Gulonolactone

?

H0 :OH OH

ÇH OH 2

(SOT

I

HOCH I HCOH I

HOCH HCOH I

UDP- glucuronote

HCOH

0-Glucuronate

Ο

Ç0 H 2

HOÇH HOC H HCOH I

[NAD]

L- Gulonote

C0 H 2

HOCH I

?-°

HCOH

UOP-glucose

HOCH -Keto-LCH OHgulonote 2

Glycogen-

• C0

^Hexose phosphate ( pentose ^\ phosphate \V pathway) \ *

2

C-0 HCOH

CH 0H

HOCH CH OH

2

C-0 HOCH I HCOH I CH OP03H 2

2

CH OH

2

L-Xylulose 4L 1NADPH| 2

CH 0H

D-Xylulose \ ^ 5-phosphate

2

i-o

A O P ^ ^ HOCH A

T

P

HCOH

C H OH


2

;

HOCH

HCOH to)

HOCH

HCOH Hué H

HioH CH OH

CHgOH j

2

y Xylitol

Figure 2. The glucuronic acid-xylulose cycle

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

128

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

and i t s considerable o x i d a t i o n to CO2 are of course i n d i c a t i o n s of e f f i c i e n t u t i l i z a t i o n . The data i n Table I I show that x y l i t o l and r i b i t o l are w e l l metabolized to CO2 and glycogen, whereas Da r a b i t o l i s not. L - A r a b i t o l i s poorly u t i l i z e d and shows a p e c u l i a r discrepancy between the low l e v e l of i n c o r p o r a t i o n of l a b e l i n t o l i v e r glycogen and a moderately good o x i d a t i o n to CO2. X y l i t o l u t i l i z a t i o n involves i t s conversion to D-xylulose. Ribit o l i s s i m i l a r l y converted to D - r i b u l o s e and then probably phosphorylated to D - r i b u l o s e 5-phosphate. X y l i t o l has been very widely studied i n the l a s t decade as a s u b s t i t u t e f o r glucose i n p a r e n t e r a l n u t r i t i o n . In animal studi e s i t was demonstrated that the a d m i n i s t r a t i o n of x y l i t o l does not increase blood glucose n e a r l y as much as glucose i t s e l f does, a point of importance i n the use of x y l i t o l f o r the treatment of d i a b e t i c s . I t was a l s o observed that the a d m i n i s t r a t i o n of x y l i t o l causes an increase i n plasma i n s u l i n f a r i n excess of that induced by glucose. However f i n d i n g does not hold f o The b a s i s f o r the current use of x y l i t o l i n n u t r i t i o n and medicine may be summarized as f o l l o w s : a) I t i s a normal metabol i t e , b) i t has a sweet t a s t e , c) i t i s inexpensive, d) i t does not undergo the M a i l l a r d r e a c t i o n when autoclaved with amino a c i d s or p r o t e i n hydrolysates, e) i t i s g e n e r a l l y considered to be non-toxic, f ) i t i s u t i l i z e d i n l a r g e doses (at l e a s t s e v e r a l hundred grams per day by man), and g) i t s u t i l i z a t i o n i s i n s u l i n independent. Some of these advantages were f i r s t pointed out by Konrad Lang; three symposium volumes may be consulted i n connect i o n with the development of x y l i t o l i n p a r e n t e r a l n u t r i t i o n (18, 19^, 20) . These d e s i r a b l e c h a r a c t e r i s t i c s of x y l i t o l have l e d to s i g n i f i c a n t pharmaceutical developments i n c o u n t r i e s other than the United S t a t e s . Intravenous x y l i t o l i s w e l l u t i l i z e d and i s an important c l i n i c a l agent i n Japan and, to some extent, i n Germany. High doses of x y l i t o l cause uricemia and b i l i r u b i n e m i a , but s i n c e t h i s e f f e c t i s a l s o shown by f r u c t o s e and s o r b i t o l , F b r s t e r (21) does not consider i t a p a r t i c u l a r c h a r a c t e r i s t i c of x y l i t o l administration. For a d d i t i o n a l references, and f u r t h e r d i s c u s s i o n of the value of x y l i t o l i n p a r e n t e r a l n u t r i t i o n , the reader i s r e f e r r e d to s e v e r a l recent reviews (21,22,23,24,25). An important i n f l u e n c e of the a c c e p t a b i l i t y of x y l i t o l has been a report from A u s t r a l i a on a number of deaths i n c u r r e d by p a t i e n t s who had been given x y l i t o l (24,26). I t has been pointed out, however, that the dosage l e v e l s exceeded those g e n e r a l l y employed, that a contaminant may have been present, and that the h i s t o p a t h o l o g i c a l observations were due to the types of p a t i e n t s involved (27). U n t i l t h i s matter i s r e s o l v e d , the use of x y l i t o l i n America i s l i k e l y to continue to be severely r e s t r i c t e d . O r a l x y l i t o l has been studied i n animals and man, with some evidence r e s u l t i n g to suggest that i t reduces dental c a r i e s (27, 28,29).

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

McCormick and Touster (17)

b

Guinea P i g Rat Rat

L-Arabitol

McCormick and Touster (9)

Rat or guinea P i g

D-Arabitol

a

31.4

Guinea Pig Rat

Ribitol

13.3

10

Rat Guinea P i g Guinea Pig

Xylitol

Oxid. to C0

Species

Pentitol 0

37.1

29.9

25

In u r i n e

Liver

3.1 0.95

2 2 24

2 24

19.75

0.28

2 2 24

Time (Hrs.)

11.8 23.8

glycogen

% of Administered Tracer Dose

TABLE I I . UTILIZATION OF FREE PENTITOLS BY THE RAT AND GUINEA PIG

130

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

I should mention that although x y l i t o l a b s o r p t i o n from the i n t e s t i n a l t r a c t i s slow and i n l a r g e amounts causes osmotic d i a r r h e a , there i s a r a p i d adaptation by humans which might m i n i ­ mize t h i s problem with o r a l x y l i t o l (27,30). IV.

S o r b i t o l and Other H e x i t o l s

From the work of Hers (31,32)> the major route of u t i l i z a ­ t i o n of administered sorbitoI^sHknown:

Route of U t i l i z a t i o n of S o r b i t o l and Fructose CH 0(f) 2

CH 0H

CH 0H

o

2

C=0 I

HCOH ι HOCH ι HCOH

HOC

-> F - l - P

HCOH

I

I

CHO I

HCOH

HCOH ι CH 0H

I

HCOH _ I

CH OH

2

CH 0H 2

Sorbitol

CHO I

CHOH C H O ®

D-Fructose

S o r b i t o l i s o x i d i z e d to f r u c t o s e , which i s then phosphorylated to fructose-l-phosphate followed by cleavage to dihydroxyacetone phosphate and glyceraldehyde. The l a t t e r i s then phosphorylated to the 3-phosphate. Both t r i o s e phosphates are members of the Embden-Meyerhof g l y c o l y t i c pathway. Gabbay (33) has r e c e n t l y observed another metabolic i n t e r ­ r e l a t i o n s h i p between f r u c t o s e and h e x i t o l s . Dietary fructose i s converted, i n about 3% y i e l d , t o u r i n a r y ^-mannitol. He has presented evidence that the enzyme aldose reductase s u r p r i s i n g l y has the c a p a c i t y to c a t a l y z e t h i s r e d u c t i o n of f r u c t o s e to mannitol. S o r b i t o l i s i n f a c t an important metabolic intermediate as shown below:

n

n

1

NADPH. _ , , -. NAD Sorbitol

v

D-Glucose

v

(aldose reductase)

^-Fructose

(sorbitol dehydrogenase)

The s o r b i t o l pathway (34,35) i n v o l v e s the conversion of glucose to s o r b i t o l through the mediation of the enzyme aldose reductase,

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

7.

TOUSTER

Polyols (Alditols)

131

with NADPH as coenzyme. S o r b i t o l i s o x i d i z e d to D-fructose by s o r b i t o l dehydrogenase. There i s some evidence that t h i s pathway of glucose u t i l i z a t i o n , as w e l l as the g l u c u r o n i c - x y l u l o s e c y c l e , are somewhat more e x t e n s i v e l y u t i l i z e d during i n s u l i n d e f i c i e n c y (35). Should glucose enter c e l l s i n higher amount than usual and be converted to s o r b i t o l and f r u c t o s e , which are p o o r l y permeable, osmotic changes may occur which are u n d e s i r a b l e . The evidence i s strong that t h i s sequence of events i s involved i n the production of d u l c i t o l from galactose i n the lens and the formation of c a t a r a c t i n i n d i v i d u a l s with galactosemia (1,36). Gabbay (37) has emphasized that p o l y o l accumulation i n nerve may be r e l a t e d to the occurrence of d i a b e t i c neuropathy. For example, galactosemia causes increases i n nerve d u l c i t o l and water content, and decreases the motor nerve conduction v e l o c i t y . If the animals are then placed on a normal d i e t , the d u l c i t o l and water content decrease d th conductio v e l o c i t i r e s t o r e d to normal. Moreover the r a t causes an increas glucose i n p e r i p h e r a l nerve. A decrease i n the nerve conduction v e l o c i t y occurs c o n c u r r e n t l y . In an attempt to modify these events, Gabbay, Dvornik, and t h e i r a s s o c i a t e s have been studying s y n t h e t i c i n h i b i t o r s of aldose reductase as p o s s i b l e agents f o r b l o c k i n g p o l y o l formation i n animals. A recent report demons t r a t e s that the i n h i b i t o r AY22284 reduces the accumulation of g a l a c t i t o l i n both the lens and s c i a t i c nerve of galactosemic r a t s , g r e a t l y reduces and delays the formation of d e t e c t a b l e c a t a r a c t s i n g a l a c t o s e - f e d r a t s , and reduces the accumulation of s o r b i t o l and of f r u c t o s e , but not of glucose, i n the s c i a t i c nerve of a r t i f i c i a l l y - d i a b e t i c r a t s (38) . Of course, l i t t l e work has been done on the human i n t h i s area, and there w i l l be continued d i f f i c u l t i e s i n doing such i n v e s t i g a t i o n s on human d i a b e t i c s . A cause and e f f e c t r e l a t i o n s h i p between p o l y o l accumulation i n nerve and decrease i n f u n c t i o n has not been d e f i n i t e l y establ i s h e d even i n experimental animals, and even i f t h i s were to be accomplished, i t would s t i l l remain to be determined whether d i a b e t i c neuropathy i n the human has the same cause. The moderate, and dose dependent, u t i l i z a t i o n of o r a l l y administered mannitol, and the e f f e c t s of t h i s h e x i t o l on i n t e s t i n a l f u n c t i o n , have been e x t e n s i v e l y studied (39) . The use of mannitol as a d i u r e t i c agent has r e c e n t l y been reviewed (40). V.

M a l t i t o l and

Isomaltitol

M a l t i t o l has been studied r e c e n t l y at Charles P f i z e r and Company because of r e p o r t s that t h i s p o l y o l i s low c a l o r i c and t h e r e f o r e might be a superior sweetening agent (41 ,42) . Dr. H. H. Rennard of P f i z e r has informed me of h i s work, some of which was done i n c o l l a b o r a t i o n with Dr. J. R. Bianchine of Johns Hopkins. The low recovery of administered l a b e l e d m a l t i t o l i n the u r i n e and feces of the r a t i n d i c a t e d that i t was e f f i c i e n t l y

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

132

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

u t i l i z e d , and s i m i l a r l y encouraging r e s u l t s were obtained i n the human, i n c l u d i n g high recovery of -^C i n expired CO2. However, the f a c t that the expired 0 ^0 peak i n r a t s occurred a t 4 t o 5 hours a f t e r feeding suggested that the l a b e l might have been ab­ sorbed i n the lower gut. I t was indeed found that the m a l t i t o l was being u t i l i z e d by i n t e s t i n a l m i c r o f l o r a , which apparently were converting the l a b e l e d p o l y o l i n t o v o l a t i l e f a t t y a c i d s . In a d d i t i o n , i n t e s t i n a l mucosal preparations of r a t s have a low c a p a c i t y to hydrolyze m a l t i t o l . Therefore, although the u t i l i z a ­ t i o n of the m a l t i t o l i s i n d i r e c t , i n v o l v i n g i t s p r e l i m i n a r y con­ v e r s i o n to f a t t y a c i d s , t h i s p o l y o l i s considered by Rennard t o be a w e l l u t i l i z e d substance. The u t i l i z a t i o n of i s o m a l t i t o l has a l s o been i n v e s t i g a t e d . From studies on r a t s i t was suggested that i t could be a u s e f u l i n g r e d i e n t i n c a l o r i e - r e d u c e d foods and beverages (43). 1Ζ

2

VI.

Concluding Remark

At the present time s o r b i t o l and x y l i t o l are the most impor­ tant a l d i t o l s i n that they are key metabolic intermediates and are being fed or administered t o humans i n considerable amounts. As f a r as the f u t u r e i s concerned, i t appears to me that the problems which w i l l most a c t i v e l y concern i n v e s t i g a t o r s are r e l a t e d t o the more general use of x y l i t o l , f u r t h e r i n v e s t i g a t i o n of the p o s s i b l e r o l e of s o r b i t o l i n diabetes, and the p o s s i b l e advantages of p o l y o l s i n decreasing d e n t a l c a r i e s . Literature Cited

1. van Heyningen, R., in "Proceedings of the International Sym­ posium on Metabolism, Physiology, and Clinical Use of Pentoses and Pentitols," Hakone, Japan, August 27-29, 1967 (B. L. Horecker, K. Lang, and Y. Takagi, eds.), pp. 109123, Springer-Verlag, Berlin, Heidelberg, New York (1969). 2. Touster, O., Fed. Proc. (1960) 19, 977-983. 3. Touster, O. and Harwell, S., J. Biol. Chem. (1958) 230, 1031-1041. 4. Pitkänen, E. and Pitkänen, Α., Ann. Med. Exp. Fenn. (1964) 42, 113-116. 5. Ingram, P., Applegarth, D. Α., Sturrock, S., and Whyte,J.N. C., Clin. Chim. Acta (1971) 35, 523-524. 6. Touster, O. and Shaw, D. R. D., Physiol. Rev. (1962) 42, 181225. 7. Hollmann, S. and Touster, O., J. Biol. Chem. (1957) 225, 87102. 8. Arsenis, C. and Touster, O., J. Biol. Chem. (1969) 244, 38953899. 9. McCormick, D. B. and Touster, O., J. Biol. Chem. (1957) 229, 451-461.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

7.

TOUSTER

Polyols

(Alditols)

133

10. Burns, J. J. and Kanfer, J., J. Am. Chem. Soc. (1957) 79, 3604-3605. 11. Touster, O., Fed. Proc. (1960) 19, 977-983. 12. Hiatt, H. H., in "The Metabolic Basis of Inherited Disease" (J. B. Stanbury, J. B. Wyngaarden, and D. S. Fredrickson, eds.), third ed., pp. 119-130, McGraw-Hill, New York (1972). 13. Gupta, S. D., Chaudhuri, C. R., and Chatterjee, I. B., Arch. Biochem. Biophys. (1972) 152, 889-890. 14. Hollmann, S. and Touster, O., "Non-glycolytic Pathways of the Metabolism of Glucose," p. 107, Academic Press, New York (1964). 15. Winegrad, A. I. and Shaw, W. Ν., Am. J. Physiol. (1964) 206, 165-168. 16. Winegrad, A. I. and Burden, C. L., New Engl. J. Med. (1966) 274, 298-305. 17. McCormick, D. B. and Touster O Biochim Biophys Act (1961) 54, 598-600 18. Horecker, B. L., Lang, Κ., and Takagi, Y., eds., "Proceed ings of the International Symposium on Metabolism, Physiology, and Clinical Use of Pentoses and Pentitols," Hakone, Japan, August 27-29, 1967, Springer-Verlag, Berlin, Heidelberg, New York (1969). 19. Lang, K. and Fekl, W., Z. Ernährungswissenschaft (1971) Suppl. 11. 20. Sipple, H. L. and McNutt, K. W., eds., "Sugars in Nutrition," Academic Press, New York, San Francisco, London (1974). 21. Förster, H., in "Sugars in Nutrition" (H. L. Sipple and K. W. McNutt, eds.), pp. 259-280, Academic Press, New York, San Francisco, London (1974). 22. Froesch, E. R. and Jakob, Α., in "Sugars in Nutrition" (H. L. Sipple and K. W. McNutt, eds.), pp. 241-258, Academic Press, New York, San Francisco, London (1974). 23. Meng, H. C., in "Sugars in Nutrition" (H. L. Sipple and K. W. McNutt, eds.), pp. 527-566, Academic Press, New York, San Francisco, London (1974). 24. Thomas, D. W., Edwards, J. B., and Edwards, R. G., in "Sugars in Nutrition" (H. L. Sipple and K. W. McNutt, eds.), pp. 567-590, Academic Press, New York, San Fran­ cisco, London (1974). 25. van Eys, J., Wang, Y. M., Chan, S., Tanphaichitr, V. S., and King, S. M., in "Sugars in Nutrition" (H. L. Sipple and K. W. McNutt, eds.), pp. 613-631, Academic Press, New York, San Francisco, London (1974). 26. Thomas, D. W., Edwards, J. B., Gilligan, J. E., Lawrence, J. R., and Edwards, R. G., Med. J. Australia (1972) 1, 12381246. 27. Brin, M. and Miller, O. N., in "Sugars in Nutrition" (H. L. Sipple and K. W. McNutt, eds.), pp. 591-606, Academic Press, New York, San Francisco, London (1974).

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

134

PHYSIOLOGICAL EFFECTS OF FOOD CARBOHYDRATES

28. Grunberg, E., Beskid, G., and Brin, M., Int. J. Vitamin and Nutrition Res. (1973) 43, 227-232. 29. Mäkinen, Κ. Κ., in "Sugars in Nutrition" (H. L. Sipple and K. W. McNutt, eds.), pp. 645-687, Academic Press, New York, San Francisco, London (1974). 30. Bässler, Κ. H., in "Proceedings of the International Sympo­ sium on Metabolism, Physiology, and Clinical Use of Pentoses and Pentitols," Hakone, Japan, August 27-29, 1967 (B. L. Horecker, K. Lang, and Y. Takagi, eds.), pp. 190-196, Springer-Verlag, Berlin, Heidelberg, New York (1969). 31. Hers, H. G., Biochim. Biophys. Acta (1960) 37, 127-138. 32. Hue, L. and Hers, H.-G., Eur. J. Biochem. (1972) 29, 268275. 33. Gabbay, Κ. H., Clin. Research (1974) XXII (3), 468A. 34. Hers, H. G., Biochim. Biophys. Acta (1956) 22, 202-203. 35. Winegrad, A. I., Clements "Handbook of Physiology, ed.), Sect. 7, Vol. I, pp. 457-471, Williams & Wilkins, Baltimore, Maryland (1972). 36. Kinoshita, J. H., Invest. Ophthalmol. (1965) 4, 786-799. 37. Gabbay, Κ. H., New Engl. J. Med. (1973) 288, 831-836. 38. Dvornik, D., Simard-Duquesne, N., Krami, M., Sestanj, Κ., Gabbay, Κ. H., Kinoshita, J. H., Varma, S. D., and Merola, L. O., Science (1973) 182, 1146-1147. 39. Nasrallah, S. M. and Iber, F. L., Am. J. Med. Sci. (1969) 258, 80-88. 40. Ginn, Η. Ε., in "Sugars in Nutrition" (H. L. Sipple and K. W. McNutt, eds.), pp. 607-612, Academic Press, New York, San Francisco, London (1974). 41. Naito, F., New Food Industry (1971) 13, 1. 42. Hosoya, Ν., Ninth Internat. Congr. Nutrition (1972) Mexico. 43. Musch, K., Siebert, G., Schiweck, H., and Steinle, G., Z. Ernährungswissenschaft (1973), Suppl. 15, pp. 3-16.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

8 Metabolism and Physiological Effects of the Pentoses and Uronic Acids OSCAR TOUSTER Department of Molecular Biology, Vanderbilt University, Nashville, Tenn. 37235

I. Introduction The importance of the pentoses needs little comment, since they are so well known to be components of DNA, RNA, coenzymes, ATP, and proteoglycans. Moreover, pentose phosphates are impor­ tant metabolic intermediates in such processes as CO fixation in photosynthesis. Similarly, uronic acids are components of proteo­ glycans, or mucopolysaccharides, and of metabolic pathways, including those leading to the pentoses. These structural and metabolic aspects will be briefly reviewed, as well as the utili­ zation of these substances and relevant pathological considera­ tions. 2

II.

The Physiologically Important Pentoses

Table I lists pentoses which are important in mammalian metabolism. TABLE I. PHYSIOLOGICALLY-IMPORTANT PENTOSES D-Ribose - in nucleotides, pentose phosphate pathway D-2-Deoxyribose - in deoxyribonucleotides D-Ribulose - in pentose phosphate pathway; Ru-1,5-DP is CO accep­ tor in photosynthesis 2

D-Xylulose - in pentose phosphate pathway, in glucuronic acidxylulose cycle L-Xylulose - in glucuronic acid-xylulose cycle, excreted in gram quantities by humans with essential pentosuria D-Xylose - in gal-gal-xyl linkage of mucopolysaccharide to protein 135 In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

136

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

D-Ribose of course is a key component of RNA and other r i b o n u c l e o t i d e s , and D-ribose 5-phosphate is a member of the pentose phosphate pathway of carbohydrate metabolism. D-2-Deoxyribose is a component of the n u c l e o t i d e s in DNA. D-Ribulose, as the 5phosphate, i s an intermediate in the pentose phosphate pathway and, as the 1,5-diphosphate, i s the CO acceptor in photosynthesis. D-Xylulose as the 5-phosphate d e r i v a t i v e is an intermediate i n the pentose phosphate pathway and occurs as the f r e e sugar in the g l u c u r o n a t e - x y l u l o s e c y c l e , as described in the preceding paper i n t h i s symposium. L - X y l u l o s e is a l s o a member of the c y c l e and is noteworthy because of its rather l a r g e e x c r e t i o n by humans with the genetic metabolic d i s o r d e r known as e s s e n t i a l p e n t o s u r i a . In proteoglycans (mucopolysaccharides) D-xylose is the sugar r e s i d u e attached to s e r i n e i n the bridge l i n k i n g the polymeric carbohydrate to the p r o t e i n core. The s t r u c t u r e of the g a l a c t o s e - g a l a c t o s e - x y l o s e bridge was mainly worked out through the work of Lennart Rodé occur i n nature and a r i n Table I are of the g r e a t e s t importance to mammals in t h e i r normal metabolism. 2

III.

U t i l i z a t i o n of Pentoses

14 Table I I summarizes some experiments i n which C-labeled pentoses were administered to animals and man and t h e i r u t i l i z a t i o n estimated. Examination of the l i v e r glycogen column shows that r i b o s e i s u t i l i z e d as e f f e c t i v e l y as glucose. I t should be borne i n mind, however, that s i n c e these a r e t r a c e r q u a n t i t i e s of pentose, i t need not be t r u e that equivalent but l a r g e amounts of pentose would be u t i l i z e d as w e l l as glucose. In man D-xylose, D-ribose and D-lyxose are o x i d i z e d t o C0 to a moderate extent, a f i n d i n g c o n s i s t e n t with the f a c t that most of the l a b e l which appears i n the u r i n e occurs i n a form that i s not the administered sugar. In these experiments the sugars were infused i n human subjects over a 15-minute p e r i o d . The C 0 and l i v e r g l y c o gen experiments i n d i c a t e that L-arabinose i s u t i l i z e d only to a n e g l i g i b l e extent. Although D-xylose may be isomerized to D-xylulose by plant and b a c t e r i a l enzymes, i n mammals t h i s aldopentose i s e i t h e r reduced to x y l i t o l , an intermediate i n the g l u c u r o n a t e - x y l u l o s e c y c l e (7), or o x i d i z e d to f^-xylonic a c i d by an NAD-linked Dxylose dehydrogenase detected i n c a l f lens by Van Heyningen (8). fJ-Ribose i s converted to r i b o s e 5-phosphate, an intermediate i n the pentose phosphate pathway. The route of u t i l i z a t i o n of D-lyxose has not been e s t a b l i s h e d ; perhaps i t i s isomerized to D-xylulose. 2

2

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

Mouse Man Rat

Mouse Man Guinea p i g

Mouse Man

Mouse Man

Man

JL-Ribose

2-Xylose

D-Arabinose

L.-Arabinose

D-Lyxose

(Table adapted

-

-

-

-0.8 14

19

35 75

72

85

57

6-24

-

-

3 6-24

3 24 0

-

0.03

-

1.0

3 6-24 24

7.1

3 6-24 2

10.0

10

Time (Hrs.)

3

glycogen

8.3

Liver

-

16 15

In u r i n e

-

2

48 --

Oxid. to C 0

% of Administered Tracer Dose

UTILIZATION OF FREE PENTOSES BY MAMMALS

from Hollmann and Reinauer (6))

H i a t t (2) Segal and F o l e y (3) McCormick and Touster (4) McCormick and Touster (5)

Mouse

D-Glucose

a b c d

Species

Pentitose

TABLE I I .

b

a b

a b

a b d

a b c

a

Ref

138

IV.

PHYSIOLOGICAL

EFFECTS

Pentoses and D-Glucuronic A c i d as Metabolic

OF

FOOD

CARBOHYDRATES

Intermediates

The pentose phosphate pathway f o r glucose u t i l i z a t i o n i s shown i n F i g u r e 1. The main feature of t h i s pathway f o r glucose o x i d a t i o n i s that 3 molecules of glucose phosphate are o x i d i z e d , i n NADP-linked steps, to form 3 molecules of pentose phosphate. A s e r i e s of i s o m e r i z a t i o n s and group t r a n s f e r s occur which have the o v e r a l l e f f e c t of converting 3 molecules of hexose to 3 molec u l e s of f r u c t o s e phosphate and 1 molecule of glyceraldehyde phosphate plus 3 molecules of C0 . T h i s i s t h e r e f o r e the oxid a t i o n pathway of glucose metabolism. I t i s the main pathway f o r the production of NADPH, which i s the n u c l e o t i d e coenzyme commonly used i n r e d u c t i v e b i o s y n t h e t i c processes, and i t i s the route by which r i b o s e i s made f o r the production of n u c l e o t i d e s and f o r deoxyribose production as w e l l . I t should be emphasized that most of the r e a c t i o n s although not the decarboxylations are r e v e r s i b l e . Most o derived from t h i s pathwa l e f t to r i g h t , i n other words, from the non-oxidative p o r t i o n of the pathway. The phosphorylation of r i b u l o s e 5-phosphate to r i b u l o s e 1,5-diphosphate provides the C0 acceptor i n p l a n t s . We would a l s o point out that a l l pentoses i n t h i s pathway are phosphorylated. From the q u a n t i t a t i v e point of view, t h i s i s a minor pathway f o r carbohydrate o x i d a t i o n , i n comparison to the EmbdenMeyerhof g l y c o l y t i c route, but a greater amount of glucose i s d i r e c t e d through t h i s pathway when there i s need f o r NADPH. Another route by which pentoses are formed i s through the conversion of D-glucuronic a c i d to the x y l u l o s e s i n the g l u c u r o n i c - x y l u l o s e pathway,in which the pentose intermediates are not phosphorylated. A pentosuric i n d i v i d u a l excretes a rather constant amount of ^ - x y l u l o s e . The feeding of D-glucuronolactone elevates the u r i n a r y L - x y l u l o s e i n an amount i n d i c a t i n g that the conversion occurs i n rather high y i e l d (9). That t h i s i s a d i r e c t conversion has been demonstrated with l a b e l e d l a c t o n e . I t i s r e l e v a n t to mention that ^-glucuronate i s poorly converted to L - x y l u l o s e i n an experiment of t h i s type because, u n l i k e the lactone, the f r e e a c i d or s a l t i s poorly absorbed or impermeable to c e l l s . 2

2

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

Pentoses and

TOUSTER

Uronic Acids

Λ u sa

QOX O-O-O X X

*2 χ κ χ ο ο ; X X Ί5Ε.

S - - Ο Χ Χ Ν X*u χ ο ό Χ ν>ο-υ-ο-ο-ο

" 3

- Χ Χ ο ο ο XXX

"Ν

ο

s i

μ

11

χ

7Γε 11

« Λ

J( -a ; ν

•Ο ι

ο ο χ

χ

<ΜΟ Χ Χ Χ Ν χ •χ ο ο ο χ υ-ο-ο-ο-ο-ο-ϋ ο χ χ χ χ

χ

1)5

—

χ Χ ? Χ ι

, ο

^

1

2*

XXX μ Ο Ο Ο Ο Χ ΙΜΗΗ)·ϋ Χ Χ Χ Χ

14

. Χ Ο.

(Μ Ο

* ϊ

Χ Χ <Μ Χ ιί ο ο χ ο-Ο-ϋ-ο-ϋ χ χ

U 2 ι ο Κ)

α. ο

•

g j

& ί

ο χ _ s χ ^» ο χ ο ο χ > χ χ

Ή.

3

lis -

Γ"—1 χ ο <

2

Χ Χ Ν ο ο χ ο χ χ Ο Χ Χ χ

9* s

s

χ οχ χ , ο , χ ο χ ο ι χ χ ο χ χ χ

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

140

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

Related t o the D-glucuronic a c i d - L - x y l u l o s e conversion i s the production of L r a s c o r b i c a c i d from Dj-glucose, a process i n which 2j-glucuronate i s an intermediate (10) .

CHO

CHO

HCOH i HOCH

HCOH I . HOCH

i

HCOH I HCOH I CH OH D-Glucose

COOH I HCOH HOCH I I -H 0 HOCH vT^ HOCH CH 0H o

NADPH

HCOH ! HCOH I COOH

2

HCOH I HCOH I COOH

^-Glucuronic aci

L-Gulonic

0 11

0 II

η

π

HOCH 3

HOCH HC I HOCH I CH„OH L-Gulono-y-lactone

acid

0

II

c

1

o=c Λ

HCOH I HOCH I CH OH^

1

0

HOCH I HC— I HOCH I CH OH

Ί

! HOC _

II spon- ^ HOC taneous ι ; HC—

1

2-Keto-L-gulonoy-lactone

c

ι HOCH 1 CH OH 2

L-Ascorbic a c i d

T h i s pathway, l a r g e l y based on the work of King and Burns i n the r a t , shows that the ]l-glucose chain i s i n v e r t e d during i t s t r a n s ­ formation i n t o L - a s c o r b i c a c i d . The same holds f o r Jl-glucuronic a c i d , but not f o r the J±-gulonic a c i d d e r i v a t i v e s . These and other s t u d i e s on the b i o s y n t h e s i s of L - a s c o r b i c a c i d and of J±x y l u l o s e l e d t o the formulation of the glucuronate-xylulose path­ way (Figure 2) Ç7,11,12,13). The f u n c t i o n s of the glucuronate-xylulose c y c l e , which occurs i n a l l mammals s t u d i e d , a r e discussed i n the previous paper i n t h i s volume and w i l l not be f u r t h e r described a t t h i s p o i n t . However, we should l i k e t o mention that there i s s t i l l u n c e r t a i n t y about the s p e c i f i c r e a c t i o n ( s ) by which f r e e glucuronate i s formed. We can accept that D-glucuronate i s the precursor of L-gulonate, and we know a good d e a l about the b i o s y n t h e s i s of

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

8.

TOUSTER

Pentoses and Uronic

141

Acids

L- Ascorbic Acid - Keto-L-gulonoloctone L- Gulonoloctone HO

ÇH OH 2

C0 H 2

HO

* HOC H

V" 'IT'HOCH 1

^

HCO èOjH

C0 H 2

0-Glucuronate

UDP- glucuronote

CH OH 2

L- Gulonote

C0 H 2

HOCH

?-° HCOH

UDP-glucose

HOCH CH OH 2

-Keto-L- gulonote Glycogen ς

^Hexose phosphate CH OH

I pentose ^\ phosphate \ V pathway) \ * CH OH

HCOH HOCH CH OH L-Xylulose 4L INADPHI

2

C-0 ι HOCH I HCOH I CH OP03H 2

CO2

2

C-0

2

2

D-Xylulose 5-phosphate ADP > ATP i r

CH OH HCOH

CH OH HOCH

2

2

tL)

HOCH H<JoH

H C 0 H

H0(|H

^ CH 0H 2

CHzOH j

Xylitol

Figure 2.

Glucuronic acid-xylulose cycle

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

142

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

UDP-glucuronic a c i d . There have been two suggestions as to D-glucuronate might be formed from UDP-glucuronic a c i d :

V

_ d

how

Glucuronic a c i d 1-phosphate \ ^

A

^

UDP-Glucuronic a c i d

Glucuronic a c i d Β

Ο

X-glucuronide

In pathway A, the enzyme UDP-glucuronic a c i d pyrophosphatase would cleave the n u c l e o t i d phosphate, which would f r e e glucuronate. An o b j e c t i o n to t h i s route i s that the pyro­ phosphatase has been l o c a l i z e d i n our l a b o r a t o r y as a c o n s t i t u e n t of the plasma membrane of l i v e r c e l l s (14). T h i s r e l a t i v e l y non­ s p e c i f i c enzyme, n u c l e o t i d e pyrophosphatase, i s the same enzyme as phosphodiesterase I. I t seems h i g h l y u n l i k e l y that an enzyme of such broad s p e c i f i c i t y and, i n p a r t i c u l a r , one l o c a t e d i n the plasma membrane plays a r o l e i n a metabolic pathway such as we are c o n s i d e r i n g here. A second o b j e c t i o n stems from the f a c t that the only phosphatases i n l i v e r which can hydrolyze g l u c u r o n i c acid-l-phosphate are l o c a t e d i n the lysosomes (15,16). These phosphatases a l s o are g e n e r a l l y not very s p e c i f i c . A second sug­ gested route (B) has UDPGlcUA donating a g l u c u r o n i c a c i d r e s i d u e to a h y p o t h e t i c a l acceptor, X, to form a glucuronide which e i t h e r hydrolyzes spontaneously because of i t s i n t r i n s i c i n s t a b i l i t y to y i e l d f r e e g l u c u r o n i c a c i d or i s hydrolyzed by the enzyme 3-glucuronidase. Although glucuronide formation i s a common r e a c t i o n i n l i v e r , there i s no b a s i s f o r s p e c u l a t i n g on the i d e n t i t y of X. More important, 3-glucuronidase i s l o c a t e d i n the endoplasmic r e t i c u l u m and, e s p e c i a l l y , i n the lysosomes. Although 3-glucuro­ nidase does produce g l u c u r o n i c a c i d from complex glucuronides, unpublished s t u d i e s i n our l a b o r a t o r y have f a i l e d to provide e v i ­ dence that t h i s enzyme produces the g l u c u r o n i c a c i d that i s the precursor of L - a s c o r b i c a c i d . Whether we are naive about some aspect of the enzymology or c e l l biochemistry i n v o l v e d , or whether there i s another route not as yet apparent, i s u n c e r t a i n . For some years i t was b e l i e v e d that i n o s i t o l , which occurs r a t h e r commonly i n animals and i s obviously a food c o n s t i t u e n t , might be metabolized through more than one metabolic r o u t e . In a d d i t i o n , a kidney enzyme was discovered which converts i n o s i t o l to Drglucuronic a c i d . Consequently, a j o i n t study by four l a b o r a ­ t o r i e s was undertaken i n which l a b e l e d i n o s i t o l was administered to normal and p e n t o s u r i c human beings (17). We found that the

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

8.

TOUSTER

Pentoses and

Uronic Acids

143

i n o s i t o l was o x i d i z e d to C0 to a considerable extent i n the normal but was much reduced i n pentosuric i n d i v i d u a l s , a r e s u l t cons i s t e n t with the formulation of a pathway from i n o s i t o l to D-glucuronic a c i d , which would then be metabolized through the glucuronate-xylulose c y c l e . Pentosuric subjects would accumulate l a b e l e d L - x y l u l o s e , whereas normal subjects would not have a block at t h i s p o i n t . The l a b e l i n g patterns i n i s o l a t e d metabolites conformed to the postulated metabolic pathways. M y o - i n o s i t o l l a b e l e d at p o s i t i o n 2 d i d i n f a c t y i e l d C-^-labeled u r i n a r y I±-xylulose i n the pentosuric i n d i v i d u a l s . There was a l s o a preponderance of radioactivity in of blood glucose, a f i n d i n g c o n s i s t e n t with the metabolism of the i n o s i t o l through the x y l u l o s e pathway and then on through the pentose phosphate pathway to glucose. 2

V.

P a t h o l o g i c a l Aspects of the Pentoses

Although e s s e n t i a order (9,18), not a metaboli e f f e c t the r e d u c t i o n of ^ - x y l u l o s e to x y l i t o l appears not to be d e l e t e r i o u s i n any way, the underlying b a s i s of t h i s d i s o r d e r w i l l be described at t h i s p o i n t . For some time t h i s enzymatic l o c a l i z a t i o n of the defect (at L - x y l u l o s e reduction) i n pentosuria was r a t h e r i n d i r e c t , pentosuric i n d i v i d u a l s being understandably r e l u c t a n t to donate a piece of l i v e r f o r enzymatic analyses. Not long ago Asakura i n Japan reported that the two x y l i t o l dehydrogenases are present i n red blood c e l l s , a f i n d i n g which made poss i b l e i n v e s t i g a t i o n of the normal and mutant enzymes i n human red c e l l s . When Wang and van Eys (19) c a r r i e d out such a study, i t was at f i r s t s u r p r i s i n g to f i n d i n pentosuric erythrocytes a moderate amount of a c t i v i t y of the NADP-linked x y l i t o l dehydrogenase c a t a l y z i n g the r e d u c t i o n of L - x y l u l o s e . However, f u r t h e r study showed that the enzyme was abnormal. I t s 1^ f o r NADP i s 10 to 20 times higher than that of the normal enzyme, that i s , i t has a much lower a f f i n i t y f o r t h i s substrate than does the normal enzyme. In other words, there i s a mutant enzyme present, and i t i s not very e f f e c t i v e . That the block i n pentosuria i s not t o t a l has i n f a c t been apparent from i n v i v o s t u d i e s . The feeding of xylose i n high amounts to r a t s causes x y l i t o l c a t a r a c t (20), but I should emphasize that x y l i t o l a d m i n i s t r a t i o n does not. The xylose i s transported i n t o the l e n s , where i t i s reduced to x y l i t o l and upsets the osmotic balance. In regard to the medical aspects of x y l o s e , I should mention that o r a l xylose doses are given to p a t i e n t s i n t e s t s of i n t e s t i n a l a b s o r p t i o n . Since the xylose i s poorly metabolized and i s t h e r e f o r e excreted to a considerable extent i n the u r i n e , the amount of u r i n a r y xylose i s a rough measure of the a b s o r p t i v e c a p a c i t y of the patients.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

144 VI.

PHYSIOLOGICAL

P h y s i o l o g i c a l l y Important Uronic

EFFECTS

OF

FOOD

CARBOHYDRATES

Acids

By f a r the most important u r o n i c a c i d i n mammals i s D-glucur o n i c a c i d . As shown i n Table I I I , i t i s important because i t i s a c o n s t i t u e n t of many mucopolysaccharides or proteoglycans and i s a c o n s t i t u e n t of glucuronide d e r i v a t i v e s of drugs and hormones. As already i n d i c a t e d above, i t i s a precursor of L - a s c o r b i c a c i d and the x y l u l o s e s and i s the d i r e c t product of the metabolic o x i ­ d a t i o n of i n o s i t o l i n man. K a r l Meyer o r i g i n a l l y discovered that some of the uronic a c i d i n mucopolysaccharides i s L - i d u r o n i c a c i d , the 5 -epimer of ^ - g l u c u r o n i c a c i d . F i g u r e 3 shows a p o r t i o n of the heparan s u l f a t e molecule c o n t a i n i n g α-linked s u l f a t e d N-acetylglucosamine residues i n t e r s p e r s e d w i t h s u l f a t e d L - i d u r o n i c a c i d and 33-glucuronic a c i d . Some time ago the e p i m e r i z a t i o n of UDP-L-iduronic a c i d by r a b b i t t i s s u e e x t r a c t s was r e p o r t e d . How­ ever, t h i s work has not been confirmed i n any other l a b o r a t o r y and there i s now evidenc occurs at the macromolecula c o n c u r r e n t l y w i t h s u l f a t i o n of the polymer (21). ?

VII.

U t i l i z a t i o n and Production of the Uronic

Acids

The routes f o r the u t i l i z a t i o n of D-glucuronate i n v a r i o u s organisms are summarized below (7):

Microorganisms fi-Glucur ona t e

^ - f rue tur ona t e

>-D-mannona t e

>-2-keto-3-deoxygluconate (KDG)

*KDG 6-phosphate

^-pyruvate + t r i o s e phosphate Plants

^ g l u c u r o n i c a c i d 1-P D-Glucuronate

UTp

'>UDPGlcUA •pectins and h e m i c e l l u l o s e s

-glucaric acid Animals ^ x y l u l o s e pathway ^*L-gulonate ^ ΓΙ-Glucuronate -^L-ascorbic a c i d ^ ^ - g l u c a r i c acid

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

8.

Pentoses and Uronic Acids

TOUSTER

145

TABLE I I I . PHYSIOLOGICALLY IMPORTANT URONIC ACIDS CHO I HCOH I HOCH I HCOH I HCOH I COOH

D-Glucuronic a c i d - i n h e p a r i n , h y a l u r o n i c a c i d , c h o n d r o i t i n s u l f a t e s , e t c . , and glucuronides (of drugs and hor­ mones) ; i n v o l v e d i n metabolism of i n o s i t o l , L-ascorbic a c i d , and the x y l u l o s e s

CHO I HCOH I HOCH

L-Iduronic a c i dermata

sulfate

i

HOCH I COOH Figure 3.

Ν S0

Portion of heparin and heparin sulfates

Ο 3

S0

Ν 3

S0

Ν 3

Biosynthesis : 1. 2. 3.

P o l y m e r i z a t i o n of GlcNAc and g l u c u r o n i c a c i d N-deacp.tylated N- and O-sulfated w i t h e p i m e r i z a t i o n of some D^-glucuronic acid to L-iduronic acid.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

146

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

The metabolism of glucuronate i n microorganisms i s q u i t e s p e c i a l , as shown by Ashwell and h i s c o l l a b o r a t o r s s e v e r a l years ago, the f i r s t step being e p i m e r i z a t i o n to D-fructuronate. In p l a n t s , Ιλglucuronate can be d i r e c t l y phosphorylated, a r e a c t i o n that does not occur i n animals. UDP-glucuronic a c i d i s a precursor of plant p o l y s a c c h a r i d e s . Glucuronate can a l s o be o x i d i z e d to the corresponding d i c a r b o x y l i c a c i d , g l u c a r i c a c i d . In animals the conversion to g l u c a r i c a c i d and the presence of the l a t t e r i n u r i n e have been demonstrated. We have already discussed the r e d u c t i o n of glucuronate to L-gulonate and i t s conversion to L ascorbate or i t s metabolism through the x y l u l o s e s . The u t i l i z a t i o n and production of g l u c u r o n i c a c i d i n v i v o are summarized below ( 7 ) :

A.

Utilization Utilization i the lactone because the a c i d probably does not enter c e l l s . High extent of u t i l i z a t i o n i s i n d i c a t e d by 14

B.

1)

high y i e l d of

CO^ from l a b e l e d lactone

2)

high y i e l d of u r i n a r y L - x y l u l o s e i n the pentosuric human

Production Glucuronic a c i d production i s increased by 1)

substances

excreted as glucuronides

2)

inducers of the microsomal P-450 system (e.g. s t e r o i d s , b a r b i t u r a t e s ) . These substances a l s o increase the production of a)

L-ascorbic acid

b)

glucaric acid

The production of g l u c u r o n i c a c i d , that i s , of UDP-glucuronate, i s responsive to some inducing agents which i n c r e a s e the produc­ t i o n of conjugated glucuronides, of L - a s c o r b i c a c i d , and of g l u c a r i c a c i d and, I might add, of L - x y l u l o s e i n the p e n t o s u r i c human. The mechanism of t h i s i n d u c t i o n has been studied f o r many years and i s probably s t i l l not very c l e a r l y understood. Various s t e r o i d s and b a r b i t u r a t e s and other drugs which induce the micro­ somal P450 system are s t i m u l a t o r s of the production of glucuronic acid derivatives.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

8.

Pentoses and Uronic Acids

TOUSTER

147

F i g u r e 4 shows that UDP-glucuronic intermediate i n metabolism:

a c i d i s somewhat of a key

Glucuronides

UDP-D-G lue ose»

>UDP-D-Glucuronic a c i d

UDP-^-Xylose

^ D-Glucuronic

acid

I

L-xylulose I

V xylitol

I D-xylulose Figure 4. Reactions of UDP-O-glucuronic acid UDP-Glucuronic a c i d i s used i n the b i o s y n t h e s i s of glucuronides and proteoglycans; i t i s the precursor of g l u c u r o n i c a c i d which goes to the x y l u l o s e s and to a s c o r b i c a c i d , and i t i s the precursor of the x y l o s e found i n the g a l - g a l - x y l bridge between mucopolysaccharide chains and polypeptide chains i n proteoglycans by v i r t u e of i t s d e c a r b o x y l a t i o n to UDP-xylose. VIII.

P a t h o l o g i c a l Aspects of D-Glucuronic and L-Iduronic A c i d s

There are now two h e r e d i t a r y lysosomal diseases s p e c i f i c a l l y a t t r i b u t a b l e to d e f i c i e n c i e s i n lysosomal uronidases. Gargoylism, or H u r l e r s d i s e a s e , a s s o c i a t e d with the accumulation of dermatan and heparan s u l f a t e s and with mental d e f i c i e n c y and a v a r i e t y of morphological changes, has r e c e n t l y been shown to be due to an L-iduronidase d e f i c i e n c y i n lysosomes (22). In the l a s t few years another type of mucopolysaccharidosis has been found that has some s i m i l a r i t y to H u r l e r ' s d i s e a s e . As a r e s u l t of the work of S l y et a l . (23) and of Neufeld and her a s s o c i a t e s (24), t h i s disease, a t y p i c a l mucopolysaccharidosis, can be a t t r i b u t e d to 3 - g l u c u r o n i dose d e f i c i e n c y i n the lysosomes. In connection with lysosomal d i s o r d e r s , i t i s r e l e v a n t to mention at a symposium such as the present one that severe lysosomal storage abnormalities have been caused by the a d m i n i s t r a t i o n of undegradable polymers, i n c l u d i n g dextrans and p o l y v i n y l p y r r o l i d o n e . Great c a u t i o n should be used i n i n j e c t i n g such m a t e r i a l s i n t o humans. 1

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

148

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

Also known a r e abnormalities i n g l u c u r o n y l t r a n s f e r a s e , the enzyme system(s) c a t a l y z i n g the t r a n s f e r of g l u c u r o n i c a c i d from UDPGlcUA t o s u i t a b l e acceptors. In C r i g 1 e r - N a j j a r disease a d e f i ciency of g l u c u r o n y l t r a n s f e r a s e i s r e s p o n s i b l e f o r i n s u f f i c i e n t conversion of the heme degradation product b i l i r u b i n to b i l i r u b i n glucuronide, the r a p i d l y e x c r e t a b l e , more water-soluble a c i d i c form. In t h i s disease the unreacted b i l i r u b i n accumulates i n the nervous system, with d e l e t e r i o u s consequences t o the i n f a n t (25). There seems t o be an animal model of t h i s disease, the Gunn r a t , i n which s i m i l a r abnormal b i l i r u b i n metabolism i s observed as w e l l as low t r a n s f e r a s e l e v e l s . IX.

Summary and Prospects

In summary, i t i s evident that pentoses and uronic a c i d s are extremely important to mammalian b i o l o g y I have not commented on c e r t a i n aspects of u t i l i z a t i o c o n t a i n i n g polymers, suc general these seem t o be p o o r l y u t i l i z e d . One area that w i l l c e r t a i n l y witness an i n t e r e s t i n g f u t u r e concerns the lysosomal storage disease because so many of the accumulated proteoglycans are r i c h i n uronic a c i d s . Indeed, s i n c e pinocytosed chemotherap e u t i c agents a r e destined to enter lysosomes, where the metabolic abnormalities a r e found, these diseases are prime t a r g e t s f o r enzyme therapy attempts. C u r r e n t l y , 3-glucuronidase i s i n f a c t being studied as a chemotherapeutic agent i n a t y p i c a l mucopolysaccharidosis .

Literature Cited

1. Rodén, L., in "Metabolic Conjugation and Metabolic Hydrolysis" (W. H. Fishman, ed.), Vol. II, pp. 345-442, Academic Press, New York (1970). 2. Hiatt, H. H., J. Biol. Chem. (1957) 224, 851-859. 3. Segal, S. and Foley, J. B., J. Clin. Invest. (1959) 38, 407-413. 4. McCormick, D. B. and Touster, O., Biochim. Biophys. Acta (1961) 54, 598-600. 5. McCormick, D. B. and Touster, O., J. Biol. Chem. (1957) 229, 451-461. 6. Hollmann, S. and Reinauer, H., Z. Ernährungswissenschaft (1971) Suppl. 11, pp. 1-7. 7. Touster, O., in "Comprehensive Biochemistry" (M. Florkin and Ε. H. Stotz, eds.), Vol. 17, pp. 219-240, Elsevier Publishing Company, Amsterdam-London-New York (1969). 8. van Heyningen, R., Biochem. J. (1958) 69, 481-491. 9. Touster, O., Fed. Proc. (1960) 19, 977-983.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

8.

TOUSTER

Pentoses

and

Uronic

Acids

149

10. Burns, J. J., in "Metabolic Pathways" (D. M. Greenberg, ed.), Vol. 1, pp. 341-356, Academic Press, New York (1960). 11. Burns, J. J. and Kanfer, J., J. Am. Chem. Soc. (1957) 79, 3604-3605. 12. Hollmann, S. and Touster, O., J. Am. Chem. Soc. (1956) 78, 3544-3545. 13. Hollmann, S. and Touster, O., J. Biol. Chem. (1957) 225, 87102. 14. Touster, O., Aronson, Ν. N., Jr., Dulaney, J. T., and Hendrickson, H., J. Cell Biol. (1970) 47, 604-618. 15. Arsenis, C., Hollmann, S., and Touster, O., Abstracts of the American Chemical Society Meeting, New York, September 1966, C-286. 16. Arsenis, C. and Touster, O., J. Biol. Chem. (1967) 242, 3400-3401. 17. Hankes, L. V., Politzer W M. Touster O., and Anderson L., Ann. Ν. Y. 18. Hiatt, H., in "Th (J. B. Stanbury, J. B. Wyngaarden, and D. S. Fredrickson, eds.), 3rd ed., pp. 119-130, McGraw Hill, New York, Toronto, London (1972). 19. Wang, Y. M. and van Eys, J., New Eng. J. Med. (1970) 282, 892-896. 20. van Heyningen, R., in "Proceedings of the International Sym­ posium on Metabolism, Physiology, and Clinical Use of Pentoses and Pentitols," Hakone, Japan, August 27-29, 1967 (B. L. Horecker, K. Lang, and Y. Takagi, eds.), pp. 109-123, Springer-Verlag, Berlin, Heidelberg, New York (1969). 21. Höök, M., Lindahl, U., Bäckström, G., Malmström, Α., and Fransson, L.-Å., J. Biol. Chem. (1974) 249, 3908-3915. 22. Matalon, R. and Dorfman, Α., Biochem. Biophys. Res. Commun. (1972) 47, 959-964. 23. Sly, W. S., Quinton, Β. Α., McAlister, W. H., and Rimoin, D. L., J. Pediat. (1973) 82, 249-257. 24. Hall, C. W., Cantz, M., and Neufeld, E. F., Arch. Biochem. Biophys. (1973) 155, 32-38. 25. Schmid, R. in "The Metabolic Basis of Inherited Disease" (J. B. Stanbury, J. B. Wyngaarden, and D. S. Fredrickson, eds.), 3rd ed., pp. 1141-1178, McGraw-Hill, New York, Toronto, London (1972).

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

9 Role of Carbohydrates in Dental Caries WILLIAM H. BOWEN Caries Prevention and Research Branch, National Caries Program, National Institute of Dental Research, National Institutes of Health, Bethesda, Md. 20014

Dental caries result specifi which colonize the tooth surface and metabolize particular compo­ nents of the diet. The action results in the rapid and sometimes prolonged production of acid on the tooth surface resulting in the dissolution of the enamel. Since the time of Aristotle it has been considered that car­ bohydrates played an essential role in the pathogenesis of dental caries. There is now an abundance of evidence accumulated from epidemiological surveys (1) and animal experimentation (2) which clearly indicates that dental caries does not develop in the absence of dietary carbohydrate. In an elegant clinical study Gustafsson, et.al. (3) showed that the incidence of caries is related to the frequency of intake of carbohydrate and not to the total amount consumed. For example, patients who in 1 year con­ sumed 94 kg. of sugar with meals had fewer new carious lesions than patients who consumed 85 kg., 15 of which was taken between meals.

The restriction in carbohydrate intake which occurred during World War II was followed by a dramatic f a l l in the prevalence in dental caries in many European countries. (4)(5)(6) Carbohydrates and sugar in particular apparently can also affect the maturation of enamel, a process which leads to i n creased mineral uptake in enamel post-eruptively. It was observed (7) that the teeth of rats exposed to high sugar diets showed delayed maturation and were therefore presumably more susceptible to decay. Rats bred on a diet (8) conducive to the formation of severe protein-calorie imbalance have been shown to have enhanced susceptibility to caries. The effect was ascribed to altered tooth size (9) and to alterations in salivary composition. The physical form in which sugar is ingested will also influence its cariogenicity. Powdered sugar is more cariogenic than similar sugars given in an aqueous solution. (10) The size of particles and the adhesiveness of the diet also influence the cariogenicity of the diet. (11) In general the longer a poten150 In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

9.

BOWEN

Carbohydrates and Dental Caries

151

t i a l l y cariogenic substance is retained in the mouth the greater is the likelihood that caries will develop. The formation of dental plaque is the earliest evidence that microorganisms and components of the diet are interacting. Dental plaque is the soft white tenacious material which occurs on tooth surfaces and is composed of microorganisms enmeshed in a matrix of carbohydrate and protein. Specific microorganisms are associated with the early formation of dental plaque and sucrose plays an important role in their establishment on the tooth surface. (12) Streptococcus mutans is a prime microbial agent in the pathogenesis of dental caries. (13)(14) It has several interesting properties; i t is found predominantly on the tooth surface and i t forms polyglucan and polyfructan from sucrose. (15)(16) Strep, mutans can become reversibly bound to the tooth surface in the absence of sucrose but following ingestion of this sugar extracellular polysaccharide is formed. (17) This substanc organism to acThere to the tooth surface and also leads to the aggregation of other microorganisms. (18) The available evidence indicates that the predominant polyglucan in plaque is composed of material possessing mainly 1-3 linkages; substantial material possessing 1-6 linkages is also present, (19) Up to 40% of the polyglucan formed in plaque is readily metaFolizable by plaque microorganisms. (20) Although polyfructan is also found i t is metabolized very rapidly(21) by substantial numbers of the microorganisms in plaque. (22T~ Apart from the contribution that extracellular polysaccharides make to the adherence of microorganisms their precise role in the pathogenesis of dental caries is unclear. Recent evidence has shown that phosphorus is tightly bound to the polysaccharide and that the carbohydrate is charged. (23) This indicates that the polysaccharide could limit the diffusion of charged substances into and out of plaque. Acid formed within plaque could not, therefore, be readily neutralized by the diffusion of such substances as bicarbonate. It is also likely that the extracellular polysaccharide protects the microorganisms from inimical influences. Whatever the precise role they have in the pathogeneses of caries there is l i t t l e doubt that the integrity of the tooth would be enhanced by either preventing their formation or their rapid removal. There is also clear evidence which indicates that microorganisms in plaque can synthesize an intracellular polysaccharide (IPS) of the amylo pectin type from a wide variety of carbohydrates. (24)(25) This material can be catabolized by plaque microorganTsms during periods when extraneous sources of carbohydrate are lacking. This catabolism is probably responsible for the comparatively low pH values observed in plaque around carious lesions even in patients who have been fasting. (26) There is some evidence to indicate that the number of intracellular polysaccharide forming organisms in plaque is positively correlated

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

152

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

with the number of carious lesions.(27) The results of research carried out by Kanap and Hamilton(28) indicate that both the synthesis and catabolism of IPS is influenced by the presence of fluoride. They have shown that the low concentrations of fluoride inhibit enolase and glucose-6-p formation without penetrating the cell significantly indicating that fluoride may affect the transport of sugar into the bacterial c e l l . This is probably one of several mechanisms through which fluoride exercises its cariostatic effect. Dental plaque also forms in the absence of dietary carbohydrate, (29)(30) e.g., in patients or animals who receive their completedetTby gastric intubation, but i t lacks several properties found in plaque formed under conventional circumstances. Animals fed in this manner do not develop caries.(31)In addition the plaque so formed lacks the ability to lower the pH value of topically applied sugar solutions in contrast to that formed normally, which may lowe or less in a matter of minutes. (32) It is generally considered that rapid démineraiization of the tooth surface occurs below pH 5.5. The types and proportions of acids formed in dental plaque are attracting an increasing amount of attention because i t is conceivable that the difference in the cariogenicity of plaques may reside in some measure in the different types or proportions of acids present. Gilmour and Poole (33) found that a constant relationship between the concentration of lactic acid in plaque and pH decrease was lacking. In some plaques i t was found that the concentration of lactic, propionic and acetic acids accounted for less than 50% of the titratable acidity. In a study carried out by Geddes (34) i t was observed that 'fasting plaque contained 3 X 10- m moles of acid/mg wet weight and that five minutes after exposure to sugar the concentration had increased to 5 Χ 10" m moles. The major change was to a five-fold increase in D (-) lactate and an eight-fold increase in D (+) lactate. There is a positive correlation between the frequency of sugar intake and the incidence of caries. (35) Each ingestion of sugar is followed by a rapid f a l l in pH value on the tooth sur­ face. The pH returns to neutrality over a 20-30 minute period. The duration for which the plaque has been allowed to accumulate will influence both the magnitude of the f a l l in pH and the time required for its recovery. (36) In general the older the plaque the greater its pathogenic potential. An extreme example of the effects of prolonged exposure to sucrose can be seen in infants who have comforters dipped in a sucrose syrup or similar solu­ tion. (37) These children develop rampant caries on the palatal surfaces of the upper molars and incisors. It is occasionally argued that much caries could be elimina­ ted i f other sugars were substituted for sucrose in the diet. Some support for this concept can be found in patients who suffer from fructose intolerance. These patients must avoid sucrose and 1

5

5

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

9.

BOWEN

Carbohydrates and Dental Caries

153

fructose. Fructose intolerant patients have substantially less caries than normal persons but they are not caries-free. (38) Clearly such patients must alter their dietary intake consTcTerably, other than merely avoiding sucrose. Sucrose has probably been blamed as the main dietary culprit in caries causation simply because i t is the sugar which is most frequently ingested. There is no evidence that its substitution by glucose or fructose would lead to a significant reduction in dental decay in humans. Results of many experiments carried in animals clearly indicate that glucose and fructose can induce significant levels of decay. (39)(40)(41) Primates which received their complete diet by gastric Intubation with the exception of glucose or fructose (i.e. plaque was formed in the presence of these sugars) formed plaque which contained significant levels of extracellular polysaccharide and in addition this plaque could lower the pH of sugar solutions rapidly. (42) The effects of polyol on plaque formation and the development of caries have been i n vestigated in animals and to a lesser extent in man. (43) It was observed in primates that the ingestion of sorbitol was followed i n i t i a l l y by the formation of plaque which had a syrupy consistency. It was also noted that the numbers of Strep, mutans in plaque declined markedly when sorbitol was substituted for sucrose even though Strep, mutans ferments sorbitol. Prolonged ingestion in man (44) or primates did not lead to the development of a plaque flora with an enhanced ability to metabolize sorbitol. All the available evidence indicates that sorbitol is substantially less cariogenic in animals than sugars. Xylitol was shown by Muhlemann, et. a l . (45) to be even less cariogenic than sorbitol. A possible explanation for the lower cariogenicity of sorbitol may be found in the manner in which i t is metabolized by microorganisms. The breakdown of sorbitol produces mainly formic acid and ethanol; in contrast, the metabolism of glucose results in the formation of primarily lactic acid. (46) It is possible that the extensive use ofpolyols would not be acceptable by the general public as in some persons even moderate doses have a cathartic effect. There is l i t t l e doubt that the incidence of caries would decline dramatically i f the general population would reduce the frequency of intake of fermentable carbohydrates. However the ingestion of candy is rarely associated in peoples minds with the formation of a carious lesion at some future time. Any disease which affects 95% of the population is unlikely to be controlled to a significant extent by individual effort. Effective control calls for public health measures and of these water fluoridation is the most effective.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

154

PHYSIOLOGICAL EFFECTS OF FOOD CARBOHYDRATES

Literature Cited

1. Read, T. and Knowles, E. Brit. Dent. J. (1938),64:185. 2. Frostell, G. and Baer, P.N. Acta Odont. Scand. (1971) 29:253. 3. Gustafson, B.E.; Quensel, C.E.; Swenander, L.; Lundquist, C.; Grahnen, H.; Bonow, B.; and Krasse, B. Acta Odont. Scand. (1954), 11: 232. 4. Toverud, K.V., Kjosnes, Ε., and Toverud, G. Odont. Tid. (1942), 50:529. 5. Sognnaes, R. and White, R. Amer. J. Dis. Child. (1940), 60: 283 6. Parfitt, G. J. Brit. Dent. J. (1954), 97:235. 7. Nikiforuk, G. J. Dent. Res. (1970), 49:1252. 8. Menaker, L. and Navia, J. J. Dent. Res. (1973), 52:680. 9. Holloway, P.J., Shaw, J.H. and Sweeney, E.A. Arch. oral Biol. (1961), 3:185. 10. Sognnaes, R.F. J. Nutr 11. Caldwell, R.C. J. Dent 12. Krasse, B. Arch. oral Biol. (1966), 11:429. 13. de Stoppelaar, J.D., van Houte, J.V. and de Moor, C.E. Arch. oral Biol. (1967), 12:1199. 14. Gibbons, R.J., De Paola, P.F., Spinell, D.M., and Skobe, Z. Infection and Immunity (1974), 9:481. 15. Gibbons, R.J. and Nygaard, M. Arch. oral Biol. (1968), 13: 1249. 16. Wood, J.M. and Critchley, P. Arch. oral Biol. (1966) 11:1039. 17. Gibbons, R.J. and van Houte, J. Infection and Immunity. (1971), 3:567. 18. Gibbons, R.J. and Fitzgerald, R.J. J. Bacteriol. (1969), 98: 341. 19. Baird, J.K., Longyear, V.M. and Ellwood, D.C. Microbios. (1973), 8:143. 20. Wood, J.M. Arch, oral Biol. (1969), 14:161. 21. van Houte, J. and Jansen, H.M. Arch, oral Biol. (1968), 13: 827. 22. da Costa, T. and Gibbons, R.J. Arch, oral Biol. (1968), 13: 609. 23. Melvaer, K.L., Helgeland, K. and Rolla, G. Arch, oral Biol. (1974), 19:589. 24. van Houte, J., Winkler, K.C. and Jansen, H.M. Arch. oral Biol. (1969), 14:45. 25. Gibbons, R.J. and Socransky, S.S. Arch, oral Biol. (1962), 7:73. 26. Stephan, R.M. J. Dent. Res. (1944), 23:257. 27. Loesche, W.J. and Henry, C.A. Arch, oral Biol. (1967), 12: 189. 28. Kanapka, J.A. and Hamilton, I.R. Arch. Biochem. and Biophys. (1971), 146:167. 29. Littleton, N.W., Carter, C.H. and Kelley, R.T. J. Amer. Dent. Assoc. (1967), 74:119.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

9.

BOWEN

Carbohydrates

and

Dental

Caries

155

30. Bowen, W.H. and Cornick, D.E.R. Int. Dent. J. (1970),20:382. 31. Kite, O.W., Shaw, J.H. and Sognnaes, R.F. J. Nutr. (1950), 42:89. 32. Stephan, R.M. and Miller, B.F. J. Dent. Res. (1943), 22:45. 33. Gilmour, M.N. and Poole, A.E, Caries Res. (1967), 1:247. 34. Geddes, D. ORCA Abstracts (1973), Zurich. 35. Zita, Α., McDonald, R.E. and Andrews, A.L. J. Dent. Res. (1959), 38:860. 36. Kleinberg, I. J. Dent. Res. (1970), 49:1300. 37. Winter, G.B., Hamilton, M.C. and James, P.M.C. Arch. Dis. Child. (1966) 41:216. 38. Marthaler, T.M. and Froesch, E.R. Brit. Dent. J. (1967), 124:597. 39. Stephan, R.M. J. Dent. Res. (1966), 45:1551. 40. Campbell, R. and Zinner, D.D. J. Nutr. (1970), 100:11. 41. Green, R. and Harltes 42. Bowen, W.H. Arch. 43. Shaw, J.H. and Griffths, D. J. Dent. Res. (1960), 39:377. 44. Cornick, D. and Bowen, W.H. Arch, oral Biol. (1972), 17:1637. 45. Muhlemann, H., Regolati, B. and Marthaler, T.M. Helv. Odont. Acta (1970), 14:48. 46. Dallmeier, E., Bestmann, H.J. and Kroncke, A. Deutsch. Zahnaerztl. Z. (1970), 25:887.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

10 The Role of Trace Elements in Human Nutrition and Metabolism R. W. TUMAN and R. J. DOISY Department of Biochemistry, State University of New York, Upstate Medical Center, Syracuse, Ν. Y. 13210

Introduction The ultimate goal of nutritionists, food chemists, and food technologists is to answer the question: What is adequate nutrition? The answer to this most important question requires know­ ledge of the nutrients required for good health, the amounts needed, and food sources which can supply the necessary nutrients. In the past, efforts to define human requirements have been preoccupied with discussions of the major essential nutrients, including: clarification of amino acid, vitamin and macro­ element requirements, recommending a proper balance of carbohy­ drate, fat and protein, and defining appropritate energy require­ ments (1). Very little attention has been given to the so-called trace elements. Only recently, however, have scientists begun to appreciate the full extent of the biochemical role of trace elements and their interactions with human health and nutrition. At the present time, the 14 trace elements listed in Table 1 have been identified as being essential for either human or animal nutrition (2). These include iron, iodine, copper, man­ ganese, zinc, cobalt, molybdenum, selenium, chromium, tin, vana­ dium, fluorine, silicon, and nickel. It i s i n t e r e s t i n g to note (Table l ) that during the f i r s t 100 p l u s years s i n c e i r o n was found to be e s s e n t i a l , only f i v e a d d i t i o n a l t r a c e elements were discovered to be r e q u i r e d f o r good h e a l t h , i n c l u d i n g i o d i n e , copper, manganese, z i n c , and c o ­ balt. In c o n t r a s t w i t h t h i s r a t h e r slow e a r l y r e c o g n i t i o n of the importance of trace elements, during the 20 year p e r i o d from 1953-1973, a t o t a l o f e i g h t a d d i t i o n a l trace elements have been found to be e s s e n t i a l , i n c l u d i n g molybdenum, selenium, chromium, t i n , vanadium, f l u o r i n e , s i l i c o n , and n i c k e l . Furthermore, no l e s s than 5 of these 8, namely, n i c k e l , t i n , s i l i c o n , f l u o r i n e , and vanadium, have emerged as e s s e n t i a l n u t r i e n t s only i n the

156 In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

10.

TUMAN

DOiSY

AND

157

Trace Elements in Nutrition TABLE 1

DISCOVERY OF ESSENTIAL TRACE ELEMENTS REQUIREMENTS 1. 2.

3.

Iodine . . . . Copper . . . .

4. 5.

Manganese.

. .

6.

Cobalt

7.

Molybdenum . .

8. 9. 10. 11.

Selenium . . ., Chromium . . ., , Tin Vanadium . . .,

12. 13.

Fluorine Silicon.

14.

N i c k e l . . . .,

. . . .

. . .. . . ..

Adapted from:

17th Century 1850 C h a t i n , A. 1928 Hart, Ε . Β . , H. Steenbock, J. Waddell, and C A. Elvehjem 1931 Kemmerer, A. R. and W. R. Todd 1934 Todd, W. R., C A. Elvehjem, and Ε . B. Hart 1935 Underwood, E. J. and J. F. F i l m e r ; Marston, H. R.; L i n e s , E. W. 1953 DeRenzo, E. C., E. K a l e i t a , P. H e y t l e r , J. J. Oleson, W i l l i a m s ; R i c h e r t , D. A. and W. W. Westerfeld Schwarz, K. and Schwarz, K. and Schwarz, K., D. Schwarz, K., D. and H. E. Mohr 1972 Schwarz, K. and 1972 Schwarz, K. and Ε . M. 1973 N i e l s e n , F. H.

1957 1959 1970 1971

Schwarz, K.,

C. W. B. B.

M. F o l t z Mertz M i l n e , and E. Vinyard M i l n e ; Hopkins, L. L.

D. B. Milne D. B. M i l n e ; C a r l i s l e ,

Federation Proceedings, ( £ ) .

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

158

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

l a s t 3-4 y e a r s . I n d i c a t i v e of the i n c r e a s e d r e c o g n i t i o n trace elements are r e c e i v i n g i n human, as w e l l as animal n u t r i t i o n , i s the r a p i d i t y with which the l i s t of e s s e n t i a l t r a c e elements i s growing. Table 2 describes the p e r i o d i c d i s t r i b u t i o n of those elements g e n e r a l l y considered to be m i c r o - n u t r i e n t elements. Examination of Table 2 r e v e a l s s e v e r a l i n t e r e s t i n g p o i n t s : l) With the recent a d d i t i o n of vanadium and n i c k e l to the l i s t of e s s e n t i a l t r a c e elements, a continuous set of 8 e s s e n t i a l e l e ments i s created i n the f i r s t t r a n s i t i o n s e r i e s , from vanadium (atomic number 23) through zinc (atomic number 30). Furthermore, when one i n c l u d e s molybdenum (atomic number 42), 9 of the Ine s s e n t i a l t r a c e elements are t r a n s i t i o n elements. Therefore, the t r a n s i t i o n s e r i e s elements, i n g e n e r a l , c o n s t i t u t e a r e g i o n of the p e r i o d i c t a b l e with s p e c i a l i n t e r e s t and importance (2). 2) With the exception of f l u o r i n e (atomic number 9) and s i l i c o n (atomic number are l o c a t e d above calcium (atomic number and none of the elements beyond i o d i n e (atomic number 53) have ever been shown to be e s s e n t i a l f o r animals or man (2). 3) More than 20 other t r a c e elements shown i n Table 2 are p o t e n t i a l l y important and are c u r r e n t l y under s p e c i a l c o n s i d e r a t i o n w i t h respect to e s s e n t i a l i t y (2). Progress i n d i s c o v e r i n g new e s s e n t i a l t r a c e elements has r e l i e d on improved research techniques. In t h i s r e s p e c t , e s t a b l i s h i n g the e s s e n t i a l i t y of the newer t r a c e elements ( t i n , vanadium, f l u o r i n e , s i l i c o n , and n i c k e l ) has r e l i e d on the development and i n t r o d u c t i o n of the u l t r a - c l e a n environment and p l a s t i c i s o l a t o r techniques, as w e l l as the use of pure c r y s t a l l i n e amino acids and vitamins i n p r e p a r i n g the d i e t s of l a b o r a t o r y animals. Recent developments i n trace element a n a l y s i s , p a r t i c u l a r l y s e n s i t i v e methods i n v o l v i n g atomic absorption spectroscopy and neutron a c t i v a t i o n , have f u r t h e r advanced the s t a t e of trace element n u t r i t i o n . At the present time, the l i s t i s composed of 14 e s s e n t i a l t r a c e elements, however, the absolute number o f r e q u i r e d trace elements i s s t i l l not known. I t i s probable that some or a l l o f the elements l i s t e d i n Table 2 as p o t e n t i a l l y important w i l l be found to p a r t i c i p a t e i n v i t a l processes as s t i l l newer experimental techniques are r e f i n e d and a p p l i e d . S e v e r a l trace elements and t h e i r d e r i v a t i v e s , f o r example, cadmium, mercury, a r s e n i c , and l e a d have been shown to be t o x i c . The d e t r i m e n t a l e f f e c t s o f l e a d and methyl-mercury on the c e n t r a l nervous system are w e l l known. B e r y l l i u m and a r s e n i c are h i g h l y carcinogenic i n l a b o r a t o r y animals, and n i c k e l carbonyls and c h r o m â t e s have been i m p l i c a t e d as a cause of lung cancer (_3)· However, i t i s obvious from past r e s u l t s , that t o x i c i t y cannot be used as a v a l i d argument a g a i n s t p o t e n t i a l e s s e n t i a l i t y . Most e s s e n t i a l elements become t o x i c at s u f f i c i e n t l y high l e v e l s and the margin between t o x i c and b e n e f i c i a l doses may be s m a l l . For example, the t o x i c i t y of selenium was demonstrated w e l l

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

C a

Mg

20

12

4jBe

IIA

IIIB

Adapted from:

55çs

37Rb

K

Na

19

n

Group IA

VIII Co

l Fe 2 7

1 2 6

^ Pd

Ni

1

6

2 8

C

I IB

7

9Au

S 0

HÊ

l 9 u l WZn 2

IB

31fe

Schwarz, K., F e d e r a t i o n Proceedings, (2^).

1 1 Elements w i t h known e s s e n t i a l i t y . — Elements p o t e n t i a l l y important. ( 3 Elements important i n normal carbohydrate metabolism.

2

h Mo|

ZhQ

23v l i ^ c r

22

T i

VI IB

VIB

VB

TVB

13M

IIIA

DISTRIBUTION OF TRACE ELEMENTS OF KNOWN AND POTENTIAL IMPORTANCE FOR HUMAN AND ANIMAL NUTRITION

PERIODIC TABLE

Table 2

8 2

51Sb Pb

S n

50

P

33AS

15

VA

3 Ge 2

^Si

IVA

34

S e

S

0 16

8

VIA

C1

F

53i

35_Br

1 7

9

VI IA

160

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

before i t s e s s e n t i a l i t y , and i t i s now known that selenium possesses both t o x i c and b e n e f i c i a l p r o p e r t i e s , depending on the dose and chemical form ( O . A more recent example of t h i s concept i s the report by Schwarz (5) of a growth-promoting e f f e c t o f very low doses of l e a d i n r a t s maintained i n an i s o l a t o r environment. Thus, i t would not be s u r p r i s i n g i f other t r a c e elements, u s u a l l y regarded as t o x i c , w i l l a l s o be found to be b e n e f i c i a l or e s s e n t i a l . The b i o l o g i c a l p r o p e r t i e s of the 14 trace elements now r e cognized to be e s s e n t i a l are very diverse (Table 3)· I n d i v i d u a l Trace Elements Iron. I t i s w e l l recognized that i r o n i s e s s e n t i a l as an i n t e g r a l component of hemoglobin and as a component of some f l a v o p r o t e i n s and the o x i d a t i v e r e s p i r a t o r y c h a i n . Suboptimal d i e t a r y i r o n intake r e s u l t wide-spread incidence o of the United S t a t e s p o p u l a t i o n was r e c e n t l y documented i n the Ten-State N u t r i t i o n Survey ( 6 ) . The RDA f o r i r o n i n the 8th e d i t i o n of Recommended Dietary Allowances published by the Food and N u t r i t i o n Board of the National Research C o u n c i l has remained at 10 mg/day f o r a d u l t males and post-menopausal females and 18 mg/day f o r women of c h i l d b e a r i n g age (7). This l a t t e r amount i s d i f f i c u l t to o b t a i n by d i e t a r y means since i t has been estimated that a balanced, average American d i e t provides only about 6 mg i r o n per 1,000 k c a l (8). Therefore, women of c h i l d b e a r i n g age would be r e q u i r e d to eat a 3,000 k c a l d i e t to meet the recommenda t i o n of 18 mg i r o n , an undesirable c a l o r i c intake f o r most women. Thus, the major change i n the 1974· RDA f o r i r o n i s a recommendation f o r supplemental i r o n intake (30-60 mg) f o r c h i l d b e a r i n g women ( 7 ) . The use of i r o n enriched foods has been suggested as a means to meet the RDA f o r i r o n . 1

Iodine. The only known f u n c t i o n of i o d i n e i n human and animal physiology i s i t s e s s e n t i a l r o l e i n formation of the t h y r o i d hormones, t h y r o x i n , and t r i i o d o t h y r o n i n e . Iodine d e f i ciency r e s u l t s i n g o i t e r . The d i e t a r y allowance f o r i o d i n e reported i n the l a t e s t RDA s has not changed. For the prevention of g o i t e r , an i o d i n e intake of 1 ug/kg body weight i s recommended w i t h a d d i t i o n a l i o d i n e intake recommended f o r growing c h i l d r e n and pregnant women ( 7 ) . The adequacy of an i n d i v i d u a l s i o d i n e intake i s d i f f i c u l t to assess and intakes vary widely. Thus, i t i s s t i l l suggested that to prevent d e f i c i e n c i e s only i o d i z e d s a l t be used. f

Zinc. Zinc plays an e s s e n t i a l r o l e i n a number of important processes i n c l u d i n g : l ) enzymatic f u n c t i o n , 2) p r o t e i n synthesis and 3) carbohydrate metabolism ( 9 ) . There are at l e a s t 18 known z i n c - c o n t a i n i n g enzymes, i n c l u d i n g l a c t a t e dehydrogenase, carbon-

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

10.

TUMAN

A N D DOISY

Trace Elements in Nutrition

161

Table 3 TRACE ELEMENTS WITH BENEFICIAL EFFECTS IN MAN AND ANIMALS

Iron

Element

Adult RDA

(Fe)

10-18 mg

Iodine

(I)

100-150 ]ig

(Cu)

Estimated 80 yg/kg

Copper

Manganese (Mh)

Zinc

(Zn)

Cobalt

^Molybdenum (Mo)

Selenium (Se )

Tin

(Cr)

(Sn)

Vanadium

(V)

Fluorine

(F)

Silicon

Nickel

i n t e g r a l component of Hb, cytochrome and enzymes. e s s e n t i a l f o r thyroxine formation and p r e v e n t i o n of g o i t e r , component of s e v e r a l enzymes, eg. cytochrome oxidase c; r o l e i n t i s s u e Fe m o b i l i z a t i o n and Hb s y n t h e s i s , r e q u i r e d by s e v e r a l enzymes, eg. glucokinase, phosphoglucomutase, a c e t y l CoA synthetase, a r g i n a s e .

15 mg

(Co)

Chromium

Estimated 2-3 mg

B i o l o g i c a l Function

(Si)

(Ni)

3 Ug as V i t . Bi2 Estimated 2 yg/kg Not e s tablished Not e s tablished Not e s tablished Not e s tablished Not e s tablished Not e s tablished

Not e s tablished

enzymes, eg. l a c t a t e dehydrogenase, carbonic anhydrase, peptidases i n t e g r a l component of V i t . B]_2 and RBC formation. i n t e g r a l component of s e v e r a l e n enzymes, eg. xanthine oxidase, a l d e hyde oxidase. i n t e g r a l component of g l u t a t h i o n e peroxidase. i n t e g r a l p a r t of Glucose Tolerance F a c t o r ; r e q u i r e d f o r normal i n s u l i n response and CHO metabolism, r e q u i r e d f o r optimal growth by r a t s (1-2 ppm of d i e t ) , r e q u i r e d f o r optimal growth by r a t s and chickens. p r e v e n t i o n of d e n t a l c a r i e s and maintenance of normal s k e l e t o n ; r e q u i r e d f o r optimal growth by r a t s . r e q u i r e d f o r optimal growth by r a t s and chickens; important r o l e i n normal bone c a l c i f i c a t i o n and s t r u c t u r a l connective t i s s u e , r e q u i r e d f o r normal l i v e r f u n c t i o n by r a t s and chickens.

C l i n i c a l evidence of d e f i c i e n c y i n ADULT man unknown.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

162

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

i c anhydrase, and s e v e r a l peptidases ( 1 0 ) . Zinc d e f i c i e n c y i n animals has r e s u l t e d i n decreased synthesis of b o t h DNA and RNA, and reduced p r o t e i n synthesis has been observed i n z i n c - d e f i c i e n t rats. The r o l e of z i n c i n carbohydrate metabolism i s s t i l l cont r o v e r s i a l ; however, impaired glucose t o l e r a n c e has been reported i n z i n c - d e f i c i e n t r a t s (11 ). More r e c e n t l y , p a t h o l o g i c a l conditions that appear to be a consequence of inadequate z i n c n u t r i t u r e have been i d e n t i f i e d i n man. Dietary d e f i c i e n c y of zinc i n man i s a s s o c i a t e d w i t h anorexia, hypogeusia (impaired t a s t e ) and hyposmia (impaired s m e l l ) , r e t a r d e d growth, delayed sexual maturation, and impaired wound h e a l i n g (12_, 13, 14_). Zinc d e f i c i e n c y was r e c e n t l y r e p o r t ed i n 8 percent of 150 c h i l d r e n from middle-income f a m i l i e s i n Denver, with another 36 percent of these c h i l d r e n probably marginal i n t h e i r z i n c i n t a k e . These d e f i c i e n t c h i l d r e n showed poor growth (below 10th p e r c e n t i l e ) , poor a p p e t i t e , impaired t a s t e a c u i t y , and low h a i vealed that the d i e t s o symptoms improved with z i n c supplementation (15_). In the Middle E a s t , zinc d e f i c i e n c y , a s s o c i a t e d w i t h hypogonadism and dwarfism, has been demonstrated i n man ( 1 6 ) . These studies suggest that marginal z i n c d e f i c i e n c y may be more widespread than p r e v i o u s l y thought and that d i e t a r y intake of z i n c i n the United States cannot be assumed to be o p t i m a l . I t i s estimated that a t y p i c a l American d i e t s u p p l i e s b e tween 10 and 15 mg z i n c per day to meet an estimated d a i l y requirement of 10 mg/day (12). Thus, the average d a i l y intake i s only s l i g h t l y more than the estimated d a i l y requirement, and does not provide a s u f f i c i e n t s a f e t y margin. Therefore, people who consume foods which are lower than average i n a v a i l a b l e z i n c , such as meat analogs made from vegetable p r o t e i n , may s u f f e r from marginal z i n c intakes ( 1 2 ) . In view of the above, the 1974 RDA's f o r the f i r s t time i n c l u d e a recommendation f o r z i n c , w i t h 15 mg b e i n g suggested f o r a d u l t men and women, and 20 to 25 mg during pregnancy and l a c t a t i o n ( 7 ) . Chromium. Chromium i s e s s e n t i a l f o r the maintenance of normal carbohydrate metabolism i n at l e a s t three species of experimental animals. T r i v a l e n t chromium i s an i n t e g r a l p a r t of Glucose Tolerance Factor (GTF), and f u n c t i o n s as a c o f a c t o r f o r the p e r i p h e r a l a c t i o n of i n s u l i n . In the r a t , the f i r s t observed consequence of m i l d chromium d e f i c i e n c y i s an impairment of glucose t o l e r a n c e , caused by a reduced s e n s i t i v i t y o f p e r i p h e r a l t i s s u e s to i n s u l i n . A more severe degree of chromium d e f i c i e n c y leads to f a s t i n g hyperglycemia, g l y c o s u r i a , and m i l d growth r e t a r d a t i o n (17, 1 8 ) . There i s considerable evidence that chromium i s a l s o e s s e n t i a l f o r man. Impaired glucose t o l e r a n c e i s the hallmark of chromium d e f i c i e n c y i n man. There has been s p e c u l a t i o n that chromium d e f i c i e n c y may c o n t r i b u t e to the development of

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

10.

TUMAN

AND

Trace Elements in Nutrition

DOiSY

163

a t h e r o s c l e r o s i s ( 1 9 ) , and i t i s of i n t e r e s t that serum c h o l e s t e r o l l e v e l s increase w i t h age as t i s s u e chromium l e v e l s decrease w i t h age ( 1 9 ) . Evidence f o r the occurrence of chromium d e f i c i e n cy i n man and the e f f e c t of d i e t a r y chromium supplementation w i l l be discussed subsequently i n t h i s paper. Cobalt. Cobalt i s e s s e n t i a l to man only through i t s f u n c t i o n as an i n t e g r a l part of Vitamin B]_ and no other f u n c t i o n s f o r c o b a l t i n human n u t r i t i o n are known. Cobalt d e f i c i e n c y has never been produced i n a nonruminant animal and the r o l e of c o b a l t i n human n u t r i t i o n i s only a question of adequate d i e t a r y intake of Vitamin B , r a t h e r than o f c o b a l t i t s e l f ( ] J ) . RDA f o r Vitamin B i s 3 ug per day ( 7 ) . 2

1 2

T

n

e

1 2

Copper. The n u t r i t i o n a l e s s e n t i a l i t y of copper derives from i t s r o l e i n the s t r u c t u r e and f u n c t i o n of s e v e r a l cuproenzymes i n c l u d i n g cytochrom oxidase and a s c o r b i c a c i d oxidase. Copper-containing enzymes and copper-containing p r o t e i n s are r e q u i r e d f o r c e l l u l a r r e s p i r a t i o n , normal hemoglobin s y n t h e s i s , and normal bone formation. The copper-containing p r o t e i n ceruloplasmin i s reported to be i n t i m a t e l y i n v o l v e d i n t i s s u e i r o n m o b i l i z a t i o n (l_, 13). Absolute copper d e f i c i e n c y has never been observed i n human a d u l t s ; however, copper d e f i c i e n c y has been described i n p a t i e n t s s u f f e r i n g from general m a l n u t r i t i o n and i n i n f a n t s i n a d v e r t a n t l y f e d formulated d i e t s low i n copper (l). The normal d i e t a r y copper intake of between 2-5 mg per day i s s u f f i c i e n t to meet the recommended d i e t a r y allowance of 2 mg per day. Thus, copper d e f i c i e n c y does not appear to be a problem i n t h i s country ( 2 0 ) . Manganese. Manganese f u n c t i o n s as a c o f a c t o r f o r s e v e r a l metallo-enzymes i n c l u d i n g : glucokinase, phosphoglucomutase, a c e t y l CoA synthetase and a r g i n a s e . The existence of manganese d e f i c i e n c y has been demonstrated i n p i g s , p o u l t r y , r a t s , mice, c a t t l e , and sheep; however, evidence of human d e f i c i e n c y has never been obtained. Manganese d e f i c i e n c y was a c c i d e n t a l l y induced i n man through the feeding of a s y n t h e t i c r a t i o n (21 ). The manifestations of manganese d e f i c i e n c y i n animals i n c l u d e growth r e t a r d a t i o n , reduced f e r t i l i t y , s k e l e t a l abnormalities and d i s o r d e r s of the c e n t r a l nervous system ( a t a x i a of the newborn) (l_, l^). In a d d i t i o n , manganese plays an important r o l e i n normal carbohydrate metabolism, as suggested by the impaired glucose t o l e r a n c e and h y p o p l a s t i c p a n c r e a t i c i s l e t c e l l s observed i n manganese d e f i c i e n t guinea p i g s ( 2 2 ) . The d a i l y manganese requirements f o r man are unknown; however, from intake and balance s t u d i e s , i t i s estimated that a manganese intake of 2-3 mg/day i s adequate f o r a d u l t s ( 7 , 20). Molybdenum.

Molybdenum plays an e s s e n t i a l r o l e as an i n t e -

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

164

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

g r a l component of s e v e r a l enzymes i n c l u d i n g , xanthine oxidase, aldehyde oxidase and s u l f i t e oxidase. Molybdenum d e f i c i e n c y has been demonstrated i n lambs, c h i c k s , and turkey p o u l t s u s i n g h i g h l y p u r i f i e d d i e t s w i t h a low molybdenum content (approximate­ l y 20 yg/kg). Feeding t h i s low molybdenum d i e t r e s u l t e d i n depressed growth (23) and decreased a c t i v i t y of the enzyme xanthine oxidase ( 2 4 ) . Molybdenum d e f i c i e n c y has never been reported i n humans. An RDA f o r molybdenum has not been e s t a b l i s h e d ; however, balance s t u d i e s i n d i c a t e that p o s i t i v e balance i n man can be maintained w i t h a molybdenum intake of about 2 pg/kg body weight per day. Selenium (13, 2 3 ) . It has r e c e n t l y been demonstrated (29 ) that selenium i s an i n t e g r a l component of the enzyme g l u t a t h i o n e peroxidase. Glutathione peroxidase i s i n v o l v e d i n the r e o x i d a t i o n of reduced g l u t a t h i o n e . The enzyme has been i s o l a t e d from sheep r e d b l o o d c e l l s ; i 80,000; and i t contains one atom of selenium per p r o t e i n subunit of approximately 20,000 molecular weight (2). Selenium d e f i c i e n c y i s i n t i m a t e l y r e l a t e d to Vitamin Ε d e f i c i e n c y d i s e a s e s . For example, the simultaneous d e f i c i e n c y of selenium and Vitamin Ε causes a v a r i e t y of p a t h o l o g i e s i n animals i n c l u d i n g : f a t a l exudative d i a t h e s i s i n chicks and turkeys, l i v e r n e c r o s i s i n the r a t and p i g , m u l t i p l e n e c r o t i c degeneration of heart, l i v e r , muscle, and kidney i n the mouse, and muscular dystrophy i n lambs, c a l v e s , and chicks (white muscle d i s e a s e ) . Recent s t u d i e s i n chicks have demonstrated t h a t simple selenium d e f i c i e n c y , uncomplicated by inadequate Vitamin E, causes impaired growth, poor f e a t h e r i n g , and f i b r o t i c degenera­ t i o n of the pancreas (25). Selenium d e f i c i e n c y i n the r a t i s manifested by impaired growth, impaired h a i r coat development, and reproductive f a i l u r e ( 2 6 ) . Furthermore, feeding a seleniumd e f i c i e n t d i e t c o n t a i n i n g adequate Vitamin Ε to subhuman primates r e s u l t s i n h e p a t i c n e c r o s i s , nephrosis, degenerative changes i n c a r d i a c and s k e l e t a l muscle, weight l o s s and death ( 2 7 ) . As stated e a r l i e r , selenium possesses b o t h b e n e f i c i a l and toxic properties. N a t u r a l l y o c c u r r i n g selenium p o i s o n i n g i n animals r e s u l t s i n the acute t o x i c i t y syndrome, "blind staggers", and the chronic t o x i c i t y syndrome of " a l k a l i disease". In adult man, no evidence of e i t h e r selenium d e f i c i e n c y or t o x i c i t y has been demonstrated, however, selenium d e f i c i e n c y has been reported i n c h i l d r e n w i t h p r o t e i n - c a l o r i e m a l n u t r i t i o n ( 2 8 ) . Due to l a c k of s u f f i c i e n t information, i t has not been p o s s i b l e to e s t a b l i s h an RDA f o r selenium i n humans. T i n , Vanadium, F l u o r i n e ,

Silicon,

Nickel

The f i v e t r a c e elements discussed below have r e c e n t l y been found to be e s s e n t i a l f o r l a b o r a t o r y animals (2, 30, 31 ).

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

10.

TUMAN

AND

DOiSY

Trace Elements in

Nutrition

165

T i n . T i n was r e c e n t l y found to be r e q u i r e d f o r o p t i m a l growth i n r a t s . T i n d e f i c i e n c y has been produced i n r a t s by housing i n an i s o l a t o r environment and be feeding h i g h l y p u r i f i e d amino a c i d d i e t s low i n t i n . S i g n i f i c a n t growth s t i m u l a t i o n was observed when the d i e t s were supplemented w i t h 1-2 mg t i n / k g (ppm) of d i e t (2, 30, 31, 32). Vanadium. Vanadium i s e s s e n t i a l i n at l e a s t two animal species, the chick and the r a t . Vanadium d e f i c i e n c y i n chickens ( d i e t s c o n t a i n i n g l e s s than 10 yg/kg) r e s u l t s i n poor f e a t h e r growth (33) and r e t a r d e d bone development (30, 31). In the r a t , d i e t s s u p p l y i n g l e s s than 100 ng vanadium/g of d i e t (100 ppb ) showed decreased body growth (3A_) and r e c e n t l y i t was shown that r a t s respond to 50-100 ng vanadium/g of d i e t w i t h s i g n i f i c a n t growth s t i m u l a t i o n (£). Fluorine. In additio as f l u o r i d e , i n the p r e v e n t i o tenance of a normal body s k e l e t o n (13), r e c e n t l y f l u o r i n e has been shown to be e s s e n t i a l f o r o p t i m a l growth i n the r a t . S i g ­ n i f i c a n t growth e f f e c t s were produced w i t h 250 yg f l u o r i n e / 1 0 0 g of d i e t (2.5 ppm) ( 2 ) . S i l i c o n . S i l i c o n has been shown to be e s s e n t i a l f o r normal growth i n animals. S i l i c o n d e f i c i e n c y i n chicks and r a t s causes depressed growth, and abnormal bone c a l c i f i c a t i o n . S i l i c o n a l s o p l a y s an e s s e n t i a l r o l e i n mucopolysaccharide metabolism and normal, connective t i s s u e development. S i l i c o n was r e c e n t l y r e ­ ported to have a growth-promoting e f f e c t i n r a t s (2_, 30). N i c k e l . N i c k e l appears to be e s s e n t i a l f o r animals, and pathology c o n s i s t e n t w i t h n i c k e l d e f i c i e n c y has been produced i n c h i c k s , r a t s , and swine. N i c k e l d e f i c i e n c y i n these animals causes metabolic a b n o r m a l i t i e s i n the l i v e r , i n c l u d i n g a decreas­ ed oxygen uptake by l i v e r homogenates i n the presence of α-gly­ cerophosphate, increased l i v e r l i p i d s , i n c r e a s e d p h o s p h o l i p i d and c h o l e s t e r o l f r a c t i o n , and hepatocyte u l t r a s t r u c t u r a l abnormali­ t i e s (30, 31). Furthermore, i n the r a t , n i c k e l d e f i c i e n c y r e s u l t e d i n abnormal r e p r o d u c t i o n as suggested by increased f e t a l m o r t a l i t y (30). At the present time, no evidence f o r human e s s e n t i a l i t y has been demonstrated f o r these f i v e newer t r a c e elements. No estimate of man* s requirements f o r these newer e s s e n t i a l t r a c e elements can be o f f e r e d i n view of the c u r r e n t i n s u f f i c i e n t evidence and knowledge of i n t a k e s i n humans. Future Considerations As brought out at a recent symposium (35.) l i t t l e i s known about the i n t e r a c t i o n s that occur between e s s e n t i a l t r a c e e l e -

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

166

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

ments. Knowledge of t r a c e element i n t e r a c t i o n s i s of utmost importance to any v a l i d recommendation o f d i e t a r y i n t a k e . That i s to say, a h i g h d i e t a r y intake of a given element may reduce a v a i l a b i l i t y o f some other element. Such known r e l a t i o n s h i p s as high calcium intake antagonizing (or reducing) copper a v a i l a b i l i t y i s but one example of such an i n t e r a c t i o n . Tungstate i s known to antagonize molybdate, and copper and zinc a l s o i n t e r a c t . For example, people who are r e c e i v i n g therapeutic doses of zinc f o r burns may have an increased copper requirement. Thus, knowing the d a i l y intake of any given element i s only part of the problem. The intake of other ( i n t e r f e r i n g ) elements and t h e i r balance i s of utmost importance. These i n t e r r e l a t i o n s are o n l y beginning to u n f o l d and much more work i s needed on t h i s aspect of t r a c e elements i n n u t r i t i o n . Role of Chromium i n Human and Animal

Nutrition

Schwarz and Mertz i d e n t i f i e d t r i v a l e n t chromium as an i n t e g r a l component of the b i o l o g i c a l l y a c t i v e Glucose Tolerance F a c t o r (GTF) i n 1959 ( 3 6 ) . Since t h a t time a l a r g e body of convincing experimental evidence has accumulated suggesting that GTF i s r e q u i r e d f o r the maintenance of normal carbohydrate metab o l i s m by both animals and man (18^). Chromium n u t r i t i o n i n man has r e c e n t l y been reviewed by Hambidge (18) and Doisy, et a l . ( 37) and w i l l not be d e a l t with i n d e t a i l i n t h i s paper. The c h a r a c t e r i s t i c s of GTF as known at t h i s time are summarized below (17): GTF i s a n a t u r a l l y o c c u r r i n g , d i a l y z a b l e , heat and a c i d s t a b l e , organic compound of low molecular weight (400-600 d a l tons). ï t can be extracted and concentrated from brewers y e a s t , while l i v e r and kidney are a l s o recognized as p o t e n t i a l l y r i c h sources. The p r e c i s e s t r u c t u r e of GTF i s not yet known; however, Mertz (39) r e c e n t l y suggested t h a t the complex contains two n i c o t i n i c a c i d molecules per chromium atom. Furthermore, g l y c i n e , c y s t e i n e , and p o s s i b l y glutamic a c i d may be r e q u i s i t e amino a c i d s . The amino a c i d s may only be r e q u i r e d to make the complex water s o l u b l e , and the b i o l o g i c a l a c t i v i t y may be due to the chromium and n i c o t i n i c a c i d s i n a unique c o o r d i n a t i o n complex. Recently, Mertz has prepared b i o l o g i c a l l y a c t i v i e s y n t h e t i c chromiumn i c o t i n i c a c i d complexes which seem to be s i m i l a r t o , but not i d e n t i c a l w i t h , the n a t u r a l l y o c c u r r i n g GTF complex ( 3 9 ) . GTF contains t r i v a l e n t chromium as the a c t i v e metal i o n . The b i o l o g i c a l e f f e c t of chromium "in v i t r o " and "in vivo", i s s o l e l y dependent on the valency s t a t e and o n l y t r i v a l e n t chromium exhibits b i o l o g i c a l a c t i v i t y . The b i o l o g i c a l e f f e c t s of GTFchromium are q u a l i t a t i v e l y s i m i l a r , but q u a n t i t a t i v e l y much g r e a t e r than simple i n o r g a n i c chromium complexes. For example, much s m a l l e r q u a n t i t i e s o f chromium are r e q u i r e d "in vivo" to r e s t o r e normal glucose t o l e r a n c e i n r a t s i f given i n the form of GTF. Furthermore, the b i o - a v a i l a b i l i t y of chromium to animals

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

10.

TUMAN

A N D DOISY

Trace Elements in Nutrition

167

and man i s dependent on the chemical form. Chromium i n the form of GTF, i s b i o l o g i c a l l y a v a i l a b l e and b e t t e r absorbed than simple i n o r g a n i c chromium s a l t s . For example, l e s s than 1 p e r ­ cent of an o r a l dose of chromium i n the form of chromic c h l o r i d e i s absorbed as compared to 10-25 percent of the chromium i n an o r a l dose of GTF (38.)· The chemical form of chromium a l s o de­ termines i t s d i s t r i b u t i o n i n t i s s u e s . Only chromium i n the form of GTF i s concentrated i n the l i v e r and only GTF chromium i s a v a i l a b l e to the fetus through p l a c e n t a l t r a n s p o r t . "In v i t r o " s t u d i e s suggest that GTF f u n c t i o n s by p o t e n t i a t ­ ing the a c t i o n of i n s u l i n and, as such, i t i s a r e q u i r e d c o f a c t o r f o r maximal i n s u l i n response i n a l l i n s u l i n - s e n s i t i v e t i s s u e s (17). As a r e s u l t of an uncorrected d e f i c i e n c y of chromium, a normal i n s u l i n response may only be achieved w i t h u n p h y s i o l o g i c a l l y high concentrations of i n s u l i n . Evidence f o r Chromium D e f i c i e n c It i s w e l l e s t a b l i s h e d that chromium d e f i c i e n c y can be i n ­ duced (both a c c i d e n t a l l y and d e l i v e r a t e l y ) i n animals by feeding a d i e t that i s low i n a v a i l a b l e chromium ( 3 6 , 40, 41., 42 ) . Evidence has accumulated which suggests that chromium d e f i c i e n c y a l s o e x i s t s i n c e r t a i n segments o f the human p o p u l a t i o n . This evidence i s i n d i r e c t and i s based on the f o l l o w i n g observations: l ) t i s s u e chromium l e v e l s decrease w i t h i n c r e a s i n g age i n the United States ( 4 3 , 4 4 ) ; 2) absence of an acute r i s e of serum chromium f o l l o w i n g an i n s u l i n or glucose challenge i n d i a b e t i c subjects ( 4 5 ) , and i n pregnant woment w i t h impaired glucose t o l e r a n c e Γ46 ); 3) diabetes i s a s s o c i a t e d w i t h low chromium l e v e l s i n h a i r (47.) and l i v e r (AS) compared to n o n d i a b e t i c con­ t r o l s ; 4 ) insulin-dependent d i a b e t i c s metabolize chromium i n a manner that i s abnormal as compared to n o n - d i a b e t i c subjects (49 ) and 5) subjects w i t h impaired glucose t o l e r a n c e , i n c l u d i n g some maturity-onset d i a b e t i c s ( 5 0 ) , middle-aged subjects (51 ), c h i l d r e n i n Jordan s u f f e r i n g w i t h kwashiorkor (52_), c h i l d r e n i n Turkey s u f f e r i n g from marasmus (22)' l subjects (37, 49, 54 ) show improved glucose t o l e r a n c e a f t e r o r a l chromium supplementation of the d i e t . Thus i t appears l i k e l y that marginal or overt chromium d e f i c i e n c y occurs i n the United States and elsewhere i n the world. a

n

d

s

o

m

e

e

d

e

r

l

y

E f f e c t s of Chromium and GTF Supplementation of the Diet The f a c t that t i s s u e chromium l e v e l s decrease w i t h age i n the United States and are e x c e p t i o n a l l y low i n e l d e r l y s u b j e c t s , i s compatible w i t h , but not proof of chromium d e f i c i e n c y . How­ ever, these data suggest a r o l e f o r chromium i n e x p l a i n i n g the e t i o l o g i c a l b a s i s f o r impaired glucose t o l e r a n c e which i s ex­ h i b i t e d by the m a j o r i t y of e l d e r l y subjects over 70 years of age. Hence, a tempting c o n c l u s i o n i s that many e l d e r l y i n d i v i d u a l s

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

168

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

have impaired glucose tolerance on the b a s i s of n u t r i t i o n a l chromium d e f i c i e n c y . Therefore, i f a d e f i c i e n c y i s suspected, i n c r e a s i n g the d i e t a r y intake of chromium should r e l i e v e the d e f i c i e n c y and normalize the glucose t o l e r a n c e . This has been done with e l d e r l y subjects thought to be chromium d e f i c i e n t . In the o r i g i n a l study by Levine, et a l . (37, 54), 86 percent of the e l d e r l y p o p u l a t i o n l i v i n g i n the Onondaga County Home d i s p l a y e d abnormal glucose tolerance t e s t s . Table 4 summarizes the r e s u l t s obtained when ten e l d e r l y s u b j e c t s , with abnormal glucose t o l e r a n c e , were t r e a t e d with 150 y g d a i l y of supplemental i n o r g a n i c chromium f o r periods up to four months. In a d d i t i o n , two young subjects o r i g i n a l l y thought to be normal and one subj e c t with hemochromatosis are i n c l u d e d . Shown are the mean two hour glucose t o l e r a n c e t e s t s on seven s u b j e c t s , before and a f t e r d i e t a r y chromium supplementation. The c r i t e r i a used f o r abnorm a l i t y i n these and subsequent s t u d i e s are a peak plasma glucose l e v e l above 185 mg/dl and/o ( c r i t e r i a adapted from the mean plasma glucose l e v e l s while on the chromium supplement, p a r t i c u l a r l y at 60, 90, and 120 minutes, are lowered c o n s i d e r ably i n a l l seven subjects compared to the pre-chromium b a s e l i n e control tests. In a d d i t i o n , the mean peak plasma glucose l e v e l of the four "responding" e l d e r l y subjects as a group was lowered from 182 to 146 mg/dl and the mean plasma sugar l e v e l 2 hours a f t e r a glucose l o a d d e c l i n e d from 156 to 115 mg/dl. Thus, i n o r g a n i c chromium supplementation of the d i e t was e f f e c t i v e i n r e s t o r i n g the impaired glucose tolerance to normal i n these seven s u b j e c t s , and i t was concluded that these subjects were, i n f a c t , chromium d e f i c i e n t . It should be noted that of the ten e l d e r l y subjects t r e a t e d , only four (40%) responded f a v o r a b l y to chromium supplementation. However, i t should a l s o be pointed out that there are many e t i o l o g i e s f o r impaired glucose tolerance, i n c l u d i n g i n f e c t i o n , emotional s t r e s s , e t c . , and only those subjects w i t h a p r e - e x i s t i n g chromium d e f i c i e n c y would be expected to b e n e f i t from chromium supplementation. On the other hand, a f a i l u r e to respond to i n o r g a n i c chromium does not exclude the p o s s i b i l i t y of a GTF d e f i c i e n c y . It i s poss i b l e that the subjects that d i d not respond to i n o r g a n i c chromium may have l o s t the a b i l i t y to convert i n o r g a n i c chromium to GTF. These subjects may have a more favorable response to d i e t a r y supplementation w i t h GTF. More r e c e n t l y , the e f f e c t of d i e t a r y GTF supplementation on abnormal glucose tolerance i n e l d e r l y subjects was s t u d i e d ( 3 7 ) . In t h i s case, 45 percent (14/31) of the subjects over the age of 65 d i s p l a y e d impaired glucose t o l e r a n c e . Each of twelve s u b j e c t s , who volunteered to go on a commercial brewers yeast e x t r a c t c o n t a i n i n g GTF, r e c e i v e d a d a i l y supplement (4^g Yeastamin/day) f o r a p e r i o d o f one to two months. Yeastamin has been See

footnote f o l l o w i n g L i t e r a t u r e C i t e d .

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

10.

TUMAN

AND

DOiSY

Trace Elements in Nutrition Table 4

MEAN GLUCOSE TOLERANCE TESTS OF RESPONDERS - Cr PLASMA GLUCOSE CONCENTRATION mg/dl Elderly Subjects/

Age

Ο

30

60

90

120

No. of Tests

143

149

79

+

84 82

138

129

163 125

170 106

2 2

78

+

67 83

122 132

186 139

197 126

167 123

2 2

88

+

82 81

153 136

180 157

165 140

153 131

2 3

98

96

+ 101

148 142

181 152

152 111

135 100

2 3

GF

HH

Ech

FW

Young Subjects

Cr

Age

22

+

90 91

161 161

192 126

151 82

115 104

2 3

24

+

81 81

146 160

194 147

127 132

120 118

2 3

-ι-

111 97

209 155

225 170

166 150

125 139

2 2

AG

LS

Subjects w i t h Hemochromatosis

Age

49

Supplement: 50 yg of Cr three times d a i l y (CrCl3'6H20 ). ^Reprinted by permission of p u b l i s h e r . Doisy, et a l . , "Effects and Metabolism of Chromium i n Normals, E l d e r l y S u b j e c t s , and D i a b e t i c s " , In: Trace Substances i n Environmental H e a l t h - I I , D. D. Hemphill, E d . , U n i v e r s i t y o f M i s s o u r i , Columbia, pp. 75-82 (37).

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

170

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

shown by Mertz to possess potent GTF a c t i v i t y . I t i s recognized that the observed responses may or may not be due to the GTF content of Yeastamin. That GTF i s the a c t i v e component must await the a v a i l a b i l i t y of pure GTF. The mean e f f e c t o f s u p p l e mentation of the d i e t on glucose t o l e r a n c e i n s i x "responding" e l d e r l y subjects i s shown i n Table 5. The subjects d i s p l a y s e v e r e l y impaired glucose t o l e r a n c e p r i o r to GTF supplementation, w i t h a peak plasma glucose l e v e l greater than 200 mg/dl and a two hour glucose value of 178 mg/dl. Following d i e t a r y GTF supplementation, however, s i x of the e l d e r l y subjects show an improved glucose t o l e r a n c e to values w i t h i n normal l i m i t s , with the one and two hour plasma glucose l e v e l s being s i g n i f i c a n t l y reduced to 162 and 132 mg/dl r e s p e c t i v e l y . In a d d i t i o n (Table 5 ) , the serum i n s u l i n l e v e l s measured during the t e s t s are decreased while on the supplement, p a r t i c u l a r l y at two hours. Thus, the work l o a d of the pancreas i s decreased and l e s s endogenou serum l e v e l s , i s neede when adequate amounts of GTF are a v a i l a b l e . This i s i n agreement w i t h the r o l e of GTF as a c o f a c t o r f o r normal i n s u l i n response. Furthermore, i n a d d i t i o n to the r e d u c t i o n i n plasma glucose and i n s u l i n l e v e l s , some subjects a l s o respond to GTF supplement a t i o n with a s i g n i f i c a n t r e d u c t i o n i n f a s t i n g serum c h o l e s t e r o l levels. C h o l e s t e r o l l e v e l s were s i g n i f i c a n t l y reduced i n these s i x subjects from a mean value of 245 to 205 mg/dl, a decrease of 40 mg/dl. In those subjects w i t h elevated t r i g l y c e r i d e l e v e l s there i s a l s o a r e d u c t i o n i n plasma t r i g l y c e r i d e . Another group of subjects d i s p l a y i n g an incidence of i m p a i r ed glucose t o l e r a n c e which i s c l e a r l y greater than that observed i n the general p o p u l a t i o n are the s i b l i n g s o f known d i a b e t i c s . The e f f e c t s of d i e t a r y GTF supplementation on the glucose t o l e r ance of a s i b l i n g o f an i n s u l i n - r e q u i r i n g d i a b e t i c are described i n Table 6 ( 3 7 ) . It i s apparent from the two i n i t i a l screening GTT s that t h i s subject d i s p l a y s " d i a b e t i c - l i k e " glucose t o l e r ance, with elevated plasma glucose l e v e l s g r e a t e r than 200 mg/dl and serum i n s u l i n l e v e l s g r e a t e r than 200 yU/ml, at two time p o i n t s during each t e s t . In a d d i t i o n , the subject i s h y p e r t r i glyceridemic. A f t e r approximately e i g h t months of d i e t a r y supplementation with 4 - 8 grams o f Yeastaiiin/day, glucose t o l e r ance i s normalized. In the l a s t t e s t (4/19/74) normal glucose l e v e l s are accompanied by a s i g n i f i c a n t r e d u c t i o n i n g l u c o s e induced plasma i n s u l i n l e v e l s . Furthermore, plasma t r i g l y c e r i d e s have been reduced to w i t h i n normal l i m i t s . As of t h i s w r i t i n g , approximately 80 other subjects with impaired glucose tolerance are on a d i e t a r y supplement c o n t a i n i n g GTF. Although not a l l subjects respond with improved glucose t o l e r a n c e , the r e s u l t s d e s c r i b e d here are more or l e s s t y p i c a l of the responses obtained i n the m a j o r i t y of s u b j e c t s . One major d i f f e r e n c e between subjects i s the v a r i a t i o n i n the length o f time on the supplement before improvement i s observed. This i s f

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

I

70 -

™ ;178 δ

δ 5

I 17

δ3 -

118

0.001

178 J 132 -

2

0.01

245 J 9t 205 - 10

Cholesterol mg/dl

NS

121 ΐ δ 112 - 12

(37).

Triglyceride mg/dl

IN

Number o f t e s t s i n parentheses. Mean - SE tUsing paired t t e s t , difference i s s i g n i f i c a n t . Supplement: 4 g of Yeastamin/day. GTT: 100 g o r a l l o a d . Reprinted by permission of p u b l i s h e r : N u t r i t i o n Foundation Monograph, Academic P r e s s , NY,

24 5 6 26 - 12

microunits/ml

Mean Serum I n s u l i n Levels

Before GTF A f t e r GTF

1 201 7 162 - 11 0.01

106 ΐ 4 9 9 - 4 NS

Before GTF ( l l ) * A f t e r GTF (9) Significance

Mean Plasma Glucose Levels mg/dl Time i n hours — 0

EFFECT OF GTF* SUPPLEMENTATION OF THE DIET ON GLUCOSE TOLERANCE TESTS ELDERLY SUBJECTS WITH IMPAIRED TOLERANCE

Table 5

172

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

Table 6 EFFECT OF GTF SUPPLEMENT ON GLUCOSE TOLERANCE/SIBLING OF DIABETIC, AGE 30, MALE Time Glucose Insulin ChoiesTriglycerDate mg/dl yunits/ml t e r o l ides 117 251 267 150 96

35 >200 >200 95 27

194

265

108 176 220 206

38 200 >200 183

198

256

158 97

138 39

0' 15» 30' 45' 60' 90' 120' ISO 180'

112 162 212 204 185 156 85 73 76

5 52 110 125 166 130 25 12 12

150

68

0' 30' 60' 90' 120' 150' 180'

92 154 131 126 117 75 63

10 57 52 44 35 22 10

206

134

0 30 ' 60» 120' 180»

8/10/73

T

0'

8/23/73

15' 30 » 45 ' 60» 90' 120' GTF 8/24/73 1/4/74

1

4/19/74

Subject gained 8 l b s . between 1/4/74 to 4/19/74. Supplement: 4 g of Yeastamin per day 8/24/73 - 11/9/73. 8 g of Yeastamin per day 11/10/73 - 4/19/74. Reprinted by permission of p u b l i s h e r : N u t r i t i o n Foundation Monograph, Academic Press, NY (37).

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

10.

TUMAN

AND

DOiSY

Trace Elements in Nutrition

173

understandable s i n c e the degree of chromium and/or GTF d e f i c i e n c y might be expected to vary w i t h each s u b j e c t . For example, the e l d e r l y subjects responded i n 1-2 months time, whereas i n some s i b l i n g s of d i a b e t i c s 6 - 8 months are r e q u i r e d before n o r m a l i z a t i o n occurs. Thus, evidence i s accumulating suggesting that chromium d e f i c i e n c y does e x i s t i n c e r t a i n segments of our p o p u l a t i o n . The r i s k and incidence of chromium d e f i c i e n c y appears to be g r e a t e s t i n the f o l l o w i n g p o p u l a t i o n groups: l ) i n o l d e r age groups, 2 ) i n c h i l d r e n w i t h p r o t e i n - c a l o r i e m a l n u t r i t i o n , 3) i n i n s u l i n - r e q u i r i n g d i a b e t i c s , 4 ) i n pregnant women, p a r t i c u l a r l y multiparae ( g e s t a t i o n a l d i a b e t e s ) , and 5) subjects maintained on formulated d i e t s f o r long periods of time may be a high r i s k f o r chromium d e f i c i e n c y . I t i s proposed that chromium d e f i c i e n c y may be caused by inadequate d i e t a r y intake and/or poor a v a i l a b i l i t y o f chromium from f o o d s t u f f s . The average America chromium, with intakes i n the USA varying from 5 yg/day to over 100 yg/day ( l , 18). The average chromium intake f o r the e l d e r l y subjects (54T was 52 yg d a i l y . The d a i l y u r i n a r y l o s s of chromium i n normal a d u l t s ranged from 3-50 yg/day ( 4 6 , 55.) thus, t h i s i s the minimal amount that must be r e p l a c e d i n order to m a i n t a i n balance i n the a d u l t . As i n d i c a t e d e a r l i e r , absorption of chromium can vary from l e s s than 1 percent to 25 percent of a given dose, depending on the form i n which i t i s p r e s e n t . Theref o r e , the d i e t a r y intake r e q u i r e d to balance the u r i n a r y l o s s could vary from 50 yg to 500 yg. D i e t s e x c e p t i o n a l l y low i n chromium which do not adequately r e p l a c e l o s s e s could l e a d to chromium d e f i c i e n c y . The importance of e v a l u a t i n g f o o d s t u f f s on the b a s i s of b i o l o g i c a l l y meaningful chromium r a t h e r than t o t a l chromium content was r e c e n t l y d i s c u s s e d (56_). Due to inadequate knowledge of the forms and b i o l o g i c a l a v a i l a b i l i t y of chomium i n foods, an RDA cannot be e s t a b l i s h e d . However, i t has been suggested that a d a i l y intake of 10-30 yg of chromium i n the form of GTF would meet our d a i l y requirement ( 2 0 ) . I t has a l s o been suggested that inadequate d i e t a r y i n t a k e of chromium may occur because of l o s s e s of chromium i n food r e f i n i n g processes (57, 58). The chromium content of various wheat and sugar products and the marked l o s s o f chromium that occurs during the r e f i n i n g of these food s t a p l e s are described below. Whole g r a i n wheat contains 1.75 yg chromium/g Dry Wgt. compared to 0.23 yg/g Dry Wgt. f o r r e f i n e d white f l o u r and 0.14 yg/ g Dry Wgt. f o r white b r e a d . This represents an 87% l o s s of chromium i n the refinement of whole wheat to white f l o u r and a 92% l o s s of chromium i n going from n a t u r a l wheat to the consumer item of white b r e a d . Whole wheat bread r e t a i n s a l i t t l e more of the o r i g i n a l chromium, but there i s s t i l l a g r e a t e r than 70% l o s s of chromium i n the r e f i n i n g p r o c e s s . S i m i l a r l o s s e s of chromium occur i n the r e f i n i n g of sugar.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

174

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

Refined white t a b l e sugars r e t a i n very l i t t l e of the o r i g i n a l chromium contained i n the raw sugars. There i s a 77-94% l o s s of chromium on a dry weight b a s i s i n going from u n r e f i n e d raw sugar to the r e s u l t i n g consumer item which the American people use much of (106-120 lbs/person/annum). Raw sugar provides 6 - 8 . 8 yg Cr/100 k c a l compared to only 0 . 5 - 2 . 5 yg Cr/100 k c a l f o r r e f i n e d sugar. Most of the chromium i s removed during the r e f i n i n g p r o c e s s , i . e . , f i n a l molasses (47 yg Cr/100 k c a l ) . Therefore, i t i s obvious from data of t h i s k i n d that modern methods o f food p r o c e s s i n g remove a l a r g e percentage of chromium from two very important food items, wheat and sugar. Thus, d i e t s h i g h i n r e f i n e d sugar and r e f i n e d wheat products could c o n t r i b u t e to marginal chromium i n t a k e s . However, chromium i s not the only e s s e n t i a l t r a c e element that i s s i g n i f i c a n t l y decreased during the process of r e f i n i n g of wheat ( 5 9 ) . The l o s s e s of s i x other e s s e n t i a l t r a c e elements that occur during the r e f i n i n In a d d i t i o n to 76% of th e s s e n t i a l trace metals are removed during the m i l l i n g of wheat to white f l o u r . The l o s s e s i n f l o u r i n c l u d e : 76% of the o r i g i n a l i r o n content, 78% of the z i n c , 86% of the manganese, 68% of the copper, 48% of the molybdenum, and 89% of the c o b a l t . As with chromium, the h i g h e s t c o n c e n t r a t i o n of these trace elements i s found i n the l e s s r e f i n e d f r a c t i o n s l i k e germ and b r a n . S i m i l a r l o s s e s of e s s e n t i a l t r a c e elements occur when other important food items are d i v i d e d i n t o t h e i r component parts by e i t h e r refinement or e x t r a c t i o n (58_). As a r e s u l t of p a r t i t i o n ing r i c e , s i g n i f i c a n t amounts of f i v e e s s e n t i a l trace elements are l o s t , i n c l u d i n g : 75% o f the chromium, 46% of the manganese, 75% of the z i n c , 27% of the copper, and 38% of the c o b a l t . In a d d i t i o n , 83% of the magnesium i s removed i n the p o l i s h i n g p r o cess. In going from corn to corn meal, there i s a marked l o s s of three e s s e n t i a l trace elements i n c l u d i n g : a 56% l o s s of chromium, a 56% l o s s o f manganese, and a 51% l o s s of z i n c . In a d d i t i o n to the a l r e a d y noted h i g h l o s s of chromium i n r e f i n e d sugar, there i s a greater than 80% l o s s of f o u r other e s s e n t i a l t r a c e elements i n the p r o d u c t i o n of white t a b l e sugar. These l o s s e s i n c l u d e 90% of the manganese, 98% of the z i n c , 83% of the copper, 88% of the c o b a l t , as w e l l as 98% of the macro-element magnesium. From these d a t a , i t i s apparent that the t r a c e mineral cont e n t of many of our major f o o d s t u f f s , i n c l u d i n g r e f i n e d f l o u r , c e r e a l products, r e f i n e d sugar and r i c e , are markedly reduced during the r e f i n i n g p r o c e s s e s . Increased consumption of h i g h l y r e f i n e d foods, snack foods, and food analogs could l e a d to American d i e t s that may be marginal w i t h respect to adequate intakes of s e v e r a l t r a c e element e s s e n t i a l f o r good h e a l t h , and i n t e n t i o n a l excessive consumption of these low n u t r i e n t foods might l e a d to n u t r i t i o n a l d e f i c i e n c y diseases through replacement of conventional food n u t r i e n t s .

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

10.

TUMAN

AND

DOiSY

Trace Elements in Nutrition

175

The N a t i o n a l Research C o u n c i l has r e c e n t l y recommended "enrichment" o f foods made from wheat, corn, and r i c e w i t h ten vitamins and m i n e r a l s , i n c l u d i n g i r o n , z i n c , calcium, and magnesium. It can be a n t i c i p a t e d that f u t u r e recommendations may i n ­ clude a d d i t i o n a l e s s e n t i a l t r a c e elements not now i n c l u d e d i n t h i s suggested enrichment program. S c i e n t i f i c proof of marginal or overt d e f i c i e n c y must be obtained before any f u t u r e a d d i t i o n s would be considered. The evidence f o r chromium d e f i c i e n c y i s slowly a c c r u i n g , but f u r t h e r work remains to be done. Acknowledgement: This i n v e s t i g a t i o n was supported by part by USPHS Grants AM 15,100 and RR 229.

Literature Cited

1. WHO Expert Committee on Trace Elements in Human Nutrition, (1973), World Health Organization Technical Report Series, No. 532, Geneva, 1-65. 2. Schwarz, K., Fed. Proc, (1974), 33 (6), 1748-1757. 3. Maugh, T. H., Science, (1973), 181, 253-4. 4. Allaway, W. H., "Trace Elements in Environmental Health (II)" (D. D. Hemphill, ed.) Columbia, Missouri: University of Missouri Press, (1969), pp. 181-206. 5. Schwarz, K., "Trace Element Metabolism in Animals - 2", University Park Press, Baltimore, (1974), pp. 355-380. 6. Ten-State Nutrition Survey 1968-1970: I. Historical Devel­ opment, and II. Demographic Data; III. Clinical, Anthropo­ metry, Dental, IV. Biochemical; V. Dietary and Highlights. DHEW Pubs. No. (HMS) 72-8130,-8131,-8132,-8133, and -8134 (1972). 7. Recommended Dietary Allowances, 1974. Food and Nutriton Board, National Academy of Sciences - National Research Council, Washington, D.C., 8th edition, Publ. 2216. 8. Mertz, W., Journ. Am. Dietet. Assoc. (1974), 64, 163-167. 9. Halsted, J. Α., Smith,J.C.,and Irwin, M. I., Jour, of Nutr., (1974), 104 (3), 347-378. 10. Parisi, A. F., and Vallee, B. L., Am. J. Clin. Nutr., (1969), 22, 1222-1239. 11. Quarterman, J., Mills, C. F., and Humphries, W. R., Biochem. Biophys. Res. Commun., (1966), 25, 354-358. 12. Sandstead, H. H., Am. J. Clin. Nutr. (1973), 26, 1251. 13. Underwood, E. J., "Trace Elements in Human and Animal Nutri­ tion", Academic Press, New York, 3rd edition (1971 ), p. 208. 14. Henkin, R. I., In: "Newer Trace Elements in Nutrition" (Mertz, W. and Cornatzer, W. E., ed.) Marcel Dekker, New York, (1971), p. 255. 15. Hambidge, K. M., Hambidge,C.,Jacobs, Μ. Α., and Baum,J.D.,

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

176

PHYSIOLOGICAL EFFECTS OF FOOD CARBOHYDRATES

Pediat. Res., (1972), 6, 868. 16. Halsted, J. Α., Ronaghy, Η. Α., Abadi, P., Haghshenass, M., Amirhakemi, G. H., Barakat, R. Μ., and Rheirihold, J. G., Am. J. Med. (1972), 53, 277. 17. Mertz, W., Physiol. Rev., (1969), 49, 169-239. 18. Hambidge, Κ. M., Am. J. Clin. Nutr., (1974), 27, 505-514. 19. Schroeder, Η. Α., Nason, A. P., and Tipton, I. H., J. Chron. Dis., (1970), 23, 123. 20. Mertz, W., Ann. N.Y. Acad. Sci. in Geochemical Environment in Relation to Health and Disease, (H. C. Hopps and H. L. Cannon Editors), (1971), 199, 191-199. 21. Doisy, Ε. Α., Jr., "Trace Element Metabolism in Animals-2", University Park Press, Baltimore, (1974) pp. 668-670. 22. Everson, G. J., and Shrader, R. E., J. Nutr., (1968), 94, 89. 23. Reid, B. L., Kurnick, Α. Α., Suacha, R. L., and Couch, J. R., Proc. Soc. Exp. Biol. (1956) 93 245 24. Higgins, E. S., Richert Nutr., (1956), 59, 25. Thompson, J. M. and Scott, M. L., J. Nutr., (1969), 97, 335. 26. McCoy, Κ. Ε. M., and Weswig, P. H., J. Nutr., (1969), 98, 383. 27. Muth, O. H., Weswig, P. H., Whanger, P. D., and Oldfield, J. E., Am. J. Vet. Res., (1971), 32, 1603. 28. Burk, R. F., Jr., Pearson, W. N., Wood, R. P., and Viteri, F., Am. J. Clin. Nutr., (1967), 20, 723. 29. Rotruck, J. T., Pope, A. L., Ganther, H. E., Swanson, A. B., Hafeman, D. G., and Hoekstra, W. G., Science, (1973), 179, 588. 30. Nielsen, F. H. and Sandstead, H. H., Am. J. Clin. Nutr., (1974), 27, 515-520. 31. Nielsen, F. H., Food Technology, (January, 1974), 38-44. 32. Schwarz, K., Milne, D. B., and Vinyard, E., Biochem. Biophys. Res. Commun., (1970), 40, 22. 33. Hopkins, L. L., Jr., and Mohr, H. E., In: "Newer Trace Elements in Nutrition", (W. Mertz and W. E. Cornatzer), Marcel Dekker, New York, (1971 ), p. 195. 34. Strasia, C. Α., Ph.D. Thesis, Ann Arbor, Michigan, University Microfilms, (1971). 35. Hill, C.H., "Interactions of Trace Elements", Symposium: Trace Elements and Human Disease, Wayne State University School of Medicine, Detroit, Michigan, July 1974, in press. Nutrition Foundation Monograph Academic Press, New York, NY. 36. Schwarz, K., and Mertz, W., Arch. Biochem. Biophys., (1959), 85, 292-295. 37. Doisy, R. J., Streeten, D. H. P., Friberg, J. M., and Schneider, A. J., "Chromium Metabolism in Man and Biochemical Effects", Symposium: Trace Elements and Hyman Disease, Wayne State University School of Medicine, Detroit, Michigan, July 1974, in press. Nutrition Foundation Monograph, Academic Press, New York, NY.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

10.

TUMAN

AND

DOISY

Trace

Elements

in

Nutrition

177

38. Mertz, W. and Roginski, Ε. E. In: "Newer Trace Elements in Nutrition", (Mertz, W. and Cornatzer, W. E., ed.), Marcel Dekker, New York, (1971), p. 123. 39. Mertz, W., Fed. Proc. (1974), 33 (3), 659 (Abstract). 40. Doisy, R. J., Endocrinology, (1963), 72, 273-278. 41. Schroeder, Η. Α., Life Sci., (1965), 4, 2057-2062. 42. Davidson, I. W. F., Lang, C. Μ., and Blackwell, W. L., Diabetes, (1967), 16, 395-401. 43. Schroeder, Η. Α., Balassa, J. J., and Tipton, I. H., J. Chronic Dis., (1962), 15, 941-964. 44. Hambidge, Κ. M. and Baum, J. D., Am. J. Clin. Nutr., (1972), 25, 376-379. 45. Glinsmann, W. H., Feldman, F. J., and Mertz, W., Science, (1966), 152, 1243. 46. Hambidge, Κ. M., In: "Newer Trace Elements in Nutrition", (Mertz, W. and Cornatzer W E. editors) Marcel Dekker New York, (1971) 47. Hambidge, Κ. M. and Rodgerson, D. O., andO'Brien,D., Diabetes, (1968), 17, 517-519. 48. Morgan, J. M., Metabolism, (1972), 21, 313-316. 49. Doisy, R. J., Streeten, D. H. P., Souma, M. L., Kalafer, M. E., Rekant, S. I., and Dalakos, T. G., In: "Newer Trace Elements in Nutrition", (mertz, W. and Cornatzer, W. E., editors), Marcel Dekker, New York, (1971), p. 155. 50. Glinsmann, W. H., and Mertz, W., Metabolism, (1966), 15, 510-520. 51. Hopkins, L. L., Jr., and Price, M. G., In: Western Hemis­ phere Nutrition Congr., Puerto Rico, (1968), 2, 40-41. (Abstract ). 52. Hopkins, L. L., Jr., Ransome-Kuti, O., and Majaj, A. S., Am. J. Clin. Nutr., (1968), 21, 203-211. 53. Gurson, C. T. and Soner, G., Am. J. Clin. Nutr., (1973), 26, 988-991. 54. Levine, R. Α., Streeten, D. H. P., and Doisy, R. J., Metabolism, (1968), 17, 114-125. 55. Wolf, W., Greene, F. E., Mitman, F. W., "Determination of Urinary Chromium by Low Temperature Ashing-Flameless Atomic Absorption", (1974), Fed. Proc, 33 (3), 59 (Abstract). 56. Toepfer, W. W., Mertz, W., Roginski, Ε. E., and Polansky, M. M., Food Chem., (1973), 21, 69. 57. Schroeder, Η. Α., Am. J. Clin. Nutr., (1968), 21, 230-244. 58. Schroeder, Η. Α., Am. J. Clin. Nutr., (1971), 24, 562-573. 59. Czerniejewski, C. P., Shank, C. W., Bechtel, W. G., and Bradley, W. B., Cereal Chemistry, (1964), 41, 65-72. *Yeastamin, a brewers yeast extract obtained from A. E. Staley Co., Vico Asmus Division, Chicago, Illinois.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

11 Oligosaccharidases of the Small Intestinal Brush Border GARY M. GRAY Division of Gastroenterology, Department of Medicine, Stanford University School of Medicine, Stanford, Calif. 94305

Carbohydrates represen of calories for man but must be digested to monosaccharides before absorption can occur in the small intestine. Until the last few years, it was commonly believed that all hydrolysis occurred within the intestinal lumen under the influence of secretions from the intestinal wall, the so-called succus entericus. However, the work of Crane and his colleagues (1-3) localizing disaccharidase activities to the brush border membrane of the intestine drew attention to the potential role of the small Table 1 intestinal cell in carbohydrate diges­ DIGESTION OF CARBOHYDRATE tion. Table 1 out­ LUMINAL INTESTINAL lines important FOOD SOURCE %OFCHO HYDROLYSIS HYDROLYSIS carbohydrates in the diet of man, STARCH 60 —•MALTOSE, —•GLUCOSE the amounts and (AMYLOPECTIN MALT0TRI0SE, ot-DEXTRINS proportion of each AMYLOSE) ingested (4), and the site of hydro­ L A C T O S E 10 NONE —GLUCOSE +GALACTOSE lysis. Notably only starch and glycogen are par­ SUCROSE —•GLUCOSE NONE 30 + FRUCTOSE tially hydrolyzed within the intestinal lumen. Hydrolysis of the residual oligo­ saccharide products of starch and of the disaccharides sucrose and lactose occurs under the influence of enzymes integral to the intestinal brush border membrane. Intraluminal Digestion of Polysaccharide Despite the common belief of 15-20 years ago that starches are hydrolyzed completely to glucose, Whelan and his colleagues 05,6) carried out extensive experiments demonstrating that the final products under physiological conditions are the oligo­ saccharides maltose, maltotriose and the α-limit dextrins 181

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

182

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

c o n t a i n i n g f i v e to nine glucose molecules and one or more α 1,6 branching l i n k s . This presumably r e f l e c t s a) the poor a f f i n i t y of α-amylase f o r α 1,6 branching l i n k s and f o r α 1,4 l i n k s a d j a ­ cent to the 1,6 l i n k s (.5,6) and b) the preference of the a c t i v e s i t e of the enzyme f o r l i n e a r o l i g o s a c c h a r i d e s of f i v e or more glucose u n i t s by cleavage of the penultimate bond a t the reducing end of the molecule (7)· Despite claims that α-amylase secreted from the pancreas may b i n d to the i n t e s t i n a l s u r f a c e p r i o r to i t s a c t i o n on p o l y s a c ­ charides ( 8 ) , i n t e s t i n a l f l u i d contains α-amylase at 10 times the c o n c e n t r a t i o n r e q u i r e d to e x p l a i n the i n v i v o s t a r c h h y d r o l y s i s i n man (9). Hence i t appears that d i g e s t i o n of s t a r c h and g l y ­ cogen to o l i g o s a c c h a r i d e s i s p r i m a r i l y an i n t r a l u m i n a l process. Oligosaccharidases

of the I n t e s t i n a l

Surface

There i s only a t r a c l u m i n a l f l u i d s of the s m a l , hydrases are concentrated i n the brush border s u r f a c e membrane of the i n t e s t i n e . Table 2 l i s t s the enzymes from human s m a l l i n t e s t i n e that have been i d e n t i f i e d and c h a r a c t e r i z e d . A l l of these are l a r g e g l y c o p r o t e i n s . Notably there i s only a s i n g l e β-galactosidase (10) but s e v e r a l a-glucosidases (11,12) i n the brush border. There are other carbohydrases w i t h i n the i n t e r i o r of the i n t e s ­ t i n a l c e l l that do not appear to have a d i g e s t i v e f u n c t i o n and these w i l l not be considered here. A l l of the a-glucosidases except t r e h a l a s e are capable of h y d r o l y z i n g maltose but i t seems p r e f e r a b l e to name them according to the s u b s t r a t e f o r which they are p e c u l i a r l y s p e c i f i c . The α 1,4 m a l t o - o l i g o s a c c h a r i d e and α-limit d e x t r i n products of amylase a c t i o n on s t a r c h are hydro­ l y z e d by glucoamylase and the α-dextrinase subunit of sucrase-ad e x t r i n a s e r e s p e c t i v e l y . The only i n t e s t i n a l carbohydrase that appears to have l i t t l e p h y s i o l o g i c a l r o l e i n d i g e s t i n g carbohy­ d r a t e i n the d i e t of modern man i s t r e h a l a s e s i n c e i t s appro­ p r i a t e s u b s t r a t e i s found only i n i n s e c t s and mushrooms. At l e a s t one of these enzymes, sucrase-a-dextrinase c o n s i s t s of a complex of two p r o t e i n s , each of which has i t s own, indepen­ d e n t l y a c t i n g enzyme s i t e (12). This p a r t i c u l a r h y b r i d enzyme has been cleaved i n t o a c t i v e , d i s t i n c t subunits of s l i g h t l y d i f ­ f e r e n t molecular s i z e that r e t a i n the same biochemical c h a r a c t e r ­ i s t i c s as found i n the n a t i v e h y b r i d (12). The α-dextrinase moiety i s commonly c a l l e d "isomaltase" because i t i s capable of h y d r o l y z i n g the 1,6 l i n k a g e s but the α 1,6 l i n k e d d i s a c c h a r i d e , isomaltose, i s not a saccharide product of amylase a c t i o n on s t a r c h and hence i s not a p h y s i o l o g i c a l s u b s t r a t e presented to the i n t e s t i n a l s u r f a c e . The reason f o r union of sucrase and α-dextrinase m o i e t i e s to form a h y b r i d molecule i s unknown s i n c e these enzymes, whether present i n h y b r i d or monomeric form, hydrolyze the a p p r o p r i a t e d i s a c c h a r i d e s u b s t r a t e i n an i d e n t i c a l

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975. 2

9

*~ 1.1 (G )

4.3 ( i s o maltose)

20

3.8 (G )

18

280,000

210,000

280,000

Mol. Wt.

tG i n d i c a t e s glucose and the s u b s c r i p t the number o f α 1,4 l i n k e d glucose u n i t s .

*Commonly c a l l e d sucrase-isomaltase although isomaltose i s not a p h y s i o l o g i c a l s u b s t r a t e i n the i n t e s t i n e .

Trehalose

50

a-Dextrins

Trehalase

25

25

Sucrose

9

Sucrase-adextrinase*

2

Malto-oligosaccharides (G t >- G )

Lactose

Glucoamylase

a-Glucosidase

Lactase

β-Galactosidase

Enzyme Type Name

HUMAN BRUSH BORDER PLIGOSACCHARIDASES % Total Principal Maltase Substrate Activity Km mM

TABLE 2

184

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

manner. However, experiments u s i n g a p u r i f i e d α-limit d e x t r i n as s u b s t r a t e have not yet been accomplished and i t i s p o s s i b l e t h a t the sucrase a c t i v e s i t e hydrolyzes adjacent α 1,4 l i n k a g e s w h i l e the α-dextrinase s i t e simultaneously hydrolyzes the α 1,6 branching p o i n t of the branched saccharide. Such c o o p e r a t i v i t y might g r e a t l y f a c i l i t a t e h y d r o l y s i s of the o l i g o s a c c h a r i d e to f r e e glucose. There i s some suggestion that l a c t a s e may a l s o e x i s t as a h y b r i d w i t h an a-glucosidase (13) and that glucoamyl a s e may be complexed w i t h an o l i g o 1,6 glucosidase (14) but s u f f i c i e n t l y pure preparations of these enzymes are not yet a v a i l a b l e to e s t a b l i s h t h i s . Development of Brush Border O l i g o s a c c h a r i d a s e s The human i n t e s t i n a l carbohydrases develop at v a r i o u s stages of u t e r i n e l i f e (15) a o u t l i n e d i F i g u r 1 Th f o th d i f f e r e n t i a l times f o r Small i n t e s t i n a l c e l l s y spa appears to be r e g u l a t e d by the r a p i d maturation and m i g r a t i o n of c e l l s from c r y p t up along the v i l l u s f o r discharge of senescent c e l l s from the v i l l u s t i p . The o l i g o s a c c h a r i d a s e s , although not present i n the immature c e l l s of the i n t e s t i n a l c r y p t s , are acquired as c r y p t c e l l s develop m o r p h o l o g i c a l l y and migrate onto the v i l l u s , as shown s c h e m a t i c a l l y i n F i g u r e 2. R e g u l a t i o n of the Oligosaccharidases The feeding of sucrose (16), f r u c t o s e (16) or glucose (17) produces a doubling i n i n t e s t i n a l sucrase a c t i v i t y . Whether t h i s occurs by v i r t u e of an i n c r e a s e i n s y n t h e s i s of the enzyme or a decrease i n degradation, i . e . s t a b i l i z a t i o n , has not been c l e a r l y d e f i n e d but i t seems l i k e l y that feeding of carbohydrates r e t a r d s degradation of the enzyme (18,19). Although the synthe­ s i s and degradation of other o l i g o s a c c h a r i d a s e s have not been shown to be d i r e c t l y r e g u l a t e d by s u b s t r a t e or products, the mono­ saccharide products r e l e a s e d i n t o the i n t e s t i n a l lumen do compete f o r the a c t i v e h y d r o l y t i c s i t e , thereby r e t a r d i n g the r a t e s of h y d r o l y s i s (20). Role of Surface O l i g o s a c c h a r i d a s e s i n D i g e s t i o n The f i n a l o l i g o s a c c h a r i d e products from glycogen and s t a r c h d i g e s t i o n and the d i e t a r y d i s a c c h a r i d e s sucrose and l a c t o s e are hydrolyzed very e f f i c i e n t l y a t the brush border s u r f a c e of the i n t e s t i n e so t h a t the r e l e a s e d monosaccharides are produced i n great abundance. Hence, n e i t h e r h y d r o l y s i s i n the i n t e s t i n a l l u m i n a l contents by α-amylase nor s u r f a c e h y d r o l y s i s by o l i g o ­ saccharidases i n t e g r a l to the i n t e s t i n e are r a t e - l i m i t i n g i n the o v e r a l l process of h y d r o l y s i s and t r a n s p o r t i n v i v o (21,22).

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

11.

185

Intestinal Oligosaccharidases

GRAY

LACTASE OTHER ouGLUCOSIDASES

TREHALASE

SUCRASE ISOMALTASE

FERTILIZATION

i

0

TERM

ι 4

ι 8

ι 12

ι 16

ι 20

ι 24

ι 28

ι 32

ι • 36

FETAL AGE (WEEKS) Figure 1. Development of human lines locate the range of fetal age during

full activity

acquired.

132 HRS

96 HRS

VILLUS

C R Y P T

ON A SYNTHESIS

PROTEIN DISACCHARIDASE SYNTHESIS ACTIVITY New England Journal of Medicine

Figure 2. Functional localization of intestinal cells from the time of their birth at the crypt base until cells migrate up the villus and are shed from the villus tip 132 hours later. Width of shaded vertical bars indicates relative amounts of the particular ac­ tivity, and vertical position denotes location of the activity in the crypt-villus unit.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

186

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

Instead, r a t e - l i m i t i n g phenomena appear to be i n v o l v e d p r i n c i p a l l y i n the f i n a l t r a n s p o r t of the released monosaccharides. The s o l e exception to t h i s i s the h y d r o l y s i s of l a c t o s e which i s much slower than h y d r o l y s i s of other o l i g o s a c c h a r i d e s , as shown i n F i g u r e 3, so t h a t h y d r o l y s i s i s even slower than transport of r e l e a s e d monosaccharides (22). Thus normal man i s at a r e l a t i v e disadvantage f o r the d i g e s t i o n of l a c t o s e as compared to other d i e t a r y saccharides, and t h i s appears to become p a r t i c u l a r l y important when i n t e s t i n a l disease i s present. D e f i c i e n c y of I n t e s t i n a l

Oligosaccharidase

Since entry i n t o the i n t e r i o r of the i n t e s t i n a l c e l l i s reserved f o r monosaccharides, absence or marked r e d u c t i o n of an o l i g o s a c c h a r i d a s e r e s t r i c t s the o f f e n d i n g o l i g o s a c c h a r i d e to the i n t e s t i n a l lumen. This can have d i r e consequences as o u t l i n e d i n F i g u r e 4 s i n c e the o l i g o s a c c h a r i d of i t s osmotic f o r c e . A small i n t e s t i n e and c o l o n , b a c t e r i a metabolize i t to two and three carbon fragments that are poorly absorbed i n lower bowel. Hence, the osmotic e f f e c t i s increased s e v e r a l f o l d so that i n g e s t i o n of only 50 grams of a d i s a c c h a r i d e may produce d i a r r h e a of 2000-3000 ml of f l u i d on an osmotic b a s i s alone. Other f a c t o r s such as the low pH produced by the metabolized fragments and s t i mulation of i n t e s t i n a l motion because of d i s t e n t i o n of the w a l l s of the hollow gut may a l s o c o n t r i b u t e to the d i a r r h e a and may s e c o n d a r i l y produce malabsorption of other n u t r i e n t s (Figure 4 ) . Primary Oligosaccharidase

Deficiencies

L a c t a s e D e f i c i e n c y . Lactase i s the i n t e s t i n a l saccharidase t h a t i s most commonly d e f i c i e n t , but the enzyme i s u s u a l l y normally a c t i v e i n c h i l d r e n and only becomes reduced i n a d o l e s cence and adulthood. I n t e r e s t i n g l y enough, most of the world's p o p u l a t i o n has a d u l t l a c t a s e d e f i c i e n c y (23-36), as shown i n Table 3. Thus, i t i s comTable 3 monly b e l i e v e d that l a c P R E V A L E N C E O F L A C T A S E DEFICIENCY tase d e f i c i e n c y i s a genGROUP % LACTASE DEFICIENT e t i c c o n d i t i o n . However, many of the r a c i a l groups WHITE w i t h a high prevalence of SCANDINAVIAN 3 adult lactase deficiency NORTH AMERICAN 5-20 s u f f e r from poor n u t r i BLACK t i o n and have an appre70 AMERICAN c i a b l e i n c i d e n c e of 50 AFRICAN OTHER small i n t e s t i n a l 80-100 disease^ c o n d i t i o n s CHINESE 55 INDIAN which are known to 95 FILIPINO depress i n t e s t i n a l 85 ABORIGINE (AUSTRALIAN) l a c t a s e out of ISRAELIS

60

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

11.

187

Intestinal Oligosaccharidases

GRAY

60 τ

or ι 50J

if)

ο

ι Q CO

40

30

20

10

Gastroenterology

Figure 3. Hydrolysis rates of lactose (L), maltose (M), and sucrose (S) from perfusion of a 30-cm segment of human jejunum in vivo (22). Brackets indicate ± 2 SE. Lactose is hydrolyzed much more slowly than the other disaccharides (P < 0.01). INTESTINAL L U M E N

SMALL INTESTINE

NO

LACTASE

LACTIC ACID

Annual Review of Medicine

WATERY DIARRHEA

MALABSORBTION FATS, PROTEINS, DRUGS

Figure 4. Schematic of the effect of disaccharidase defi­ ciency on the fate and action of a dietary disaccharide (see text for elaboration). If intes­ tinal lactase were present in normal concentrations, hydrol­ ysis would occur on the surface of the intestine and the mono­ saccharide products would be assimilated (41).

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

188

PHYSIOLOGICAL

p r o p o r t i o n to that found f o r other

EFFECTS OF

FOOD CARBOHYDRATES

oligosaccharidases.

Sucrase-a-Dextrinase D e f i c i e n c y . This h y b r i d enzyme has been found t o be markedly depressed o r absent i n about 100 docu­ mented cases (37-40). The malady appears t o be i n h e r i t e d as an autosomal r e c e s s i v e . A l l p a t i e n t s w i t h sucrase d e f i c i e n c y appear to have markedly depressed l e v e l s of α-dextrinase when isomaltose i s used as the s u b s t r a t e , but some d e x t r i n a s e a c t i v i t y u s u a l l y p e r s i s t s . This f i n d i n g , coupled w i t h t h e f a c t that sucrase and α-dextrinase a r e d i s t i n c t p r o t e i n s (12), suggests that the p r i ­ mary genetic defect c o n s t i t u t e s the a l e r a t i o n o r absence o f the sucrase subunit w i t h secondary r e d u c t i o n o f i t s α-dextrinase partner. Symptoms produced upon i n g e s t i o n of sucrase a r e i d e n t i c a l w i t h those discussed above f o r l a c t a s e d e f i c i e n c y . Amylopectin i s u s u a l l y w e l l t o l e r a t e d even though a p p r e c i a b l e q u a n t i t i e s o f α-dextrins a r e released the r e l a t i v e l y l a r g e molecula f a c t that a p p r o p r i a t e b a c t e r i a i n colon may not be present i n s u f f i c i e n t numbers to metabolize them to small o s m o t i c a l l y a c t i v e fragments; a l s o , some h y d r o l y s i s of these α-dextrins may occur by a c t i o n of the r e s i d u a l α-dextrinase subunits o r perhaps other surface α-glucosidases such as glucoamylase. Treatment o f Disaccharidase

Deficiencies

Although i t appears t o be p o s s i b l e t o administer enzymes along w i t h a d i e t a r y o l i g o s a c c h a r i d e t o promote d i g e s t i o n , t h e expenseusually makes t h i s i m p r a c t i c a l . By f a r t h e simplest form of therapy i s the e l i m i n a t i o n o f the o f f e n d i n g carbohydrate s i n c e no s i n g l e carbohydrate c o n s t i t u t e s an o b l i g a t e source o f c a l o r i e s . References

1. Miller, D. and Crane, R.K. Biochim. Biophys. Acta (1961) 52:281-293. 2. Eichholz, A. and Crane, R.K. J. Cell Biol. (1965) 26:687-691. 3. Maestracci, D., Schmitz, J., Preiser, H. and Crane, R.K. Biochim. Biophys. Acta (1973) 323:113-124. 4. Hollingsworth, D.F. and Greaves, J.P. Am. J. Clin. Nutr. (1967)20:65-72. 5. Roberts, P.J.P. and Whelan, W.J. Biochem. J. (1960) 76: 246-253. 6. Bines, B.J. and Whelan, W.J. Biochem. J. (1960) 76:253-263. 7. Robyt, J.F. and French, D. J. Biol. Chem. (1970) 245:39173927. 8. Ugolev, A.M. Physiol. Rev. (1965) 45:555-595. 9. Fogel, M.R. and Gray, G.M. J. Appl. Physiol. (1973) 35: 263-267.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

11.

GRAY

Intestinal

Oligosaccharidases

189

10. Gray, G.M. and Santiago, Ν.A. J. Clin. Invest. (1969) 48: 716-728. 11. Kelly, J.J. and Alpers, D.H. Biochim. Biophys. Acta (1973) 315:113-120. 12. Conklin, K.A., Yamashiro, K.M. and Gray, G.M. J. Biol. Chem. (1975) in press. 13. Lorenz-Meyer, Η., Blum, A.L., Haemmerli, H.P. and Semenza, G. Europ. J. clin. Invest. (1972) 2: 326-331. 14. Eggermont, E. and Hers, H.G. Eur. J. Biochem. (1969) 9:488496. 15. Dahlqvist, A. and Lindberg, T. Clin. Sci. (1966) 30:517. 16. Rosensweig, N.S. and Herman, R.H. J. Clin. Invest. (1968) 47:2253-2262. 17. Deren, J.J., Broitman, S.A., Zamcheck, N. J. Clin. Invest. (1967) 46:186-195 18. Das, B.C. and Gray 19. Das, B.C. and Gray, G.M. In preparation. 20. Alpers, D.H. and Cote, M.N. Amer. J. Phys. (1971) 221:865868. 21. Gray, G.M. and Ingelfinger, F.J. J. Clin. Invest. (1966) 45:388-398. 22. Gray, G.M. and Santiago, N.A. Gastro. (1966) 51-489-498. 23. Bayless, T.M. and Rosensweig, N.S. J. Am. Med. Assoc. (1966) 197:968. 24. Huang, S.-S. and Bayless, T.M. Science (1968) 160:83. 25. Newcomer, A.D. and McGill, D.B. Gastroenterology (1967) 53:881. 26. Littman, Α., Cady, A.B., Rhodes, J. Is. J. Med. Sci. (1968) 4:110. 27. Gudmand-Höyer, E., Dahlqvist, Α., Jarnum, S. Scand. J. Gastroenterol. (1969) 4:377. 28. Sheehy, T.W. and Anderson, P.R. Lancet (1965) 2:1. 29. Welsh, J.D., Rohrer, V., Knudsen, K.B., Paustian, F.F. Arch. Int. Med. (1967) 120:261. 30. Littman, Α., Cady, A.B., Rhodes, J. Is. J. Med. Sci.(1968) 4:110. 31. Cook, G.C., Kajubi, S.K. Lancet (1966) 1:725. 32. Chung, M.H. and McGill, D.B. Gastro. (1968) 54:225. 33. Elliott, R.B., Maxwell, G.M., Vawser, N. Med. J. Aust. (1967) 1:46. 34. Davis, A.E. and Bolin, T. Nature (1967) 216:1244. 35. Desai, H.G., Chitre, A.V., Parekh, D.V., Jeejeebhoy, K.N. Gastro. (1967) 53:375. 36. Gilat, T., Kuhn, R., Gelman, E. and Mizrahy, O. Dig. Dis. (1970) 15:895-904. 37. Burgess, E.A., Levin, B., Mahalanabis, D., Tonge, R.E. Arch. Dis. Childhood (1964) 39:431. 38. Prader, A. and Auricchio, S. Ann. Rev. Med. (1965) 16:345. 39. Sonntag, W.M. et al. Gastro. (1964) 47:18.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

190

PHYSIOLOGICAL EFFECTS OF FOOD CARBOHYDRATES

40. Auricchio, S. et al. J. Pediat. (1965) 66:555. 41. Gray, G.M. Annual Review Medicine (1971) 22:391-404. *Research supported by USPHS Grant AM 11270 and Research Career Development Award AM 47443 from the N a t i o n a l I n s t i t u t e s o f Health.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

12 Lactose Intolerance and Lactose Hydrolyzed Milk DAVID M. PAIGE, THEODORE M. BAYLESS, SHI-SHUNG HUANG, and RICHARD WEXLER Johns Hopkins Medical Institutions, 615 North Wolfe St., Baltimore, Md. 21205

Prevalence

Low levels of intestinal lactase activity have been found in many otherwise healthy adults and children in populations of American Negroes (1-5), Asians (6-10), Bantu tribes (11-12), South American Indians (13-14), Thais (15-17), and other population groups (18-20). Current evidence would indicate these low levels to be the norm for most populations of the world with notable exceptions being Scandinavians and those of northern European extraction. Approximately 70% of the world's adult population is lactose intolerant (21). It appears that those who have genetically acquired low lactase levels as adults are able to drink milk as infants; but gradually become increasingly lactose-intolerant after infancy. The onset of acquired lactose intolerance depends on the population studied. In developing countries, as our data from Peru indicates, 50% of the population is intolerant by 3 years of age (13). In a more technologically developed country such as the United States, 40% of the black population is lactose intolerant by the end of the first decade (Figure I). The difference between the accelerated loss of enzyme activity in children in developing areas, contrasted with the slower decline in blacks in this country, may reflect a relatively better nutritional state influencing and retarding the genetic expression of this event. Normally, i n g e s t e d l a c t o s e i s h y d r o l y z e d by t h e enzyme l a c t a s e found w i t h i n t h e b r u s h b o r d e r o f m i c r o v i l l o u s a r e a o f t h e j e j u n a l mucosa, s p l i t t i n g t h e l a c t o s e i n t o g l u c o s e and g a l a c t o s e , which a r e then absorbed. I f enzyme a c t i v i t y i s low, t h e i n g e s t e d lactose i s not hydrolyzed. F l u i d then e n t e r s t h e i n t e s t i n a l lumen t o d i l u t e t h i s h y p e r t o n i c l o a d , c a u s i n g 191 In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

Figure I.

Normal lactose tolerance in U.S. white and black children and Peruvian mestizo children

C/5

g

S

>

S

Ο *1 *1 Ο ο σ ο

H (/)

a

M "J M

> r

ο η

ε

><

δ

CD Κ)

X

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

12.

PAIGE

ET

AL.

Lactose Intolerance

193

abdominal d i s t e n t i o n , cramps and i n c r e a s e d p e r i s t a l s i s . In a d d i t i o n , the unabsorbed l a c t o s e i s fermented t o l a c t i c a c i d which s e r v e s as a c a t h a r t i c . Carbon d i o x i d e and hydrogen i s a l s o produced by t h i s f e r m e n t a t i o n and c o n t r i b u t e s t o the f r o t h y d i a r r h e a . Milk

Drinking

There are v a r i a t i o n s i n how much l a c t o s e w i l l cause symptoms i n persons w i t h low l a c t a s e l e v e l s . Some note symptoms o f i n t o l e r a n c e a f t e r one g l a s s o f m i l k , w h i l e o t h e r s a r e not t r o u b l e d u n l e s s they d r i n k three o r f o u r g l a s s e s a t one time. I t s h o u l d be s t r e s s ed t h a t not everyone who has low l a c t a s e l e v e l s and d e v e l o p s symptoms a f t e r a l a c t o s e t o l e r a n c e t e s t l o a d (50 g/m2) w i l l be awar f symptom a f t e d r i n k i n 8 ounce g l a s s o f m i l Data r e c e n t l y r e p o r t e d by M i t c h e l l , B a y l e s s , Paige e t a l (22), however, i n d i c a t e s t h a t o v e r h a l f o f 13 t e e n a g e r s e x p e r i e n c i n g a f l a t t o l e r a n c e c u r v e and symptoms w i t h a t e s t dose o f 50g o f l a c t o s e w i l l a l s o e x p e r i e n c e symptoms w i t h 12g o f l a c t o s e as w e l l as the consumption o f 8 ounces o f m i l k ( F i g u r e I I ) . A low l e v e l o f m i l k consumption i s o f t e n i m p l i c a t e d as the cause of low l a c t a s e l e v e l s . Yet, m i l k d r i n k i n g does not seem t o a f f e c t l a c t a s e l e v e l s . A l though some d e f i c i e n t i n d i v i d u a l s can g r a d u a l l y increase t h e i r milk i n t a k e , prolonged l a c t o s e or m i l k feeding w i l l not i n c r e a s e l a c t a s e a c t i v i t y . Keusch and h i s workers f e d T h a i a d u l t s l a c t o s e f o r 14 weeks and were u n a b l e t o e f f e c t any change i n the l e v e l s o f i n t e s t i n a l l a c t a s e o r the i n d i v i d u a l ' s l a c t o s e t o l e r a n c e . In a n o t h e r attempt to a l t e r l a c t a s e a c t i v i t y , Rosensweig and Herman (!23) were unable t o b r i n g about any changes a f t e r 14 days. C u a t r e c a s a s attempted t o a l t e r low l a c t o s e l e v e l s by l a c t o s e f e e d i n g s f o r 45 days and was unsuccesful. Newcomer and M c G i l l (24) were unable t o a l t e r l a c t a s e l e v e l s i n two s u b j e c t s a f t e r l a c t o s e loadi n g f o r t e n days. G i l a t e t a l were u n a b l e t o induce l a c t a s e a c t i v i t y i n ten lactose d e f i c i e n t adult I s r a e l i s a f t e r g i v i n g them more than 1 l i t e r o f m i l k per day f o r 6-14 months (25) . On the o t h e r hand, p r o l o n g e d a b s t i n e n c e from l a c t o s e i n t h o s e i n d i v i d u a l s d e s t i n e d t o have h i g h l a c t a s e l e v e l s does not appear t o i n h i b i t l a c t a s e a c t i v i t y . Nine w h i t e c h i l d r e n w i t h g a l a c t o s e m i a , 7 t o 17 y e a r s , had normal l a c t o s e t o l e r a n c e t e s t s , d e s p i t e t h e i r avoidance of a l l l a c t o s e - c o n t a i n i n g foods s i n c e e a r l y i n f a n c y (26). In a d u l t s , complete l a c t o s e d e p r i v a t i o n f o r 42 days was not a s s o c i a t e d w i t h a s i g n i f i c a n t change

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

194

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

100

Diarrhea

Abdom i naI Cramps

SYMPTOMS Abdom i naI

50

Cramps

Loos*

Abdominal C ra mps

Looso

Loos* Stools

Stools

Stools 25

Abdo mi η a I Bloating

Abdomi nai B l o a t i ng

Abdominal Bloat i n g

509m. Lactose

1 2 gm. Lactoso

8 oi. Milk

Figure II. Symptoms in lactose-intolerant bhcks with the consumption of 50 g and 12 g of lactose as well as 8 ounces of milk (N = 13)

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

12.

PAIGE

ET AL.

Lactose Intolerance

195

i n l a c t a s e a c t i v i t y o r i n l a c t o s e t o l e r a n c e (21). As t h e r e i s a g r a d i e n t o f symptoms w i t h l a c t o s e i n g e s t i o n , t h e f a c t t h a t some i n d i v i d u a l s a r e i n t o l e r a n t o f a l a c t o s e l o a d e q u i v a l e n t t o 3-4 g l a s s e s o f m i l k does n o t n e c e s s a r i l y mean he w i l l e x p e r i e n c e symptoms w i t h s m a l l e r q u a n t i t i e s o f m i l k . There i s e v i dence t h a t even though i n t o l e r a n t s u b j e c t s may n o t m a n i f e s t symptoms w i t h t h i s s m a l l e r q u a n t i t y o f l a c t o s e , i t i s s t i l l not being digested. Individuals experie n c i n g i n c o m p l e t e l a c t o s e d i g e s t i o n may, t h e r e f o r e , c o n t i n u e consuming m i l k d e s p i t e t h e f a c t t h a t they a r e not r e a l i z i n g i t s f u l l n u t r i t i o n a l v a l u e . M i l k taken w i t h o u t a meal seems t o cause more symptoms than i f taken w i t h f o o d . The f o o d may p o s s i b l y d i l u t e t h e milk; o r i t may d e l a y g a s t r i c emptying, and p e r m i t o n l y s m a l l amounts o f l a c t o s e t o e n t e r t h e s m a l l i n t e s t i n e enzyme system a t any one time Absorption We have r e p o r t e d (20) t h a t t h e r e appears t o be l i t t l e d i f f e r e n c e i n the a b i l i t y to p h y s i o l o g i c a l l y d i g e s t a l a c t o s e l o a d when s m a l l e r amounts a r e i n g e s t e d (29). Our d a t a suggests t h a t i n l a c t o s e i n t o l e r a n t c h i l d r e n i r r e s p e c t i v e o f t h e l a c t o s e l o a d ; whether i t i s t h e t e s t dose o f 2 g/kg o r t h e commonly consumed amount o f a p p r o x i m a t e l y 0.5 g o r t h e i n t e r m e d i a t e l e v e l o f 1.0 g/kg o f body weight, t h e r e i s an inadequate blood glucose r i s e i n the l a c t o s e i n t o l e r a n t subjects. In t h e l a c t o s e t o l e r a n t c o n t r o l , t h e b l o o d sugar r i s e i s normal and exceeds 26 mg% over t h e f a s t i n g g l u c o s e l e v e l with a l l three doses. I n a d d i t i o n , a l l 8 subj e c t s had symptoms w i t h 2.0 g/kg o f l a c t o s e . When 1.0 and 0.5 g/kg o f l a c t o s e were g i v e n , 5 o f t h e 8 s u b j e c t s were symptomatic ( F i g u r e I I I ) . Beyond t h e inadequate r i s e i n g l u c o s e , o t h e r nut r i e n t s appear t o be l e s s than a d e q u a t e l y absorbed when the r e s u l t s o f o u r s h o r t term b a l a n c e s t u d i e s o f 4 l a c t o s e i n t o l e r a n t and 2 l a c t o s e t o l e r a n t P e r u v i a n c h i l d r e n a r e a n a l y z e d (30-31). On a d i e t o f c a s e i n , c o t t o n s e e d o i l , and c a r b o h y d r a t e as s u c r o s e o r l a c t o s e , t h e r e i s i n c r e a s e d l o s s o f water, n i t r o g e n , potassium and sodium i n t h e s t o o l when t h e l a c t o s e i n t o l e r a n t s u b j e c t s a r e s w i t c h e d from a n i n e day p e r i o d on s u c r o s e t o a n i n e day p e r i o d on l a c t o s e . These v a l u e s r e t u r n t o c o n t r o l l e v e l s when t h e s u c r o s e r e p l a c e s l a c t o s e i n t h e d i e t (Figure I V ) .

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

1.0

Level of Lactose Load (g/kg)

0.5

-1-

!·

Ο ι

Symptoms No Symptoms Mean ± 1 standard deviation

2.0

ο

normal control

Figure III. Blood sugar rise and symptoms with a lactose load of 0.5, 1.0, and 2.0 g lactose/kg in one lactose-tolerant and eight lactose-intolerant subjects

10+

20+

30+

40+

• ο f I

0.5

1.0

20

ο ο σ η >

CO CT>

12.

PAIGE

ET AL.

Intolerant Figure IV.

197

Lactose Intolerance

Tolerant

Stool water, nitrogen, potassium, and sodium of lactose-intolerant and lactose-tolerant children consuming sucrose (S) or lactose (L)

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

198

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

L a c t o s e I n t o l e r a n c e and M i l k R e j e c t i o n What then i s the e f f e c t o f t h e s e p h y s i o l o g i c a l d i f f e r e n c e s on the p a t t e r n o f m i l k consumption i n l a c t o s e i n t o l e r a n t groups? Does l a c t o s e t o l e r a n c e p l a y any r o l e i n d e t e r m i n i n g m i l k d r i n k i n g h a b i t s ? The d a t a i n d i c a t e s t h a t m i l k r e j e c t i o n by b l a c k elementary s c h o o l c h i l d r e n i n an o r g a n i z e d s c h o o l f e e d i n g program i s s i g n i f i c a n t l y h i g h e r than i n s i m i l a r l y matched w h i t e c h i l d r e n (_5_) . O v e r a l l , the f r e q u e n c y o f f a i l u r e t o consume moderate amounts o f m i l k i n b l a c k s i s 20% as compared t o 10% i n w h i t e s . More i m p o r t a n t , however, a s i g n i f i c a n t a s s o c i a t i o n e x i s t s i n the b l a c k c h i l d r e n between nonmilk d r i n k i n g and l a c t o s e i n t o l erance. Seventy-seven p e r c e n t o f t h i s group had an abnormal l a c t o s e t o l e r a n c t e s t w i t h 85% f thes child r e n e x h i b i t i n g symptom Indeed, when on plot rejectio i n b l a c k and w h i t e c h i l d r e n and a d u l t s a g a i n s t the l e v e l o f l a c t o s e i n t o l e r a n c e i n t h e s e same groups, a c l o s e r e l a t i o n s h i p i s noted; m i l k r e j e c t i o n i n b o t h b l a c k and w h i t e c h i l d r e n and a d u l t s p a r a l l e l s the p r e v a l e n c e o f l a c t o s e i n t o l e r a n c e i n the p o p u l a t i o n studied (32)(Figure V). T h i s d a t a would suggest t h a t s o l u t i o n s be sought f o r t h i s s i t u a t i o n inasmuch as m i l k i s an i m p o r t a n t f o o d w i t h a p o t e n t i a l f o r p r o v i d i n g n u t r i e n t s and o t h e r much needed p r o t e i n t o d i s a d v a n t a g e d p o p u l a t i o n s , dom e s t i c a l l y and i n t e r n a t i o n a l l y . I t i s , t h e r e f o r e , imp o r t a n t t o have l a c t o s e i n t o l e r a n t i n d i v i d u a l s b o t h consume m i l k w i t h o u t symptoms and u t i l i z e i t s n u t r i e n t s w i t h o u t i n t e r f e r e n c e and l o s s . While i t i s apparent t h a t l a c t o s e i n t o l e r a n t p o p u l a t i o n s a r e heterogeneous w i t h r e s p e c t t o t h e i r m i l k d r i n k i n g h a b i t s , w i t h many e x p e r i e n c i n g l i t t l e o r no symptoms w i t h the consumption o f commonly consumed amounts o f m i l k , i t i s d o u b t f u l that a l l n u t r i e n t s contained t h e r e i n are being s a t i s factorily utilized. Obvious c o n t r o v e r s y e x i s t s as t o the p r e c i s e i n f e r e n c e s t o be drawn from the experimental d a t a on the c l i n i c a l consequences o f l a c t o s e i n t o l erance, y e t many agree t h a t v i a b l e o p t i o n s s h o u l d be c o n s i d e r e d f o r approaching t h i s p r e v a l e n t c o n d i t i o n . S u g g e s t i o n s have c e n t e r e d on c o n t i n u e d m i l k consumption d e s p i t e the p r e s e n c e o f known i n t o l e r a n c e . Human e x p e r i m e n t a l d a t a has f a i l e d , however, t o demons t r a t e i n d u c t i o n o f l a c t o s e a c t i v i t y through c o n t i n u e d m i l k consumption i n l a c t o s e i n t o l e r a n t i n d i v i d u a l s . Others have c o n s i d e r e d the c o m b i n a t i o n o f m i l k and other l i q u i d s , such as orange j u i c e , t o lower t h e l a c t o s e cont e n t t o more a c c e p t a b l e l e v e l s . R e c e n t l y , emphasis has

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

Drinkers

Drinkers

B l a c k Non M i l k

Black Milk

Drinkers

Drinkers

(0) (3) (7) (12)

20% 17% 35.7% 76.5%

(2) (5) (13)

12 14 17

(50g/m^) Symptoms

(3)

Lactose Tolerance Test Abnormal B l o o d Sugar R i s e

15

Ν

Lactose Tolerance Test Results in White and Black Milk Drinking and Non-milk Drinking School Children

White Non M i l k

White M i l k

Table I.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975. GROUPS—IN

YEARS

Figure V. Lactose intolerance and milk rejection in black and white children (A) (4, 5) and adults (B) (3)

AGE

MILK REJECTION IN WHITES

MILK REJECTION IN NEGROES

LACTOSE INTOLERANCE IN WHITES

LACTOSE INTOLERANCE IN NEGROES

δ r

X

•β

12.

PAIGE

ET

AL.

Lactose Intolerance

201

been g i v e n t o the p r e h y d r o l y s i s o f l a c t o s e i n t o i t s a b s o r b a b l e monosaccharides p r i o r t o g e n e r a l d i s t r i b u tion. Lactose Hydrolyzed

Milk

A comparison o f c a r b o h y d r a t e a b s o r p t i o n i n l a c t o s e t o l e r a n t and i n t o l e r a n t a d o l e s c e n t s who consumed unt r e a t e d whole m i l k and l a c t o s e h y d r o l y z e d m i l k was undertaken by our group (3_4) . The p o p u l a t i o n s t u d i e d c o n s i s t e d o f 32 B a l t i m o r e C i t y a d o l e s c e n t v o l u n t e e r s r a n g i n g i n age from 13-19 y e a r s w i t h a mean age o f 15.5 years. They were e q u a l l y d i v i d e d by sex w i t h a l l o f the s u b j e c t s b e i n g b l a c k . The y o u n g s t e r s were drawn from the lower s o c i o - e c o n o m i c census t r a c t s w i t h i n the c i t y w i t h o v e r 80% coming from the lowest d e c i l e cen sus t r a c t . A l l subject performed t o c a t e g o r i z t o s e l o a d . They were g i v e n 50 g o f l a c t o s e per meter square o f body s u r f a c e a f t e r an o v e r n i g h t f a s t . Twenty-two o f the 32 s u b j e c t s (69%) e v i d e n c e d a f l a t l a c t o s e t o l e r a n c e c u r v e w i t h a mean peak b l o o d sugar r i s e i n t h i s group o f 9.3 mg% and were c o n s i d e r e d lactose intolerant. The 10 l a c t o s e t o l e r a n t s u b j e c t s (31%) had a mean peak b l o o d sugar r i s e o f 36.5 mg%. Twenty o f the 22 l a c t o s e i n t o l e r a n t s u b j e c t s e x p e r i enced symptoms a l o n g w i t h the t e s t ; 3 o f the t o l e r a n t s u b j e c t s e x p e r i e n c e d symptoms w i t h the l a c t o s e l o a d . Lactose Tolerance Test Results (N = 32)

%

Mean Peak B l o o d Sugar R i s e (Mg/100ml)

Standard Deviation

Lactose Intolerant (N=22)

69

9.3

7.2

Lactose Tolerant (N=10)

31

36.5

7.4

A f t e r the i n i t i a l l a c t o s e t o l e r a n c e t e s t , the study p o p u l a t i o n , u s i n g a double b l i n d design, was g i v e n on a l t e r n a t e days; 8 ounces o f u n t r e a t e d whole m i l k c o n t a i n i n g a p p r o x i m a t e l y 12 g o f l a c t o s e , 8 ounces o f 50% h y d r o l y z e d whole m i l k c o n t a i n i n g 6 g o f l a c t o s e , and 8 ounces o f 90% l a c t o s e h y d r o l y z e d whole m i l k con-

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

202

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

t a i n i n g a p p r o x i m a t e l y 1.2 g o f l a c t o s e . The t e s t m i l k was p r e p a r e d and p r o v i d e d by t h e U. S. Department o f A g r i c u l t u r e , D a i r y P r o d u c t s L a b o r a t o r y p i l o t p l a n t i n Washington, D.C. F r e s h mixed raw whole m i l k was t r e a t e d w i t h t h e f i x e d enzyme l a c t a s e i s o l a t e d from t h e y e a s t saccharomyces l a c t i s t o h y d r o l y z e up t o 90% o f t h e l a c t o s e i n m i l k i n t o g l u c o s e and galactose. The mean maximum b l o o d sugar r i s e i n t h e 22 l a c t o s e i n t o l e r a n t s u b j e c t s d r i n k i n g 8 ounces o f u n t r e a t e d whole m i l k was 4.4 mg%; w i t h 50% h y d r o l y z e d m i l k , 8.8 mg%; and w i t h 90% l a c t o s e h y d r o l y z e d m i l k , 14.5 mg%. In t h e 10 l a c t o s e t o l e r a n t s u b j e c t s , t h e mean peak b l o o d sugar r i s e was n o t a p p r e c i a b l y d i f f e r e n t whether they consumed u n t r e a t e d whole m i l k o r l a c t o s e h y d r o lyzed milk. B l o o d Sugar

Lactose Intolerant (N=22) Lactose Tolerant (N=10)

Untreated

50%

90%

4.4

8.8

14.5

12.2

13.3

13.7

The d i f f e r e n c e between t h e b l o o d sugar r i s e i n the l a c t o s e i n t o l e r a n t and t o l e r a n t s u b j e c t s w i t h t h e consumption o f 8 ounces o f u n t r e a t e d whole m i l k i s s i g n i f i c a n t a t t h e .01 l e v e l . No s i g n i f i c a n t d i f f e r e n c e s a r e noted when t h e t o l e r a n t y o u n g s t e r s consumed l a c t o s e hydrolyzed milk. S i g n i f i c a n t i n t r a g r o u p d i f f e r e n c e s are observed between t h e peak b l o o d sugar r i s e a f t e r t h e consumption o f 8 ounces o f t h e t e s t m i l k s . I n t h e i n t o l e r a n t group the d i f f e r e n c e between u n t r e a t e d and 90% h y d r o l y z e d m i l k i s s i g n i f i c a n t a t t h e .001 l e v e l ; and between 50% and 90% h y d r o l y z e d m i l k a t t h e .02 l e v e l . No s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s a r e noted between 50% h y d r o l y z e d and u n t r e a t e d whole m i l k . In the l a c t o s e t o l e r a n t t e e n a g e r s , no s i g n i f i c a n t i n t r a g r o u p d i f f e r ences a r e noted i r r e s p e c t i v e o f t h e t e s t m i l k s t u d i e d .

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

12.

PAIGE

ET

AL.

203

Lactose Intolerance

Intra-Group S i g n i f i c a n c e Untreated/9 0 %

90%/50%

5 0 %/Untreated

Lactose Intolerant

.001

.02

N.S.

Lactose Tolerant

N.S.

N.S.

N.S.

Due t o the presence o f g l u c o s e and g a l a c t o s e w i t h the h y d r o l y s i s o f l a c t o s e , a l t e r a t i o n i n the t a s t e o f m i l k were c o n s i d e r e d i m p o r t a n t . Eighty-eight percent o f the 32 s u b j e c t s c o n s i d e r e d the u n t r e a t e d whole m i l k t o be " j u s t l i k e o r s i m i l a r t o m i l k " . Twelve p e r c e n t r e p o r t e d t h a t the u n t r e a t e d m i l k "sweete tha milk" The 50% h y d r o l y z e the respondents t o be " j u s t l i k e o r s i m i l a r t o m i l k " . N i n e t e e n p e r c e n t i n d i c a t e d t h a t i t was "sweeter than milk". The 9 0% l a c t o s e h y d r o l y z e d m i l k was r e p o r t e d as "sweeter than m i l k " by t h e m a j o r i t y o f the s u b j e c t s , 56%. The r e s t o f the s u b j e c t s d e s c r i b e d the m i l k as being " j u s t l i k e other milk". T a s t e Response (N = 32) Sweeter Than M i l k %

Just Like

Milk/Other %

U.W.M. (32)

12

88

19

81

50% (27) 90% (32)

56 44 F a c i a l e x p r e s s i o n s chosen by the s u b j e c t s t o r e f l e c t t h e i r o p i n i o n o f the v a r i o u s t e s t m i l k s p a r a l l e l e d and r e i n f o r c e d the v e r b a l assessment o f the t e s t milk. Discussion I t appears t h a t low l a c t a s e a c t i v i t y i s the more common s i t u a t i o n i n p o p u l a t i o n s around the w o r l d . Data f u r t h e r suggests t h a t the absence o f symptoms w i t h the

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

204

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

consumption o f 8 ounces o f m i l k does n o t a s s u r e t h e presence o f s u f f i c i e n t l a c t a s e a c t i v i t y t o h y d r o l y z e l a c tose. Indeed, a d o l e s c e n t s w i t h l a c t o s e i n t o l e r a n c e consuming 8 ounces o f u n t r e a t e d whole m i l k c o n t a i n i n g 12 g o f l a c t o s e have a s i g n i f i c a n t l y lower peak b l o o d sugar r i s e than s i m i l a r y o u n g s t e r s w i t h normal l a c t o s e absorption. The d i f f e r e n c e s i n c a r b o h y d r a t e a b s o r p t i o n are e l i m i n a t e d when l a c t o s e m a l a b s o r b e r s a r e g i v e n m i l k i n which 90% o f t h e l a c t o s e has been c o n v e r t e d by hyd r o l y s i s t o g l u c o s e and g a l a c t o s e . A more modest improvement i n a b s o r p t i o n i s noted w i t h h y d r o l y s i s o f 50% of the l a c t o s e i n milk. Youths w i t h normal l a c t o s e a b s o r p t i o n have a comparably h i g h peak b l o o d sugar r i s e i r r e s p e c t i v e o f the l a c t o s e content of the milk. The d e c r e a s e d b l o o d sugar r i s e o b s e r v e d o c c u r r e d even i n s u b j e c t s who d i d n o t complain o f symptoms w i t h the m i l k . Diarrhea i n the s i g n i f i c a n t l as none o f t h e a d o l e s c e n t s i n t h i s study e x p e r i e n c e d t h i s symptom. The i n t o l e r a n t s u b j e c t s s t u d i e d do n o t , t h e r e f o r e , u t i l i z e a s i g n i f i c a n t p o r t i o n o f the carboh y d r a t e and c a l o r i c c o n t e n t o f a g l a s s o f whole m i l k , i r r e s p e c t i v e o f t h e p r e s e n c e o r absence o f symptoms. Furthermore, i t appears t h a t an a p p r o p r i a t e l e v e l o f l a c t o s e h y d r o l y s i s s h o u l d approach 90% inasmuch as s i g n i f i c a n t d i f f e r e n c e s i n b l o o d sugar r i s e a r e noted between 50 and 90% h y d r o l y s i s . The p r e h y d r o l y s i s o f l a c t o s e d i d r e s u l t i n a r e p o r t e d i n c r e a s e i n sweetness o f t h e 90% h y d r o l y z e d m i l i ^ b u t d i d n o t seem t o i n t e r f e r e w i t h t h e acceptance o f the p r o d u c t i n t h i s s t u d y . The r e s u l t s suggest t h a t 90% l a c t o s e h y d r o l y z e d m i l k s h o u l d be more b r o a d l y e v a l u a t e d as a more a p p r o p r i a t e m i l k f o r major p o p u l a t i o n groups w i t h low l a c t a s e l e v e l s . The p r o v i s i o n o f l a c t o s e h y d r o l y z e d m i l k , may p r o v i d e a more r a t i o n a l answer t o p r o v i d i n g much needed n u t r i e n t s t o many i n t o l e r a n t groups b o t h d o m e s t i c a l l y and i n t e r n a t i o n a l l y i n need o f n u t r i t i o n a l r e i n f o r c e m e n t .

Supported i n p a r t by g r a n t s MC-R-240278-01-0 from t h e M a t e r n a l and C h i l d H e a l t h S e r v i c e s and HD-07179-01 from the N a t i o n a l I n s t i t u t e o f H e a l t h .

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

12.

PAIGE

ET

AL.

Lactose

Intolerance

205

Literature Cited

1. Bayless, T.M. and Rosensweig, Ν.S., Journal of the American Medical Association, (1966), 197, 968-972. 2. Paige, D.M., Bayless, T.M. and Graham, G.G., American Journal of Clinical Nutrition, (1973), 26, 238-240. 3. Cuatrecasas, P., Lockwood, D.H. and Caldwell, J.R., Lancet, (1965), 1, 14-18. 4. Huang, S.S. and Bayless, T.M., New England Journal of Medicine, (1967), 276, 1283-1287. 5. Paige, D.M., Bayless, T.M., Ferry, G.D. and Graham, G.G., Johns Hopkins Medical Journal, (1971), 129(3) 163-169. 6. Huang, S.S. and Bayless, T.M., Science, (1968), 160, 83-84. 7. Chung, M.H. and (1968), 54, 225-226. 8. Bolin, T.D., David A.E., Seah, C H . , et al, Gastroenterology, (1970), 59, 76-84. 9. Bolin, T.D., Crane, G.G. and Davis, A.E., Austra­ lian Annals of Medicine, (1968), 17, 300-306. 10. Davis, A.E. and Bolin, T.D., Nature, (1967), 216, 1244-1245. 11. Cook, G.C. and Kajubi, S.K., Lancet, (1966), 1, 725-729. 12. Jersky, K. and Kinsley, R.H., South African Medical Journal, (1967), 41, 1194-1196. 13. Paige, D.M., Leonardo, E., Cordano, Α., Nakashima, J., Adrianzen, B. and Graham, G.G., American Journal of Clinical Nutrition, (1972), 25, 297-301. 14. Alzate, Η., Gonzales, H. and Guzman, J., American Journal of Clinical Nutrition, (1969), 22, 122-123. 15. Keusch, G.T., Troncale, F.J. and Miller, L.G., Pediatrics, (1969), 43, 540-545. 16. Keusch, G.T., Troncale, F . J . , Thavaramara, G., et al., American Journal of Clinical Nutrition, (1969) 22, 638-641. 17. Flatz, G., Saengudom, C. and Sanguanbhokai, T., Nature, (1969), 221, 758-759. 18. Paige, D.M., Bayless, T.M. and Graham, G.G., American Journal of Public Health, (1972), 62(11), 1486-1488. 19. Gudmand-Hoyer, E. and Jarnum, S., Acta Medica Scandia, (1969), 186, 235-237. 20. Gilat, T., Kuhn, R., Gelman, E . , et a l . , American Journal of Digestive Diseases, (1970), 15, 895-904. 21. Bayless, T.M., Paige, D.M. and Ferry, G.D., Gastroenterology, (1971), 60, 605-608.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

206

22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.

PHYSIOLOGICAL EFFECTS OF FOOD CARBOHYDRATES

Mitchell, K.J., Bayless, T.M., Paige, D.M., Goodgame, R.W., Huang, S.S., "Intolerance of 8 Ounces of Milk in Healthy Teenagers", Pediatrics, (1974), In Press. Rosensweig, N.S. and Herman, R.H., The Journal of Clinical Investigation, (1968), 47, 2253-2262. Newcomer, A.D. and McGill, D.B., Gastroenterology, (1967) , 53, 881-889. Lancet, (1969), 1, 613, "Off Milk", Editorial. Kogut, M.S., Donnell, G.N. and Shaw, K.N.F., Journal of Pediatrics, (1967), 71, 75-81. Knudsen, K.B., Welsh, J.D., Kronenberg, R.S., et al., American Journal of Digestive Diseases, (1968), 13, 593-597. Leichter, J., American Journal of Clinical Nutrition, (1973) 26 393-396 Paige, D.M., Leonardo Adrianzen T., Β., , , Journal of Clinical Nutrition, (1972), 25, 467-469. Paige, D.M. and Graham, G.G., Pediatric Research, (1972), 6, 329. Graham, G.G., Paige, D.M., Symposia of Swedish Nutrition Foundation XI, (1972), 45-51. Paige, D.M., Graham, G.G., American Journal of School Health, (1974), 44(1). Paige, D.M., Bayless, T.M., Huang, S.S. and Wexler, R., "Lactose Hydrolyzed Milk", American Journal of Clinical Nutrition, (1974), In Press.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

13 Oligosaccharides of Food Legumes: AlphaGalactosidase Activity and the Flatus Problem JOSEPH J. RACKIS Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Ill. 61604

The raffinose famil stachyose and verbascose--occurs in seeds of food legumes at levels that cause flatulence in man and animals. These carbohydrates escape digestion be­ cause there is no α-galactosidase activity in mammalian intestinal mucosa and because they are not absorbed into the blood. Consequently, bacteria in the lower intestinal tract metabolize them to form large amounts of carbon dioxide and hydrogen and to lower the pH. The amount and pattern of expelled gases reflect differences in type, location, and abundance of intestinal microorganisms possessing agalactosidase activity in a favorable nutrient environment. Water and aqueous alcohol extraction, as well as enzymatic hydrolysis, can be used to process food legumes into products having low flatus activity. According to in vitro and in vivo studies, antibi­ otics and naturally occurring substances can inhibit bacterial activity in the intestinal tract; however, it is unlikely such additives would be approved for human consumption. Breeding soybeans low in oligo­ saccharide content holds little promise. Food legumes, which include oilseeds, peas, and beans, pro­ vide important sources of protein and calories. While population i s expanding worldwide, and i s expected to double i n the next 30 years, more and more reliance i s being placed on the direct consumption of these sources of protein. The Protein Advisory Group of the United Nations (1) recommends urgent research atten­ t i o n to eight species of food legumes. A challenging s c i e n t i f i c problem, flatulence, i s one of the p r i o r i t i e s i n t h i s research and w i l l become important as food legumes are consumed i n ever increasing quantities.

207 In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

208

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

There are many causes for the formation i n the alimentary canal of gas (2, 3) which may lead to nausea, cramps, diarrhea, abdominable rumbling, and s o c i a l discomfort with ejection of excessive r e c t a l gas. Here, I s h a l l r e s t r i c t my discussion of flatulence associated with the consumption of foods to those containing the oligosaccharides--raffinose, stachyose, and verbascose. These oligosaccharides are related by having one or more α-β-galactopyranosyl groups i n their structure. Figure 1 i l l u s t r a t e s their structural interrelationships, where the agalactose units are bound to the glucose moiety of sucrose, aGalactosyl groups are also found i n nature joined to other sugars and nonsugars (4), but whether these constituents cause f l a t u ­ lence i s unknown. Oligosaccharides i n Food Legumes The content of th various types of food legume give Soybean t a i n the highest levels of raffinose and stachyose, whereas other legumes have the highest levels of verbascose. Dehulled, de­ fatted soybean meal contains 15-18% polysaccharide and 13% o l i ­ gosaccharide (Table I I ) . There i s no starch i n mature soybeans, whereas i n other food legumes, starch i s the predominant carbo­ hydrate. The oligosaccharide content reported by Kawamura (5) represents average values f o r s i x U.S. and three Japanese soy­ bean v a r i e t i e s . Oligosaccharide content of v a r i e t i e s and strains of soybeans was determined by Hymowitz et a l . (6) i n establishing whether these carbohydrates could be eliminated genetically (Table I I I ) . Since there i s a high degree of s t a b i l i t y i n the oligosaccharide content of soybeans, elimination of flatulence by breeding holds l i t t l e promise unless new soybean strains are discovered which contain l i t t l e or no oligosaccharides. Oligosaccharides : Metabolism i n the Intestinal Mucosa Gitzelmann and Auricchio (7_) found no α-galactosidase (EC 3.2.1.22, α-Q-galactoside galactohydrolase) a c t i v i t y i n human i n t e s t i n a l mucosa. They demonstrated that a normal c h i l d and a galactosemic c h i l d were unable to digest raffinose and stachyose since there was no absorption of galactose i n the blood. Only trace amounts of raffinose and stachyose were excreted into the urine of the two children. R u t t l o f f et a l . (8) a l s o found no enzymatic hydrolysis of r a f f i n o s e i n tKe i n t e s t i n a l mucosa of

r a t s , pigs, and humans. Other studies on the absorption and deg­ r a d a t i o n of oligosaccharides containing a-galactosyl groups show that less than 1% of the administered dose was able to pass through the i n t e s t i n a l wall of man and animals (£, 10) (Fig­ ure 2). In the absence of α-galactosidase a c t i v i t y i n the mucosa, these oligosaccharides remain intact and enter the lower intestine where they can be metabolized by existing microflora.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

13.

RACKis

Oligosaccharides of Food Legumes

209

RAFFINOSE

Γ

α-0-Gal-ll—^6)-a-o-Glu(1-*2)-/S-D-Fru

STACHYOSE

I

I

a - D - G a l - ( 1 — 6 ) - a - D - G a l - | 1 — 6 ) - a - o - G l u - ( 1 — - 2)-0-o-Fru

VERBASCOSE

ι

1 a-D-Gal-(1—* 6 ) - a - o - G a l - ( 1 - » 6 ) - a - D - G a l ( 1 — 6)aD-Glu(1—2)-0-D-Fru

Figure 1.

Structural relationships between the raffinose family of oligosaccharides

0.35

__i 5-15

ι 35-45

i_l 70-80

Distance from Pylorus, c m . Ernaehrungsforschung

Figure 2.

Absorption rates of sugars and oligosaccharides in the small intestine of the rat (10)

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

210

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

Table I. Oligosaccharides i n Leguminous Seeds Raffinose ""a ΈΓ

Legume Beans, black mung, dry Beans, green mung, dry Chick peas Cow peas F i e l d beans Horse gram Lentils Lima beans, canned Peas, green, dry Peas, yellow, dry Pigeon peas Soybeans, dry

0.5 0.8 1.0 0.4 0.5 0.7 0.6

— — 1.1 0.4 0.2

— 0.9 — — — 0.6 — 0.3 —

Stachyose "a b

— — 2.5

1.8 2.5 2.5 2.0 2.1 2.0 2.2 0.2

4.8 1.2

— —

1.7

— 2.7 — 1.9 —

Verbascose "ΊΓ ïï~ 3.7 3.8 4.2 3.1 3.6 3.1 3.0

— — — —

— Hardinge et a l . (30), % of edible portion.

——

—

— Cristofaro (17), % of dry matter.

Table I I . Carbohydrate Constituents of Dehulled, Defatted Soybean Meal Constituent

% of Meal

Polysaccharide content, t o t a l Acidic polysaccharides Arabinogalactan C e l l u l o s i c material , Oligosaccharide content, t o t a l Sucrose Stachyose Raffinose Verbascose

15-18 8-10 5 1-2 13.3 6.6 5.3 1.4 Trace

- A s p i n a l l et a l . (31). — Kawamura (5).

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

— — — 0.5 4.0

— — 2.2

1.4

2.2

— —

13.

RACKis

211

Oligosaccharides of Food Legumes

Table I I I . Strain, Maturity Groups, and Average Weight of Total Sugar, Sucrose, Raffinose, and Stachyose i n Soybean Seed Weight i n g/100 g seed Strain

Maturitygroups

Total sugar

Ada Flambeau Morsoy

00 00 00

9.68 9.86 10.11

6.33 6.43 6.38

0.54 0.74 0.95

2.80 2.69 2.78

Clay Merit M60-92

0 0 0

10.04 10.07 10.22

6.78 6.33 6.76

0.77 0.69 0.78

2.49 3.05 2.68

Chippewa 64 M59-120 Hark

I I

9.33 9.41

5.71 6.11

0.60 0 68

3.02 2.62

1

Sucrose

Raffinose Stachyose

Beeson Corsoy Amsoy

II II II

9.04 9.25 9.79

5.64 5.76 6.28

0.72 0.59 0.65

2.68 2.89 2.86

SL-9 Wayne Calland

III III III

8.57 9.08 9.44

5.52 5.71 5.90

0.82 0.88 0.94

2.23 2.48 2.60

IV IV IV

8.30 8.37 8.65

5.06 5.10 5.28

0.80 0.76 0.78

2.46 2.48 2.59

9.36

5.96

0.75

2.65

L66-1359 Kent Cutler Mean Range of means

8.30-10.11 5.06-6.78 0.54-0.95 2.23-3

Hymowitz et a l . (6). - For maturity groups 00, 0, I, I I , I I I , and IV average values are f o r 10, 12, 23, 33, 27, and 24 locations, respectively. Flatulence i n Humans When a human experiences flatulence following a meal containing cooked dry beans, there i s a sudden increase i n passage of flatus that reaches a maximum i n about 4-5 hr, whereas with a diet containing soy f l o u r , the gas peak occurs i n about

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

212

PHYSIOLOGICAL

EFFECTS OF

FOOD

CARBOHYDRATES

8-10 hr. The increase i n flatus i s due primarily to increased amounts of two gases--carbon dioxide and hydrogen. Appreciable amounts of methane may also be produced. Composition of gases i n various segments of the i n t e s t i n a l tract under different dietary and physiological conditions has been compiled (3). Analy s i s o f o r a l l y and r e c t a l l y expelled gases r e f l e c t s différences i n type, location, and abundance of i n t e s t i n a l microorganisms that possess α-galactosidase a c t i v i t y (11). Four male graduate students volunteered to determine the flatus a c t i v i t y of various commercially manufactured, toasted soy protein products [Table IV, (12)]. The gas-producing factor resides mainly i n two products, wKëy solids and 80% ethanol extractives. These fractions contain about 55% carbohydrate, primarily as sucrose, raffinose, and stachyose. Glucosides of sapogenins, sterols, and isoflavones are also present i n small amounts. Table IV.

Effects of Soy Products on Flatus i n Man

Product—

Carbohydrate content, %

F u l l - f a t soy f l o u r Defatted soy f l o u r Soy protein concentrate Soy proteinate Water-insoluble residueWhey solids^80% Ethanol extractives— Navy bean meal Basal diet

Flatus volume, Daily intake, cc/hr g Average Range

27 33

146 146

30 71

0-75 0-290

25 <1

146 146

36 2

0-98 0-20

75 56

146 48

1 3

0-30

3002:

55

27 146 146

240 179 13

d

—

220-260 5-465 0-28

Steggerda et a l . (12). — A l l products were toasted with l i v e steam at 100° C f o r 40 min. — Fed at a l e v e l three times higher than that present i n the defatted soy flour diet. — Amount equal to that present i n 146 g of defatted soy flour. — One subject, otherwise four subjects per test.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

13.

RACKIS

Oligosaccharides of Food Legumes

213

In contrast, soy protein concentrate (72% protein and 25% polysaccharide) and the water-insoluble residue (251 protein and 75% polysaccharide) have low flatus a c t i v i t y . Soy proteinate, with a protein content of at least 92% and less than 1% oligosaccharide, i s devoid of flatus a c t i v i t y . A basal diet produces on the average 13 ml flatus/hr. On a navy bean d i e t , flatus volume increases to about 179 ml/hr. Some volunteers produce less than 5 ml/hr on a test meal of navy beans and others nearly 500 ml. Usually soy f l o u r diets are less f l a t u l e n t , but occasionally some subjects produce amounts of gas comparable to a navy bean diet. Even though defatted soy flour and soybean meal contain about 30% t o t a l carbohydrate, i t s metabolic a v a i l a b i l i t y ranges from 14% i n chicks to 40% i n rats. On the basis of r a t feeding, c a l o r i c value of soy carbohydrates i s 1.68 Cal/g compared to a value of about 4.0 f o highl digestibl (13) Sucros accounts f o r the c a l o r i As a r e s u l t , raffinose stachyos polysaccharide not metabolized by mammalian enzymes, but only the oligosaccharides are u t i l i z e d f o r f l a t u s production by the i n t e s t i n a l microflora. Richards et a l . (14) demonstrated that anaerobic bacterial cultures isolated"Tronfthe dog colon can metabolize raffinose and stachyose to produce large amounts of carbon dioxide and hydrogen, the major gases i n f l a t u s . This i n v i t r o technique was used by Rackis et a l . (15) to determine tEë gas-producing activi t y of the same procTucts used i n the human studies. As shown i n Table V, there i s good agreement between i n v i t r o tests and results obtained by human tests (Table IV). Table V. Anaerobic Fermentation i n v i t r o of Soybean Products. Anaerobic Cultures Isolated from Dog Colon Biopsies— Sample Soybean meal (dehulleddefatted) Whey solids 80% Alcohol extractives Water-insoluble residue Sodium soy proteinate Sodium caseinate

Gas volume, cc/24 hr

40 40 39 3 0 0

44 51 47 48 34 61 No gas for analysis No gas for analysis No gas for analysis

Rackis et a l . (15). — To 10 ce of thioglycollate-anaerobic bacteria media, 0.5 g of sample was added.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

214

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

Raffinose, when added to a soy protein isolate diet at a l e v e l equivalent to the t o t a l amount of raffinose plus stachyose i n a soy g r i t diet (defatted soybean meal), produced j u s t as much gas as the soy g r i t control diet [Figure 3 (16)]. Sucrose consumed at a l e v e l twice that found i n soy g r i t s did not cause flatulence. Animal Tests In a series of detailed experiments with rats, Cristofaro et a l . (17) found that diets containing stachyose and verbascose ëS&Tïïiteathe highest flatus a c t i v i t y . Carbon dioxide and hydrogen were the primary gases collected. Raffinose and lactose, incorporated at a l e v e l to equal the galactose l e v e l of the stachyose d i e t , induced an i n s i g n i f i c a n t increase i n flatus (Figures 4 and 5). As shown i n Figur matter of dogs fed a f l a t u l e n y gri greate than the amount formed on a nonf latulent all-meat diet (18). Heat resistant bacteria i n fecal matter heated to 80° C Tor 20 min before incubation account for more than 75% of the t o t a l gas produced. Carbon dioxide content ranged from 76 to 85%, hydrogen content averaged about 10%, with hydrogen s u l f i d e , nitrogen, and other gases accounting for the remaining portion (5-14%). Relationship Between Intestinal Bacteria and Flatus Richards and Steggerda (19) reported that 80% of the gas produced i n surgically prepare? i n t e s t i n a l segments of the dog when incubated with a navy bean homogenate occurred i n the ileum and colon (Table VI). In our studies with soybean meal homogenates, almost a l l the gas was also produced i n the ileum and colon. Carbon dioxide and hydrogen, the major constituents i n f l a t u s , were again the primary gases produced i n i n t e s t i n a l segments. P r a c t i c a l l y no gas was produced i n the intestines containing methyl cellulose. Under these conditions of l i t t l e or no flatus production, nitrogen was the primary gas. Based on studies of anaerobic cultures of the dog colon, Richards et a l . (14) concluded that Clostridium nerfringens, normally present i n the gastro-intestinal tract i n man and animals, was primarily responsible for the production of flatus i n the ileum and colon. Rockland et a l . (20) published data to show that the growth of ClostriaTa i s stimulated by a substrate containing beans. Carbon dioxide and hydrogen were the major gases formed. When dejecta from human i l e a l and colonic segments of the intestine are cultured with stachyose, large amounts of gas are produced (21). The dominant gases were: 58-62% carbon dioxide and 28-36%""nydrogen. About 12% methane was produced only

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

13.

RACKIS

Oligosaccharides

of Food Legumes

500

Isolate

+ + Raffinose Sucrose

Grits

Journal of the American Oil Chemists Society

Figure 3. Flatus activity of soybean oligosaccharides in humans. Raffinose intake, 13 g/day; sucrose intake, 23 g/day (16).

Control

2.4%

3.6%

2.1%

"Sugars in Nutrition"

Figure 4. Effect of various sugars on gas volume and composition in the rat intestine (17)

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

216

PHYSIOLOGICAL

Control

EFFECTS

OF FOOD

CARBOHYDRATES

Stachyose "Sugars in Nutrition"

Figure 5.

Fhtus activity of oligosaccharides in the rat intestine (17)

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

13.

RACKIS

Oligosaccharides of Food Legumes

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

217

218

PHYSIOLOGICAL

EFFECTS OF

FOOD

CARBOHYDRATES

with colon dejecta. These data suggest that the i n t e s t i n a l f l o r a may contain an a-l,6-galactosidase. Table VI. Volume and Composition of Gas i n Intestinal Segments of the Dog

Substrate

Intestinal segments

Mean gas volume, ml/3 hr

0.

Gas composition, co

2

°2

N

h

2

2

Duodenum Jejunum Ileum Colon

0 1.5 1.5 1.5

9.3 9.3 9.3

13.9 13.9 13.9

76.8 76.8 76.8

0 0 0

Navy bean^ homogenate

Duodenu Jejunu Ileu Colon

5.7

21.3

6.5

38.3

33.9

31.9

34.3

3.8

28.7

33.2

Soy f l o u r homogenate

Duodenum Jejunum Ileum Colon

5.9 4.8 11.0 12.0

21.5 21.5 32.0 33.0

3.8 3.6 3.7 3.8

46.7 47.8 33.1 26.9

28.0 27.1 31.2 36.3

Methyl celluloses-

— Richards and Steggerda (19). Table VII. Anaerobic Gas Production with Carbohydrates. Anaerobic Cultures Isolated from Dog Colon Biopsies

Substrate^

ΓΈτ

Monosaccharides Glucose Maltose Fructose Galactose Oligosaccharides Sucrose Raffinose Stachyose Control

Total gas produced, ml

Gas compo­ sition, %

6 hr

12 hr

2ΤΈτ

C0

0 0 0 0

23 19 10 8

38 38 20 20

51 43 37 36

37 38 41 38

62 61 58 61

0 0 0 0

19 9 10 1.5

27 25 27 1.5

30 30 31 2.0

32 35 37

68 65 63

2

Rackis et a l . (15). - To 10 ml of thioglycollate anaerobic bacteria medium, 0.1 g of substrate was added.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

H

2

13.

RACKis

Oligosaccharides of Food Legumes

219

Time, Figure 7. Rehtionship between the enzymatic hydrolyses of stachyose and gas produc­ tion. Anaerobic culture isolated from the dog colon. Curve A, stachyose; curve B, diand trisaccharides; curve C., monomer sugars; curve Ό, gas production.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

220

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

Rackis et a l . (15) confirmed that anaerobic cultures iso­

lated from dog colonTiopsies can metabolize raffinose and stach­ yose to produce the characteristically high concentrations of carbon dioxide and hydrogen. Glucose gave the most gas and at a rate greater than for either fructose or galactose (Table VII). With raffinose and stachyose, gas production occurred at the slowest rate. Evidently, the oligosaccharides break down to monosaccharides before the gas-producing mechanism can take place. It i s understandable then that a longer delay must occur before the onset of gas production with raffinose and stachyose, particularly i f glucose i s the preferred substrate (see Fig­ ure 1). Gas production was related to the degree of enzymatic hy­ drolysis of stachyose and the corresponding intermediate break­ down products consisting of d i - and trisaccharides (Figure 7). As a result, rate of gas production paralleled the formation of monosaccharides. Pape drolysis of stachyose by g Relative concentrations were determined by photodensitometry. Elimination of Flatulence Since removal of oligosaccharides through genetic means does not look promising (Table III), various processing techniques such as those used for the manufacture of isolates and concen­ trates are required to remove them. These techniques include hot water treatment and aqueous alcohol extraction which insolub i l i z e protein. Soaking and hot water extraction have been used to prepare tempeh, a fermented soy product, having l i t t l e flatus a c t i v i t y (22). Enzymatic processes that hydrolyze oligosaccharides have been developed (23, 24, 25, 26). An immobilized α-galactosidase continuous flow reactor nâs Been used to reduce the raffinose content i n beet sugar molasses (27). About 70% of the raffinose plus stachyose was removed from soybeans by a combination of various treatments that involved pH adjustment, soaking, and germination (28). Germinating soybeans contain an α-galactosid­ ase since rafTïhose and stachyose decrease rapidly during the f i r s t 3 days and disappear after 5 days of germination (17). However, soybean and mung bean sprouts retain most of the flatulence-inducing a c t i v i t y of the intact seed when tested i n humans (22). In contrast, autolysis of California small white beans, as measured by the disappearance of α-oligosaccharides, significantly reduces rat hydrogen production (29). The reason for this anomaly i s unclear and requires further investigation. Possibly the soybean polysaccharides, which normally do not have flatulent properties, are degraded during sprouting to form i n ­ termediate products that could then be fermented by intestinal bacteria into large amounts of gas, whereas, the starch i n white beans would be digested and absorbed.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

13.

RACKIS

Oligosaccharides

of

Food

Legumes

221

Literature Cited

1. Protein Advisory Group of the United Nations System, PAG Bulletin, Vol. III, No. 2, 1 (1973). 2. Beck, J. Ε., Ann. N.Y. Acad. Sci. U.S.A. (1968) 150, 1. 3. Calloway, D. Η., Handb. Physiol. Alimentary Canal (1968) 5, 2839. 4. French, D., Advan. Carbohyd. Chem. (1954) 9, 149. 5. Kawamura, S., Tech. Bull. Fac. Agr. Kagawa Univ. (1967) 18, 117. 6. Hymowitz, T., Walker, W. Μ., Collins, F. I., Panczer, P., Commun. Soil Sci. Plant Anal. (1972) 3, 367. 7. Gitzelmann, R., Auricchio, S., Pediatrics (1965) 36, 231. 8. Ruttloff, H., Taeufel, Α., Krause, W., Haenel, H., Taeufel, Κ., Nahrung (1967) 11, 39. 9. Taeufel, K., Krause W. G. Ruttloff H. Maune R. Z Gesamte Exp. Med 10. Krause, W. G., Taeufel, K., Ruttloff, H., Maune, R., Ernaehrungsforschung (1968) 13, 161. 11. Calloway, dT H., Burroughs, S. E., Gut (1969) 10, 180. 12. Steggerda, F. R., Richards, Ε. Α., Rackis, J. J., Proc. Soc. Exp. Biol. Med. (1966) 121, 1235. 13. Liener, I. E., in "Soybeans: Chemistry and Technology," Vol. 1, "Proteins," Edited by A. K. Smith and S. J. Circle, Avi Publishing Co., Westport, Conn. (1972). 14. Richards, Ε. A., Steggerda, F. R., Murata, Α., Gastroenter­ ology (1968) 55, 502. 15. Rackis, J. J., Sessa, D. J., Steggerda, F. R., Shimizu, J., Anderson, J., Pearl, S. L., J. Food Sci. (1970) 35, 634. 16. Rackis, J. J., J. Amer. Oil Chem. Soc. (1974) 51, 161A. 17. Cristofaro, E., Mottu, F., Wuhrmann, J. J., in "Nutrition Monogram, Sugars in Nutrition," Edited by H. L. Sipple, International Conference Sugars in Nutrition, Vanderbilt University, Nashville, Tennessee, November 1972. 18. Shimizu, T., Steggerda, F. R., Rackis, J. J., unpublished data. 19. Richards, Ε. Α., Steggerda, F. R., Proc. Soc. Exp. Biol. Med. (1966) 122, 573. 20. Rockland, L. B., Gardiner, B. L., Pieczarka, D., J. Food Sci. (1969) 34, 411. 21. Calloway, D. H., Colasito, D. J., Mathews, R. D., Nature (1966) 212, 1238. 22. Calloway, D. Η., Hickey, C. Α., Murphy, E. L., J. Food Sci. (1971) 36, 251. 23. Sherba, S. Ε., U.S. Patent 3,632,346 (1972). 24. Sugimoto, H., Van Buren, J. P., J. Food Sci. (1970) 35, 655. 25. Ciba-Geigy, A. G., French Patent 2,137,548 (1973). 26. Yamane, T., La Sucrerie Belge. (1971) 90, 345. 27. Reynolds, J. H., Biotechnol. Bioeng. (1974) 16, 135.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

222

PHYSIOLOGICAL EFFECTS OF FOOD CARBOHYDRATES

28. Kim, W. J., Smit, C. J. B., Nakayama, Τ. O. Μ., Lebensm.Wiss. Technol. (1973) 6, 201. 29. Becker, R., Olson, A. C., Frederick, D. P., Kon, S., Gumbmann, M. R., Wagner, J. R., J. Food Sci. (1974) 39, 767. 30. Hardinge, M. G., Swarner, J. Β., Crooks, H., J. Amer. Diet Ass. (1965) 46, 197. 31. Aspinall, G. O., Bigbie, R., McKay, J. Ε., Cereal Sci. Today (1967) 12, 233.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

14 The Lysosomal α-Glucosidases of Mammalian Tissues BARBARA I. BROWN, A L L E N K. MURRAY, and DAVID H. BROWN Department of Biological Chemistry, Division of Biology and Biomedical Sciences, Washington University, St. Louis, Mo. 63110

This Symposium has focused attention on various dietary car­ bohydrates, their absorption, interconversions and physiological fates. As has been pointed out, one of the criteria for utiliza­ tion of [ C]-labeled sugars has been the extent of isotope in­ corporation into glycogen as well as the formation of CO . The average 70 kg adult may have stored in his tissues half a kilogram of glycogen. However, a 7 kg infant with Type II Glycogen Storage Disease (Pompe's disease), which is one of the more common forms of glycogen storage disease, also may have stored a like quantity of glycogen. This fact serves to emphasize the importance of the α-glucosidase which has an acidic pH optimum in the catabolism of glycogen in normal human tissues, since patients with Type II disease have been shown to have a congenital and generalized de­ ficiency of this glucosidase (1). The purified enzyme has both α-1,4 and α-1,6 glucosidase activity and can convert glycogen totally to glucose (2). The activity in normal tissue homogenates is such that if the enzyme were in contact with its substrate, it would have the capacity of totally degrading the polysaccharide present in liver in 3 to 5 hours and that in muscle in 8 to 12 hours. 14

14

2

By comparison o f i t s p r o p e r t i e s w i t h those o f an a - g l u c o s i dase p u r i f i e d t o homogeneity from r a t l i v e r lysosomes ( 3 ) , the human enzyme i s assumed t o be i n lysosomes a l s o . E l e c t r o n micro­ graphs o f l i v e r samples obtained from p a t i e n t s w i t h Type I I g l y ­ cogen storage disease show t h a t a l a r g e q u a n t i t y o f glycogen i s present w i t h i n membrane enclosed vacuoles and t h i s o b s e r v a t i o n i s c o n s i s t e n t w i t h the hypothesis t h a t the α-glucosidase has a l y s o ­ somal l o c a l i z a t i o n ( 4 ) . S i m i l a r enzymes have been p u r i f i e d from a v a r i e t y of sources such as r a t C2,_5,6) and beef l i v e r ( 7 ) , rab­ b i t muscle ( 8 ) , and human p l a c e n t a (6) and l i v e r . Most of the p r e p a r a t i o n s have taken advantage o f the property o f the enzyme to be s p e c i f i c a l l y r e t a r d e d by a d s o r p t i o n to Sephadex as an im­ p o r t a n t a i d i n i t s p u r i f i c a t i o n . F i g u r e 1 i l l u s t r a t e s the behav­ i o r o f the human l i v e r enzyme upon Sephadex G-100 chromatography. Previous steps i n the p u r i f i c a t i o n procedure had i n c l u d e d repeated 223 In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

224

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

f r e e z i n g and thawing of the l i v e r homogenate to rupture the l y s o ­ somes, a heat treatment at 55° and ammonium s u l f a t e f r a c t i o n a t i o n between 35 and 55% s a t u r a t i o n . The d i a l y z e d , concentrated ammo­ nium s u l f a t e f r a c t i o n a p p l i e d to the Sephadex column u s u a l l y con­ t a i n e d 60% of the s t a r t i n g u n i t s of enzyme a c t i v i t y and had a s p e c i f i c a c t i v i t y of 0.15 u n i t per mg p r o t e i n corresponding to i t s being 10 to 1 2 - f o l d p u r i f i e d at t h i s stage. However, the en­ zyme recovered from the Sephadex column u s u a l l y had an a c t i v i t y of about 30 u n i t s per mg corresponding to an o v e r a l l p u r i f i c a t i o n of more than 2000-fold w i t h a recovery of approximately 40% of the t o t a l u n i t s . From 1 to 2 mg of enzyme could be obtained from 100 grams of human l i v e r ( 9 ) . As i n d i c a t e d i n F i g u r e 1, maltose or glycogen can be used as a s u b s t r a t e i n measuring the a c t i v i t y of the enzyme. The s p e c i f ­ i c a c t i v i t i e s given above r e f e r to ymoles of maltose hydrolyzed per minute per mg of p r o t e i n at pH 4 and 37° and at a s u b s t r a t e c o n c e n t r a t i o n of 10 mM. c a r r i e d out w i t h 1% s u b s t r a t e expressed as ymoles of glucose formed per minute at 37°. The p l a ­ c e n t a l enzyme p u r i f i e d by de Barsy et a l . (6) had a s p e c i f i c ac­ t i v i t y of 7.3 u n i t s per mg when assayed at 3.7 mM maltose. How­ ever, these i n v e s t i g a t o r s found that the 1^ f o r the human p l a c e n ­ t a l enzyme was 11 mM f o r maltose and 2% f o r glycogen. Even though glycogen i s the n a t u r a l s u b s t r a t e f o r the en­ zyme, f o r convenience many i n v e s t i g a t o r s have used maltose as a s u b s t r a t e i n assaying f o r the α-glucosidase. We had p r e v i o u s l y shown t h a t the r a t l i v e r enzyme e x h i b i t e d s u b s t r a t e i n h i b i t i o n by maltose and by m a l t o s i d i c a l l y l i n k e d o l i g o s a c c h a r i d e s when the i n i t i a l s u b s t r a t e concentrations exceed 5 to 10 mM ( 2 ) . Studies w i t h the enzyme p u r i f i e d from human l i v e r have revealed no sub­ s t r a t e i n h i b i t i o n a t concentrations as h i g h as 100 mM maltose. The Kflj f o r maltose f o r the human l i v e r enzyme appears to be 9 mM, w h i l e f o r isomaltose a Km of 33 mM was found. Polyacrylamide g e l e l e c t r o p h o r e s i s of the p u r i f i e d human l i v e r enzyme r e s u l t e d i n a s i n g l e peak of a c t i v i t y as shown i n F i g u r e 2. In t h i s experiment a f t e r e l e c t r o p h o r e s i s the g e l was f r o z e n , s l i c e d i n t o 1 mm s l i c e s and the a c t i v i t y of the e l u t e d en­ zyme determined i n each s l i c e . E s s e n t i a l l y 100% of the loaded u n i t s were recovered. Of g r e a t e r i n t e r e s t i s the f a c t t h a t a good correspondence was found between t o t a l absorbance at 280 nm, pro­ t e i n s t a i n i n g w i t h Coomassie b l u e , enzymatic a c t i v i t y revealed by i n c u b a t i o n w i t h methyl u m b e l l i f e r y l glucopyranoside (another sub­ s t r a t e of the α-glucosidase), and PAS p o s i t i v e m a t e r i a l i n the same or p a r a l l e l g e l s . Since PAS s t a i n i n g i n d i c a t e s the presence of carbohydrate, the α-glucosidase i s o l a t e d from human l i v e r ap­ pears to be a g l y c o p r o t e i n ( 9 ) . In i s o e l e c t r i c f o c u s i n g g e l s there always was evidence of the presence of s e v e r a l isozymes as i n d i c a t e d i n F i g u r e 3 where the e l u t e d enzyme showed s i m i l a r p a t t e r n s of a c t i v i t y w i t h the two s u b s t r a t e s . The pH values were obtained by measuring the pH of

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

14.

BROWN

ET AL.

Lysosomal

a-Glucosidases

225

water i n which groups of four consecutive s l i c e s of a d u p l i c a t e g e l were e x t r a c t e d f o l l o w i n g overnight e l e c t r o p h o r e s i s o f a 17 cm g e l c a s t w i t h pH 3 t o 6 ampholine. D u p l i c a t e g e l s were a l s o s t a i n e d f o r p r o t e i n and PAS r e a c t i v e m a t e r i a l . The scans of t h e s t a i n e d g e l s (Figure 4) i n d i c a t e d correspondence between p r o t e i n and carbohydrate throughout the r e g i o n c o n t a i n i n g the charge i s o ­ zymes. F o l l o w i n g column chromatography o f the glucosidase on B i o g e l P-200, some d i f f e r e n c e i n the isozyme p a t t e r n o f i n d i v i d u a l f r a c t i o n s could be a s c e r t a i n e d i n i s o e l e c t r i c focused g e l s such t h a t the more a c i d i c isozymes were more prominent i n the e a r l i e r f r a c t i o n s e l u t e d from the column w h i l e the l e s s a c i d i c ones emerged l a t e r . The p o s i t i o n o f the main peak i s compatible w i t h a molecular weight on the order of 100,000 s i m i l a r t o the v a l u e of 114,000 found f o r the r a t l i v e r lysosomal α-glucosidase (3) and of 107,000 found f o r the bovine l i v e r enzyme ( 7 ) . One o f the main purpose purifyin live was t o make i t p o s s i b l i n v e s t i g a t e whether, u s i n g such an antibody, evidence could be found f o r c r o s s - r e a c t i v e m a t e r i a l i n the t i s s u e s o f i n d i v i d u a l s a f f l i c t e d w i t h Type I I glycogen storage d i s e a s e . The evidence t h a t the α-glucosidase has an e s s e n t i a l r o l e i n normal metabolism has been i n f e r r e d from the apparent s e r i o u s e f f e c t s o f i t s ab­ sence i n the t i s s u e s o f the i n f a n t w i t h t h i s disease who f r e ­ quently d i e s a t an e a r l y age w i t h massive accumulation o f g l y ­ cogen p a r t i c u l a r l y i n the heart and s k e l e t a l muscles. However i t should be pointed out t h a t a d u l t forms o f the d e f i c i e n c y a l s o e x i s t (10) and, w h i l e these a d u l t s have e s s e n t i a l l y no demonstra­ b l e α-glucosidase a c t i v i t y a t a c i d pH i n e x t r a c t s o f t h e i r t i s ­ sues, the content o f glycogen may be o n l y s l i g h t l y above normal and the c l i n i c a l symptoms may be confined t o a m i l d muscular weak­ ness. Since the a c t i v i t y of the α-glucosidase i s demonstrable i n leukocytes and c u l t u r e d s k i n f i b r o b l a s t s o f normal i n d i v i d u a l s , f i b r o b l a s t s may be u t i l i z e d as an experimental t i s s u e (11,12). Rabbits were immunized by subcutaneous i n j e c t i o n of 0.5 t o 1 mg o f enzyme i n 1 ml o f complete Freunds adjuvant i n m u l t i p l e s i t e s a t monthly i n t e r v a l s f o l l o w e d by 0.1 mg of enzyme i n t r a ­ venously. The t i t e r o f antibody obtained v a r i e d w i t h the r a b b i t and the time o f b l e e d i n g d u r i n g the immunization schedule. A t y p i c a l p r e p a r a t i o n o f antibody was 5 t o 10 times as e f f e c t i v e i n i n h i b i t i n g the a c t i v i t y of the enzyme toward glycogen as toward e i t h e r maltose o r isomaltose when a l l a c t i v i t i e s were assayed a t c o n c e n t r a t i o n s equal t o the Km o f the r e s p e c t i v e s u b s t r a t e s . Ac­ t i o n on glycogen could be completely i n h i b i t e d by antibody, w h i l e i n general no more than 80% o f maltose h y d r o l y s i s could be blocked even by l a r g e amounts o f antibody. S i m i l a r e f f e c t s were observed by deBarsy e t a l . (6). In g i v i n g a t t e n t i o n next t o some of the experimental r e s u l t s obtained u t i l i z i n g f i b r o b l a s t s , i t i s s i g n i f i c a n t t o recognize t h a t u n i t s o f a c t i v i t y are expressed as nanomoles o f maltose h y d r o l y z e d , o r glucose formed from glycogen, per minute per mg of

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

10.0

GLYCOGEN.

rw

/

X. \

w

/' 7 / /

JJf^ 1000

500

EFFLUENT VOLUME

(ml)

Figure 1. Chromatography of human liver a-glucosidase on Sephadex G-100. See text for assay conditions.

75|

θ

θ MALTOSE

IO

ο

X


To

50

"E

>25

Ο <

!\ 0

20

GEL SLICE

Tracking ^Dye

40

60

80

NUMBER

Figure 2. Elution of a-glucosidase activity fol­ lowing polyacrylamide gel electrophoresis of the purified enzyme on a 6% gel (1% crosslinked) according to the method of Orr et al. (19)

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

14.

BROWN

227

Lysosomal a-Glucosidases

ET AL.

4.4 4.3 4.2 4.1

PH

ι—I I

0 20

MALTOSE

4.4 4.3 4.2 4.1

<

SLICE NUMBER

Figure 3. Elution of α-glucosidase activity follow­ ing electrophoresis on an isoelectric focusing gel cast with pH 3-6 ampholine and run for 11 hrs. Gel slices extracted overnight with pH 4, 0.05M acetate buffer before assay. Measurements of pH made on water extracts of slices from gels run in parallel. The 17-cm gels were cut into 170 slices; only the area of recovered enzyme activity is shown.

PROTEIN

CARBOHYDRATE

Θ

θ

Figure 4. Coomassie Blue staining (protein) and PAS staining (carbohydrate) of other isoelectric focused gels run in parallel with the gel shown in Figure 3

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

228

PHYSIOLOGICAL

EFFECTS OF

FOOD

CARBOHYDRATES

p r o t e i n i n f i b r o b l a s t s o n i c a t e s . Assays u s u a l l y have i n v o l v e d i n c u b a t i o n a t pH 4 o f about 50 yg o f s o n i c a t e p r o t e i n f o r periods of up t o 3 hours i n a f i n a l volume o f 0.10 t o 0.15 ml. S l i g h t i n ­ h i b i t i o n o f α-glucosidase a c t i v i t y a t 200 mM maltose was sometimes seen. F i b r o b l a s t s as w e l l as other t i s s u e s a l s o c o n t a i n an α-glu­ cosidase w i t h an a l k a l i n e pH optimum. Whether the t r a c e o f a c t i v ­ i t y measurable a t pH 4 i n f i b r o b l a s t s c u l t u r e d from Type I I g l y ­ cogen storage disease p a t i e n t s i s due t o the p e r s i s t e n c e o f an isozyme o f the a c i d α-glucosidase o r i s due t o a low a c t i v i t y of the n e u t r a l glucosidase a t t h i s pH could not be a s c e r t a i n e d . A l ­ though the glucosidase a c t i v i t y a t a c i d pH present i n f i b r o b l a s t s o n i c a t e s could be i n h i b i t e d by exposure t o the antibody t o the p u r i f i e d human l i v e r enzyme, the f i b r o b l a s t sonicates contained i n s u f f i c i e n t p r o t e i n t o produce a p r e c i p i t i n r e a c t i o n i n double d i f f u s i o n t e s t c a r r i e d out i n Ouchterlony p l a t e s . F r a c t i o n s p r e ­ pared from l i v e r s o f Type I I glycogenosis p a t i e n t s f o l l o w i n g the steps o u t l i n e d f o r the l i v e r , f a i l e d t o produc obtained w i t h comparable f r a c t i o n s from c o n t r o l l i v e r s . Attempts were a l s o made t o detect c r o s s - r e a c t i n g p r o t e i n i n a f f e c t e d t i s s u e s v i a antibody consumption experiments. That i s , a l i q u o t s o f an antibody s o l u t i o n were preincubated w i t h f r a c t i o n s prepared from Type I I glycogen storage disease t i s s u e s o r f i b r o ­ b l a s t s and then added t o p u r i f i e d enzyme o r , i n other experiments, to t i s s u e e x t r a c t s o r f i b r o b l a s t s from non-Type I I i n d i v i d u a l s , p r i o r t o the a d d i t i o n of s u b s t r a t e . The i n h i b i t i o n curves were not i n f l u e n c e d by the c o n d i t i o n s o f p r e i n c u b a t i o n i n any of these experiments. F i b r o b l a s t c u l t u r e s were a v a i l a b l e from s e v e r a l a d u l t s who were d e f i c i e n t i n α-glucosidase a c t i v i t y a t a c i d pH and s o n i c a t e s o f these c e l l s a l s o f a i l e d t o a l t e r the antibody i n ­ h i b i t i o n curves o f the p u r i f i e d human l i v e r enzyme. An example of the e f f e c t o f a d d i t i o n of i n c r e a s i n g amounts of antibody t o a f i b r o b l a s t s o n i c a t e i s shown i n the upper p o r t i o n of Table I . As i n d i c a t e d i n the legend, the numbers given a r e measurements o f s p e c i f i c a c t i v i t y . I n h i b i t i o n was found t o be p r o p o r t i o n a l t o the amount of antibody added. The lower p o r t i o n of the Table r e p o r t s the r e s u l t s obtained w i t h s i x d i f f e r e n t con­ t r o l f i b r o b l a s t c u l t u r e s each of which was incubated w i t h a n t i ­ body which e i t h e r had o r had not been preincubated w i t h other s o n i c a t e s prepared from f i b r o b l a s t s c u l t u r e d from p a t i e n t s w i t h Type I I glycogen storage disease. Up t o 3 times as much p r o t e i n from the Type I I s o n i c a t e s were present as i n the c o n t r o l s o n i ­ c a t e s . Under these c o n d i t i o n s there was no demonstrable d i f f e r ­ ence i n the amount o f a c t i v i t y i n h i b i t e d by added antibody.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

14.

BROWN

ET AL.

229

Lysosomal a-Glucosidases

Table I Glycogen H y d r o l y s i s By F i b r o b l a s t Sonicates I n The Presence Of An Antibody P r e p a r a t i o n (nanomoles o f glucose formed/min/mg s o n i c a t e p r o t e i n ) A Control Activity 8.87

B

C Antibody*

A-B

a

5.44 3.72b

3.43 5.15 6.48

2.39C

4.79 5.40 6.69 7.25 10.77 10.87

A-C

Antibody + Type I I S o n i c a t e * *

2.36a 4.23d 4.86d 3.79a 5.58c 7.07a

2.68a 4.10d

2.43 1.17

2.11 1.27

5.58c 6.9ia

4.05C

4.00C

5.19 3.80 6.82

5.19 3.96 6.87

*

The antibody p r e p a r a t i o n was d i l u t e d 1:250 i n 0.9% NaCl and the volumes added per 0.1 ml r e a c t i o n mixt u r e were as f o l l o w s : (a) 0.01 m l ;(b) 0.015 m l ; (c) 0.02 ml. Experiments l a b e l e d (d) contained 0.01 ml o f a 1:300 d i l u t i o n of the antibody.

** Sonicates o f f i b r o b l a s t s c u l t u r e d from p a t i e n t s w i t h Type I I glycogen storage d i s e a s e preincubated w i t h antibody p r i o r t o a d d i t i o n of c o n t r o l s o n i c a t e s . Another approach t o the problem o f a s s e s s i n g the importance of t h e α-glucosidase i n the glycogen c a t a b o l i s m has i n v o l v e d the p r o d u c t i o n o f glycogen storage i n normal f i b r o b l a s t s by growth o f the c u l t u r e s i n the presence o f an i n h i b i t o r o f the a - g l u c o s i d a s e . In t h e course o f experiments on the metabolism o f the f i b r o b l a s t c e l l l i n e s d e r i v e d from p a t i e n t s w i t h Pompe s d i s e a s e , i t had been observed t h a t the glycogen content o f such c e l l s tended t o be h i g h e r than t h a t found i n normal c e l l l i n e s and t h a t the h a l f l i f e of glycogen which had been formed d u r i n g c u l t u r e i n [ ^ C ] - g l u c o s e tended t o be prolonged. D i n g l e , F e l l , and co-workers had shown t h a t c u l t u r e o f s e v e r a l c e l l types i n sucrose o r other non-metabo l i z a b l e sugars l e d t o e x t e n s i v e v a c u o l a t i o n o f t h e cytoplasm and the appearance o f lysosomal a c i d phosphatase w i t h i n the vacuoles (13,14). On t h e assumption t h a t other enzymes o f the primary lysosome o f the c e l l might a l s o appear w i t h i n such p e r s i s t e n t vacuoles and t h a t some glycogen might be sequestered there as 1

1

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

230

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

w e l l , we added a p o t e n t i a l i n h i b i t o r of the α-glucosidase to the medium i n which normal f i b r o b l a s t s were growing. I t was thought p o s s i b l e t h a t a normal c e l l l i n e grown i n the presence of such a d i s a c c h a r i d e i n h i b i t o r might become s i m i l a r to Type I I f i b r o ­ b l a s t s , i n which the glucosidase i s m i s s i n g , i n that the glycogen content of the t r e a t e d c e l l s might be s i g n i f i c a n t l y elevated and the turnover r a t e of p r e - l a b e l l e d polysaccharide might a l s o be markedly a f f e c t e d (15). P r e l i m i n a r y experiments e s t a b l i s h e d that the glycogen content of a given c e l l l i n e was r e l a t i v e l y constant during a number of s u b c u l t u r e s and that i t was independent of the glucose concentra­ t i o n of the medium w i t h i n the range of 1 to 20 mM (15). Several d i s a c c h a r i d e s were considered f o r use i n these experiments. Hers and co-workers had found that D-(+)- turanose i s a s p e c i f i c i n ­ h i b i t o r of the lysosomal glucosidase (16), and we had shown by k i n e t i c a n a l y s i s that t h i s d i s a c c h a r i d e i s almost p u r e l y a non­ competitive i n h i b i t o r o l i v e r lysosomal enzyme that turanose might be w e l l s u i t e d to produce glycogen storage i n f i b r o b l a s t c u l t u r e s , p r e l i m i n a r y experiments i n d i c a t e d t h a t there was a s i g n i f i c a n t i n h i b i t i o n of c e l l growth i n i t s presence. We had a l s o shown t h a t t r e h a l o s e i s an i n h i b i t o r of α-glucosidase of human l i v e r and heart a c t i n g on glycogen at pH 4 (11). When i t was found t h a t t r e h a l o s e could be added to the c u l t u r e media i n which human f i b r o b l a s t s were growing a t c o n c e n t r a t i o n up to 0.15 M without s e r i o u s e f f e c t on growth r a t e , i t s e f f e c t on the α-glu­ cosidase of f i b r o b l a s t s was i n v e s t i g a t e d . Trehalose proved to be a potent, non-competitive i n h i b i t o r w i t h a Kj_ of from 5 to 8 mM depending on the c e l l l i n e i n v e s t i g a t e d (17). When f i b r o b l a s t s were grown i n the presence of added t r e h a l o s e , the glycogen con­ t e n t increased to an extent which was dependent upon both the c o n c e n t r a t i o n of the d i s a c c h a r i d e i n the medium and the time of exposure to i t . In view of the presumed mode of a c t i o n of t r e ­ halose i n inducing glycogen storage mentioned p r e v i o u s l y , i t was of i n t e r e s t to measure the t o t a l a c t i v i t y of some enzymes which are presumably c h i e f l y of lysosomal o r i g i n , a f t e r the f i b r o b l a s t s and any s u b c e l l u l a r o r g a n e l l e s which they might c o n t a i n were bro­ ken by s o n i c treatment. Table I I shows r e s u l t s obtained w i t h 3 normal c e l l l i n e s and i n c l u d e s a comparison of the a c t i o n of t r e h a l o s e w i t h that of sucrose - which has a l s o been reported to be an i n h i b i t o r of lysosomal α-glucosidase - and of l a c t o s e which i s supposed to be i n e f f e c t i v e i n t h i s r e s p e c t . I t i s apparent that t r e h a l o s e and sucrose induce glycogen storage and t h a t l a c ­ tose does not. Of i n t e r e s t was the f i n d i n g that N - a c e t y l - 3 - g l u cosaminidase showed a r e g u l a r and s u b s t a n t i a l i n c r e a s e i n a c t i v ­ i t y , s i n c e i t has been suggested t h a t t h i s glucosaminidase i s f i r m l y bound to the lysosomal membrane. We have shown by growing f i b r o b l a s t s i n 0.06 M [ ^ C ] - t r e h a l o s e f o r three days that the i n t r a c e l l u l a r content of t h i s d i s a c c h a r i d e becomes as great as 0.4 to 0.6 ymole per mg of p r o t e i n . I f t h i s t r e h a l o s e were 1

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

14.

BROWN

ET

AL.

231

Lysosomal a-Glucosidases

l o c a l i z e d w i t h i n lysosomes, i t s c o n c e n t r a t i o n would be s u f f i c i e n t to i n h i b i t lysosomal α-glucosidase d u r i n g growth. However, i n s o n i c a t e s of such c e l l s , the t r e h a l o s e c o n c e n t r a t i o n would not exceed 5% of i t s K-^ v a l u e as an i n h i b i t o r of glycogen h y d r o l y s i s . Table I I E f f e c t of D i s a c c h a r i d e A d d i t i o n During C u l t u r e On The R a t i o Of Experimental To C o n t r o l Values For Glycogen Content And Enzyme A c t i v i t y In F i b r o b l a s t s C e l l Line

Disaccharide Added

Glycogen Content

a-Glucosidase (Glycogen, pH 4.2)

N-Acetyl-3Glucosaminidase

1

Trehalose Sucrose Lactose

1.50 1.43

1.09 0.82

1.81 1.95

2

Trehalose Sucrose Lactose

1.78 2.01 0.68

1.18 1.14 0.86

1.8 1.82 1.14

3

Trehalose Sucrose Lactose

2.0 2.2 1.0

0.68 0.75 1.10

1I+

F i g u r e 5 compares the l o g a r i t h m i c r a t e of d e c l i n e of [ C ] glycogen i n normal f i b r o b l a s t s maintained i n the presence and ab­ sence of 40 mM t r e h a l o s e added to the c u l t u r e media. The f i b r o ­ b l a s t s were preloaded w i t h [ C ] - g l y c o g e n by c u l t u r e of the c e l l s to c o n f l u e n c y i n the presence of [ **C]-glucose i n the media. At zero time, the c e l l s were washed w i t h u n l a b e l l e d media and c u l t u r e continued i n the absence of l a b e l l e d glucose. The glycogen con­ tent of the c o n t r o l c e l l s averaged 0.82 ymole of polymeric g l u ­ cose per mg of p r o t e i n and increased to 1.34 ymoles/mg i n the c e l l s maintained i n t r e h a l o s e . The time r e q u i r e d f o r the d i s ­ appearance of 50% of the r a d i o a c t i v i t y from glycogen was about t r i p l e d (from 1.4 to 4.6 days) i n the c e l l s maintained i n t r e ­ h a l o s e . As p r e v i o u s l y mentioned, some c e l l l i n e s from type I I glycogen storage d i s e a s e p a t i e n t s have shown a remarkable p r e s e r ­ v a t i o n of [ C ] - g l y c o g e n i n t h e i r c u l t u r e d f i b r o b l a s t s . Data from one such c e l l l i n e i s a l s o shown where an apparent h a l f - l i f e of 7 days was measured f o r i t s glycogen. The average glycogen con­ tent of the 12 samples g i v i n g r i s e to t h i s curve was 1.3 ymoles of polymeric glucose per mg of p r o t e i n . Note that no t r e h a l o s e was present. Other Type I I f i b r o b l a s t s have had apparent h a l f - l i v e s of t h e i r glycogen of 4 t o 5 days. In summary, the α-glucosidase a c t i v e a t pH 4 has been p u r i ­ f i e d t o homogeneity from human l i v e r . I t appears to be a g l y c o ­ p r o t e i n . Antibody t o the enzyme has been produced i n r a b b i t s . 14

1

lk

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

PHYSIOLOGICAL

EFFECTS O F FOOD

CARBOHYDRATES

TIME (DAYS) "Biochemistry of the Glycosidic Linkage" 14

Figure 5. Turnover of [ C] glycogen in fibroblasts of a child with Type II glycogen storage disease (J.A.) and in control fïbrobhsts (D.B.) cultured in the presence and absence of 40 mM trehalose (18, p. 727)

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

14.

BROWN

ET AL.

Lysosomal a-Glucosidases

233

No evidence f o r c r o s s r e a c t i v e p r o t e i n has been obtained u s i n g f i b r o b l a s t c u l t u r e s d e r i v e d from p a t i e n t s w i t h e i t h e r the i n f a n ­ t i l e or a d u l t forms of the d e f i c i e n c y . Glycogen storage and de­ layed turnover of t h i s p o l y s a c c h a r i d e can be induced i n normal f i b r o b l a s t s by c u l t u r e of the c e l l s i n the presence of an i n h i b ­ i t o r o f the α-glucosidase a c t i v i t y . Acknowledgments : A p a r t of t h i s work was c a r r i e d out w h i l e one o f us (A.K.M.) was a Post D o c t o r a l Fellow of the N a t i o n a l I n s t i t u t e s of Health. Present address (AKM) : Department of P e d i a t r i c s , C a l i f o r n i a C o l ­ lege of Medicine, U n i v e r s i t y of C a l i f o r n i a , I r v i n e , C a l i f o r n i a 92664. This work has been supported i n p a r t by Grant GM-04761 from the N a t i o n a l I n s t i t u t e s o f Health and Grants 71-973 and 74-928 from the American Heart A s s o c i a t i o n . Communications r e ­ garding t h i s p u b l i c a t i o Brown, Department of B i o l o g i c a School of Medicine, 660 South E u c l i d Avenue, S a i n t L o u i s , M i s s o u r i 63110. Literature Cited

1. Hers, H.G., Biochem. J., (1963), 86, 11. 2. Jeffrey, P.L., Brown, D.H. and Brown, B.I., Biochemistry, (1970), 9, 1416. 3. Jeffrey, P.L., Brown, D.H. and Brown, B.I., Biochemistry, (1970), 9, 1403. 4. Baudhuin, P., Hers, H.G. and Loeb, H., Lab. Invest., (1964), 13, 1139. 5. Auricchio, F., Bruni, C.B. and Sica, V., Biochem. J., (1968), 108, 161. 6. deBarsy, T., Jacquemin, P., Devos, P., and Hers, H.G., Eur. J. Biochem., (1972), 31, 156. 7. Bruni, C.B., Auricchio, F. and Covelli, I., J. Biol. Chem., (1969), 244, 4735. 8. Palmer, T.N., Biochem. J., (1971), 124, 701. 9. Murray, A.K., Brown, D.H. and Brown, B.I., In preparation. 10. Engel, A.G., Brain, (1970), 93, 599. 11. Brown, B.I., Brown, D.H. and Jeffrey, P.L., Biochemistry, (1970), 9, 1423. 12. Angelini, C ., Engel, A.G. and Titus, J.L., New Eng. J. Med., (1972), 287, 948. 13. Dingle, J.T., Fell, H.B. and Glauert, A.M., J. Cell Science, (1969), 4, 139. 14. Nyberg, E. and Dingle, J.T., Exper. Cell Res., (1970), 63, 43. 15. Brown, B.I. and Brown, D.H., Biochem. Biophys. Res. Commun., (1972), 46, 1292. 16. Lejeune, N., Thines-Sempoux, D. and Hers, H.G., Biochem. J., (1963), 86, 16.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

234

PHYSIOLOGICAL EFFECTS OF FOOD CARBOHYDRATES

17. Brown, D.H. and Brown, B.I., In "Biochemistry of the Glyco­ sidic Linkage," PAABS Symposium, editors, R. Piras and H.G. Pontis, p. 687, Academic Press, New York and London, (1972). 18. Brown, B.I., In "Biochemistry of the Glycosidic Linkage," PAABS Symposium, editors, R. Piras and H.G. Pontis, p. 725, Academic Press, New York and London, (1972). 19. Orr, M.D., Blakley, R.L. and Panagou, D., Anal. Biochem., (1972), 45, 65.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

15 Enzymes of Glycogen Metabolism and Their Control HAROLD L. SEGAL Division of Cell and Molecular Biology, State University of New York, Buffalo, N.Y. 14214

While there is a continua expenditur energy g organisms, albeit of variable degree above the basal level essential to physiological function, the dietary intake of utilizable energy is sporadic. Therefore, the ability to store energy resources and meter their release in a manner consistent with need is an attribute of living organisms essential to their survival. Thus, there must be a post-prandial flow of nutrients into storage forms, and a reverse flow out of storage forms between meals. The two major dietary energy resources are carbohydrate and fat, with protein normally comprising a significantly smaller contribution. Carbohydrates are stored as such or converted to fat, dietary fat is stored only as such, and protein is convertible to both carbohydrate and fat (Fig. 1). It is an important point to emphasize that of the total fat and carbohydrate energy stores, the latter represents less than 10% by weight and half that by caloric content. However, carbohydrate is ubiquitously distributed and readily available, whereas fat is stored primarily at remote depots and must be mobilized, transported, and transformed before entering the c a t a b o l i c p a t h w a y . Therefore, carbohydrate r e p r e sents a more immediately a v a i l a b l e energy s t o r e , w h i l e fat c o n s t i t u t e s a much l a r g e r , long term s t o r e . S i n c e a m i n i m a l l e v e l of c i r c u l a t i n g g l u c o s e is e s s e n t i a l to life and s i n c e the amount of stored carbohydrate is r e l a t i v e l y s m a l l , the c o n v e r t i b i l i t y of protein to carbohydrate is v i t a l for the maintenance of blood g l u c o s e l e v e l s during prolonged food d e p r i v a t i o n or on s e v e r l y c a r b o h y d r a t e - l o w d i e t s . C o n s o n a n t with the purposes of this s y m p o s i u m , my d i s c u s s i o n w i l l d e a l with the r e g u l a t i o n of the f o r m a tion and u t i l i z a t i o n of g l y c o g e n , the storage form of carbohydrate in a n i m a l t i s s u e s . The predominant sugar of the d i e t is g l u c o s e , w h i c h is c o n verted to g l y c o g e n without c l e a v a g e of the h e x o s e c h a i n . As s e e n 235 In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

236

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

in F i g . 1/ this a p p l i e s to g a l a c t o s e a s w e l l , w h i c h i s e p i m e r i z e d to a s u b s t i t u t e d form of g l u c o s e that i s on the d i r e c t pathway of g l u c o s e m e t a b o l i s m . F r u c t o s e , on the other hand, i s for the most part not c o n v e r t e d to a g l u c o s e d e r i v a t i v e , e x c e p t i n d i r e c t l y v i a t r i o s e s , a n d therefore r e g u l a t i o n of its m e t a b o l i s m w o u l d be e x p e c t e d to r e s e m b l e more c l o s e l y that of g l y c o g e n i c amino a c i d s or g l y c e r o l , a s w e l l a s to i n v o l v e unique f e a t u r e s of its own. F i g . 2 shows the immediate s t e p s i n the formation a n d u t i l i z a t i o n of g l y c o g e n , c a t a l y z e d by g l y c o g e n s y n t h e t a s e a a n d p h o s p h o r y l a s e a., r e s p e c t i v e l y , and the i n t e r c o n v e r t i n g s y s t e m s w h i c h produce a n d remove these forms. S e v e r a l p o i n t s s h o u l d be noted i n these p a t h w a y s . a) A t l e a s t three e n z y m e s i n this scheme e x i s t in a c t i v e a n d i n a c t i v e forms, d e s i g n a t e d a. and b, r e s p e c t i v e l y . These are glycogen synthetase, phosphorylase The a_ and b forms d i f f e a b s e n c e of phosphate groups c o v a l e n t l y l i n k e d to s e r i n e r e s i d u e s of the p r o t e i n . Some e v i d e n c e has been reported s u g g e s t i n g the e x i s t e n c e of a c t i v e a n d i n a c t i v e forms of p h o s p h o r y l a s e p h o s phatase and glycogen synthetase phosphatase a s w e l l . b) The a c t i v e form c a n be e i t h e r the p h o s p h o r y l a t e d form, a s is the c a s e w i t h p h o s p h o r y l a s e a n d p h o s p h o r y l a s e k i n a s e , or the d e p h o s p h o r y l a t e d form, a s i s the c a s e w i t h g l y c o g e n s y n t h e t a s e . c) The a c t i v e a n d i n a c t i v e forms i n a s e t are i n t e r c o n v e r t i b l e by a k i n a s e - c a t a l y z e d , ATP-dependent p h o s p h o r y l a t i o n a n d a p h o s p h a t a s e - c a t a l y z e d h y d r o l y s i s of e s t e r i f i e d phosphate. d) The p h o s p h o r y l a s e a n d g l y c o g e n s y n t h e t a s e systems are l i n k e d by the common c y c l i c A M P - s t i m u l a t e d protein k i n a s e w h i c h a c t i v a t e s the former a n d d e a c t i v a t e s the latter; p h o s p h o r y l a s e k i n a s e i s a d i s t i n c t enzyme. e) The p o t e n t i a l for f u t i l e c y c l i n g e x i s t s between g l y c o g e n and g l u c o s e - l - p h o s p h a t e ( G - l - P ) . Each c y c l e c o s t s one h i g h energy phosphate bond; c y c l i n g i s h e l d i n c h e c k by the o p p o s i t e e f f e c t s of the c y c l i c A M P - s t i m u l a t e d protein k i n a s e on the two s y s t e m s , a s w e l l a s other f a c t o r s to be d i s c u s s e d s u b s e q u e n t l y . f) C o n f l i c t i n g reports e x i s t r e g a r d i n g the i d e n t i t y or d i s t i n c t i v e n e s s of the three p h o s p h a t a s e s i n these s y s t e m s . In one c a s e a p r e p a r a t i o n from m u s c l e w a s d e s c r i b e d w h i c h p o s s e s s e d d e p h o s p h o r y l a t i n g a c t i v i t y toward p h o s p h o r y l a s e k i n a s e a n d g l y c o g e n s y n t h e t a s e , but not p h o s p h o r y l a s e q j ; i . e . , that the e n z y m e s w h i c h were common s u b s t r a t e s f o r protein k i n a s e were a l s o common s u b s t r a t e s f o r the same p h o s p h a t a s e . In another report, however, a p r e p a r a t i o n from heart w a s s t a t e d to d e p h o s p h o r y l a t e a l l three of the i n t e r c o n v e r t i b l e enzymes (2).

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

15.

237

Glycogen Metabolism Enzymes

SEGAL

DIETARY CARBOHYDRATE

t

r

1

GLUCOSE

GALACTOSE Gal-1-P I UDPGal

FRUCTOSE

G-6-Pl

F-1-P

it

FAT

1

^G-1-P*

^TRIOSES<

ïAMINO ACIDS

UDPG ] 'GLYCOGEN Figure 1. Interconversions of carbohydrates, fats, and amino acids

phosphorylose kinase phosphatase

phosphorylose _b

ι

phosphorylose kinase b

phosphorylose kinase a

ATP

Iphosphorylose P| /phosphatase

y

phosphorylose g

GLYCOGEN

protein jinose ATP glycogen synthetase _b_

UTP glycogen synthetase a.

UDPG^

2 Pi

glycogen synthetase phosphatase Figure 2. Components involved in glycogen formation and utilization

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

238

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

The c o n t r o l of both the p h o s p h o r y l a s e and g l y c o g e n s y n t h e t a s e s y s t e m s i s c o m p l e x . P h o s p h o r y l a s e a., the p h o s p h o r y l a t e d form, is h i g h l y a c t i v e under a l l c o n d i t i o n s l i k e l y to e x i s t in t i s s u e s . The d e p h o s p h o r y l a t e d form, p h o s p h o r y l a s e b, i s c o m p l e t e l y i n active in liver. M u s c l e phosphorylase b is also inactive except in the p r e s e n c e of high l e v e l s of A M P , a s i t u a t i o n w h i c h c a n o c c u r i n v i v o under c o n d i t i o n s of o x y g e n d e f i c i e n c y . In r e s t i n g t i s s u e p h o s p h o r y l a s e i s present m a i n l y or e n t i r e l y i n the i n a c t i v e b f o r m , and, e x c e p t i n the state of r e l a t i v e a n o x i a referred to a b o v e , where the s y s t e m c a n be turned on by e l e v a t e d A M P l e v e l s , its a c t i v a t i o n depends upon the c o n v e r s i o n of the b form to the a_ form. This i s a c c o m p l i s h e d by p h o s p h o r y l a s e k i n a s e . This enzyme, however, a l s o i s p r e s e n t p r i m a r i l y in the i n a c t i v e or b form and must be a c t i v a t e d to becom phosphorylase itself, thi accomplishe ways P h o s p h o r y l a s e k i n a s e b c a n be s t i m u l a t e d by e l e v a t e d C a 2 l e v e l s , w h i c h o c c u r s as a c o n s e q u e n c e of m u s c l e s t i m u l a t i o n and membrane d e p o l a r i z a t i o n . W i t h this there i s a very r a p i d a c t i v a t i o n of the k i n a s e and i n c o n s e q u e n c e a c o n v e r s i o n of p h o s p h o r y l a s e to the a c t i v e form and an i n i t i a t i o n of r a p i d g l y c o l y s i s . Thus energy p r o d u c t i o n i s p r o v i d e d s y n c h r o n o u s l y w i t h its need to maintain muscle contraction. Alternatively, phosphorylase kinase c a n be a c t i v a t e d by c o n v e r s i o n to the a c t i v e , or a, form. T h i s r e q u i r e s a p h o s p h o r y l a t i o n and i s a c c o m p l i s h e d by the c y c l i c A M P - d e p e n d e n t p r o t e i n k i n a s e . T h i s k i n a s e , i n turn, i s a c t i v a t e d by e l e v a t i o n of c y c l i c A M P l e v e l s . Recent r e v i e w s of the p h o s p h o r y l a s e s y s t e m have been p u b l i s h e d (3,4). R e g u l a t i o n of the p h o s p h o r y l a s e r e a c t i o n i s e f f e c t e d by a number of hormones w h i c h e l e v a t e c y c l i c A M P l e v e l s ; n o t a b l y g l u c a g o n i n the l i v e r , e p i n e p h r i n e i n the m u s c l e , and both of these p l u s A C T H and other hormones i n a d i p o s e t i s s u e . Thus, i n r e s p o n s e to e x t e r n a l s i g n a l s i n the form of t i s s u e - s p e c i f i c hormones, p h o s p h o r y l a s e i s c o n v e r t e d to a form w h i c h i s f u l l y a c t i v e , and i n t h i s way the r e s t r a i n t s w h i c h e x i s t to s e r v e i n t r a c e l l u l a r h o m e o s t a s i s are o v e r r i d e n to meet a more urgent need of the w h o l e organism (5). Another regulatory f a c t o r of more d i r e c t r e l a t i o n s h i p to the s u b j e c t of this s y m p o s i u m i s g l u c o s e i t s e l f . G l u c o s e has been shown to s t i m u l a t e the p h o s p h o r y l a s e p h o s p h a t a s e r e a c t i o n w h i c h c o n v e r t s p h o s p h o r y l a s e from the f u l l y a c t i v e a. form to the i n a c t i v e b form (6). Thus, in r e s p o n s e to an adequate l e v e l of g l u c o s e to meet m e t a b o l i c n e e d s , the r e l e a s e of g l u c o s e units from g l y c o g e n is b l o c k e d and the carbohydrate stores are p r e s e r v e d . G l u c o s e +

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

15.

SEGAL

Glycogen Metabolism Enzymes

239

a l s o i n h i b i t s g l u c a g o n r e l e a s e from the p a n c r e a s , thereby r e d u c i n g the c y c l i c A M P - m e d i a t e d s t i m u l a t i o n of the p h o s p h o r y l a s e k i n a s e branch w h i c h turns p h o s p h o r y l a s e on. Thus g l u c o s e produces a two-pronged e f f e c t i n s t i m u l a t i n g p h o s p h o r y l a s e p h o s phatase a c t i v i t y and r e d u c i n g p h o s p h o r y l a s e k i n a s e a c t i v i t y , both of w h i c h d i m i n i s h the l e v e l of a c t i v e p h o s p h o r y l a s e a n d spare g l y c o g e n u t i l i z a t i o n . These patterns of r e g u l a t i o n are summarized i n F i g . 3. S i m i l a r patterns of c o n t r o l e x i s t w i t h the g l y c o g e n s y n t h e t a s e s y s t e m , often by the same mediators a s w i t h p h o s p h o r y l a s e , o p e r a t i n g in the r e v e r s e s e n s e , a n d therefore i n the same d i r e c t i o n from the p o i n t of v i e w of m e t a b o l i c f l o w . A s w i t h p h o s p h o r y l a s e , the a_ form of s y n t h e t a s e , w h i c h i n t h i s c a s e i s the d e p h o s p h o r y l a t e d form i s f u l l y a c t i v e under p h y s i o l o g i c a l c o n d i t i o n s . The b form i n a c t i v e e x c e p t i n the p r e s e n c e of h i g h l e v e l s of g l u c o s e - 6 phosphate (G-6-P) - a s i t u a t i o n u n l i k e l y to e x i s t i n l i v e r , but w h i c h may o c c u r i n p o s t - t e t a n i c m u s c l e . Thus a c t i v a t i o n of the s y s t e m depends p r i m a r i l y on c o n v e r s i o n of the b to a form be dephosphorylation (Fig. 2). In t h i s c a s e , the e f f e c t of c y c l i c A M P - d e p e n d e n t p r o t e i n k i n a s e i s the r e v e r s e of that w i t h p h o s p h o r y l a s e , c o n v e r t i n g s y n t h e t a s e to the i n a c t i v e form. Thus the e f f e c t of c y c l i c A M P e l e v a t i n g hormones i s to turn off s y n t h e t a s e s y n c h r o n o u s l y w i t h the turning on of p h o s p h o r y l a s e , and thereby d i m i n i s h i n g f u t i l e c y c l i n g of G - l - P into and out of g l y c o g e n . S i n c e the " o f f " form of s y n t h e t a s e i s the p h o s p h o r y l a t e d form and the r e v e r s e i s true for p h o s p h o r y l a s e , a n d s i n c e the " o f f " forms are n o r m a l l y the predominant o n e s , i t i s apparent that the k i n a s e i s a c r i t i c a l f a c t o r i n c o n v e r t i n g p h o s p h o r y l a s e to a n a c t i v e s t a t e , but i s of s i g n i f i c a n c e i n the c a s e of s y n t h e t a s e o n l y in promoting its return to a n i n a c t i v e state o n c e a c t i v a t e d . This s u g g e s t s that it i s a g a i n the a c t i v a t i n g p r o c e s s , i n t h i s c a s e c a t a l y z e d by the p h o s p h a t a s e branch, w h i c h i s the c r i t i c a l one in r e g u l a t i n g the t i s s u e l e v e l of s y n t h e t a s e a c t i v i t y . Furthermore, a number of f a c t o r s w h i c h s t i m u l a t e h e p a t i c g l y c o g e n s y n t h e s i s have now been shown to be e f f e c t o r s of the p h o s p h a t a s e branch of the g l y c o g e n s y n t h e t a s e s y s t e m . S i n c e there i s a c l o s e c o r r e l a t i o n in i n d i v i d u a l l i v e r s between the l e v e l of g l y c o g e n s y n t h e t a s e a. and the rate of g l y c o g e n s y n t h e s i s , i t c a n be c o n c l u d e d that promotion of the p h o s p h a t a s e r e a c t i o n , w i t h the c o n s e q u e n t e l e v a t i o n of s y n t h e t a s e a_ l e v e l s a n d g l y c o g e n s y n t h e s i s , u n d e r l i e s i n large measure the g l y c o g e n i c e f f e c t of these factors.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

240

PHYSIOLOGICAL EFFECTS OF FOOD CARBOHYDRATES

Thus the fundamental q u e s t i o n i n t h i s c a s e i s , what are the regulatory p r o c e s s e s of the p h o s p h a t a s e branch w h i c h c o n v e r t s y n t h e t a s e to the a c t i v e form a n d turn on g l y c o g e n s y n t h e s i s ? At present at l e a s t 4 f a c t o r s appear to be i n v o l v e d i n r e g u l a t i o n of the p h o s p h a t a s e s y s t e m , namely, g l u c o c o r t i c o i d s , i n s u l i n , glucose, and glycogen itself. The l o n g - k n o w n e f f e c t of g l u c o c o r t i c o i d s i n promoting l i v e r g l y c o g e n d e p o s i t i o n can be a t t r i b u t e d , i n part a t l e a s t , to a d i r e c t or i n d i r e c t i n f l u e n c e of these hormones on the p h o s p h a t a s e branch of the g l y c o g e n s y n t h e t a s e s y s t e m . In a d r e n a l e c t o m i z e d rats d e p r i v e d of f o o d , g l y c o g e n l e v e l s , s y n t h e t a s e a. l e v e l s , a n d p h o s p h a t a s e a c t i v i t y are v i r t u a l l y n i l . S e v e r a l hours after g l u c o c o r t i c o i d a d m i n i s t r a t i o n there i s a r e s t o r a t i o n of p h o s p h a t a s e a c t i v i t y , s y n t h e t a s e ^ l e v e l s , a n d rates of g l y c o g e n s y n t h e s i s i n a sequence consisten This e f f e c t , w h i c h appear by c y c l o h e x i m i d e or a c t i n o m y c i n D, a n d thus may be c o n c l u d e d to depend upon a n i n d u c t i o n of the p h o s p h a t a s e or some other component w h i c h l e a d s i n d i r e c t l y to a n e l e v a t i o n of p h o s p h a t a s e a c t i v i t y (7). A d i f f e r e n t type of g l u c o c o r t i c o i d e f f e c t has a l s o been o b s e r v e d . In t h i s c a s e pretreatment of a n i m a l s w i t h the hormone l e d to a p h o s p h a t a s e a c t i v i t y i n l i v e r e x t r a c t s where the l a g period w h i c h i s n o r m a l l y o b s e r v e d prior to the e x p r e s s i o n of f u l l a c t i v i t y w a s g r e a t l y r e d u c e d . P h o s p h o r y l a s e a. i s a n i n h i b i t o r of s y n t h e t a s e p h o s p h a t a s e , a n d g l u c o c o r t i c o i d s i n c r e a s e the a c t i v i t y of p h o s p h o r y l a s e p h o s p h a t a s e , w h i c h removes p h o s p h o r y l a s e .a. Therefore, it w a s proposed that the l a g p e r i o d r e p r e s e n t s the time r e q u i r e d for r e m o v a l of p h o s p h o r y l a s e a. and that the g l u c o c o r t i c o i d r e s p o n s e i n t h e s e experiments depends o n the i n c r e a s e d rate of p h o s p h o r y l a s e a. removal (8). Two types of e f f e c t of g l u c o s e on the p h o s p h a t a s e system have been o b s e r v e d . In the s t a r v e d , a d r e n a l e c t o m i z e d a n i m a l s referred to a b o v e , i n t u b a t i o n of g l u c o s e r e s t o r e d s y n t h e t a s e p h o s phatase a c t i v i t y s i m i l a r l y to but i n d e p e n d e n t l y of the g l u c o c o r t i c o i d e f f e c t (7). In this c a s e the r e s p o n s e w a s a l s o b l o c k e d by c y c l o h e x i m i d e , but not by a c t i n o m y c i n . A p o s s i b l e e x p l a n a t i o n for the g l u c o c o r t i c o i d a n d g l u c o s e e f f e c t s i s that i n the s t a r v e d , a d r e n a l e c t o m i z e d l i v e r , a f a c t o r r e q u i r e d for p h o s p h a t a s e s t a b i l i z a t i o n ( g l y c o g e n ? ) i s l a c k i n g , a n d that t h i s f a c t o r i s r e s t o r e d by g l u c o s e a d m i n i s t r a t i o n , or by g l u c o c o r t i c o i d s v i a a p r o c e s s d e p e n d i n g upon enzyme i n d u c t i o n . In c o n t r a s t to the g l u c o s e e f f e c t referred to a b o v e , w h i c h r e q u i r e s a minimum of about 1 1/2 hours to become m a n i f e s t e d , a

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

15.

Glycogen Metabolism Enzymes

SEGAL

phosphorylase 9 £ k , n

s e

241

adenyl cyclase + HORMONES

phosphorylase kinase phosphatase

Co

4

phosphorylase b

phosphorylas + GLUCOSE

\MP phosphorylase a

GLYCOGENFigure 3.

phosphorylase b_

G-1-P

The phosphorylase system and its regulation

GLUCOCORTICOIDS, GLUCOSE phosphorylase a. phosphorylase phosphatase •

synthetase b.

-x-

protein kinase (c-AMP)

GLUCOCORTICOIDS. INSULIN. GLUCOSE (activation?, induction?)

synthetase phosphatase (g?)

G-6-P G-1-P-—UDPG-

synthetase a

GLYCOGEN

Figure 4. The glycogen synthetase system and its regulation

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

242

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

very r a p i d r e s p o n s e to g l u c o s e of a different sort has a l s o been o b s e r v e d . In normal a n i m a l s or perfused l i v e r s therefrom, w i t h i n a few minutes after g l u c o s e a d m i n i s t r a t i o n there was a r i s e in the l e v e l s of a c t i v e s y n t h e t a s e (a form), together with a f a l l in p h o s p h o r y l a s e a. l e v e l s (9). G i v e n the i n h i b i t i o n of s y n t h e t a s e p h o s phatase by p h o s p h o r y l a s e a. d i s c u s s e d above and the stimulatory effect of g l u c o s e on the p h o s p h o r y l a s e p h o s p h a t a s e r e a c t i o n w h i c h removes p h o s p h o r y l a s e a_ (6), this acute g l u c o s e effect on s y n t h e t a s e a c t i v a t i o n appears to be e x p l a i n a b l e by its effect on the latter r e a c t i o n . In l i v e r s from s t a r v e d , a d r e n a l e c t o m i z e d a n i m a l s referred to above or in d i a b e t i c a n i m a l s , where p h o s phatase a c t i v i t y has d i s a p p e a r e d , the acute e l e v a t i o n of s y n t h e t a s e a in r e s p o n s e to g l u c o s e does not o c c u r . The r e d u c t i o n in p h o s p h a t a s e a c t i v i t y in the d i a b e t i c state is restored s e v e r a l hours evidence exists which i n t e r c o n v e r t i b l e forms d i f f e r i n g in their s e n s i t i v i t y to M g ^ and that i n s u l i n promotes c o n v e r s i o n to the more a c t i v e form (10), but the m e c h a n i s m of the i n s u l i n effect s t i l l remains l a r g e l y u n defined. F i n a l l y , there appears to be a type of f e e d - b a c k r e g u l a t i o n by g l y c o g e n i t s e l f , s i n c e , although low c o n c e n t r a t i o n s of g l y c o g e n promote the p h o s p h a t a s e r e a c t i o n in v i t r o , at higher c o n c e n t r a tions it is i n h i b i t o r y . Furthermore, there is a c o r r e l a t i o n between the s e n s i t i v i t y of l i v e r and m u s c l e p h o s p h a t a s e to g l y c o g e n and the l e v e l of g l y c o g e n w h i c h these t i s s u e s c a n a c c u m u l a t e . That i s , g l y c o g e n a c c u m u l a t i o n is cut off at a lower l e v e l in i n u s c l e than in l i v e r , and its p h o s p h a t a s e is i n h i b i t e d by a lower l e v e l of g l y c o g e n than is the l i v e r p h o s p h a t a s e . +

The foregoing regulatory elements are summarized in F i g . 4. Recent r e v i e w s of the g l y c o g e n s y n t h e t a s e s y s t e m and its c o n t r o l have appeared ( 5 , 1 1 ) . The r e g u l a t i o n of g l y c o g e n m e t a b o l i s m is o b v i o u s l y complex and not a l l its i n t r i c a c i e s have been r e l a t e d to the p h y s i o l o g i c a l role they s u b s e r v e . N e v e r t h e l e s s , it seems c l e a r that the key to u n d e r s t a n d i n g the a d a p t a b i l i t y p r o v i d e d by the r a p i d i n t e r c o n v e r t i b i l i t y between a c t i v e and i n a c t i v e forms l i e s in the c a p a b i l i t y it p r o v i d e s to respond to external s i g n a l s w h i c h override the internal r e s t r a i n t s that maintain internal h o m e o s t a s i s . A number of c r i t i c a l a s p e c t s of the nature of the operation and c o n t r o l of these s y s t e m s have not yet been s o l v e d . T h e s e i n c l u d e the s p e c i f i c i t y of the r e l e v a n t p h o s p h a t a s e s , whether the latter e x i s t in i n t e r c o n v e r t i b l e f o r m s , and the m e c h a n i s m s of their r e g u l a t i o n .

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

15.

SEGAL

Glycogen

Metabolism

Enzymes

243

Literature Cited

1. Zieve, F. J., and Glinsmann, W. H., Biochem. Biophys. Res. Commun. (1973), 50, 872-878. 2. Nakai, C., and Thomas, J. Α., J. Biol. Chem. (1974), 249, 6459-6467. 3. Fischer, Ε. Η., Pocker, Α., and Saari, J. C., Essays in Biochem., ed. by P. N. Campbell and F. Dickens (1970), 6, 23-68. 4. Fischer, E. H., Heilmeyer, L. M. G., Jr., and Haschke, R. Η., Current Topics in Cellular Regulation, ed. by B. L. Horecker and E. R. Stadtman (1971), 4, 211-251. 5. Segal, H. L., Scienc (1973) 180 25-32 6. Holmes, P. Α., an , , Biophys Acta (1968), 156, 275-284. 7. Gruhner, Κ., and Segal, H. L., Biochim. Biophys. Acta (1970), 222, 508-514. 8. Stalmans, W., De Wulf, Η., and Hers, H. G., Europ. J. Biochem. (1971), 18, 582-587. 9. Glinsmann, W. Η., Pauk, G., and Hern, E., Biochem. Biophys. Res. Commun. (1970), 39, 774-782. 10. Bishop, J. S., Biochim. Biophys. Acta (1970), 208, 208-218. 11. Larner, J., and Villar-Palasi, C., Current Topics in Cellular Regulation, ed. by B. L. Horecker and E. R. Stadtman (1971), 3, 195-233.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

16 α-Amylase Inhibitors from Plants J. JOHN MARSHALL Laboratories for Biochemical Research, Howard Hughes Medical Institute and Department of Biochemistry, University of Miami School of Medicine, Miami, Fla. 33152

Introduction

Many plants contain substances which are inhibitory to enzyme action and among the most widespread of such compounds are inhibitors of hydrolytic enzymes. In this article, the nature and distribution of enzyme inhibitors in plants is reviewed, and possible in vivo functions of these inhibitors are discussed. Particular consideration will be given to the proteinaceous inhibitors of the important digestive enzyme, α-amylase, including the detection, assay, specificities and other properties, and physiological effects of these inhibitors. Distribution and Function of Hydrolytic Enzyme Inhibitors A survey of the literature, summarized in Table 1, has reveal­ ed reports of inhibitors of proteases, ribonuclease, invertase, polygalacturonase, cellulase and α-amylase from plant sources. These inhibitors are of two general types - those which are proteins and are rather specific for a particular enzyme, and those which are non-proteinaceous, usually polyphenolic or tannin in nature. Inhibitors of the latter type are much less specific, often inhibiting a number of quite different enzymes. Proteinaceous i n h i b i t o r s o f p r o t e o l y t i c enzymes have been i s o l a t e d from many p l a n t s , and t h e i r chemistry has been examined in d e t a i l . Primary s t r u c t u r e s o f many o f these i n h i b i t o r s a r e known, c r e d i b l e mechanisms o f a c t i o n have been p o s t u l a t e d , and the nature o f p r o t e a s e - i n h i b i t o r i n t e r a c t i o n has been examined by Xray c r y s t a l l o g r a p h y (1-4). The r o l e o f protease i n h i b i t o r s i n p l a n t s has not been e s t a b l i s h e d unequivocally; p o s s i b l e functions include t h e i r involvement i n the r e g u l a t i o n o f the a c t i v i t y o f plant proteases, p r o t e c t i o n o f p l a n t s against m i c r o b i a l a t t a c k o r insect prédation, o r that they serve as storage p r o t e i n s (3)· I n h i b i t o r s o f other h y d r o l y t i c enzymes have been examined i n much l e s s d e t a i l than the protease i n h i b i t o r s and, with the exception o f α-amylase i n h i b i t o r s , only b r i e f mention o f them w i l l 244 In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

16.

MARSHALL

245

α-Amylase Inhibitors from Plants

TABLE 1 NATURALLY OCCURRING INHIBITORS OF HYDROLYTIC ENZYMES

Enzyme

Source o f Inhîbî t o r

Nature o f Inhibitor

References

Protease

Many p l a n t s

Protein

I- 3

Invertase

Potato Maize Ipomoea p e t a l s

Protein Protein Protein

9 10

Polygalacturonase

Bean hypocotyls tomat culture eel 1 s

Protein

I I - 13

Cellulase

Many p l a n t s

Polyphenol

14-17

Rîbonuclease

L i l a c leaves

Probably protein

19

or Amylase

Many p l a n t s

Polyphenol and p r o t e i n

See Table 2

5-8

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

246

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

be made. Invertase i n h i b i t o r s , which are p r o t e i n s , probably serve to regulate sucrose breakdown i n p l a n t s . This can be s a i d w i t h some degree o f c e r t a i n t y because they do i n h i b i t the corresponding plant invertases (5~8). I t has been suggested (11-13) that poly­ galacturonase i n h i b i t o r s , which are a l s o p r o t e i n s , are involved i n p r o t e c t i n g p l a n t s against invasion by plant pathogens, which u s u a l l y gain entry i n t o p l a n t s by breakdown o f the c e l l w a l l s under the a c t i o n o f p e c t i c enzymes. C e l l u l a s e i n h i b i t o r s (14-17) are, apparently without e x c e p t i o n , polyphenolic and may play a r o l e i n p r o t e c t i n g the s t r u c t u r a l m a t e r i a l of p l a n t s against degradation by c e l l u l o l y t i c micro-organisms. It i s probable that they a l s o a c t to i n h i b i t the c e l l u l o l y t i c enzymes o f rumen micro­ organisms, so that such i n h i b i t o r s may have an undesirable e f f e c t on the growth c h a r a c t e r i s t i c s o f animals fed on m a t e r i a l s r i c h i n such i n h i b i t o r s (15,17,18). There i s only one report (19) o f an i n h i b i t o r o f ribonuclease i n p l a n t s It was suggested that t h i s i n h i b i t o r i s proteinaceous L i k e the protease i n h i b i t o r s , α-amylas appea to be widely d i s t r i b u t e d throughout the plant kingdom, and because of t h e i r a b i l i t y to i n a c t i v a t e α-amylase in vivo, they may be o f c o n s i d e r a b l e n u t r i t i o n a l s i g n i f i c a n c e . For t h i s reason, much a t t e n t i o n i s now being focussed on these i n h i b i t o r s . Their f u n c t i o n i n p l a n t s i s not y e t known with c e r t a i n t y . However, con­ s i d e r a t i o n o f the s p e c i f i c i t i e s o f α-amylase i n h i b i t o r s (vide infra) suggests that they might serve to p r o t e c t the seeds i n which they occur against prédation by i n s e c t s and animals, by v i r t u e o f t h e i r I n h i b i t o r y a c t i v i t y towards the d i g e s t i v e amylases o f the predators. Most α-amylase i n h i b i t o r s are i n ­ a c t i v e towards m i c r o b i a l and plant α-amylases, i n d i c a t i n g that they are not a n t i m i c r o b i a l agents, nor do they serve t o regulate the l e v e l s o f α-amylase i n the p l a n t s . The remainder o f t h i s a r t i c l e reviews the current s t a t u s o f our knowledge regarding i n h i b i t o r s of α-amylase. Detection

and Assay of α-Amylase

Inhibitors

A v a r i e t y o f methods f o r determination o f α-amylase i n h i b i t o r a c t i v i t i e s i n p l a n t e x t r a c t s have been used, and v i r t u a l l y a l l workers have expressed i n h i b i t o r a c t i v i t i e s i n d i f f e r e n t ways. However, with only a few exceptions i t has been r e a l i z e d that i t i s necessary to pre-incubate α-amylase and i n h i b i t o r f o r i n h i b i t ­ ion t o take place, rather than simply t o incorporate i n h i b i t o r d i r e c t l y Into a d i g e s t c o n t a i n i n g a mixture o f enzyme and s u b s t r a t e . For the most p a r t , the d u r a t i o n , pH, temperature and other c o n d i t i o n s o f pre-incubât ion appear t o have been a r b i t r a r i l y chosen. A f t e r p r e - i n c u b a t i o n , u n i n h i b i t e d amylase a c t i v i t y remaining has been determined by a v a r i e t y o f methods, most u s u a l l y based on the increase o f reducing power [measured w i t h dlnltrosalîcylic a c i d (20) o r an a l k a l i n e copper reagent ( 2 1 ) ] , o r by the decrease i n iodine s t a i n i n g power (22), during a c t i o n on

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

16.

MARSHALL

α-Amylase Inhibitors from Plants

247

s t a r c h . Amylase i n h i b i t o r a c t i v i t i e s have in many instances been expressed in u n i t s , based on the amount o f i n h i b i t o r which w i l l I n h i b i t a c e r t a i n p r o p o r t i o n o f a f i x e d amount o f α-amylase. I t has not u s u a l l y been made c l e a r whether the measured extent o f i n h i b i t i o n represents the maximum o b t a i n a b l e w i t h the amount o f i n h i b i t o r used, o r whether i t i s l e s s than the maximum o b t a i n a b l e w i t h that amount o f i n h i b i t o r . In the l a t t e r case, i n h i b i t o r a c t i v i t y i s being measured i n terms o f the r a t e o f amylase i n h i b i t i o n , rather than r e f l e c t i n g the s t o i c h i o m e t r y o f amylaseinhibitor interaction. During recent s t u d i e s (23) on the amylase i n h i b i t o r from kidney beans Phaseolus vulgaris, p a r t i c u l a r a t t e n t i o n was given t o the s e l e c t i o n o f pre-incubation c o n d i t i o n s which express optimal i n h i b i t o r a c t i v i t y . The f i n d i n g s h i g h l i g h t e d the importance o f c a r e f u l choice of the p r e - i n c u b a t i o n c o n d i t i o n s . Assays based on determination o f the t o t a l t f α-amylas which sampl f the i n h i b i t o r can rende pre-incubation times wer require , and l o s s o f α-amylase a c t i v i t y tended t o occur during the pre­ incubation p e r i o d , even i n the absence o f i n h i b i t o r . Measurement of the r a t e o f i n h i b i t i o n o f α-amylase was found t o be much more convenient and accurate,and required only short p r e - i n c u b a t i o n times. The c o n d i t i o n s found s u i t a b l e f o r assay o f the kidney bean i n h i b i t o r are summarized in Scheme I. A temperature o f 37°C and a pH o f 5.5 are used f o r the p r e - i n c u b a t i o n , these c o n d i t i o n s being f a v o r a b l e f o r r a p i d i n h i b i t i o n o f hog p a n c r e a t i c α-amylase by the kidney bean i n h i b i t o r (23). Calcium c h l o r i d e and human serum albumin are included i n the p r e - i n c u b a t i o n d i g e s t , t o pro­ t e c t the α-amylase against spontaneous i n a c t i v a t i o n . Amylase a c t i v i t y i s measured, a f t e r p r e - i n c u b a t i o n , by a d d i t i o n o f s t a r c h buffered a t pH 6.9, and determination o f the decrease i n i o d i n e s t a i n during incubation f o r 5 min. One u n i t o f i n h i b i t o r a c t i v i t y i s defined as the amount which causes 50% i n h i b i t i o n o f the aamylase during p r e - i n c u b a t i o n f o r 20 min. The extent o f i n h i b i t ­ ion i s p r o p o r t i o n a l t o the amount o f i n h i b i t o r present and t o the d u r a t i o n o f p r e - i n c u b a t i o n , provided the extent o f incubation does not exceed 50%. Thus the amount o f i n h i b i t o r and the d u r a t i o n o f pre-incubation must be such as t o g i v e no more than t h i s degree o f i n h i b i t i o n in order t o ensure a v a l i d assay. A simple method f o r d e t e c t i o n o f amylase i n h i b i t o r s i n e x t r a c t s o f b i o l o g i c a l m a t e r i a l s has r e c e n t l y been described (24). A narrow c e l l u l o s e s t r i p saturated w i t h the s o l u t i o n being exam­ ined f o r i n h i b i t o r , i s placed on a buffered starch-agar gel p l a t e for 2 hours a t 37°C. The s t r i p i s then removed and another narrow s t r i p saturated w i t h amylase s o l u t i o n i s placed on the gel a t r i g h t angles across the p o s i t i o n occupied by the f i r s t s t r i p . A f t e r incubation f o r 6-18 hr a t 37°C., the second s t r i p i s removed and the s l a b i s flooded w i t h i o d i n e s o l u t i o n , amylase a c t i v i t y being shown by c l e a r zones on a purple background. The presence

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

248

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

SCHEME I ASSAY OF Q.-AMYLASE INHIBITOR ACTIVITY (23) Ppe-inoubation:

A 1.5 ml d i g e s t c o n t a i n i n g

and

hog pancreatic α-amylase (1 yg) calcium c h l o r i d e (1.5 mg) human serum albumin (1.5 mg) acetate b u f f e r 6.7 mM, pH 5-5) inhibitor

i s incubated a t 37°C f o r a s u i t a b l e period o f time ( u s u a l l y 20 min). The amount o f i n h i b i t o r present should be

period. Assay of Residual ^.-Amylase Activity

Controls:

:

Buffered s o l u b l e s t a r c h s o l u t i o n (1.0 ml c o n t a i n i n g 10 mg s t a r c h i n 100 mM glycerophosphate b u f f e r pH 6.9 and 20 mM calcium c h l o r i d e ) i s added to the pre­ incubation mixture. The r e s u l t i n g d i g e s t i s then incubated a t 37°C f o r e x a c t l y 5 min. The absorbance o f the s t a r c h - i o d i n e complex obtained on a d d i t ­ ion o f a sample o f the d i g e s t (0.1 ml) to 5.0 ml o f 0.02? iodine - 0.2? potassium iodide s o l u t i o n , i s measured at 680 nm. α-Amylase a c t i v i t y i s expressed as the d i f f e r e n c e i n the absorbance from that o f a substrate blank d i g e s t c o n t a i n i n g everything ex­ cept α-amylase. (1) α-Amylase pre-incubated without i n h i b ­ i t o r (gives the u n i n h i b i t e d a c t i v i t y ) . (2) In cases where the i n h i b i t o r preparation a l s o contains amylase a c t i v i t y , a sample of the i n h i b i t o r preparation i s preincubated w i t h a l l the other d i g e s t c o n s t i t u e n t s except α-amylase, then incubated w i t h s t a r c h f o r the usual time. This c o n t r o l i s u s u a l l y necessary when crude e x t r a c t s are being assayed.

Unit of Inhibitor:

1 Unit o f i n h i b i t o r i s the amount which causes 50? i n h i b i t i o n o f α-amylase i n 20 min under the above c o n d i t i o n s .

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

16.

MARSHALL

ct-Amyhse Inhibitors from Fiants

249

o f i n h i b i t o r i s i n d i c a t e d by i n t e r r u p t i o n o f the l y s i s zone where the i n h i b i t o r - c o n t a i n i n g and amylase-containing s t r i p s crossed. A v a r i a t i o n o f the method, i n v o l v i n g measurement o f the diameter of c l e a r e d zones produced by a m y l a s e - i n h i b i t o r mixtures placed i n s ^ a l l w e l l s cut i n the starch-agar gel s l a b , was a l s o d e s c r i b e d . \x\ view o f the extended time required t o o b t a i n r e s u l t s , and t h e i r e s s e n t i a l l y q u a l i t a t i v e nature, there appears t o be l i t t l e advant­ age gained by the use o f t h i s method, rather than a more conventtonal i n h i b i t o r assay, such as that described above. Properties

of ^Amylase

Inhibitors

General Properties. α-Amylase i n h i b i t o r s have been known f o r a c o n s i d e r a b l e time, but compared with the protease i n h i b i t o r s , l i t t l e i s understood about t h e i r s t r u c t u r e s and mechanism o f action, their nutritiona significance thei in vivo. Proteinaceous and ηοη-prote been discovered (Table 2). Insoluble α-amylase i n h i b i t o r s , such as the "sisto-amylase" described by Chrzaszcz and J a n i c k i (30,31) may f u n c t i o n simply by adsorption o f the enzyme, preventing i t s i n t e r a c t i o n w i t h sub­ s t r a t e , rather than by formation o f a s p e c i f i c enzyme-inhibitor complex. An e t h e r - s o l u b l e substance from navy beans (54) i n t e r ­ f e r e s w i t h α-amylase a c t i o n on s t a r c h probably by i n t e r a c t i n g w i t h the s u b s t r a t e r a t h e r than the enzyme. Neither o f these substances can be considered a true i n h i b i t o r o f α-amylase. Potato tubers and c e r t a i n v a r i e t i e s o f sorghum contain d i a l y z a b l e , nonproteinaceous, α-amylase i n h i b i t o r s (25-28). The i n h i b i t o r from sorghum i s the b e t t e r c h a r a c t e r i z e d ; i t i s d i a l y z a b l e , heats t a b l e (even on a u t o c l a v i n g ) , and o r g a n i c - s o l v e n t s o l u b l e (2 7). It i n h i b i t s many enzymes besides α-amylase, and appears t o be a tannin o f the leucocyanidin group (28). The p r o p e r t i e s o f the i n h i b i t o r from sorghum c o n t r a s t w i t h those o f the proteinaceous α-amylase i n h i b i t o r s , which a r e s p e c i f i c f o r α-amylase, a r e n o n - d i a l y z a b l e , and may be i n a c t i v a t e d by heating. Proteinaceous α-amylase i n h i b i t o r s , although suscept­ i b l e t o heat i n a c t i v a t i o n , a r e g e n e r a l l y rather h e a t - s t a b l e p r o t e i n s , o f t e n r e q u i r i n g prolonged heating a t elevated temperat­ ures, o r even a u t o c l a v i n g , t o destroy t h e i r a c t i v i t y completely. Of a l l the proteinaceous α-amylase i n h i b i t o r s which have been i s o l a t e d from p l a n t s (Table 2), the best known i s that from wheat, f i r s t studied by Kneen and h i s co-workers (36,37) i n the 1940's, although the i n h i b i t o r s i n rye (36,37), and i n navy beans (51) were f i r s t detected a t about the same time. Only those α-amylase i n h i b i t o r s o f the proteinaceous type w i l l be considered i n d e t a i l . In the f o l l o w i n g s e c t i o n s the s p e c i f i c i t i e s o f i n h i b i t o r s from d i f f e r e n t sources w i l l be con­ s i d e r e d , then the p r o p e r t i e s o f the two b e s t - c h a r a c t e r i z e d proteinaceous i n h i b i t o r s o f α-amylase, those from wheat and kidney beans, w i l l be d i s c u s s e d .

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

250

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

TABLE 2 NATURALLY OCCURRING INHIBITORS OF a-AMYLASE

Source o f inhibitor

Nature o f Inhibitor

Potato tubers

Characteristics

References

Non-proteinaceous

Ether-soluble; dialyzable

25,26

Sorghum

Tannins o f the leucocyanidin group

Organic-solvent soluble; general enzyme i n a c t i v a t o r

27,28

Acorn

Not

Buckwheat mal t

Protein

Water-insoluble; h e a t - l a b i l e (500C)

30,31

Oat seeds

Protein

Non-dialyzable; h e a t - s t a b l e (100°C); also inhibited 3-amylase; i n h i b i t i o n reversed by s u l f h y d r y l compounds

32

Mangoes

Protein

Heat-labile (unspecî f i e d temperature); non-dialyzable

33

Colooasia esculenta tubers

Protein

Heat-stable (100°C); destroyed by autoclaving; non-dialyzable

34,35

Wheat

Protein

Refer t o t e x t

36-49

Rye

Protein

Heat-stable (100°C); non-dialyzable

45,50

Phaseolus vulgaris

Protein

Refer t o t e x t

23,51-53

identifie

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

16.

MARSHALL

α-Amylase Inhibitors from Plants

251

Specificities of α-Amylase Inhibitors. Of the α-amylase I n h i b i t o r s which are protelnaceous In nature, only two have been reported t o i n h i b i t enzymes other than α-amylase. An i n h i b i t o r preparation from navy beans a l s o i n h i b i t e d t r y p s i n ( 5 1 ) , but the a c t i v i t y towards the l a t t e r enzyme was probably due t o contamin­ a t i o n by a s p e c i f i c t r y p s i n i n h i b i t o r , a l s o known t o be present In these beans ( 5 5 ) . An I n h i b i t o r preparation from oats (32) i n h i b i t s both β-amylase and α-amylase. The a b i l i t y t o r e s t o r e the a c t i v i t y of these enzymes by a d d i t i o n o f s u l f h y d r y l reagents, such as g l u t a ­ t h i o n e , i n d i c a t e d that the i n h i b i t i o n was the r e s u l t o f I n t e r ­ a c t i o n o f the polypeptide i n h i b i t o r w i t h t h i o l groupings i n the enzymes, rather than a s p e c i f i c p r o t e i n - p r o t e i n i n t e r a c t i o n as i s considered t o take place w i t h other i n h i b i t o r s . Thus the I n h i b i t o r from oat seeds, although apparently protelnaceous, belongs t o a d i f f e r e n t c l a s s from the s p e c i f i c proteinaceous α-amylase i n h i b i t ­ o r s , In which we are p r i m a r i l y Interested In t h i s a r t i c l e α-Amylases from d i f f e r e n s u s c e p t i b i l i t y to inhibitio ors o f α-amylase (Table 3 ) . Furthermore, these i n h i b i t o r s do not a l l have the same s p e c i f i c i t y . The I n h i b i t o r from Colocasia esculenta ( 3 4 , 3 5 ) has the simplest s p e c i f i c i t y ; s a l i v a r y aamylase i s the only enzyme i t has been found t o i n h i b i t . Kneen gnd Sandstedt reported that the wheat i n h i b i t o r was a c t i v e towards s a l i v a r y and p a n c r e a t i c α-amylases, and a l s o the s a c c h a r i f y i n g type (56) o f b a c t e r i a l α-amylase; i t was without a c t i o n on the more common b a c t e r i a l α-amylase, the l i q u e f y i n g type, o r on p l a n t α-amylase ( 3 6 , 3 7 ) . In a more recent study o f the wheat i n h i b i t o r ( 4 0 , 4 2 ) , Shainkln and B i r k separated two p r o t e i n s w i t h I n h i b i t o r y a c t i o n . One o f these (designated Aml ) had s p e c i f i c i t y s i m i l a r t o that reported by Kneen and Sandstedt f 3 6 , 3 7 ) and was a l s o found to i n h i b i t Insect (Tenebrio molitor l a r v a l midgut) α-amylase ( 5 7 ) . The second form (Amlj) i n h i b i t e d only the i n s e c t α-amylase. S i m i l a r f i n d i n g s were reported by S i l a n o and co-workers ( 4 7 ) . The α-amylase i n h i b i t o r from rye ( 3 6 , 3 7 , 4 5 ) i n h i b i t s s a l i v a r y and p a n c r e a t i c α-amylases and, l i k e that from wheat, Is without a c t i o n on b a c t e r i a l l i q u e f y i n g α-amylase and plant α-amylase ( 5 0 ) . Jaffé and co-workers reported that a p a r t l y p u r i f i e d i n h i b i t o r from kidney beans (Phaseolus vulgaris) was a c t i v e towards s a l i v a r y and p a n c r e a t i c α-amylases, and a l s o t o a small extent against b a c t e r i a l l i q u e f y i n g α-amylase ( 5 3 ) . Studies i n our Laboratory (23) d i d not support the suggestion that b a c t e r i a l α-amylase i s i n h i b i t e d by the Phaseolus vulgaris i n h i b i t o r . The most l i k e l y explanation o f these d i f f e r e n t f i n d i n g s Is that the Venezuelan workers may not have Included adequate c o n t r o l s , t o c o r r e c t f o r the spontaneous l o s s o f α-amylase a c t i v i t y during p r e - i n c u b a t i o n . In the case o f the b a c t e r i a l enzyme, such losses are p a r t i c u l a r l y large because o f the presence o f traces o f p r o t e o l y t i c enzymes, even i n c r y s t a l l i n e preparations o f the amylase ( 5 8 ) . The i n h i b i t o r from kidney beans a l s o i n h i b i t s Helix pomatia ( s n a i l ) α-amylase, but a t about only h a l f the rate i t i n h i b i t s s a l i v a r y 2

9

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

+ +

+ +

+

ND

ND ND

+

ND

ND

ND

ND +

ND ND ND

.ND

ND

ND ND

ND + +

ND

ND

Insect {Tenebrio molitor)

a

s i m i l a r r e s u l t s reported i n Refs. 47 and 48.

+ indicates i n h i b i t i o n ; - i n d i c a t e s no i n h i b i t i o n ; ND i n d i c a t e s not determined. The r e l a t i v e a f f i n i t i e s o f a p a r t i c u l a r i n h i b i t o r f o r the d i f f e r e n t α-amylases which i t i n h i b i t s , when these a r e known, are discussed i n the t e x t .

52,53 23

ND

-

-

+

+

Phaseolus vulgaris

a

-

42

+

36,37

Wheat Ami j

-

-

+

+

-

-

-

-

50

Rye

α-Amylase Plant Helix Bacterial Bacterial pomatia (1iquefying) (sacchari f y i n g )

Fungal

Pancreatic

+

34,35

Colooasia esculenta

Salivary

OF INHIBITORS TOWARDS a-AMYLASES FROM DIFFERENT SOURCES

Ami 2

Reference

Source o f Inhibitor

SPECIFICITIES

TABLE 3

16.

MARSHALL

a-Amylase Inhibitors from Plants

253

and p a n c r e a t i c α-amylases, the l a t t e r two enzymes beinq i n a c t i v a t e d a t almost i d e n t i c a l rates ( 2 3 ) . In t h i s respect the bean i n h i b i t o r d i f f e r s markedly from one cereal (rye) α-amylase i n h i b i t o r , which acts on the s a l i v a r y enzyme about 10-times f a s t e r than on the p a n c r e a t i c enzyme ( 5 0 ) . One p o s s i b l e e x p l a n a t i o n f o r the d i f f e r e n c e i n rate of i n h i b i t i o n of these two α-amylases i s that the carbohydrate present i n the l a t t e r ( 5 9 ) , but apparently absent from the former, i n t e r f e r e s w i t h enzyme-inhibitor i n t e r ­ a c t i o n . Decrease in s u s c e p t i b i l i t y to i n h i b i t i o n by n a t u r a l l y o c c u r r i n g proteinaceous i n h i b i t o r s has p r e v i o u s l y been observed a f t e r chemical attachment of carbohydrate to p a n c r e a t i c α-amylase and p a n c r e a t i c t r y p s i n (60). At the present time, the d i f f e r e n c e s in s u s c e p t i b i l i t y of amylases to i n h i b i t i o n are not c l e a r l y under­ stood. However the p o s s i b i l i t y does e x i s t that s t u d i e s o f enzymei n h i b i t o r i n t e r a c t i o n w i l l e v e n t u a l l y be of value in examinations of the s t r u c t u r e s and a c t i v s i t topograph f α-amylases α-Amylase Inhibitors from Wheat. The α-amylase i n h i b i t o r from wheat has received more a t t e n t i o n than any of the other aamylase i n h i b i t o r s . I n i t i a l s t u d i e s on the i n h i b i t o r ( 3 6 , 3 7 ) , prepared by e x t r a c t i o n of wheat f l o u r w i t h water, followed by b o i l i n g to destroy endogenous amylase a c t i v i t y , showed i t to have the p r o p e r t i e s of a p r o t e i n . Thus the i n h i b i t o r was p r e c i p i t a t e d by ammonium s u l f a t e , and by high concentrations of e t h a n o l . Although i t was q u i t e thermostable, being l i t t l e a f f e c t e d by prolonged storage at 70°C., or on b o i l i n g f o r short p e r i o d s , autoc l a v i n g at 121°C f o r 30 min caused complete l o s s of i n h i b i t o r a c t i v i t y . As w e l l as being w a t e r - s o l u b l e , the i n h i b i t o r was a l s o s o l u b l e in aqueous ethanol at concentrations up to 70? e t h a n o l . The ethanol s o l u b i l i t y was used to demonstrate that amylase i n h i b i t i o n was r e v e r s i b l e , a d d i t i o n of ethanol to a f i n a l c o n c e n t r a t i o n of 70? p r e c i p i t a t i n g α-amylase from a mixture of amylase and i n h i b i t o r , l e a v i n g the i n h i b i t o r i n s o l u t i o n . Kneen and Sandstedt ( 3 6 , 3 7 ) reported that complete i n h i b i t i o n of aamylase w i t h the wheat i n h i b i t o r could not be achieved; this observation may i n d i c a t e that amylase i n h i b i t i o n i s p a r t l y r e v e r s i b l e by s u b s t r a t e . Treatment w i t h pepsin rendered the inhibitor inactive. The production of wheat i n h i b i t o r w i t h kernel development was f o l l o w e d , and showed that the i n h i b i t o r was produced j u s t as the kernels reached m a t u r i t y , approximately 15 days a f t e r f l o w e r i n g (36,37). It d i d not disappear on germination. Since the l e v e l of i n h i b i t o r was markedly lower in wheat brans than in whole ground wheat, the suggestion was made that the m a j o r i t y of the i n h i b i t o r i s located in the endosperm. The i n h i b i t o r was present i n s i m i l a r amounts i n a l l wheats t e s t e d , namely hard red w i n t e r wheat, s o f t red w i n t e r wheat, and hard red s p r i n g wheat. Subsequent work (38) on the wheat i n h i b i t o r r e s u l t e d i n a 7 5 0 - f o l d p u r i f i c a t i o n from wheat e x t r a c t s by a 70°C heat treatment, ethanol f r a c t i o n a t i o n and adsorption on alumina. The a b i l i t y to

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

254

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

i n a c t i v a t e the i n h i b i t o r by t r e a t i n g with n i t r o u s a c i d , which reacts with p r o t e i n amino groups, and by d i g e s t i o n with the p r o t e o l y t i c enzyme, f i c i n , was presented as f u r t h e r evidence that the i n h i b i t o r i s a p r o t e i n . Papain, however, did not destroy i t s a c t i v i t y . These l a t e r s t u d i e s (39) showed some d i f f e r e n c e s from the e a r l i e r f i n d i n g s (36,37). Thus, p u r i f i c a t i o n involved p r e c i p i t a t i o n with 70% e t h a n o l ; p r e v i o u s l y the i n h i b i t o r was reported to be s o l u b l e in t h i s concentration of a l c o h o l , and no reason was suggested f o r the apparent discrepancy. A f u r t h e r d i f f e r e n c e was in the e f f e c t of d i a l y s i s on the a c t i v i t y of the i n h i b i t o r . While a c t i v i t y was retained on d i a l y s i s against tap water or s a l t s o l u t i o n s , the a c t i v i t y of the p u r i f i e d material was l o s t on d i a l y s i s against d i s t i l l e d water. This observation was taken to i n d i c a t e the requirement f o r a d i a l y z a b l e inorganic c o f a c t o r f o r a c t i v i t y . The less-pure preparation used e a r l i e r d i d not lose a c t i v i t y on d i a l y s i s against d i s t i l l e d water, and i t was suggested that contaminating p r o t e i n vented exhaustion of c o f a c t o The heat s t a b i l i t y p r o p e r t i e s of the p u r i f i e d i n h i b i t o r preparation were examined under a v a r i e t y of c o n d i t i o n s , from which i t was found that a c t i v i t y was l o s t f a s t e r in a l k a l i n e than in a c i d i c s o l u t i o n , at any p a r t i c u l a r temperature (38). Denaturation was achieved in 10 min at 95 C and pH 9.0. At pH 5.3, more than 60 min was r e q u i r e d . The s t a b i l i t y of the i n h i b i t o r , even in a c i d i c s o l u t i o n , appeared to be lower than that reported p r e v i o u s l y (36,37), but again the reason may have been the s t a b i l i z i n g e f f e c t of contaminating p r o t e i n s in the l e s s pure préparât ion. The i n h i b i t o r was i n a c t i v a t e d by a number of reducing agents, i.e. sodium s u l f i t e , hydrogen s u l f i d e and a s c o r b i c a c i d (39). O x i d i z i n g agents - c h l o r i n e , bromine, sodium c h l o r i t e and hydrogen peroxide - were even more e f f e c t i v e in causing loss of a c t i v i t y . A strong p o s i t i v e t e s t f o r tryptophan in the i n h i b i t o r p r e p a r a t i o n , together with the knowledge that o x i d i z i n g agents r e a d i l y a t t a c k tryptophan, led to the suggestion that tryptophan plays an important part in the a c t i v i t y of the i n h i b i t o r (39). Further support f o r the important r o l e of tryptophan came from the observation that aldehydes, such as acetaldehyde and benzaldehyde, which react w i t h indole r i n g s , a l s o caused i n a c t i v a t i o n . Freshly prepared s o l u t i o n s of the p u r i f i e d i n h i b i t o r increased in i n h i b i t o r y power on standing, t h i s being explained on the basis of d i s s o c i a t i o n to reveal the a c t i v e s i t e s responsible f o r combination w i t h α-amylase (39). It was a l s o shown that i n h i b i t ­ ion of α-amylase d i d not take place instantaneously on a d d i t i o n of i n h i b i t o r , but increased during a period of pre-incubation of enzyme and i n h i b i t o r , before a d d i t i o n of s t a r c h . A study of the nature of the I n h i b i t i o n showed t h i s to be non-competitive. This l a t t e r f i n d i n g was considered to be in accord w i t h the d i f f e r e n c e In the e f f e c t of the i n h i b i t o r towards d i f f e r e n t α-amylases (vide supra). If the i n h i b i t o r acted by competing w i t h s t a r c h f o r the

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

16.

255

α-Amylase Inhibitors from Plants

MARSHALL

a c t i v e s i t e o f the enzyme, a l l amylases would have been expected to be a f f e c t e d . Since the r e a c t i o n i s non-competitive, i t f o l l o w s that c e r t a i n amylases have groupings which can combine w i t h the i n h i b i t o r , w h i l e others do not, although a l l the amylases have the c a t a l y t i c groupings necessary f o r s t a r c h h y d r o l y s i s . The i n h i b i t o r from wheat received l i t t l e f u r t h e r a t t e n t i o n u n t i l r e c e n t l y ; i t i s now being i n v e s t i g a t e d by several groups o f workers. Shainkin and B i r k (40-42) p u r i f i e d two forms o f the aamylase i n h i b i t o r from wheat f l o u r by ammonium s u l f a t e f r a c t i o n ­ a t i o n , and chromatography on DEAE-cel1ulose and CM-cel1ulose. These two i n h i b i t o r s , designated Ami] and A m l 2 , d i f f e r e d i n s p e c i f i c i t y (Table 3 ) , i n molecular weight (18,000 f o r Ami] and 26,000 f o r A m l ) , and i n many other p r o p e r t i e s (42). D i l u t e s o l u t i o n s o f Ami] were not a f f e c t e d when b o i l e d (at u n s p e c i f i e d pH) f o r 10 min, whereas A m l l o s t 10-15? o f i t s a c t i v i t y a f t e r 1 min, and 70-80? a f t e r 10 min Treatment w i t h 6.4 M urea f o r 24 hr had no a f f e c t on but reduction w i t h mercaptoethano 6.4 M urea r e s u l t e d i n t o t a l loss o f a c t i v i t y i n both cases. The d i f f e r e n c e s between the two i n h i b i t o r s were apparent from t h e i r s u s c e p t i b i l i t y t o d i g e s t i o n w i t h proteases. Ami] was i n a c t i v a t e d by t r y p s i n and chymotrypsin, whereas A m l was r e s i s t a n t t o these enzymes. Pronase completely destroyed Ami] and 60? o f the Aml£ in 3 hr; pepsin r e a d i l y i n a c t i v a t e d both forms o f the i n h i b i t o r . Ami] and A m l a l s o reacted i n a d i f f e r e n t manner when subjected t o chemical m o d i f i c a t i o n s such as e s t e r i f i c a t i o n w i t h methanol/HCl, and on carboxymethylation a t d i f f e r e n t pH values a f f e c t i n g s e l e c t ­ i v e l y methionine, h i s t i d i n e and aromatic amino a c i d s , as w e l l as on cleavage w i t h cyanogen bromide (42). On the whole, Aml2 was more s e n s i t i v e t o these treatments than was Ami]. Cyanogen bromide treatment, performed i n a c o n t r o l l e d manner, removed a l l the a c t i v i t y o f Am12 towards s a l i v a r y α-amylase, but had l i t t l e e f f e c t on the a c t i v i t y o f A m l , o r the more s p e c i f i c i n h i b i t o r Ami], towards Tenebrio molitor α-amylase. On the basis o f t h i s f i n d i n g i t was suggested that Aml2 i s comprized o f Ami] plus an a d d i t i o n a l peptide fragment which c o n t a i n s the b i n d i n g s i t e f o r s a l i v a r y α-amylase. I n h i b i t i o n o f α-amylase by the wheat i n h i b i t o r s was markedly greater a f t e r pre-incubât ion o f enzyme w i t h i n h i b i t o r , followed by a d d i t i o n o f s u b s t r a t e , than a f t e r p r e - i n c u b a t i o n o f s u b s t r a t e w i t h i n h i b i t o r , followed by a d d i t i o n o f enzyme (41,42). A s i m i l a r s i t u a t i o n i s observed i n the case o f α-amylase i n h i b i t o r from kidney beans (vide infra). Although a d d i t i o n o f s u b s t r a t e cannot reverse i n h i b i t i o n , when α-amylase i s added t o a mixture of kidney bean i n h i b i t o r and excess s u b s t r a t e , the enzyme a c t s a t the same rate as i t does i n the absence o f i n h i b i t o r (23). Shainkin and B i r k suggested that pre-incubât ion o f s u b s t r a t e and i n h i b i t o r gives a complex i n which the amylase b i n d i n g s i t e i n the i n h i b i t o r i s masked. However, i n the absence o f any good evidence t o show that i n h i b i t o r and s u b s t r a t e do a c t u a l l y 2

2

2

2

2

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

256

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

a s s o c i a t e , the low extent o f i n h i b i t i o n observed when α-amylase is added l a s t can be explained simply on the basis o f the a f f i n i t y o f the enzyme f o r i t s s u b s t r a t e , and the high concentration o f the s u b s t r a t e compared with i n h i b i t o r i n the mixture, so that sub­ s t r a t e p r o t e c t s the enzyme from i n a c t i v a t i o n by the i n h i b i t o r . The data on s o l u b i l i t y , charge p r o p e r t i e s , amino a c i d composition and molecular weights o f the i n h i b i t o r s (42) suggested a resemblance o f these p r o t e i n s to the g l i a d i n s (43) > wheat p r o t e i n s without any p r e v i o u s l y known b i o l o g i c a l a c t i v i t y . The p o s s i b l e r e l a t i o n between wheat α-amylase i n h i b i t o r and the wheat g l i a d i n s was a l s o recognized by Strumeyer, who prepared the i n h i b i t o r by water e x t r a c t i o n o f wheat f l o u r , followed by heat treatment a t 70°C., then ammonium s u l f a t e and ethanol p r e c i p i t a t i o n C45). M o l e c u l a r - s i e v e chromatography gave a value o f 55,000 f o r the molecular weight o f the i n h i b i t o r (46), t h i s being rather d i f f e r e n t from that reported by Shainkin and B i r k (42), and others (47-49). Recent s t u d i e s i Strumeyer's value. Dis showed two major p r o t e i n bands, w i i c h corresponded i n e l e c t r o p h o r e t i c m o b i l i t y t o the α-glîadîns (46). The amino a c i d composition o f the i n h i b i t o r preparation showed that i t contained high amounts o f glutamine (30%) and p r o l i n e (50%) but a low l e v e l of l y s i n e ( l e s s than ] % ) . None o f these f i g u r e s i s s i m i l a r t o those i n the amino a c i d a n a l y s i s o f the two forms o f i n h i b i t o r i s o l a t e d by Shainkin and B i r k . Strumeyer and F i s h e r demonstrated complex formation between i n h i b i t o r and α-amylase by gel f i l t r a t i o n (46). S i l a n o and co-workers (47), l i k e Shainkin and B i r k (42), have separated two types o f α-amylase i n h i b i t o r from wheat, by using chromatography on columns o f Sephadex G-100. They have a l s o shown (48) that albumins from the kernels o f hexaploid wheat can be separated e l e c t r o p h o r e t i c a l l y i n t o s i x f r a c t i o n s . Five o f these albumins had very s i m i l a r p r o p e r t i e s , and were considered t o belong to a f a m i l y o f c l o s e l y r e l a t e d p r o t e i n s ; a l l i n h i b i t e d Tenebrio molitor α-amylase, but no other α-amylase. The s i x t h albumin, designated 0.19, was d i s t i n c t from the o t h e r s , and i n h i b i t e d s a l i v a r y and p a n c r e a t i c α-amylases i n a d d i t i o n t o Tenebvio molitor α-amylase. The 0.19 albumin contained two noni d e n t i c a l subunits (48). One subunît, responsible f o r the i n h i b i t o r y a c t i v i t y towards Tenebrio molitor α-amylase, was considered to be a member o f the f i v e albumin f a m i l y s p e c i f i c f o r t h i s amylase. The nature o f the second subunît, responsible f o r the i n h i b i t o r y a c t i v i t y o f 0.19 albumin towards s a l i v a r y α-amylase was not c l e a r . It was a l s o proposed (48) that 0.19 albumin c o r r e s ­ ponded t o the Am12 i n h i b i t o r i s o l a t e d by Shainkin and B i r k (42). While these l a t t e r workers had suggested (42) that the polypeptide chain o f Aml2 c o n s i s t e d o f Ami], plus an a d d i t i o n a l peptide segment, S i l a n o and co-workers proposed (48) that Ami2, l i k e 0.19 albumin, contained two s u b u n i t s , one o f which was Aml|. The s i t u a t i o n regarding the number o f α-amylase i n h i b i t o r s i n 9

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

16.

MARSHALL

α-Amylase Inhibitors from Pfonts

257

wheat, and t h e i r r e l a t i o n s h i p , has become more confused f o l l o w i n g a recent report from Saunders and Lang (49). These workers separated two albumin p r o t e i n s w i t h I n h i b i t o r y a c t i v i t y , termed i n h i b i t o r s I and 11, by chromatography o f a heat-treated wheat e x t r a c t on DEAE-Sephadex. I n h i b i t o r s I and II had almost i d e n t i c ­ al p h y s i c a l p r o p e r t i e s ( pi 6.7, molecular weight 20,000 f o r I; pi 6.5, molecular weight 21,000 f o r 11) and i t i s not c l e a r whether they a r e d i s t i n c t p r o t e i n s , o r whether the f r a c t i o n a t i o n was a r t e f a c t u a l . I n h i b i t o r s I and II were d i s t i n c t from S i l a n o ' s 0J9 albumin (pi 7.3, molecular weight 23,800). Each i n h i b i t o r , I, U and 0.19 was reported t o a c t i n an uncompetitive f a s h i o n against p a n c r e a t i c α-amylase, w i t h Kj 5 x 10"8m. Further work w i l l be necessary t o c l a r i f y the exact number o f α-amylase i n h i b i t o r s present i n wheat, and t o d e f i n e t h e i r r e l a t i o n s h i p a t the molecular l e v e l . (x-Amylase Inhibitor from f i r s t detected i n the 1940 (51), fn beans d i d not r e c e i v e f u r t h e r a t t e n t i o n u n t i l r e c e n t l y . The observation that r a t s fed on d i e t s o f raw kidney beans excreted undigested s t a r c h (61) prompted Jaffé and h i s co-workers t o examine e x t r a c t s o f these beans f o r the f a c t o r s r e s p o n s i b l e . They p u r i f i e d an α-amylase i n h i b i t o r 3-fold from a crude e x t r a c t , by heat treatment a t 60°C f o r 30 min (52). The p a r t l y p u r i f i e d i n h i b i t o r , which was n o n - d l a l y z a b l e and was destroyed by heating at 100°C f o r 15 min, had the p r o p e r t i e s o f a p r o t e i n . I t s molecular weight was judged, by molecular-sieve chromatography on Sephadex, t o be greater than 50,000. The nature o f the i n h i b i t i o n was reported t o be non-competItive. I n h i b i t o r s w i t h apparently s i m i l a r p r o p e r t i e s are present i n many v a r i e t i e s o f beans and other legumes (53)· Kidney bean α-amylase i n h i b i t o r has r e c e n t l y been prepared in a high s t a t e o f p u r i t y i n t h i s Laboratory (23), by using a s e r i e s o f conventional f r a c t i o n a t i o n techniques (summarized i n Table 4). The c l o s e c o r r e l a t i o n between p r o t e i n and i n h i b i t o r a c t i v i t y In the f r a c t i o n from the CM-cellulose column ( F i g . 1), the f i n a l step i n the p u r i f i c a t i o n scheme, was taken as i n d i c a t i v e of p u r i f i c a t i o n t o homogeneity. This was confirmed by p o l y acrylamide gel e l e c t r o p h o r e s i s and a n a l y t i c a l u l t r a c e n t r i f u g a t l o n . P u r i f i c a t i o n can a l s o be achieved (23) by t a k i n g advantage o f the a f f i n i t y o f the enzyme f o r Sepharose-bound α-amylase, the i n h i b i t o r being recovered by washing w i t h pH 3.0 b u f f e r - probably as a r e s u l t o f d e s t r u c t i o n o f the a c i d - l a b i l e α-amylase. This method i s , however, not used r o u t i n e l y because p u r i f i c a t i o n can be achieved so r e a d i l y by the conventional f r a c t i o n a t i o n tech­ niques. Examination o f the p r o p e r t i e s o f the i n h i b i t o r (23) showed that the i n h i b i t i o n was pH dependent - a phenomenon which has not been observed, nor apparently even i n v e s t i g a t e d , i n the case o f s t u d i e s on other α-amylase i n h i b i t o r s . The maximum r a t e o f

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

770

142,500

150,100

116,700

Step 4 : DEAE-cellulose chromatography

Step 5 : Sephadex G-100 chromatography

Step 6 : CM-cellulose chromatography

A c t i v i t i e s determined as i n Scheme I.

1,150

203,200

Step 3 : d i a l y s i s

a

34,000

222,600

Step 2 : heat treatment (70°C., 15 min)

3,100

10,100

64,600

Total p r o t e i n (mg)

312,800

Total u n i t s

152

131

46

20.1

6.5

4.8

Specific activity (units/mg)

OF PHASEOLUS VULGARIS OL-AMYLASE INHIBITOR

Step 1 : crude e x t r a c t (500g beans)

Steps and procedures

PURIFICATION

TABLE 4

37

48

46

65

71

100

Recovery (%)

(23)

31.7

27.3

9.6

4.2

1.4

Purification

16.

MARSHALL

259

α-Amylase Inhibitors from Fiants

i n h i b i t i o n o f α-amylase i s observed a t pH 5-5, the rate dropping o f f sharply a t higher pH values. I n h i b i t o r a c t i v i t y i s a l s o s t r o n g l y dependent on the temperature o f p r e - i n c u b a t i o n ; inhibit­ ion o f α-amylase takes place about 20-times f a s t e r a t 37°C than a t 25°C., and i s n e g l i g i b l e a t 0°C. However, once i n h i b i t i o n has occurred, i t cannot be reversed by c o n d i t i o n s which a r e themselves unfavorable f o r i n h i b i t o r a c t i v i t y - low temperature o r a l k a l i n e pH. Neither can substrate reverse the i n h i b i t i o n , showing that aamylase has g r e a t e r a f f i n i t y f o r the i n h i b i t o r than i t has f o r i t s s u b s t r a t e . I n t e r a c t i o n o f α-amylase and i n h i b i t o r i s reversed a t low pH, presumably as a r e s u l t o f α-amylase d e s t r u c t i o n as i n d i c a t e d above. I n v e s t i g a t i o n o f the s t o i c h i o m e t r y o f i n t e r ­ a c t i o n has shown that hog-pancreatic α-amylase and i n h i b i t o r combine in a 1:1 f a s h i o n . Complex formation has been demonstrated d i r e c t l y by molecular-sieve chromatography on Sephadex G-100 a f t e r pre-incubation o f s t o i c h i o m e t r i c amounts o f α-amylase and i n h i b i t o r ( F i g . 2). Phaseolus molecular weight o f approximatel lower than the value reported by Jaffé and co-workers (52). At the present time nothing i s known about the r o l e o f f u n c t i o n a l group(s) c o n t r i b u t i n g t o the a c t i v i t y o f the i n h i b i t o r . Physiological Inhibitors

Effects

and Nutritional

Significance

of v.-Amylase

The presence o f high l e v e l s o f α-amylase i n h i b i t o r s i n common plant f o o d s t u f f s , p a r t i c u l a r l y legumes and c e r e a l s , r a i s e s the question whether these i n h i b i t o r s can have any e f f e c t on aamylases in vivo and, i f so, whether t h i s r e s u l t s i n impaired starch digestion. M i l l e r and Kneen (27) suggested that the polyphenolic i n h i b i t o r i n sorghum i s u n l i k e l y t o have an e f f e c t in vivo, because i t i s r a t h e r r e a d i l y rendered i n e f f e c t i v e as a r e s u l t o f i t s a b i l i t y t o combine w i t h many p r o t e i n s besides α-amylase. However, brown sorghums, which contain high l e v e l s o f t a n n i n s , have been shown t o r e t a r d growth o f c h i c k s , and t o i n t e r f e r e w i t h dry matter d i g e s t i b i l i t y i n r a t s , more than does white (low tannin) sorghum (62-66). The e f f e c t on growth, and f o o d s t u f f d i g e s t i b i l i t y , may be caused by the t a n n i n s , and might w e l l e x p l a i n the b i r d - r e s i s t a n t p r o p e r t i e s o f brown sorghums (62). I t might a l s o be noted that Mandels and Reese (15) b e l i e v e that t a n n i n - c o n t a i n i n g p l a n t s a r e l i k e l y t o be undesirable foods f o r ruminants because o f the i n h i b i t i o n o f rumen c e l l u l a s e by tannins (17). Grazing animals, and even s n a i l s , avoid p l a n t s high i n t a n n i n s , perhaps because o f t h e i r i n h i b i t o r y e f f e c t on h y d r o l y t i c enzymes (67). The s u s c e p t i b i l i t y t o p r o t e o l y s i s o f the proteinaceous aamylase i n h i b i t o r from wheat has been mentioned above. On the b a s i s o f t h i s f i n d i n g , Kneen and Sandstedt (37) concluded that i t would be i n a c t i v a t e d by pepsin i n the stomach, and that the only

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

260

PHYSIOLOGICAL

< 7L

EFFECTS

O F FOOD

CARBOHYDRATES

1.0

0.5 ι

0.8 -

0.4

0.6

0.3

0.4

Ο 0.2 >-

Ο &f 0.2 -Jo

_J_

10 FRACTION NUMBER Figure 1. Purification of Phaseolus vulgaris α-amylase inhibitor by chromatogra­ phy on CM-cellulose (23)

15

10 v

o

20

25

30

35

FRACTION NUMBER

Figure 2. Molecular sieve chromatography on Sephadex G-100 of hog pancreatic α-amylase (Δ), Phaseolus vulgaris a-amylase inhibitor (•), and a-amylase / inhibitor complex ( O ) ( ) 23

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

16.

MARSHALL

α- Amyhse Inhibitors from Phnts

261

e f f e c t t h e i n h i b i t o r w o u l d have in vivo w o u l d be on s a l i v a r y a a m y l a s e . They t h e r e f o r e s u g g e s t e d t h a t t h e wheat I n h i b i t o r w o u l d have l i t t l e o r no s i g n i f i c a n c e i n normal human p h y s i o l o g y , and t h a t o n l y i n cases o f impaired p r o t e o l y s i s would i n t e r f e r e n c e w i t h s t a r c h h y d r o l y s i s be a n t i c i p a t e d . S h a i n k i n and B i r k (42) came t o t h e same c o n c l u s i o n . E v i d e n c e I s , however, now a c c u m u l a t i n g t o s u g g e s t t h a t t h e p r o t e i n a c e o u s i n h i b i t o r s o f α-amylase may p l a y an i m p o r t a n t r o l e i n m o d u l a t i n g s t a r c h d i g e s t i o n in vivo. I t h a s been s u g g e s t e d t h a t wheat a m y l a s e I n h i b i t o r s may be, a t l e a s t i n p a r t , r e s p o n s ­ i b l e f o r the d i f f e r e n c e s In n u t r i t i o n a l v a l u e o f d i f f e r e n t v a r i e t i e s o f w h e a t , a s measured by t h e r e t a r d a t i o n o f d e v e l o p m e n t o f Tribolium oastaneum ( H e r b s t ) l a r v a e , compared w i t h t h e o p t i m a l development a c h i e v e d on a s t a n d a r d minimal a r t i f i c i a l d i e t ( 6 8 ) . Recent work has shown In a more c o n v i n c i n g f a s h i o n t h a t t h e a a m y l a s e I n h i b i t o r I n wheat w i l l I n t e r f e r e w i t h s t a r c h m e t a b o l i s m in vivo. In t h i s L a b o r a t o r and a n i m a l f o o d s t u f f s c o n t a i a c t i v i t y (69). R a t s f e d on a d i e t o f o n e l a b o r a t o r y a n i m a l f o o d ­ s t u f f ( R a l s t o n P u r i n a r a t chow) I n w h i c h t h e i n h i b i t o r had been I n a c t i v a t e d by a u t o c l a v i n g , grew a t a r a t e 15-20% f a s t e r t h a n d i d r a t s f e d on t h e u n t r e a t e d f e e d . S i m i l a r l y , when g r o u p s o f r a t s w e r e f e d on a c a s e i n / s t a r c h d i e t i n t h e p r e s e n c e and a b s e n c e o f p u r i f i e d wheat I n h i b i t o r , r e d u c t i o n o f g r o w t h r a t e and I n c r e a s e d f e c a l s t a r c h l e v e l s w e r e c a u s e d by t h e p r e s e n c e o f i n h i b i t o r , t h e m a g n i t u d e o f t h e s e changes b e i n g d e p e n d e n t o n t h e i n h i b i t o r d o s a g e ( 7 0 ) . Added I n h i b i t o r d i d n o t have a n y e f f e c t when s u c r o s e r e p l a c e d s t a r c h a s d i e t a r y c a r b o h y d r a t e ; neither did Inactivated Inhibitor a f f e c t starch u t i l i z a t i o n . Strumeyer (45) has r e c e n t l y s u g g e s t e d t h a t a m y l a s e I n h i b i t o r s may be r e s p o n s i b l e f o r t h e s e n s i t i v i t y t o wheat a n d r y e f l o u r In c e l i a c disease - a gluten-induced enteropathy. Individuals with this d i s o r d e r e x h i b i t i m p a i r e d a b i l i t y t o m e t a b o l i z e s t a r c h and a b s o r b fats. These n u t r i e n t s a r e e x c r e t e d and t h e p a t i e n t s s u f f e r f r o m v i t a m i n d e f i c i e n c y and m a l n u t r i t i o n . A c c o r d i n g t o S t r u m e y e r (45) t h e c e r e a l a m y l a s e i n h i b i t o r s m i g h t be r e s p o n s i b l e f o r t h e s e n s i t i v i t y t o wheat and r y e f l o u r s by d e p r e s s i o n o f an a l r e a d y d e f i c i e n t p a n c r e a t i c α-amylase s u p p l y . I t i s p r o b a b l e t h a t bean α-amylase i n h i b i t o r i s a l s o a c t i v e in vivo. V e n e z u e l a n w o r k e r s have d e t e c t e d l a r g e amounts o f u n ­ d i g e s t e d s t a r c h , and a m y l a s e i n h i b i t o r y a c t i v i t y , i n t h e f e c e s o f r a t s f e d on d i e t s o f raw k i d n e y beans ( 6 1 , 7 1 ) . Evans and c o ­ w o r k e r s have s t u d i e d t h e e f f e c t o f d i f f e r e n t p r o t e i n f r a c t i o n s f r o m Phaseolus vulgaris on t h e g r o w t h o f r a t s . They c o n c l u d e d t h a t t h e r e I s p r e s e n t i n beans a g r o w t h i n h i b i t o r y f a c t o r d i s t i n c t from the o t h e r t o x i c f a c t o r s , phytohemagglutInins and p r o t e a s e I n h i b i t o r s ( 7 2 , 7 3 ) . Many o f t h e p r o p e r t i e s o f t h i s g r o w t h i n h i b i t o r y p r o t e i n (73) r e s e m b l e t h o s e o f t h e Phaseolus vulgaris α-amylase i n h i b i t o r p u r i f i e d and c h a r a c t e r i z e d I n t h i s Laboratory (23).

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

Δ BG (mg/100 ml) +40

+20

h or—

-20 Δ IRI (juU/ml) +40 h

•*

/A

+20

L

0

15 30 45 60

90

180

120 minutes

Diabetologia

Figure 3. Elevation of blood glucose (ABG) and serum insulin (MRI) levels in healthy human subjects following intake of starch (100 g) in the absence of wheat a-amylase inhibitor (*) and in the presence of inhibitor (φ, 350 mg; A, 700 mg). Results shown are the mean for seven subjects (75).

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

16.

MARSHALL

a-Amyhse Inhibitors from Phnts

263

In view o f t h e i r in vivo a c t i o n , the presence o f α-amylase i n h i b i t o r s in f o o d s t u f f s prepared from p l a n t s such as wheat, rye and bean&, i s c l e a r l y undesirable when maximum n u t r i t i o n a l value of the f o o d s t u f f i s required. Wheat amylase i n h i b i t o r has been reported t o p e r s i s t through bread baking, being found i n large amounts in the centers o f loaves (37, 7*0. In t h i s Laboratory i t has been shown that a number o f common l a b o r a t o r y animal feeds, of which wheat i s a major c o n s t i t u e n t , contain i n h i b i t o r a c t i v i t y (69). The same i s true o f a number o f wheat-based human breakfast c e r e a l s (69). It would seem l i k e l y that the true c a l o r i c value o f these f o o d s t u f f s w i l l be lower than expected on the basis o f t h e i r s t a r c h contents, because o f the i n t e r f e r e n c e with s t a r c h d i g e s t i o n caused by endogeneous i n h i b i t o r . The r e l a t i v e l y s t a b l e nature o f α-amylase i n h i b i t o r s means that s p e c i a l a t t e n t i o n w i l l be required to ensure t h e i r d e s t r u c t i o n o r removal during food processing. F i n a l l y , i t i s appropriate t o consider α-amylase i n h i b i t o r s as p o s s i b l e t h e r a p e u t i c to block s t a r c h degradatio p o s s i b l e use as novel d i e t e t i c agents, o f use in the therapy o f o b e s i t y in subjects who f i n d d i f f i c u l t y in completely e l i m i n a t i n g s t a r c h from t h e i r d i e t s . A second a p p l i c a t i o n would be t o a l l o w d i a b e t i c i n d i v i d u a l s t o consume a moderate amount o f s t a r c h , i f ingested in the presence o f i n h i b i t o r . The e f f e c t o f the i n h i b i t ­ or on s t a r c h d i g e s t i o n w i l l help prevent the elevated blood sugar l e v e l which normally r e s u l t s from s t a r c h i n t a k e , and which i s undesirable in the d i a b e t i c . Puis and h i s co-workers {kh) have patented a wheat α-amylase I n h i b i t o r p r e p a r a t i o n , and have proposed i t s use as an antihyperglycemlc agent. Their s t u d i e s w i t h t h i s I n h i b i t o r provide the most convincing evidence o f the in vivo e f f e c t o f an α-amylase i n h i b i t o r , and support i t s p o s s i b l e use as a t h e r a p e u t i c agent. On a d m i n i s t r a t i o n o f the wheat i n h i b i t o r t o r a t s , dogs and healthy human s u b j e c t s , the hyperglycemia and hyperînsulinemla r e s u l t i n g from s t a r c h loading could be reduced dose dependently by the i n h i b i t o r (75). The e f f e c t o f i n h i b i t o r on the l e v e l s o f blood glucose and i n s u l i n in human subjects f o l l o w i n g i n g e s t i o n of raw s t a r c h Is shown in F i g . 3. When cooked, as opposed t o raw, s t a r c h was used, the smoothing e f f e c t on hyperglycemia was somewhat reduced. The suggestion was made that t h i s might be because the a f f i n i t y o f amylase f o r g e l a t i n i z e d s t a r c h i s greater than the a f f i n i t y f o r the i n h i b i t o r , and i t was suggested that an i n h i b i t o r w i t h g r e a t e r a f f i n i t y f o r α-amylase might be advantageous f o r t h e r a p e u t i c use. Acknowledgements Helpful d i s c u s s i o n with Dr. W.J. Whelan i s g r a t e f u l l y acknow­ ledged. J.J. Marshall i s an I n v e s t i g a t o r o f Howard Hughes Medical Insti tute.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

264

PHYSIOLOGICAL EFFECTS OF FOOD CARBOHYDRATES

Literature Cited

1. FRITZ, H. and TSCHESCHE, H. (eds.), Proceedings of the International Conference on Proteinase Inhibitors, Munich, Nov. 1970, Walter de Gruyter, Berlin, 1971. 2. WERLE, E. and ΖICKGRAF-RÜDEL, G., Z. Klin. Chem. u. Klin. Biochem. (1972) 4, 139-150. 3. RYΑΝ, C.A., Ann. Rev. Plant Physiol. (1973) 24, 173-196. 4. RÜHLMANN, Α., KUKLA, D., SCHWAGER, P., BARTELS, K. and HUBER, R., J. Mol. Biol. (1973) 77, 417-436. 5. SCHWIMMER, S., MAKAVER, R.U. and ROREM, E.S., Plant Physiol. (1961) 36, 313-316 6. PRESSEY, R., Arch. 7. PRESSEY, R., Plant Physiol. (1967) , 8. PRESSEY, R. and SHAW, R., Plant Physiol. (1966) 4l, 1657-1661. 9. JAYNES, T.A. and NELSON, O.E., Plant Physiol. (1971) 47, 629-634. 10. WINKENBACH, F. and MATILE, P., Z. Pflanzenphysiologie (1970) 63, 292-295. 11. ALBERSHEIM, P. and ANDERSON, A.J., Proc. Nat. Acad. Sci. U.S. (1971) 68, 1815-1819. 12. ANDERSON, A.J. and ALBERSHEIM, P., Physiol Plant Pathol. (1972) 2, 339-346. 13. FISHER, M.L., ANDERSON, A.J. and ALBERSHEIM, P., Plant Physiol. (1973) 51, 489-491. 14. MANDELS, M., HOWLETT, W. and REESE, E.T., Can.J.Microbiol. (1961) 7, 957-959. 15. MANDELS, M. and REESE, E.T., in REESE, E.T. (ed.), Advances in Enzymic Hydrolysis of Cellulose and Related Materials, pp.115-157,Pergamon Press, Oxford, 1963. 16. MANDELS, M. and REESE, E.T., Ann. Rev. Phytopathol. (1965) 3, 85-102. 17. SMART, W.W.G., BELL, T.A., STANLEY, N.W. and COPE, W.A., J. Dairy Sci. (1961) 44, 1945-1946. 18. KING, K.W., Va. Agric. Exp. Sta. Tech. Bull. 127, December 1956. 19. BERNHEIMER, A.W. and STEELE,J.M.,Proc. Soc. Exp. Biol. Med. (1955) 89, 123-126. 20. BERNFELD, P., in COLOWICK, S.P. and KAPLAN, N.O. (eds.), Methods in Enzymology, Vol. 1, pp. 149-158, Academic Press, New York, 1955. 21. NELSON, Ν., J. Biol. Chem. (1944) 153, 375-380. 22. HOPKINS, R.H. and BIRD, R., Biochem.J.(1954) 56, 86-99. 23. LAUDA, C.M. and MARSHALL, J.J., in preparation (1975). 24. F0SSUM, K. and WHITAKER, J.R., J. Nutr. (1974) 104, 930-936. 25. HEMBERG, T. and LARSS0N, I., Physiol. Plant(l961)14, 861-867. In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

16.

MARSHALL

α-Amylase

Inhibitors

from

Plants

265

26. SUKHORUKOV, I., KLING, E. and OVCHAROV, Κ., Compt. Rend. Acad. Sci. URSS (1939) 18, 597-602, via Chem. Abs. (1938) 32, 6686 . 27. MILLER, B.S. and KNEEN, E., Arch. Biochem. (1947) 15., 251-264. 28. STRUMEYER, D.H. and MALIN, M.J., Biochim. Biophys. Acta (1969) 184, 643-645. 29. STANKOVIC, S.C. and MARKOVIC, N.D., Glasnik Hem. Drustva, Beograd (1960-1961) 25-26, 519-525, via Chem. Abs. (1963) 59, 3084d. 30. CHRZASZCZ, T. and JANICKI, J., Biochem.Z.(1933) 260, 354368. 31. CHRZASZCZ, T. and JANICKI, J., Biochem. Z. (1933) 264_, 192208, 32. ELLIOTT, B.B. and LEOPOLD, A.C., Physiol. Plant (1953) 6, 6577. 33. MATTOO, A.K. and MODI 34. RAO, M.N., SHURPALEKAR Biochem. (1967) 4., 185. 35. RAO, M.N., SHURPALEKAR, K.S. and SUNDARAVALLI, O.E., Ind. J. Biochem. (1970) 7, 241-243. 36. KNEEN, E. and SANDSTEDT, R.M., J. Amer. Chem. Soc. (1943) 65, 1247. 37. KNEEN, E. and SANDSTEDT, R.M., Arch. Biochem. (1946) 9, 235249. 38. MILITZER, W., IKEDA, C. and KNEEN, E., Arch. Biochem. (1947) 15, 309-320. 39. MILITZER, W., IKEDA, C. and KNEEN, E., Arch. Biochem. (1947) 15, 321-329. 40. SHAINKIN, R. and BIRK, Y., Israel J. Chem. (1966) 3, 96. 41. SHAINKIN, R. and BIRK, Y., Israel J. Chem. (1967) 5, 129 p. 42. SHAINKIN, R. and BIRK, Y., Biochim. Biophys. Acta(1970)221, 502-513. 43. POMERANZ, Y. (ed.) Wheat Chemistry and Technology, 2nd edn. American Association of Cereal Chemists, St.Paul, Minn.,1971. 44. SCHMIDT, D. and PULS, W., German Patent 2,003,934, Aug. 5, 1971, via Chem. Abs. (1971) 75, 91296 p. 45. STRUMEYER, D.H., Nutr. Rep. Intern. (1972) 5, 45-52. 46. STRUMEYER, D.H. and FISHER, B.R., Fed. Proc, (1973) 31, 624. 47. SILANO, V., MINUTTI, M., PETRUCCI, T., TOMASI, M. and POCCHIARI, F., Abs. 9th Intern. Congr.Biochem.,Stockholm 1973, Abs. 2pl2. 48. SILANO, V., POCCHIARI, F. and KASARDA, D.D. Biochim. Biophys. Acta (1973) 317, 139-148. 49. SAUNDERS, R.M. and LANG, J.Α., Phytochem. (1973) 12, 12371241. 50. LAUDA, C.M. and MARSHALL, J.J., unpublished work (1974). 51. BOWMAN, D.E., Science (1945) 102, 358-359. 52. HERNANDEZ, A. and JAFFÉ, W.G., Acta Cient. Venezolana (1968) 19, 183-185. 4

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

266

PHYSIOLOGICAL EFFECTS OF FOOD CARBOHYDRATES

53. J A F F É , W.G., MORENO, R. and WALLIS, V., Nutr. Rep. Intern. (1973) 7, 169-174. 54. BOWMAN, D.E., Science (1943) 98, 308-309. 55. PUSZTAI, Α., Europ.J.Biochem. (1968) 5, 252-259. 56. KNEEN, E. and BECKFORD, L.D., Arch. Biochem. (1946) 10, 41-54. 57. APPLEBAUM, S.W., JANKOVIC, M. and BIRK, Y., J. Insect Physiol. (1961) 7, 100-108. 58. FISCHER, E.H. and STEIN, E.A., In BOYER, P.D., LARDY, H. and MYRBÄCK, K. (eds.), The Enzymes, 2nd edn., Vol. 4, pp.313" 343, Academic Press, New York, 1960. 59. BEAUPOIL-ABADIE, B., RAFFALI, M., COZZONE, P. and MARCH ISMOUREN, G., Biochim. Biophys. Acta (1973) 297, 436-440. 60. MARSHALL, J.J., In preparation (1975). 61. JAFFÉ, W.G. and LETTE, C.L.V., J. Nutr. (1968) 94., 203-210. 62. MAXSON, E.D. and ROONEY, L.W., Cer. Sci. Today (1972) 7, 260. 63. FULLER, H.L., POTTER Univ. Georgia, Coll. Agr. Exp. Sta., Athens, Ga., Nov. 1966. 64. MAXSON, E.D., R00NEY, L.W., LEWIS, R.W., CLARK, L.E. and JOHNSON,J.W.,Nutr. Rep. Intern. (1973) 8, 145-152. 65. CONNOR,J.K.,HARWOOD, I.S., BURTON, H.W. and FUELLING, D.E., Aust.J.Exp. Agr. Anim. Rusb. (1969) 9, 497-501. 66. CHANG, S.I. and FULLER, H.L., Poultry Sci. (1964) 43, 30-36. 67. NIERENSTEIN, Μ., The Natural Organic Tannins: History, Chemistry, Distribution, p. 287, Churchill, London, 1934. 68. APPLEBAUM, S.W. and KONIJN, A.M., J. Stored Prod. Res. (1967) 2, 323-329. 69. LAUDA, C.M., MARSHALL, J.J. and WHELAN, W.J., unpublished work (1974). 70. LANG,J.A.,CHANG-HUM, L.E., REYES, P.S. and BRIGGS, G.M., Fed. Proc. (1974) 33, 718. 71. MORENO, G.R., Thesis, Central University of Caracas (1971). 72. KAKADE, M.L. and EVANS, R.J., J. Agric. Ed. Chem. (1965) 13, 450-452. 73. EVANS, R.J., PUSZTAI, Α., WATT, W.B. and BAUER, D.H., Biochim. Biophys. Acta (1973) 303, 175-184. 74. BESSH0, H. and KUROSAWA, S., Eiyo To Shokuryo (1967) 20, 317319, via Chem. Abs. (1968) 68, 113474e. 75. PULS, W. and KEUP, U., Diabetologia (1973) 9, 97-101.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

17 The Physiological Effects of Alginates and Xanthan Gum W. H. McNEELY and PETER KOVACS Kelco Company, San Diego, Calif.

Alginates

Algin, found in all species of brown algae where it functions as an important constituent of cell walls, was discovered by Stanford in 1881. (1) The largest source of algin for actual commercial extraction is Macrocystis pyrifera, although other species are also used. (2) After 1935, alginates became widely used in the food industry as stabilizers, emulsifiers, and viscosity-adjusting agents. Alginates are commercially supplied to the food industry as the sodium, potassium, ammonium, and calcium salts of alginic acid, along with propylene glycol alginate. As indicated in Figure 1, alginic acid is a linear glycuronan consisting of ß-D-mannuronic acid and α-L-guluronic acid units linked throughC and C . For Macrocystis pyrifera, the alginic acid is composed of approximately 60 percent mannuronic acid and 40 percent guluronic acid. (2) The fine structure of the alginic acid molecule has been shown by graded acid hydrolysis and p.m.r. spectroscopy to consist of blocks of polymannuronic acid units and blocks of polyguluronic acid units linked by segments in which the two uronic acid residues alternate. (3,4) 1

4

The most recent acute o r a l t o x i c i t y study conducted by Woodard and co-workers on r a t s i n d i c a t e d t h a t the maximum amount o f sodium a l g i n a t e t h a t could be administered by o r a l i n t u b a t i o n was 5 g/kg f o r one day. T h i s feeding l e v e l caused no m o r t a l i t i e s . Gross necropsy f i n d i n g s , coupled with general observations, i n d i c a t e d t h a t sodium a l g i n a t e i s non-toxic with respect to acute t o x i c i t y by the o r a l route. (5) A number o f acute t o x i c i t y studies have been conducted on a v a r i e t y o f animals by intravenous o r i n t r a p e r i t o n e a l injections of alginate solutions. Arora and co-workers reported that i n t r a p e r i t o n e a l i n j e c ­ t i o n s o f sodium a l g i n a t e t o r a t s a t up t o 1000 mg/kg caused no m o r t a l i t y , while some m o r t a l i t y occurred among the

269 In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

270

PHYSIOLOGICAL EFFFCTS OF FOOD CARBOHYDRATES

I i

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

17.

MCNEELY

AND

KOVACS

Alginates and Xanthan

271

Gum

mice upon 500 mg/kg dosage a d m i n i s t r a t i o n . (6) Intravenous i n j e c t i o n s o f sodium a l g i n a t e performed by Solandt to mice a t the 200-500 mg/kg l e v e l proved f a t a l i n 1 minute to 12 hours.(7) The LD-50 t o r a t s by t h i s method was determined by Sokow as 1000 mg/kg. (8) The LD-50 of sodium a l g i n a t e by the intravenous route to r a b b i t s was found t o be 100 mg/kg. I t was p o s t u l a t e d that the t o x i c i t y caused by the intravenous a d m i n i s t r a t i o n of sodium a l g i n a t e was due to the formation and p r e c i p i t a t i o n of the i n s o l u b l e calcium a l g i n a t e . (J) Due to i t s known r e a c t i v i t y with calcium ions to form i n s o l u b l e calcium a l g i n a t e , a l g i n has never been recommended f o r use i n intravenous i n j e c t i o n s . The LD-50 o f propylene g l y c o l a l g i n a t e determined by o r a l i n t u b a t i o n was found t o be above the 5 g/kg l e v e l . No mort a l i t y occurred and no signs of t o x i c i t y or changes i n the v i s c e r a of the t r e a t e d r a t s were found (5) The o r a l a d m i n i s t r a t i o n of 10 g/kg propylen found t o be harmless b of some t r a n s i e n t depression. (9) Intravenous a d m i n i s t r a t i o n of a l g i n i c a c i d by Thienes and co-workers (10) and calcium a l g i n a t e by Sokow (8) produced r e s u l t s s i m i l a r t o t h a t o f sodium a l g i n a t e ; namely, low LD-50 l e v e l s and s i g n i f i c a n t m o r t a l i t y . I t was suggested that the m o r t a l i t y was caused by embolism, as calcium a l g i n a t e as w e l l as a l g i n i c a c i d i s i n s o l u b l e i n water. A number o f short-term s t u d i e s have been conducted on sodium a l g i n a t e . A 10-day study was conducted by V i o l a and co-workers on r a t s where 0, 5, and 10 percent of the t e s t animals d i e t c o n s i s t e d of sodium a l g i n a t e . No apparent e f f e c t was found a t the 5 percent l e v e l , but at 10 percent l e v e l s , depressions i n calcium absorption were noted, while the u t i l i z a t i o n of p r o t e i n was not s i g n i f i c a n t l y a f f e c t e d . (11) In a 10-week experiment, the d a i l y d i e t s o f four groups of r a t s , each c o n s i s t i n g of s i x animals, were supplemented with 5, 10, 20, and 30 percent sodium a l g i n a t e . The t e s t s r e s u l t s are shown i n Table I. 1

Table I Short-Term, High-Level Sodium A l g i n a t e Feeding Studies with Rats Sodium A l g i n a t e Level 5% 10% 20% 30%

Mean D a i l y Weight Gain 3.81 3.47 2.91 2.35

g g g g

Food Consumed Gain i n Weight 3.20 3.31 3.48 3.87

g g g g

Water Consumed Gain i n Weight 5.84 6.28 8.83 13.47

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

g g g g

272

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

At the h i g h e s t feeding l e v e l , only two r a t s s u r v i v e d . The m o r t a l i t y was a t t r i b u t e d t o the low n u t r i t i o n a l q u a l i t y o f the b a s a l d i e t and not t o any t o x i c e f f e c t s of the sodium a l g i n a t e . (12) In an unpublished i n v e s t i g a t i o n by Woodard and co-workers, the e f f e c t of feeding 5 and 15 percent sodium a l g i n a t e and propylene g l y c o l a l g i n a t e t o purebred beagles f o r a p e r i o d o f one year was determined, A f t e r one year the animals were s a c r i f i c e d , and gross h i s t o p a t h o l o g i c a l examinations were made on the important organs. The r e s u l t s o f t h i s study i n d i cated t h a t the dogs t o l e r a t e d l e v e l s as high as 15 percent sodium a l g i n a t e and propylene g l y c o l a l g i n a t e . Animals even at the h i g h e s t amount o f a l g i n a t e gained weight, as d i d the c o n t r o l s . V a r i a b l e s t o o l c o n s i s t e n c i e s at the 15 percent feeding l e v e l s i n d i c a t e d the presence o f unabsorbed c o l l o i d . Hemograms and blood chemistry values were g e n e r a l l y w i t h i n normal l i m i t s and showe the a d m i n i s t r a t i o n o f th above, i t was concluded that both sodium a l g i n a t e and propylene g l y c o l a l g i n a t e were devoid of any harmful o r d e l e t e r i o u s e f f e c t s . (13) For a p e r i o d o f one year, propylene g l y c o l a l g i n a t e was fed t o mice by N i l s o n and Wagner a t 0, 5, 10, 15, and 25 percent by weight of the d i e t . No s i g n s o f t o x i c i t y were noted, but a t the two h i g h e s t feeding l e v e l s , smaller weight gains and i n c r e a s e d m o r t a l i t y r a t e s were noted. T h i s was a t t r i b u t e d t o the water a b s o r p t i o n q u a l i t y o f the d i e t l i m i t i n g the e s s e n t i a l n u t r i e n t intake. (12) By the same workers, propylene g l y c o l a l g i n a t e was added t o the d i e t o f guinea p i g s at 0, 5, 10, and 15 percent l e v e l s . A f t e r 26 weeks, no untoward e f f e c t s were noted which could be a t t r i b u t e d t o the propylene g l y c o l a l g i n a t e . (12) In a feeding study by N i l s o n and Wagner, c a t s were f e d 0, 5, 10, and 15 percent propylene g l y c o l a l g i n a t e as p a r t o f t h e i r d i e t f o r up to 111 days. Although the c a t s r e c e i v i n g propylene g l y c o l a l g i n a t e i n t h e i r d i e t had apparent problems i n swallowing and e a t i n g and consequently l o s t weight when compared t o the c o n t r o l animals, no i n d i c a t i o n s o f c h r o n i c t o x i c i t y were noted. Gross and h i s t o p a t h o l o g i c a l examinations of organs o f these animals revealed no l e s i o n s which could be a t t r i b u t e d t o any s p e c i f i c problems. (12) Table I I i n d i c a t e s the r e s u l t s o f a study where, f o r a p e r i o d o f two months, a l g i n i c a c i d was f e d t o r a t s a t 0, 5, 10, and 20 percent o f t h e i r d i e t . As shown i n t h i s t a b l e , at up t o 10 percent c o n c e n t r a t i o n s , no s i g n i f i c a n t e f f e c t on growth o r food consumption was noted. At the 20 percent l e v e l , however, a s i g n i f i c a n t decrease i n weight g a i n was observed. (10)

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

17.

M C NEELY

AND

KOVACS

Alginates and Xanthan

273

Gum

Table II Short-Term A l g i n i c A c i d Feeding Studies with Rats

Dietary Algin % 0 5 10 20

Average Weight Gain Initial Final 66 66 64 63

Average D a i l y Food Consumption g/100 g.b.w. 10.8 12.6 12.1 8.8

156 154 156 116

In a d e t a i l e d two-year, three-generation reproduction study by Morgan, et a l . 5 percent sodium a l g i n a t When the animals were f i v age, y mated, and the o f f s p r i n g , the F-^ generation, were grouped, as were the parent animals, and fed the same d i e t s . When the F i generation r a t s were about four months o l d , they were a l s o mated, and the r e s u l t i n g o f f s p r i n g , the F generation, were d i v i d e d , as the parent generations were, and p l a c e d on the same d i e t . Table III shows the body weight data f o r the three generations of c o n t r o l animals and those r e c e i v i n g 5 percent sodium a l g i n a t e and propylene g l y c o l a l g i n a t e . As i n d i c a t e d i n t h i s t a b l e , there were no s i g n i f i c a n t d i f ferences between the c o n t r o l and t r e a t e d groups or between the e f f e c t s of sodium a l g i n a t e and propylene g l y c o l a l g i n a t e . The c o n c l u s i o n s of t h i s study revealed t h a t the feeding of sodium a l g i n a t e and propylene g l y c o l a l g i n a t e t o r a t s f o r the p e r i o d o f two years at 5 percent l e v e l s of t h e i r d i e t s d i d not a f f e c t the growth r a t e of the parent generation and t h e i r progeny f o r two generations when compared t o the c o n t r o l group. No gross p a t h o l o g i c a l or hematological changes were noted i n the t e s t animals. A s l i g h t change i n the b a c t e r i a l f l o r a of the g a s t r o i n t e s t i n a l t r a c t was observed, but these were not s i g n i f i c a n t enough t o cause changes i n the d i g e s t i v e process or t o r e t a r d the h e a l t h of the animals. (15) Pregnant mice, r a t s , and hamsters i n d i c a t e d no signs of t e r a t o g e n i c e f f e c t s when they were administered by o r a l i n t u b a t i o n propylene g l y c o l a l g i n a t e i n doses of 780, 720, and 700 mg/kg during g e s t a t i o n . (16) While i n v e s t i g a t i n g the c a r c i n o g e n i c i t y of s e v e r a l c o l l o i d s , E p s t e i n reported t h a t no change i n the frequency o f tumors i n i n f a n t mice occurred a f t e r repeated subcutaneous i n j e c t i o n s o f a l g i n i c a c i d . (17) E a r l i e r p u b l i c a t i o n s on the d i g e s t i b i l i t y of a l g i n c a r r i e d out on v a r i o u s animal species by N i l s o n and co-workers (12, 20) tended t o show t h a t a l g i n was p a r t i a l l y d i g e s t i b l e . 2

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

243 234

204

304

243 202

204 236

402

323 292

287 268

403

32 3 270

287 272

761

202 202

212 212

761

202 202

212 212

5% A l g i n a t e

Control

5% A l g i n a t e

Control

5% A l g i n a t e

(S A)

291

298

401

401

761

761

Control

209

298

S A

PGA

S A

PGA

Mean Body Weight Female

PGA

Mean Body Weight Male

S A

Diet Level

Propylene G l y c o l A l g i n a t e (PGA)

Sodium A l g i n a t e

Parent

Generation

Day of Termination

Long-Term Sodium A l g i n a t e and Propylene G l y c o l A l g i n a t e Feeding Studies with Rats

Table I I I

a ο

&

Ο

ο

ο r

17.

M C NEELY

AND

KOVACS

Alginates and Xanthan

Gum

275

A n a l y t i c a l methods used were based on i s o l a t i o n of the a l g i n from the f e c e s . These methods are not very r e l i a b l e . Recent s t u d i e s by the more accurate r a d i o a c t i v e C method by Humphreys and T r i f f i t t (18) f o r sodium a l g i n a t e and by S h a r r a t t and Dearn (19) f o r propylene g l y c o l a l g i n a t e found n e g l i g i b l e or no absorption of the a l g i n . Numerous published r e p o r t s d e a l i n g with the e f f e c t of sodium a l g i n a t e on the g a s t r o i n t e s t i n a l absorption of r a d i o a c t i v e strontium and s e v e r a l other m e t a l l i c ions are l i s t e d i n a review by Tanaka and Skoryna. (21) While s e v e r a l authors reported t h a t a l g i n i n h i b i t s the r a d i o a c t i v e strontium absorption, much l e s s e r e f f e c t s have been produced on the absorption of calcium, magnesium, i r o n , copper, and z i n c . The current p u b l i c i n t e r e s t created by the a l l e g e d d e t o x i f i c a t i o n p r o p e r t i e s of sodium a l g i n a t e with respect to lead does not appear to be j u s t i f i e d In a recent stud b H a r r i s o n , et a l . , huma sodium a l g i n a t e , which t h e i r systems.(22) Based on a human feeding study, Carr, et a l . , reported t h a t sodium a l g i n a t e does not a f f e c t the abs o r p t i o n of sodium, potassium, magnesium, and phosphorous i n the g a s t r o i n t e s t i n a l t r a c t . (23) The e f f e c t of a l g i n i c a c i d on the c a t i o n absorption i n humans was reported e a r l i e r by Feldman and co-workers. Four a d u l t males were maintained on a c o n t r o l d i e t c o n t a i n i n g 500 mg sodium per day f o r seven days. A f t e r that time, 15 g of a l g i n i c a c i d , three times d a i l y , was administered f o r seven days. In three p a t i e n t s t h i s study was repeated with 1500 mg d a i l y intake of sodium. The a l g i n i c a c i d was w e l l t o l e r a t e d by the subjects with the exception of m i l d l a x a t i v e e f f e c t s . (14) In a calciumbalance experiment performed on s i x a d u l t humans by M i l l s and Reed, the r e s u l t s i n d i c a t e d that sodium a l g i n a t e does not i n t e r f e r e with the calcium absorption of the normal v a r i e d d i e t . (24) 1 4

Xanthan

Gum

Xanthan gum, a r e l a t i v e l y r e c e n t l y developed p o l y saccharide produced by the b a c t e r i a l fermentation of glucose with the organism Xanthomonas campestris, was approved by the FDA as a food a d d i t i v e i n 1969. Since t h a t time, xanthan gum has been widely accepted by the food i n d u s t r i e s of many c o u n t r i e s as a general purpose s t a b i l i z e r - t h i c k e n e r . The commercial success of xanthan gum was r e c e n t l y manifested by the p r e s e n t a t i o n of the 1974 IFT Food Technology I n d u s t r i a l Achievement Award to Kelco Company and the Northern Regional Research Laboratory of the USDA. (25) As i n d i c a t e d i n Figure 2, xanthan gum i s a heteropolysaccharide with main b u i l d i n g b l o c k s c o n s i s t i n g of D-glucose, Dj-mannose, and D-glucuronic a c i d r e s i d u e s . (26) The polymer

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

Generally accepted xanthan gum structure Figure 2. In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

17.

MCNEELY

AND

KOVACS

Alginates and Xanthan

Gum

a l s o contains pyruvate attached t o a glucose s i d e chain at an, as yet, undetermined l o c a t i o n . The molecule i s a very long l i n e a r chain with a molecular weight of 5 to 10 m i l l i o n . Xanthan gum i s perhaps the most e x t e n s i v e l y i n v e s t i g a t e d polysaccharide from the standpoint of t o x i c o l o g i c a l and safety properties. The r e s u l t s of the acute t o x i c i t y s t u d i e s are summarized i n Table IV. Table

IV

Acute T o x i c i t y Studies with Xanthan

Animal

Route

Mouse

Ora Intraperitoneal Intravenous

Gum

Maximum mg/kg Body Weight Administered

150 100-250

(27) (28)

Rat

Oral

5000

(29)

Dog

Oral

20000

(30)

In the r a t and dog s t u d i e s , the LD-50 l e v e l s are a c t u a l l y r e p r e s e n t a t i v e of the maximum l e v e l s the animals were able t o consume w i t h i n the t e s t p e r i o d . Since no m o r t a l i t i e s or t o x i c manifestations occurred, the true o r a l LD-50 l e v e l s are above those shown i n t h i s t a b l e . There were two short-term feeding s t u d i e s conducted on both r a t s and dogs by Booth, et a l . No untoward e f f e c t s were noted upon the extensive i n v e s t i g a t i o n of r a t s when they were fed xanthan gum f o r a p e r i o d of 99-110 days at 7.5 percent and 10 percent as p a r t of t h e i r d a i l y d i e t . (29) In a 91-day feeding study, normal weight gains were recorded at 3 percent and 6 percent xanthan gum l e v e l s i n the d i e t , while some weight gain r e d u c t i o n was noted at 7.5 percent and 15 percent l e v e l s . No d i f f e r e n c e s i n organ weights, hemog l o b i n concentration, and white and red c e l l counts were found. At the highest dosage l e v e l s , the animals d i d produce abnormally high f e c a l p e l l e t s , but no occurrence of d i a r r h e a was noted. In a p a i r e d feeding t e s t study at 7.5 percent feeding l e v e l , no g r o w t h - i n h i b i t i n g f a c t o r a t t r i b u t a b l e t o xanthan gum was found. (29) Feeding dogs 2 g/kg xanthan gum f o r a p e r i o d of two weeks produced d i a r r h e a i n the t e s t group, while the same d i d not occur at the 1 g/kg feeding l e v e l . Gross h i s t o p a t h o l o g i c a l examination d i d not r e v e a l any organ damage a t t r i b u t a b l e to the i n g e s t i o n of xanthan gum. (31) T h i s occurrence i s not unexpected, as most h y d r o p h i l i c c o l l o i d s , due to t h e i r

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

278

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

e f f i c i e n t water-absorbing p r o p e r t i e s , are known t o cause d i a r r h e a a t high feeding l e v e l s . In long-term feeding s t u d i e s by Woodard, e t a l . , a group of 30 male and 30 female r a t s was fed xanthan gum at 0, 0.25, 0.5, and 1.0 g/kg as p a r t of t h e i r d a i l y d i e t f o r a p e r i o d of 104 weeks. No abnormalities were found which could be a t t r i b u t e d to the i n g e s t i o n of the product. S u r v i v a l r a t e , body weight gain, food consumption, behavior, and appearance were normal when compared to the c o n t r o l group. Hemat o l o g i c a l values, organ weights, and tumor occurrence showed no s i g n i f i c a n t v a r i a t i o n . S l i g h t l y s o f t e r s t o o l s were noted i n the middle- and h i g h - l e v e l t e s t animals, but the d i f f e r e n c e from the c o n t r o l was not s t a t i s t i c a l l y s i g n i f i c a n t . (32) In a 107-week-long study on a group of four male and four female beagle dogs, 0, 0.25, 0.37, and 1 g/kg xanthan gum was fed to the animals as p a r t of t h e i r d a i l d i e t No adverse e f f e c t s were note to s u r v i v a l , food intake blood pressures, hemograms, organ weights, gross necropsy observations, and h i s t o p a t h o l o g i c a l observations. At the h i g h e s t feeding l e v e l , a d o s e - r e l a t e d increase i n f e c a l weights and a measurable increase i n the s p e c i f i c g r a v i t y of the u r i n e and more frequent presence of u r i n a r y albumin were noted. T h i s t e s t revealed no untoward e f f e c t s caused by the treatment of xanthan gum a t any dosage l e v e l s . (32) The feeding of xanthan gum was a l s o examined i n a threegeneration reproduction study by Woodard, et a l . , using 10 male and 20 female r a t s i n the f i r s t generation and 20 male and 20 female r a t s i n subsequent generations. Dosage l e v e l s were 0, 0.25, and 0.5 g/kg as p a r t of the animals' d i e t . The t e s t r e s u l t s were evaluated f o r s u r v i v a l , body weights, general appearance, behavior, reproductive performance, p h y s i c a l c o n d i t i o n of the o f f s p r i n g , and the s u r v i v a l of the o f f s p r i n g . With respect to a l l c r i t e r i a , no adverse e f f e c t s were noted i n t h i s study which would be a t t r i b u t a b l e to the presence of xanthan gum i n the d i e t of the animals. (32) According to a t e s t conducted by the c a l o r i c a v a i l a b i l i t y method, the d i g e s t i b i l i t y of xanthan gum was found to be zero. T h i s c o n c l u s i o n was s u b s t a n t i a t e d i n a r e p o r t by Booth and coworkers which found that p r a c t i c a l l y a l l xanthan gum fed f o r a p e r i o d of seven days could be recovered i n the s t o o l of the animals. (27) In another study, conducted by the more accurate r a d i o a c t i v e t r a c e r method, the d i g e s t i b i l i t y of xanthan gum was found to be approximately 15 percent. The p o l y s a c c h a r i d e c o n s t i t u e n t s d i d not accumulate i n the t i s s u e s , and they were metabolized by the expected route as carbohydrates. In v i t r o t e s t s i n d i c a t e d t h a t non-enzymatic h y d r o l y s i s and the a c t i o n of microorganisms were r e s p o n s i b l e f o r the i n i t i a l breakdown of the molecule. Based on the above s t u d i e s , the approximate c a l o r i c value of xanthan gum i s 0.5 k i l o -

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

17.

MCNEELY

Alginates and Xanthan Gum

A N D KOVACS

279

c a l o r i e s per gram. Conclusion The foregoing, o f course, i s an abridged summary o f the t o x i c o l o g i c a l studies conducted on a l g i n a t e s and xanthan gum. There i s a b s o l u t e l y no information a v a i l a b l e i n d i ­ c a t i n g t h a t the consumption o f these products a t t h e i r normal usage l e v e l s would present any h e a l t h hazards. Furthermore, i t should be emphasized t h a t the f u n c t i o n a l l e v e l s and the per c a p i t a consumption o f a l g i n a t e s and xanthan gum are orders of magnitude below the feeding l e v e l s used t o e s t a b l i s h t h e i r s a f e t y as food a d d i t i v e s . "Literature Cited"

1 Stanford, E. C. C 2 McNeely, W. H. and Pettitt, D. J., in "Industrial Gums," 2nd Ed., pp. 49-81, Academic Press, New York, New York, 1973. 3 Haug, Α., Larsen, G., Smidsrød, O., Acta. Chem. Scand., (1967) 21, 691. 4 Penman, A. and Sanderson, G., Carb. Res., (1972) 25, 273. 5 Knott, W. B. and Johnston, C. D., "Sodium and Propylene Glycol Alginate Acute Oral Toxicity to Rats," Woodard Research Corporation unpublished report, 1972. 6 Arora, C. Κ., Chandhury, S. K., Chanha, P. S., Indian J. Physiol. Pharmacol., (1968) 12 (3) 129-30. 7 Solandt, Ο. Μ., Quart. J. Exp. Physiol., (1941) 31, 25-40. 8 Sokov, L. Α., Radioactivnye Izotopy Vo Vneshnei Srede i Organizme (Russian), (1970) 247-51. 9 Stanford Research Institute, "Study of Mutagenic Effects of Propylene Glycol Alginate (71-18)," PB 221 826, Ν.T.I.S., 1972. 10 Thienes, C. H., Skillen, R. G., Meredith, Ο. Μ., Fairchild, M. D., McCandless, R. S., Thienes, R. P., Arch. Intern. Pharmacodyn., (1957) 111 (2) 167-81. 11 Viola, S., Zimmerman, G., Mokady, S., Nutr. Rep. Int., (1970) 1 (6) 367-76.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

280

PHYSIOLOGICAL EFFECTS OF FOOD CARBOHYDRATES

12 Nilson, H. W. and Wagner, J. Η., Proc. Soc. Exp. Biol. Med., (1951) 76 (4) 630-5. 13 Dardin, V. J., "Feeding of KELGIN or KELCOLOID to Dogs for One Year," Woodard Research Corporation unpublished report, 1959. 14 Feldman, H. S., Urbach, K., Naegle, C. F., Regan, F. D., Doerner, Α. Α., Proc. Soc. Exp. Biol. Med., (1952) 79, 439-41. 15 Morgan, C. F., Farber, J. E. Jr., Dardin, V.J.,"The Effects of Algin Products on the Rat," Georgetown Univer­ sity Medical School unpublished report, 1959. 16 Food and Drug Research Laboratories, "Teratological Evaluation of Propylen Ν.T.I.S., 1972. 17 Epstein, S. S., Toxicol. Appl. Pharmacol., (1970) 16, 321-4. 18 Humphreys, Ε. R. and Triffitt, J. T., Nature, (1968) 219, 1172-3. 19 Sharratt, M. and Dearn, P., Food Cosmet. Toxicol., (1972) 10 (1) 35-40. 20 Nilson, H. W. and Lemon, J. Μ., "Metabolism Studies with Algin and Gelatin," pp. 1-9, U.S. Fish and Wildlife Serv. Research Report No. 4, 1942. 21 Tanaka, Y. and Skoryna, S. C., in "Intestinal Absorption of Metal Ions, Trace Elements, and Radionuclides," pp. 10114, Pergamon Press, New York, New York, 1971. 22 Harrison, G. E., Carr, T. E. F., Sutton, Α., Humphreys, E. R., Nature, (1969) 224, 1115-6. 23 Carr, T. E. F., Harrison, G. Ε., Humphreys, E. R., Sutton, Α., Int. J. Radiat. Biol., (1968) 14 (3) 225-33. 24 Millis, J. and Reed, F. Β., Biochem J., (1947) 41, 273-5. 25 Anonymous, Food Technol., (1974) 28 (6) 18-21. 26 Sloneker, J. H., Orentes, D. G., Jeanes, Α., Can.J.Chem., (1964) 42, 1261-9.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

17.

MC

NEELY

AND

KOVACS

Alginates

and

Xanthan

Gum

21 Booth, A. N., Hendrickson, A. P., DeEds, F., Toxicol. Appl. Pharmacol., (1963) 5, 478-84. 28 Hendrickson, A. P. and Booth, Α. Ν., "Supplementary Acute Toxicological Studies of Polysaccharide B-1459," Western Reaional Research Laboratory Research Report, Albany, California, 1964. 29 Jackson, N. N., Woodard, M. W., Woodard, G., "Xanthan Gum Acute Oral Toxicity to Rats," Woodard Research Corporation unpublished report, 1968. 30 Jackson, Ν. Ν., Woodard, M. W., Woodard, G., "Xanthan Gum Acute Oral Toxicity to Dogs," Woodard Research Cor­ poration unpublished report, 1968. 31 Robbins, D. J., Moulton Cosmet. Toxicol., (1964)2, 32 Woodard, G., Woodard, M. W., McNeely, W. Η., Kovacs, P., Cronin, M. T. I., Toxicol. Appl. Pharmacol., (1973) 24, 30-6.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

18 Physiological Effects of Carrageenan DIMITRI J. STANCIOFF and DONALD W. RENN Marine Colloids, Inc., Rockland, Maine 04841

Abstract

Breakdown absorption and toxicity of carrageenan in the gas­ trointestinal tract is reviewed. Carrageenan, a linear sulfated galactan (molecular weight 250,000) derived from red seaweeds, is used as a food stabilizer, particularly in dietetic and dairy products. Breakdown in the gut appears to be insignificant and there is no evidence that it is absorbed by test animals with the possible exceptions of guinea pigs and rabbits. In large doses it inhibits pepsin activity (in vitro), depresses gastric juice secretion and reduces food absorption in rats. There is no evi­ dence of such effects with the low levels used in food. Depolymerized carrageenan (molecular weight < 20,000), used in France for treatment of peptic ulcers, has shown no toxicity in man but caused mucosal erosions in the cecum of guinea pigs and at high doses also effected cell changes in the colon of rats and monkeys. Degraded carrageenan was demonstrated as being partly absorbed by the epithelial cells, deposited in the Kupffer cells of the liver and found in the urine. A broad margin of safety for food grade carrageenan in the diet is assured by low func­ tional use levels and a minimum molecular weight of 100,000. Introduction E x c e l l e n t reviews on the p h y s i o l o g i c a l e f f e c t s o f carrageenan have been p u b l i s h e d i n the l a s t s i x years: two of them by Anderson (1,2) and the most recent by DiRosa ( 3 ) . Although these r e views are very comprehensive, t h e i r emphasis i s on the pharmacolo g i c a l p r o p e r t i e s o f carrageenan administered by p a r e n t e r a l routes. For example, carrageenan introduced subcutaneously i n duces c o l l a g e n p r o l i f e r a t i o n w i t h the r e s u l t a n t formation o f a granuloma (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14). When i n j e c t e d i n t o a rodent's paw, a r e p r o d u c i b l e inflammatory edematous condit i o n r e s u l t s which has been s u c c e s s f u l l y used i n screening compounds f o r anti-inflammatory a c t i v i t y (15, 16, 17, 18, 19, 20). 282 In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

18. STANciOFF

AND RENN

Carrageenan

283

A n t i - c o a g u l a n t (21, 22, 23 24) and hypotensive p r o p e r t i e s (25) have a l s o been observed. I n a d d i t i o n , v a r i o u s immunological r e sponses have been reported which i n c l u d e i n v i t r o and i n v i v o complement d e p l e t i o n and delayed h y p e r s e n s i t i v i t y (26, 27, 28, 29, 30, 31, 32, 33). Although these p h y s i o l o g i c a l m a n i f e s t a t i o n s o f carrageenan may seem somewhat foreboding, 99.9% o f the carrageenan consumed by humans i s i n food r a t h e r than i n drugs, and the emphasis of t h i s symposium i s on d i e t a r y r a t h e r than pharmacological aspects of carbohydrates. I n t h i s context the e f f e c t s o f p a r e n t e r a l l y administered carrageenan i s about as r e l e v a n t as the e f f e c t s o f an intravenous i n j e c t i o n o f beef stew. Though the l a t t e r i s a wholesome food, i t s p h y s i o l o g i c a l e f f e c t i n the bloodstream would, no doubt, be q u i t e d e v a s t a t i n g . We w i l l , t h e r e f o r e , r e s t r i c t the s u b j e c t o f t h i s review to carrageenan taken o r a l l y . The use o f p u r i f i e d carrageenan i n modern food technology dates back to the e a r l t i o n s by O r i e n t a l and Europea e r a l c e n t u r i e s o l d (34). In s p i t e of t h i s long h i s t o r y o f food use, or perhaps because of i t , f a r more has been p u b l i s h e d on i t s p h y s i o l o g i c a l e f f e c t as an experimental drug than as a food i n g r e d i e n t . I n f a c t p u b l i c a t i o n s a r e i n i n v e r s e r a t i o to use: 10,000,000 l b s / y e a r used i n food - 20 papers 10,000 l b s / y e a r a n t i - u l c e r drug - 70 papers 1 l b / y e a r p a r e n t e r a l use - 150 papers N e v e r t h e l e s s , we can glean enough from the l i t e r a t u r e to est a b l i s h that carrageenan i s not broken down o r absorbed and that at the low l e v e l s used i n food has no adverse p h y s i o l o g i c a l e f f e c t s i n humans. Source P r o p e r t i e s and Uses o f Carrageenan But f i r s t l e t ' s say a few words about the o r i g i n , chemistry, and p h y s i c a l p r o p e r t i e s o f carrageenan. Carrageenan can be obtained from about 250 species o f c l o s e l y r e l a t e d red algae o f the order G i g a r t i n a l e s but only about h a l f a dozen o f them are used commercially: These i n c l u d e species o f Chondrus, Eucheuma and G i g a r t i n a . I t i s the major i n t e r c e l l u l a r c o n s t i t u e n t o f these p l a n t s and represents about 607o o f t h e i r s a l t - f r e e dry weight. Carrageenan i s g e n e r a l l y prepared by hot aqueous e x t r a c t i o n of the seaweed, f o l l o w e d by f i l t r a t i o n to remove i n s o l u b l e matter, c o a g u l a t i o n i n a l c o h o l , vacuum d r y i n g , and g r i n d i n g . Chemically, carrageenan i s a s t r a i g h t c h a i n s u l f a t e d g a l a c t a n w i t h a backbone o f a l t e r n a t i n g 1-4 l i n k e d ^ - D - g a l a c t o s e and 1-3 l i n k e d ^ - D - g a l a c t o s e o r i t s 3,6 anhydride. Commercial e x t r a c t s u s u a l l y have a weight average molecular weight 100,000 to 500,000.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

284

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

This simple p i c t u r e i s somewhat complicated by the f a c t that the number and p o s i t i o n of the e s t e r s u l f a t e groups and the r a t i o of g a l a c t o s e to 3,6-anhydrogalactose may v a r y c o n s i d e r a b l y . Greek l e t t e r p r e f i x e s are used f o r c a t e g o r i z i n g the v a r i o u s com­ binations . Two major groups of carrageenan are recognized. In the f i r s t , the 1 , 3 - l i n k e d u n i t s are s u l f a t e d i n the 4 - p o s i t i o n w h i l e i n the second, the s u l f a t e i s i n the 2 - p o s i t i o n (Figure 1). The f i r s t group i s s u b d i v i d e d a c c o r d i n g to the nature of the 1- 4 l i n k e d u n i t s . These may be present as g a l a c t o s e 6 - s u l f a t e (>i-carrageenan) or g a l a c t o s e 2-6 d i s u l f a t e ( ^-carrageenan) or as the corresponding 3,6-anhydrides i n fC- and L -carrageenan. In nature the 3,6-anhydrides of /(- and £-carrageenan are formed by enzymatic e l i m i n a t i o n of the 6 - s u l f a t e from the μ- and p-forms, but the c o n v e r s i o n i s not always complete. In some seaweeds these carrageenan types can be i s o l a t e d i n almost pure form w h i l e i n others they e x i s t a In the second group 2- p o s i t i o n . In λ-carrageenan the 6 - p o s i t i o n i s a l s o s u l f a t e d w h i l e i n ξ-carrageenan i t i s not. Aqueous s o l u t i o n s of carrageenan are h i g h l y v i s c o u s . Kappa and C -carrageenan form heat r e v e r s i b l e gels i n the presence of potassium and c a l c i u m i o n s . Carrageenans a l s o r e a c t s t r o n g l y w i t h l a r g e p o s i t i v e l y charged i o n s , notably w i t h p r o t e i n s below their i s o e l e c t r i c point. The main uses of carrageenan are as t h i c k e n e r s , s t a b i l i z e r s , and g e l l i n g agents i n food. I t s strong i n t e r a c t i o n w i t h c a s e i n i s u t i l i z e d i n d a i r y products such as c h o c o l a t e m i l k , i c e cream and puddings. The g e l l i n g p r o p e r t i e s are u s e f u l f o r making j e l l i e s , r e l i s h e s , and p i e f i l l i n g s . The l i s t of a p p l i c a t i o n s a l s o i n c l u d e s whipped t o p p i n g , non-dairy c o f f e e w h i t e n e r s , and a host of convenience foods. The f u n c t i o n a l p r o p e r t i e s of carrageenan are h i g h l y dependent on molecular weight. When the m o l e c u l a r weight i s below 100,000 the s t a b i l i z i n g p r o p e r t i e s are almost completely l o s t . More de­ t a i l e d i n f o r m a t i o n on the sources, p r o p e r t i e s , and uses of c a r r a ­ geenan has been p u b l i s h e d i n reviews by Rees ( 3 5 ) , by Glicksman (36), Sand and Glicksman ( 3 7 ) , and by Towle ( 3 8 ) . Degraded Carrageenan When speaking o f p h y s i o l o g i c a l p r o p e r t i e s of ingested c a r r a ­ geenan we must d i s t i n g u i s h between food grade carrageenan, which has a m o l e c u l a r weight of 100,000 to 500,000 and s o - c a l l e d de­ graded carrageenan. Degraded carrageenan i n which the g l y c o s i d i c l i n k a g e s are h y d r o l i z e d to reduce the molecular weight to l e s s than 20,000 i s used i n France f o r p e p t i c u l c e r treatment. I t i s i n e f f e c t i v e as a food s t a b i l i z e r and a l s o has d i f f e r e n t p h y s i o ­ l o g i c a l p r o p e r t i e s . We w i l l come back to i t l a t e r , but f i r s t l e t s t a l k about food grade carrageenan. 1

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

18.

STANCiOFF

AND RENN

3 lg A

285

Carrageenan

4 lo B

Β

3 1J A

4 ia B

3 ^

A

UNITS:

UNITS

D-GAUVCTOSE 6-SULFATE

mu

D-GALACTOSE

nu

2J6-DISULFATE

D-GALACTOSE 4-SULFATE 3,6-ANHYDRO-D-GALACTOSE

kappa

3,6-ANHYDRO-D-GALACTOSE 2-SULFATE

iota

D-GALACTOSE 2-SULFATE

xi

D-GALACTOSE 2J6-DI SULFATE

lambda

D-GALACTOSE 2-SULFATE

3,6-ANHYDRO-D-GALACTOSE 2-SULFATE

theta

Figure 1. Repeating units of carrageenans

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

286

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

P h y s i o l o g i c a l E f f e c t s of Food Grade Carrageenan Breakdown i n the I n t e s t i n a l T r a c t . Breakdown of carrageenan i n the d i g e s t i v e system i s probably minimal, f o r i t seems that n e i t h e r man nor the experimental animals t e s t e d so f a r possess the necessary enzymes to h y d r o l y z e i t . In the stomach, where pH i s very low, a small amount o f a c i d h y d r o l y s i s undoubtedly does occur. However, i n v i t r o experiments w i t h simulated g a s t r i c j u i c e at pH 1.2 and 37°C showed that i n three hours (which i s about the maximum residence time i n the stomach) the breakdown of g l y c o s i d i c linkages was l e s s than 0.1% (39). Information about what happens i n the lower gut i s n o t o r i o u s l y l a c k i n g . I n c u b a t i o n of a carrageenan s o l u t i o n w i t h the c e c a l contents of r a t s f o r s e v e r a l hours at 37°C d i d not a l t e r i t s v i s c o s i t y , which i n d i c a t e s that the m i c r o b i a l f l o r a of the r a t gut a t any r a t e , w i l l not brea There i s always th feeding large doses of carrageenan, the m i c r o b i a l f l o r a would be changed enough to cause some breakdown. However, there i s so f a r no suggestion that t h i s might occur. I n f a c t there seem to be very few b a c t e r i a , other than those of marine o r i g i n , that can decompose carrageenan. Our own waste d i s p o s a l d i f f i c u l t y w i t h d i g e s t i n g carrageenan w i t h a c t i v a t e d sewage sludge a t t e s t s to t h a t . A b s o r p t i o n . I f carrageenan i s not broken down, i s i t absorbed without breakdown? In three species of monkey (41, 42, 43), dog (44), p i g ( 4 5 ) , r a t (41, 46, 47, 48, 49), mouse ( 4 1 ) , f e r r e t ( 4 1 ) , hamster ( 4 1 ) , i t a p p a r e n t l y i s not. Houck (44) obtained 100% r e covery i n the feces of dogs. In a 2-year feeding study by N i l s o n and Wagner ( 4 8 ) , r a t s r e c e i v e d from 1 to 25% carrageenan mixed w i t h the dry d i e t . Growth r a t e s and i n t e r n a l organs of the a n i mals were normal a t dose l e v e l s up to 10%. At high doses growth was r e t a r d e d , and some animals fed 25% carrageenan showed l i v e r a b n o r m a l i t i e s . N i l s o n and Wagner a t t r i b u t e d the l a t t e r to a d i e t a r y d e f i c i e n c y caused by the large bulk of the ingested c a r r a geenan. Recovery i n the feces was 50% i r r e s p e c t i v e of the l e v e l fed. This r a t h e r s u r p r i s i n g r e s u l t was, however, obtained by an a n a l y t i c a l method which l e f t something to be d e s i r e d . Two l a t e r s t u d i e s , a l s o w i t h r a t s , r e s u l t e d i n 90-100% recovery (46, 47). In the case of guinea pigs and r a b b i t s , r e s u l t s have been cont r a d i c t o r y . Watt and Marcus (50) found that h i g h molecular weight carrageenan caused u l c e r a t i o n of the cecum and c o l o n of guinea p i g s . Grasso, S h a r r a t t , C a r p a n i n i and Gangoli (41) confirmed these r e s u l t s and a l s o showed that r a b b i t s were s i m i l a r l y a f f e c t ed. Although the bulk of the carrageenan was e x c r e t e d i n the f e c e s , a c e r t a i n amount was absorbed by the macrophages i n the s u b e p i t h e l i a l l a y e r s of the cecum and c o l o n . On the other hand, a study a t the Albany M e d i c a l College showed n e i t h e r a b s o r p t i o n nor l e s i o n s (49, 51). Anderson and Soman ( 5 2 ) , l i k e w i s e , showed

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

18.

STANCiOFF

AND

RENN

Carrageenan

287

that high molecular weight carrageenan was not absorbed through the gut of guinea pigs and could not be detected i n the blood or u r i n e unless a d m i n i s t e r e d i n t r a v e n o u s l y . Grasso et a l . hypothes i z e that a b s o r p t i o n by macrophages may be p e c u l i a r to guinea pigs and r a b b i t s , herbivorous rodents, both o f which possess an unu s u a l l y large cecum, and suggest that a b s o r p t i o n may be due to i n complete neonatal c l o t u r e of the i n t e s t i n a l b a r r i e r to macromolecules . Other E f f e c t s . Now, i f carrageenan i s not digested and not absorbed, j u s t what p h y s i o l o g i c a l e f f e c t s does i t have? So f a r , s e v e r a l e f f e c t s have been reported i n the l i t e r a t u r e : Reduction of p e p t i c a c t i v i t y Reduced flow of g a s t r i c s e c r e t i o n s i n the stomach Antilipemic a c t i v i t Increase i n wate A l l these e f f e c t s can be a t t r i b u t e d to the h y d r o p h i l i c and p o l y a n i o n i c p r o p e r t i e s of the macromolecule, however, they a l l r e s u l t only from very h i g h dosages of carrageenan and cannot be detected at the low l e v e l s a t which carrageenan i s used i n food. A n t i p e p t i c a c t i v i t y . The f i r s t and the best documented of these e f f e c t s i s the a n t i p e p t i c a c t i v i t y . Carrageenan i n t e r f e r e s w i t h the p r o t e o l y t i c a c t i v i t y of p e p s i n , both ixi v i t r o and i n v i v o (44, 52, 53). Several researchers have concluded that the i n h i b i t i o n i s due to the i n t e r a c t i o n of carrageenan w i t h the subs t r a t e and not w i t h the p e p s i n . This view i s supported by the f a c t that the degree of i n h i b i t i o n depends on the r a t i o of c a r r a geenan to s u b s t r a t e and not on the amount of p e p s i n (54, 55, 56). Anderson ( 1 , 54) has pointed out that a t pH 1.3 most p r o t e i n s become p o s i t i v e l y charged and form i n s o l u b l e complexes w i t h c a r r a geenan, whereas pepsin w i t h an i s o e l e c t r i c p o i n t of 1.0 remains a n i o n i c under these c o n d i t i o n s and i s u n l i k e l y to r e a c t . The noni n t e r a c t i o n of carrageenan and p e p s i n has a l s o been shown e l e c t r o p h o r e t i c a l l y (57). You may ask then, i f carrageenan i n h i b i t s p e p t i c a c t i v i t y , w i l l i t not then i n t e r f e r e w i t h d i g e s t i o n and cause reduced prot e i n intake? This c e r t a i n l y would have been a p o s s i b i l i t y i f carrageenan i n t e r a c t e d w i t h the p e p s i n , because then, even small doses should i n t e r f e r e . However, since i t r e a c t s w i t h the subs t r a t e , the amount of p r o t e i n d i g e s t e d depends on how much of i t remains unreacted w i t h the carrageenan. Since the amount of proe i n ingested i n a normal d i e t i s c o n s i d e r a b l e , very l a r g e doses of carrageenan would be needed to i n a c t i v a t e a l l the s u b s t r a t e . Indeed, such seems to be the case. Hawkins and Yaphe (47) found that young r a t s gained weight more s l o w l y only i f t h e i r d i e t contained more than 107 carrageenan (10,000 mg/kg). Such an excess i s , however, completely u n r e a l i s t i c and the work of o

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

288

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

Vaughan, F i l e r and C h u r e l l a (56) shows what occurs under condit i o n s c l o s e r to r e a l i t y . They examined p e p t i c i n h i b i t i o n of c a r rageenan w i t h s e v e r a l p r o t e i n s i n v i t r o and a l s o measured i n v i v o d i g e s t i b i l i t y of m i l k p r o t e i n and carrageenan mixtures i n r a t s . They found that i n h i b i t i o n depended on the r a t i o of carrageenan to p r o t e i n . In a l l cases there was no d e t e c t a b l e i n h i b i t i o n a t r a t i o s s m a l l e r than 0.1 and i n some cases r a t i o s were as h i g h as 0.3 before i n t e r f e r e n c e occurred. In the i n v i v o s t u d i e s the d i g e s t i b i l i t y was 100% a t r a t i o s below 0.1. Even a t the h i g h r a t i o of 0.3 p e p t i c a c t i v i t y was reduced only 10 to 20%. The g r e a t e s t use of carrageenan i s i n the d a i r y i n d u s t r y , however, the l e v e l s a t which i t i s used are extremely low. In puddings the carrageenan to p r o t e i n r a t i o i s about 0.03, i n chocolate m i l k i t i s 0.01, and i n i c e cream, evaporated m i l k and i n f a n t f o r mulas i t i s about 0.005. Products, such as r e l i s h e s or low c a l o r i e j e l l i e s which may c o n t a i n up to 0.77 carrageena ceeds the amount of p r o t e i n the d i e t and are u s u a l l y eaten a t the same time as other foods which have a h i g h p r o t e i n content. Therefore, unless someone dec i d e d to go on a pure j e l l y and r e l i s h binge the l i k e l i h o o d of p r o t e i n d e f i c i e n c y due to a s u r f e i t of carrageenan i s q u i t e n e g l i g i b l e . In f a c t the average per c a p i t a consumption o f carrageenan i n the U n i t e d States i s l e s s than 0.5 mg/kg of body weight per day. The h i g h e s t l e v e l s are consumed by i n f a n t s r e c e i v i n g s p e c i a l d i e t a r y formulas during the f i r s t 2-3 months a f t e r b i r t h . The d a i l y l e v e l s average 25 to 50 mg/kg of body weight, but the r a t i o of carrageenan to p r o t e i n i s o n l y about 0.005. o

G a s t r i c s e c r e t i o n . Carrageenan diminishes the volume and a c i d i t y of h i s t a m i n e - s t i m u l a t e d g a s t r i c s e c r e t i o n , but on the other hand, i t a l s o r e s t o r e s normal s e c r e t i o n i n cases where supramaximal histamine s t i m u l a t i o n has caused submaximal a c i d output. According to Anderson ( 5 8 ) , carrageenan complexes w i t h mucin on the stomach w a l l and i t s a b i l i t y to i n h i b i t g a s t r i c s e c r e t i o n (59, 60) could be r e l a t e d by t h i s i n t e r a c t i o n . I t s a b i l i t y to r e store normal flow i s , however, more d i f f i c u l t to e x p l a i n (61). These phenomena are u n l i k e l y to take p l a c e w i t h carrageenan that i s ingested w i t h food -- f i r s t because of the small amounts present and, second, because the carrageenan would be complexed w i t h the food p r o t e i n . Lipemia c l e a r i n g . The t h i r d e f f e c t of carrageenan i s i t s hypocholesterolemic a c t i v i t y . A g a i n , large dosages are necessary. Fahrenbach et a l (62) obtained s i g n i f i c a n t r e d u c t i o n i n blood c h o l e s t e r o l of white Leghorn c h i c k s which were fed 1 to 3% c a r r a geenan i n the d i e t . E r s h o f f and W i l l s (63) obtained a s i m i l a r e f f e c t w i t h 10% carrageenan i n the d i e t of r a t s fed 1% c h o l e s t e r o l . L i v e r c h o l e s t e r o l remained a t normal l e v e l and t o t a l l i v e r

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

18.

STANCiOFF

AND RENN

Carrageenan

289

l i p i d were g r e a t l y reduced. The mechanism o f lipemia c l e a r i n g i s not known. Perhaps, carrageenan, l i k e other n o n - d i g e s t i b l e f i b e r may i n t e r f e r e w i t h c h o l e s t e r o l a b s o r p t i o n . I t i s unfortunate t h a t , i n order to take advantage o f the a n t i - l i p e m i c p r o p e r t i e s of carrageenan, the o r a l dosage has to be so h i g h . Water content o f the bowel. There i s nothing unusual about the f o u r t h e f f e c t o f carrageenan which increases the water content o f the bowel and causes s o f t e n i n g o f the s t o o l . This e f f e c t i s common to a l l h y d r o p h i l i c polymers s e v e r a l o f which, notably agar, p s y l l i u m gum, and p e c t i n , are used as m i l d l a x a t i v e s because o f t h e i r w a t e r - h o l d i n g p r o p e r t i e s . P h y s i o l o g i c a l E f f e c t s o f Degraded

Carrageenan

Now a few words about degraded o r depolymerized carrageenan This product, which ha veloped as a more convenient treatment o f p e p t i c and duodenal u l c e r s i n humans. I t i s produced by p a r t i a l h y d r o l y s i s o f the g l y c o s i d i c l i n k a g e s w i t h a c i d . Carrageenan, both i n the degraded and undegraded form, prevents o r diminishes histamine-induced experimental p e p t i c u l c e r a t i o n (44, 60) and a l l e v i a t e s p e p t i c and duodenal u l c e r s i n humans (64). Anderson (58) a t t r i b u t e s t h i s e f f e c t to the a b i l i t y o f carrageenan t o complex w i t h g a s t r i c mucin, thus p r o t e c t i n g the stomach w a l l a g a i n s t a t t a c k by the g a s t r i c j u i c e s . The degraded product i s as e f f e c t i v e i n p r o t e c t i n g the stomach w a l l as undegraded carrageenan and i s much e a s i e r f o r a pat i e n t t o take because o f i t s very low v i s c o s i t y . In c o n t r a s t to food grade carrageenan, the degraded product i s p a r t l y absorbed through the gut. I t has been detected i n the u r i n e o f baboons (42) and guinea pigs ( 5 2 ) . The amount absorbed was l e s s than 1%. I n Rhesus monkeys i t accumulated i n the l y s o somes o f the r e t i c u l o e n d o t h e l i a l c e l l s o f the l i v e r , s p l e e n , and lymph nodes, and could s t i l l be detected s i x months a f t e r t r e a t ment (43). Degraded carrageenan a l s o causes u l c e r a t i o n of the cecum and c o l o n o f s e v e r a l t e s t s p e c i e s . G e r b i l s and mice were not a f f e c t ed (51). Guinea pigs and r a b b i t s were p a r t i c u l a r l y s u s c e p t i b l e (40, 41, 50, 51, 65). Lesions were a l s o produced i n Rhesus monkeys (43) a t high dose l e v e l s (3,000 mg/kg/day), but not i n s q u i r r e l monkeys (41). C l i n i c a l t e s t s by B o n f i l s (64) on 200 p a t i e n t s r e c e i v i n g 5 grams per day (100/mg/kg/day) o f degraded carrageenan i n p e p t i c u l c e r treatment showed no adverse e f f e c t s on the c o l o n a f t e r s i x months to two years of treatment. The a b s o r p t i o n and u l c e r a t i o n a p p a r e n t l y are h i g h l y dependent on dose l e v e l and molecular weight. Above a molecular weight o f 50,000 no a b s o r p t i o n can be detected (51, 52).

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

18.

STANciOFF

AND

RENN

Carrageenan

291

Despite the apparent l a c k of t o x i c i t y to humans we must, n e v e r t h e l e s s , be c a u t i o u s i n the event that degraded carrageenan might have adverse e f f e c t s a f t e r prolonged i n g e s t i o n . For t h i s reason the Food and Drug A d m i n i s t r a t i o n r e q u i r e s a minimum molecu l a r weight of 100,000 f o r carrageenan i n food. This i s no problem because the s t a b i l i z i n g p r o p e r t i e s of carrageenan depend on high molecular weight. However, carrageenan i s p o l y d i s p e r s e and there i s always the p o s s i b i l i t y that low molecular weight m a t e r i a l may be present. At Marine C o l l o i d s we have developed a r a p i d method f o r determining molecular weight and molecular weight d i s t r i b u t i o n of c a r rageenan. The method i s based on the r e l a t i v e e l e c t r o p h o r e t i c m o b i l i t y of d i f f e r e n t s i z e carrageenan molecules i n a t h i n f i l m agarose g e l (66). S e p a r a t i o n i s p r i m a r i l y according to molecular weight, w i t h charge d e n s i t y e x e r t i n g o n l y a minor e f f e c t . The separated species can be q u a n t i t a t e d b t r a n s m i s s i o n densitometry By comparing a s e r i e s o whose molecular weights y ultracentrifuga t i o n , a l i n e a r r e l a t i o n s h i p was found between e l e c t r o p h o r e t i c m o b i l i t y and the cube root of the molecular weight. The method has the advantage that o n l y about 50 micrograms of sample i s r e q u i r e d , s e v e r a l samples can be t e s t e d at the same time, r e s u l t s are obtained i n a day, and s e p a r a t i o n of high and low molecular weight carrageenan i s very good. The densitometer scan of a pherogram of a mixture of high and low molecular weight carrageenan i s shown i n F i g u r e 2. Our work has shown that o n l y minute amounts of low molecular weight m a t e r i a l i s present i n food grade carrageenan. C u r r e n t l y we are t e s t i n g carrageenan processed under c o n d i t i o n s used i n food p r o c e s s i n g . Our r e s u l t s to date show l i t t l e or no change i n mol e c u l a r weight d i s t r i b u t i o n under normal p r o c e s s i n g c o n d i t i o n s . Conclusion In c o n c l u s i o n , the s a f e t y o f carrageenan as a food a d d i t i v e has been the t a r g e t of many in-depth s t u d i e s supported by government agencies here and abroad as w e l l as by the v a r i o u s c a r r a geenan producers. From the r e s u l t s of these s t u d i e s , we can conclude that food grade carrageenan -- an i s o l a t e d component of n a t u r a l food products used e x t e n s i v e l y f o r over two c e n t u r i e s -has no adverse p h y s i o l o g i c a l e f f e c t s and that i t s s a f e t y i n foods i s assured. Addendum Development since September, 1974. Recently d u r i n g the course of a current t h r e e - g e n e r a t i o n feeding study, researchers at the Food and Drug A d m i n i s t r a t i o n noted changes i n the s u r f a c e appearance of the l i v e r s of animals being fed 5% and 2.5% carrageenan i n the dry d i e t (67). L i v e r s u r f a c e s appeared to be lumpy and i r r e g -

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

292

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

u l a r . A t the 5% carrageenan l e v e l the frequency o f t h i s occur­ rence was 93% while a t the 2.5% l e v e l i t was 40%. Male animals fed the highest l e v e l gained l e s s weight than normal. In other respects the r a t s were q u i t e h e a l t h y and r e p r o d u c t i o n was normal. The l i v e r changes are d i f f i c u l t to e x p l a i n i n the l i g h t o f so many previous s t u d i e s , p a r t i c u l a r l y since the changes were very obvious and could hardly have been missed i f they had occurred before. On the other hand, a very thorough six-month feeding study at Wyeth Labs., Inc. (68), with 4% carrageenan i n the d i e t o f r a t s , revealed no a b n o r m a l i t i e s whatsoever. In t h i s case the carrageenan had been mixed i n t o skim m i l k a t a c o n c e n t r a t i o n equal to that o f the p r o t e i n a f t e r which the mixture was spraydried or lyophilized. The carrageenan had no i n f l u e n c e on growth r a t e , d i e t energy e f f i c i e n c y , a b s o r p t i o n o f p r o t e i n , f a t , or c a l ­ cium, u t i l i z a t i o n o f p r o t e i n f o r growth o r the u t i l i z a t i o n o f i r o n . Gross examinatio malities. Further test crepancies between the v a r i o u s s t u d i e s . Literature Cited

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Anderson, W., Can. J. Pharm. Sci., (1967), 2, 81-90. Anderson, W., Proc. Int. Seaweed Symp., (1969), 6, 627-635. DiRosa, Μ., J. Pharm. Pharmacol., (1972), 24, 89-102. Benitz, K.F. and Hall, L.M., Proc. Soc. Exp. Biol. Med. (1959), 108, 442-445. DiRosa, Μ., Giroud,J.P.and Willoughby, D.A., J. Pathol., (1971), 104, 15-29. DiRosa, M. and Sorrentino, L., Eur. J. Pharmacol., (1968), 4, 340-342. Garg, Bhagwan D. and McCandless, Esther L., Anat. Rec., (1968), 162, (1), 33-40. Hurley,J.V.and Willoughby, D.A., Pathol. (1973), 5, 9-21. Jackson, D.S., Biochem. J., (1957), 65, 277-284. Jackson, D.S., Biochem. J., (1957), 65, 459-464. McCandless, E.L., Ann. N.Y. Acad. Sci., (1965), 118, (22), 867-882. Monis, Β., Weinberg, T. and Spector, G.J., Brit.J.Exp. Pathol., (1968), 49, 302-310. Robertson, W., Hiwett, J. and Herman, L.C., J. Biol. Chem., (1959), 234, 105-108. Salvaggio, J. and Kundur, V., Proc. Soc. Exp. Biol. Med., (1970), 134, 1116-1119. Winter, C.A., Risley, E.A. and Nuss, G.W., Proc. Soc. Exp. Biol. Med., (1962), 111, 544-547. Van Arman, C.G., Begany, A.J., Miller, L.M., and Pless, H.H., J. Pharmacol. Exp. Ther., (1965), 150, 328-334.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

18. STANciOFF AND RENN 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35.

36. 37.

38. 39. 40.

Carrageenan

293

Niemegeers, C.J.E., Verbrugger, F.J. and Janssen, P.Α., J. Pharm. Pharmacol., (1964), 16, 810-816. Bush, J.E. and Alexander, R.W., Acta Endocrinol., (1960), 35, 268-276. Benitz, K.F. and Hall, L.M., Arch. int. Pharmacodyn., (1963), 144, (1-2), 185-195. Schwartz, H.J. and Kellermeyer, R.W., Proc. Soc. Exp. Biol. Med., (1969), 132, 1021-1024. Anderson, W. and Duncan, J.G.C., J. Pharm. Pharmacol., (1965), 17, 647-654. Hawkins, W.W. and Leonard, V.G., Can. J. Biochem. Physiol., (1963), 41, 1325-1327. Houck, J.C., Morris, R.K. and Lazaro, E.J., Proc. Soc. Exp. Biol. Med., (1957), 96, 528-530. Schimpf, Κ., Lenhard, J. and Schaaf, G., Thrombos. Diath. Haemorrh., (1969), 21, 525-533. Noordhoek, J. and Ther., (1972), 197, (2), Bice, D.E., Gruwell, D.G., Salvaggio, J.E. and Hoffman, E.O., Immunol. Commun. (1972), 1, (6), 615-625. Bice, D., Schwartz, Η., Lake, W. and Salvaggio, J., Int. Arch. Allergy, (1971), 41, 628-636. Borsos, T., Rapp, H.J. and Crile, C., J. Immunol., (1965), 94, 662-666. Davies, G.E., Immunology, (1965), 8, 291-299. Davies, G.E., Immunology, (1963), 6, 561-568. Johnston, K.H. and McCandless, E.L., J. Immunol., (1968), 101, 556-562. Mizushima, Y. and Noda, Μ., Experientia, (1973), 29, (5), 605-606. Schwartz, H.J. and Leskowitz, S., J. Immunol., (1969), 103, 87-91. Sauvageau, C., "Utilisation des Algues Marines," 268-335, Gaston Doin et Cie, Paris, (1920). Rees, D.A., Structure Conformation and Mechanism in the Formation of Polysaccharide Gels and Networks, "Advances in Carbohydrate Chemistry and Biochemistry," 24, 267-332, Academic Press, New York, (1969). Glicksman, Μ., "Gum Technology in the Food Industry," 214-235, Academic Press, New York (1969). Sand, R.E. and Glicksman, Μ., Seaweed Extracts of Potential Economic Importance, "Industrial Gums," Second Edition, 147-194, Academic Press, New York, (1973). Towle, G.A., Carrageenan, "Industrial Gums," Second Edition, 83-114, Academic Press, New York (1973). Stancioff, D.J. Unpublished results. "The Biological Activity of Native and Degraded Carra­ geenan," The British Industrial Biological Research Associa­ tion, Carshalton, Surrey, unpublished report (1971).

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

294

PHYSIOLOGICAL EFFECTS OF FOOD CARBOHYDRATES

41. Grasso, P., Sharratt, Μ., Carpanini, F.M.B., and Gangolli, S.D., Food Cosmet. Toxicol., (1973), 2, 555-564. 42. Beattie, I.Α., Blakemore, W.R., Dewar, E.T. and Warwick, M.H., Food Cosmet. Toxicol., (1970), 8, 257-266. 43. Abraham, R., Golberg, L. and Coulston, F., Exp. Mol. Pathol., (1972), 17, 77-93. 44. Houck, J.C., Bhayana, J. and Lee, T., Gastroenterology, (1960), 39, 196-200. 45. Poulsen, Ε., Food Cosmet. Toxicol., (1973), 2, 219-227. 46. Dewar, E.T. and Maddy, M.L., J. Pharm. Pharmacol., (1970), 22, 791-793. 47. Hawkins, W.W. and Yaphe, W., Can. J. Biochem., (1965), 43, 479-484. 48. Nilson, H.W. and Wagner, J.A., Food Res., (1959), 24, 235-239. 49. Abraham, R., Golberg, L., and Coulston, F., Paper No. 192, presented at the Annua March 10-14, 1974 50. Watt, J. and Marcus, R., J. Pharm. Pharmacol., (1969), 21, Suppl. 187S. 51. "Safety Evaluation of Carrageenan," Albany Medical College, Institute Experimental Pathology and Toxicology, Albany, New York, (Unpublished report 1971). 52. Anderson, W., and Soman, P.D., J. Pharm. Pharmacol., (1966), 18, 825-827. 53. Anderson, W. and Harthill, J.E., J. Pharm. Pharmacol., (1967), 17, 647-654. 54. Anderson, W., and Baillie, A.J., J. Pharm. Pharmacol., (1967), 19, 720-728. 55. Anderson, W., Baillie, A.J. and Harthill, J.E., J. Pharm. Pharmacol., (1968), 20, 715-722. 56. Vaughan, O.W., Filer, L.J. Jr., and Churella, Μ., J. Agr. Food Chem., (1962), 10, 517-519. 57. Martin, F., Vagne, A.B., and Lambert, R., C.R. Soc. Biol., (1965), 159, 1582-1585. 58. Anderson, W. and Watt, J., J. Pharm. Pharmacol., (1959), 11:318. 59. Watt, J., Eagleton, J.B. and Marcus R., Nature, (1966), 211, 989. 60. Anderson, W., Marcus, R. and Watt, J., J. Pharm. Pharmacol., (1962), 14, 119-121. 61. Anderson, W. and Soman, P.D., Nature, (1967), 214, 823-824. 62. Fahrenbach, M.J., Riccardo, B.A. and Grant, W.C., Proc. Soc. Exp. Biol. Med., (1966), 123, 321. 63. Ershoff, B.H. and Wills, A.F., Proc. Soc. Exp. Biol. Med., (1962), 110, 580-582. 64. Bonfils, S., Lancet, (1970), 2, 414. 65. Watt, J. and Marcus, R., J. Pharm. Pharmacol., (1970), 22, 130-131.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

18. STANCIOFF AND

RENN

Carrageenan

295

66. Stanley, N.F. and Renn, D.W., Proc. Int. Seaweed Symp., 8th, (1974), Bangor, Wales (In press). 67. Food Chemical News, (1974), 16, (29), 3-5. 68. Tomarelli, R.M., Tucker, W.D. Jr., Bauman, L.M., Savini, S. and Weaber, J.R., J. Agr. Food Chem., 22, 819-824.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

19 Physiological Effects of Mannans, Galactomannans, and Glucomannans S. E. DAVIS and B. A. LEWIS Division of Nutritional Sciences, New York State College of Human Ecology, Cornell University, Ithaca, Ν. Y. 14850

Galactomannans in the form of the ground endosperm of cer tain leguminous seeds have been consumed as foodstuffs since ancient times. In the last two decades these gums together with konjac glucomannan and the yeast mannans have come to be associ­ ated with certain physiological activities which have stimulated nutritional and physiological research. Many of the studies are contradictory and several questions remain unanswered. This paper attempts to review the recent literature on the nutritional value of the mannans and the physiological activities which they possess. Occurrence in Foods Few natural foods have been subjected to complete analysis for their component carbohydrates. For the most part, analyses have been directed toward the soluble low molecular weight sugars, starch, pectin and crude fiber (consisting of cellulose and smaller amounts of associated but usually unidentified cell wall hemicelluloses). Thus, the infrequent reports of mannans in foods could reflect their rarity or merely that they have been overl ooked. Pure mannans (homopolymer) are particularly rare in nature. The most familiar sources are the yeasts, wherein Saccharomyces

cerevisjae, for example, mannose occurs as the branched α - ( 1 · * 3 , l-»6)-linked yeast mannan Q ) . Galactomannans are much more common since they are important as industrial gums for a variety of uses (1-1). Found most fre­ quently in the endosperm of seeds of leguminous plants, these gums find their way into processed foods as additives for control of texture, rheological behavior and as binders. The basic structure (Figure 1 ) is that of an essentially linear 3-mannan with single unit branches of α-galactopyranose units. The galactomannans from different sources vary in molecular weight and in the ratio of the two sugars. Thus in guaran (guar gum) the ratio of mannose to galactose is 2 : 1 ; whereas in locust bean 296 In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

19.

DAVIS

A N D LEWIS

297

Galactomannans

α-D-Gal 1

P

\

6 -3-D-Man -(l^)-3-D-Man -(l+4)-3-D-Man r

r

r

Figure 1. Guar and locust bean galactomannans

gum (carob gum) i t is variously reported as 3:1 to 6:1. This difference in degree of branching is reflected in the physical properties of the gums; guar galactomannan readily hydrates in cold water to form a colloidal solution while locust bean gum dispersions must be heated to develop maximum viscosity. Historically the guar plant (Cyamopsis tetragonolobis) has been grown for centuries in India and Pakistan as a food crop for humans as well as animals. The plant was introduced into the United States for agricultura the galactomannan became commercially available in the 1950's. Commercial guar gum, which is milled free from the hull and germ, consists of about 78-82% galactomannan, 4-5% protein, 1.5-2.0% crude fiber, and a small amount of ash and ether-soluble material. Food usage of the pods of the locust bean tree (Ceratonia siliqua L.) probably dates back for thousands of years. Since Biblical times the locust bean or carob has been immortalized as St. John's bread. Like guar, the commercial gum is obtained from the seeds of the pods by removing the germ and husk. The compo­ sition of the typical gum of commerce is not unlike that of guar and is dependent on the efficiency of separation of the galactomannan-containing endosperm. Other mannose-containing heteropolysaccharides include the glucomannans which occur most commonly in woody plants in associ­ ation with cellulose. One of the few identified food sources is konnyaku powder obtained by grinding the tubers of the plant Amorphophalis konyac. Extraction of konnyaku powder with water affords konjac mannan, a 3-l->4-linked glucomannan (Figure 2) with sequences of at least three mannose units. -3-D-Man -(l-^)-3-D-Man -(l->4)-3-D-G -(l->4)-3-D-Man -(l->4)p

p

p

p

Figure 2. Konjac mannan

Mannose is also a common constituent of glycoproteins where i t occurs in variable amounts from less than 1% in some proteins to a significant proportion of other proteins. Thus, glycopro­ teins such as the soy globulins contain up to 5% carbohydrate. The carbohydrate moiety of glycoproteins most frequently consists of chains of varying length of α-linked D-mannopyranose units attached successively through 3-D-mannosyl and 3-D-glucosaminyl

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

298

PHYSIOLOGICAL

EFFECTS

O F FOOD CARBOHYDRATES

(usually N-acetylated) units to the protein chain. Linkage to the protein occurs through an N-glycosidic bond to the 3-amide of asparagine or less frequently by an O-glycosidic bond to the hydroxyl of serine or threonine. For a number of proteins inclu­ ding ovalbumin, ribonuclease Β and taka amylase the inner core of the glycoprotein has the following structure (4,5): 3-D-Man-(1^4)-D-GNAc-(l->4)-3-D-GNAc->L-ASN Little is known of the significance of the mannan moiety of glycoproteins or of its effect on the digestibility of proteins. Intracellular mannose-containing glycoproteins are catabolized by lysosomal a- and 3-mannosidases which display maximal activity at slightly acid pH (.5-8). However, i t should be noted that non-lysosomal α-mannosidases are known (5., 9_ 10). 9

Digestion of Mannans Although l i t t l e is known about the fate of dietary mannans and mannose-containing glycoproteins, i t is possible that some mannose might be released by action of mannosidases in the gas­ trointestinal tract ( υ ) . McMaster et a l ^ (1_2) reported an a-Dmannosidase in canine pancreatic juice. The acidic pH optimum of this enzyme, however, suggests its probable origin in pancreatic tissue lysosomes. While the activity of pancreatic juice a-mannosidase was low compared to trypsinogen or amylase, the output of a l l these enzymes increased similarly in response to pancre­ atic stimulation by secretin-cholecystokinin or by secretin-pentagastrin (13). α-Mannosidase activity has been detected also in the pan­ creas of sheep and ox, in the wall of the stomach, ileum, and co­ lon of sheep, ox, pig, rabbit and rat ( ϋ ) , and in the small intestine of monkey. The enzyme from monkey small intestine is dependent on Zn (15), a characteristic of α-mannosidases from several sources (16, J_7). The role of gastrointestinal α-manno­ sidases in vivo is not known. Mannans (including gluco- and galactomannans) are relatively resistant to mannosidase attack but are degraded readily by exo- (5_, 18) and endo-mannanases (19-22). Reports of in vivo digestion of mannans are contradictory. It is generally assumed that digestion does not occur to a sig­ nificant extent, yet some studies in animals suggest that partial digestion occurs (23-26). Hypocholesterolemic Activity of Mannans Plasma cholesterol levels in experimental animals respond to supplemental dietary cholesterol in a manner that is remarkably sensitive to the specific dietary carbohydrate. With sucrose as the sole carbohydrate in the diet, plasma cholesterol levels in chickens were shown to be double those displayed when glucose

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

19.

DAVIS

A N D LEWIS

Galactomannans

299

was the source of carbohydrate (27). However, galactomannans and glucomannans, like a number of other mucilaginous polysaccharides (28) , e l i c i t a significant hypocholesterolemic response when incorporated at low levels (o.5-3% of diet) into cholesterol-supplemented diets; an effect that has been noted in chickens and rats (29). Of the 16 crude polysaccharides evaluated by Fahrenbach et a l . , guar gum (galactomannan) and carrageenan were most effective in lowering plasma cholesterol, while pectin was much less effective. Guar gum, incorporated at 2% of diet, also lowered endogenous plasma cholesterol in chickens fed a basal casein-sucrose diet without cholesterol supplementation. Similar results have been obtained with rats although much higher dietary levels (510%) of guar gum or pectin were required to significantly lower serum and liver cholesterol (29). Total rat liver lipids were also lowered by these high levels of guar. Supplementation of cholesterol did not cause the dramatic increases in serum and liver cholesterol and total liver lipids in the rat compared with the levels observed with the casein-sucrose diet. Nevertheless dietary guar gum lowered liver cholesterol and total lipid levels (29) and was more effective in this respect than pectin. Studies to determine the mechanism of this hypocholesterolemic activity of mannans have been limited to konjac mannan (glucomannan), an important foodstuff of the Japanese. The mechanism and the structural requirements, however, probably apply as well to the galactomannans and other polysaccharides which show such activity. In vivo studies with rats (30) demonstrated that konjac mannan decreases intestinal absorption of bile salts by interference with the active transport mechanism. Bile acid transport in everted sacs prepared from rat ileum, the site of active transport, was decreased up to 50% by the presence of 0.25% konjac mannan in the mucosal medium (Table I). Active transport of cholic acid is very low in the proximal jejunum of the rat intestine and passive diffusion of cholate through everted sacs prepared from this region was not altered by konjac mannan. In vitro studies (30) also f a i l to give evidence of binding of bile salts by konjac mannan. Thus the diffusion rate of cholate and taurocholate across a cellophane membrane was not altered by either the glucomannan or pectin. With adult rats on a hypercholesterolemia diet, 4-'C-cholesterol transport into the plasma and liver was significantly lowered by dietary konjac mannan (31). Infusion studies with anesthetized rats demonstrated the same effect. Thus the hypocholesterolemic activity of konjac mannan in the rat appears to be due to an inhibition of cholesterol absorption in the jejunum and bile salt absorption in the ileum. Inhibition of bile salt absorption is reversed when the konjac mannan concentration drops below a minimum level.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

ι αυ ι c

ι

10183 + 678 10550 + 790 13120 + 640 11030 + 590 11070 + 1620

216 + 37 1300 + 130 470 + 30 1140 + 110 680 + 80

613 + 97 2970 + 210 930 + 80 2340 + 520 1360 + 180

6

5

5

5

5

+KM

Control

+KM

Control

+KM

4

1 Female

2

3

4

0.25| Konjac mannan (KM) added to mucosal medium. Mucosal fluid (5ml) was labeled with 0.01 yCi of C-cholate. Incubation time, 40 min. Journal of Nutrition

8900 + 473

402 + 41

1241 + 302

6

Control

3

a

11268 + 238

161 + 36

403 + 86

6

+KM

3

2

1 Male

Sac no.

•ι Μ

Effect of Konjac Mannan on C-Cholic Acid Transport in Everted Sacs from the Distal One-fourth of the Small Intestine of Male Rat (30) Total radioactivity re­ Total radioactivity maining in mucosal transported to the No. of fluid (A)/100 mq sac serosal fluid (A) sacs Addition (dpm) (dpm) (dpm) Control 6 1132 + 96 378 + 22 9285 + 1162

19.

DAVIS

A N D LEWIS

Galactomannans

301

Certain structural and physical characteristics of the polysaccharide are required for cholesterol-depressing activity. Native konjac mannan is a water-soluble high molecular weight glucomannan and both features are required for activity (32). Hypocholesterolemic activity was increased by purification, demonstrating that the polysaccharide and not a contaminant is the active agent (.33). Hydrolysis of the glucomannan with cellulase or acid completely eliminated the hypocholesterolemic effect; this effect was evident even when cellulase was incorporated in the diet containing the native konjac mannan (33). Hypocholesterolemic activity is also lost by irreversibly coagulating native water-soluble polysaccharide with lime water (32). Konnyaku, a popular food in Japan, is prepared by such an alkaline treatment. Edible water-insoluble konnyaku has no hypocholesterolemic activity in rats. It has also been shown with locust bean gum preparations (29) that viscosity and water solubility influenced th high viscosity preparations were more effective. Thus the variations in hypocholesterolemic activity of certain crude gums reported by different investigators may reflect differences in the molecular weight, water-solubility and amount of polysaccharide in the crude gum as affected by plant species, growing conditions or processing methods. From studies with konjac glucomannan and guar and locust bean galactomannan, i t is apparent that certain high molecular weight, water-soluble polysaccharides incorporated into hypercholesterolemic diets have the effect of lowering serum and liver cholesterol in certain experimental animals (rat, chicken, rabbit). In the rat konjac glucomannan activity derives from an as yet unexplained interference with active transport of cholesterol in the jejunum and bile acids in the ileum. Growth Inhibition and Toxicity Early studies showed that growth of chickens was inhibited when their diet contained locust bean or guar meal (34-36). Vohra and Kratzer (36) reported growth in chicks of only 61-68% of the controls after 20 days on a diet containing 2% guar gum, and growth of 75% of the controls on diets containing 2% locust bean gum. No growth depression was observed when the guar gum was predigested with enzymes before feeding. Although other investigators have also noticed some reduction in body weight gain of chicks on diets containing mucilaginous polysaccharides, Fahrenbach et aj^ (28) were unable to repeat the results of Vohra and Kratzer. In these studies (28) chicks fed 2% guar flour incorporated into diets similar to those used previously (36) showed an insignificant increase in body weight compared with controls. However, levels of 5-10% guar in either a 1% cholesterol-supplemented casein-sucrose or commercial basal diet did cause a reduction in weight gain in rats (29).

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

302

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

Ershoff and Wells (37) found no significant depression in growth of rats fed a casein-sucrose basal diet containing 10% guar or locust bean gum and 1% cholesterol for 28 days. In summary, some depression of growth is observed in animals fed high levels of guar and locust bean gum but the results are widely variable (25, 38, 39). This is a general phenomenon, however, which is typical of a wide variety of mucilaginous polysaccharides. With increased interest in nutrition and the safety of food additives the pertinent literature on guar (25) and locust bean gum (38, 39) have been reviewed and animal studies have been carried out to evaluate the toxicological status of guar (40-43) and locust bean gum (44-46). Ninety-day toxicity studies were conducted with rats fed diets containing 1, 2 and 5% guar gum (40). Gross and microscopic examination did not reveal pathological changes which could be attributed to ingestio and survival of the rats were not adversely affected. Serum enzyme activities (glutamic-oxaloacetic transaminase, glutamicpyruvic transaminase and alkaline phosphatase), hematology values, blood sugar levels and urine composition were unaltered compared with controls. Blood urea nitrogen was slightly elevated. At a l l levels of dietary guar, the relative weight of the caecum was increased. This appears to be a phenomenon common to most mucilaginous polysaccharides as well as some starches and can be attributed to the hydration properties of the gums. As in previous studies, body weights tended to be somewhat lower in the guar-fed animals compared with the controls. A similar ninety-day toxicity study of locust bean gum was also conducted (44). The results were generally the same as with guar. The only significant changes in the test animals compared with the controls were in the increased caecum weight and the somewhat lower body weights of females on the 1 and 5% locust bean diets. Other Physiological Effects Polysaccharides when injected into the body e l i c i t various physiological effects. Two such activities are interferon-stimulation and tumor inhibition. Previous studies (47, 48) have shown that various agents such as bacteria and their endotoxins stimulate interferon release from leucocytes. Since both the bacteria and their endotoxins contain polysaccharides, i t has been suggested that the constituent polysaccharides are responsible for the stimulation. In vitro studies with mouse peritoneal cells demonstrated that purified mannans and mannan-protein complexes from Candida albicans when incorporated into the medium stimulated interferon release. However, the mannan from !S. cerevisiae was inactive (49). Lower molecular weight mannans (MW 20,000) showed the

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

19.

DAVIS

A N D LEWIS

Galactomannans

303

greatest activity which could explain why the higher molecular weight Saccharomyces mannan was inactive. Interferon-stimulation also occurred in in vivo studies although the results were less reproducible than with in vitro studies. The mechanism by which interferon release is activated by the mannans has not been established. The role of a variety of polysaccharides in tumor inhibition has been a subject of continuing interest. Mannans from baker's yeast (50) and from Candida util is (51, 52) injected intraperitoneal ly inhibited growth of implanted tumor cells such as sarcoma-180 in mice. More recently baker's yeast mannan has been shown to inhibit development of 3-methylcholanthrene-induced epithelial tumors in mice when daily intraperitoneal injections of mannan were started 10 days prior to application of the carcinogen to the skin (53). Acute toxicity, which was evident in intravenous but not in intraperitoneal injectio tion of the carboxymethyl derivative of the mannan (54). Enhancement of the cellular antibody response of the host animal has been considered to be the general mechanism by which the mannans act. Labeled carboxymethyl mannan accumulated to some extent in the solid tumor (sarcoma-180) and in various organs but was most strongly accumulated by the liver and spleen (52). Mannose Absorption and Metabolism Absorption and Reabsorption. When mannose was administered to rats by stomach tube, i t was absorbed at only 12.3% the rate of glucose (55). Mannose was weakly accumulated in everted hamster intestinal slices and accumulation was not stimulated by Na , indicating that mannose js passively absorbed rather than actively transported by the Na -dependent carrier. Presumably the failure of mannose to be actively transported can be attributed to its deviation from the D-glucopyrano configuration at C-2 (56). In a study with everted sacs of rat intestine i t was also found (57) that mannose was not actively transported but i t was metabolized in the serosal fluid. In contrast to the low absorption of mannose from the small intestine, its reabsorption by the luminal membrane of the proximal tubule of dog kidney was comparable to that of glucose, which is well conserved. The evidence suggests the presence of two separate sets of binding sites on the luminal surface, one for glucose and a different one for mannose (58). +

Mannose Metabolism. It appears that mannose is metabolized almost as readily as glucose. Indeed, mannose has even been shown (59) to be a suitable substrate for metabolism by isolated rat brain, an organ which is usually thought to have an absolute requirement for glucose. The metabolism of mannose has been reviewed by Herman (11, 60). Pathways of mannose metabolism

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

304

PHYSIOLOGICAL

EFFECTS O F FOOD

CARBOHYDRATES

are outlined in Figure 3 . Mannose is phosphorylated to mannose 6-phosphate (Man-6-P) by hexokinase in an ATP-requiring reaction. Phosphorylation coefficients for mannose by rat brain ( 6 1 ) and rat adipose tissue ( 6 2 ) hexokinases were 0 . 6 5 and 1 . 0 , respectively, compared to glu­ cose as 1 . 0 , indicating that the affinity of hexokinase for man­ nose is high. In human erythrocytes ( 6 3 ) mannose is phosphory­ lated by an enzyme that is electrophoretically indistinguishable from the glucose-phosphorylating enzyme. Mannose phosphorylation is competitively inhibited by glucose, and glucose phosphoryla­ tion is competitively inhibited by mannose. Furthermore, Man-6P ( 1 mM) ηoncompetitive!y inhibits the phosphorylation of mannose. This finding is interesting in view of the earlier report ( 6 4 ) that Man-6-P ( 8 . 5 mM), unlike glucose 6-phosphate (G-6-P), had no inhibitory effect on mannose phosphorylation by rat brain hexo­ kinase. Recently, Arnold et a l . ( 6 5 ) noted that bee hexokinase was inhibited by G-6-P bu the failure of Man-6-P t sulted in a severe reduction in the ATP level, accounts for mannose toxicity in honeybees. Man-6-P is converted to fructose 6-phosphate (Fru-6-P), an intermediate in glycolysis, by phosphomannose isomerase. This enzyme has been shown {66_ 6 7 ) to be distinct from phosphogj^cose isomerase. Interestingly, phosphomannose isomerase is a Zn metalloenzyme and inhibited by EDTA ( 6 8 - 7 0 ) . There is evidence that the enzyme may play an important role in the regulation of glycolysis. Thus, when isolated rat brain was perfused with either mannose or glucose, there was a build-up of Man-6-P rela­ tive to Fru-6-P ( 5 9 J . When human erythrocytes were incubated with mannose, Man-6-P also accumulated (Figure 4 ) , indicating that phosphomannose isomerase rather than hexokinase is the rate limiting enzyme ( 6 3 ) . Conversion of Man-6-P to mannose 1-phosphate (Man-l-P) is catalyzed by phosphomannomutase, which is apparently distinct from phosphoglucomutase. Either glucose 1,6-diphosphate or man­ nose 1,6-diphosphate must be present for the reaction to occur ( 7 1 ) . Man-l-P is transformed into guanosine diphosphate mannose (GDP-Man), a sugar nucleotide, by reaction with guanosine t r i ­ phosphate. GDP-Man is of physiological importance since (a) i t serves as substrate for mannosyl transferase and (b) i t can be transformed into guanosine diphosphate fucose, the substrate for fucosyl transferase ( 7 2 ) . It appears that mannosyl transferase^ from |çveral sources require a divalent cation, particularly Mn or Mg , for their activity ( 7 3 - 7 6 ) . Glycosyl transferases, which are probably located along the endoplasmic reticulum, are responsible for the incorporation of glycosyl residues, one at a time, into various glycoprotein, glycolipid and mannan acceptors 9

( 7 7 ) .

There is considerable evidence that polyprenyl mannosyl phosphates are involved as intermediates in the transfer of mannose

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

19.

DAVIS

A N D LEWIS

305

Galactomannans

Hexokinase

Phosphomanno> Man-6-P _

Man-

ATP

Fru-6-P isomerase Zn

Lysosomal a-mannosidase

Phosphomannomutase

Zn

Man-l-P

Glycoproteins

A

4V

Guanosine diphosphomannose phosphorylase

Mannosyl transferase

GDP-Fuc

GDP-Man.

Mannol ipid
NADPH

Man-l-P-CH CH CHMeCH (CH CH=CMeCH ) H 2

2

2

2

2 lg

Mannopyranosyl dolichyl phosphate Glycoproteins Figure 3. Mannose metabolism

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

306

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

5r

Journal of Clinical Investigation

Figure 4. Incubation of human erythrocytes with 10 mM mannos (63)

residues from GDP-Man to both mannans and glycoproteins. The participation of mannolipid intermediates in glycosylation reactions was i n i t i a l l y observed in bacterial systems (78-80). Thus, Lennarz and his colleagues (78, 79) reported the formation of mannolipid during incubation of cell-free extracts of Micrococcus jlysodeiktieus with GDP-Man in the presence of Mn or of Mg . They subsequently demonstrated its role as an intermediate in the transfer of mannose from GDP-Man to the non-reducing termini of a membrane-associated mannan (80). It now appears that mannolipids are also involved as intermediates in glycoprotein biosynthesis in mammalian systems (81-84). Indeed, in a c e l l free system from hamster liver, mannolipid was five times more effective than GDP-Man in donating mannose to endogenous proteins (84)· Recent studies (85, 86) with a S. cerevisiae enzyme, which mediated mannosyl transfer to endogenous proteins, indicated that only those mannose residues which were linked to serine or threonine were incorporated via a lipid intermediate, with subsequent mannose units transferred directly from GDP-Man. The role of mannolipid intermediates in mannan biosynthesis in microbial systems as well as the occurrence of mannolipids in plant and animal systems has been reviewed (87). Evans and Hemming (88]T~identified mannolipid isolated from pig liver endoplasmic reticulum as dolichyl mannosyl phosphate. Herscovics et a l . (89) recently found that polyprenyl mannosyl phosphates from calf pancreatic microsomes and human lymphocytes contained a 3-mannosidic linkage. An earlier report (90) indicated that the calf pancreas mannolipid was similar to dolichyl (

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

19.

DAVIS

A N D LEWIS

Galactomannans

307

α-mannosyl phosphate, but at that time synthetic dolichyl β-mannosyl phosphate was not available for comparison. The prepara­ tion of the latter was recently described (91). Effect of Mannose on Glycogen Formation. The glycogenic ability of mannose was f i r s t noted by Deuel et j l L (92.) in 1938. They reported a small increase in liver glycogen following oral administration of mannose to rats. However, glucose was more efficiently converted into glycogen even when i t was given in an amount comparable to the rate of mannose absorption. Bailey and Roe (93) also showed that mannose was much less glycogenic than glucose when given orally to rabbits but that when given paren­ te rally the rate of conversion was similar to that of glucose. Incorporation of label from C-mannose into glycogen by rat adi­ pose tissue has been demonstrated in vitro (94, 95.). Effect of Mannose o (93) that oral administration of mannose led to elevated blood glucose and the appearance of mannose in the peripheral venous blood of the rabbit. However, data for only one animal were given. More recently, i t was found (96) that following oral in­ fusion of mannose in rabbits there was no elevation in blood glu­ cose and only a slight increase in the blood mannose level. In­ travenously injected mannose also failed to produce an elevation in blood glucose. Effect of Mannose on Insulin Secretion. The ability of man­ nose to stimulate release of insulin from the pancreas in vi tro is well known. Grodsky et ajk_ (97) claimed that mannose was as effective as glucose in stimulating insulin secretion in isolated rat pancreas. However, in studies with fetal rat pancreatic expiants (98) and with pieces of rabbit pancreas (99), the stim­ ulatory effect of mannose was only half that of glucose. Using fragments of rat pancreas, Malaisse et aJN_ (100) found that man­ nose at various concentrations induced insulin secretion to an extent that was somewhat less marked than that produced by glu­ cose. Recently i t was shown (101) that insulin release from iso­ lated rat pancreas during a 60-minute period of constant mannose infusion was diphasic, with an i n i t i a l spurt in insulin output followed by a gradual decline. This suggested that mannose trig­ gers insulin secretion but not its biosynthesis. Fructose in­ creased the insulin response elicited by mannose. In an in vivo study with rabbits, Nijjar et; aJL_ (96_) found that the serum insu­ lin level was greatly elevated immediately following an intrave­ nous injection of mannose. Furthermore, i t appears that orally administered mannose elicited a slow, very slight, and probably insignificant insulin response despite the authors' conclusion that oral mannose failed to stimulate insulin release. In sup­ port of the hypothesis that only metabolizable sugars stimulate insulin secretion, Jarrett and Keen (102) reported that C-man-

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

308

PHYSIOLOGICAL

EFFECTS

O F FOOD CARBOHYDRATES

nose was readily metabolized by isolated rat islets of Langerhans as determined by C0« recovery. Several studies nave shown that insulin exerts a stimulatory effect on mannose utilization in vitro by rat adipose tissue. Thus, Wood et a l . (94) reported that insulin increased the incorporation of radioactivity from either C-labeled mannose or glucose into fatty acids, glyceride-glycerol, and glycogen to a simi l a r extent. Goodman (103) found that the uptake of C-mannose by adipose tissue, the-, in corporation of C into fatty acids, and the production of C0 was enhanced by insulin and, to a lesser extent, by growth hormone. Finally, Kuo and Dill (95) showed that insulin as well as several proteolytic enzymes that mimic certain of its effects stimulated the incorporation of label from C-mannose into C0 , fatty acids, glyceride-glycerol, protein and glycogen. 2

2

Literature Cited

1. Smith, F. and Montgomery, R. "The Chemistry of Plant Gums and Mucilages," Reinhold, New York (1959). 2. Glicksman, M. "Gum Technology in the Food Industry," Aca­ demic Press, New York (1969). 3. Whistler, R. L., ed. "Industrial Gums," 2nd ed., Academic Press, New York (1973). 4. Lee, Y.C. Fed. Proc. (1971) 30, 1223. 5. Snaith, S.M. and Levvy, G.A. in Advan. Carbohyd. Chem. Bio­ chem. (1973) 28, 401. 6. Conchie, J. and Hay, A.J. Biochem. J. (1963) 87, 354. 7. Sukeno, T., Tarentino, A.L., Plummer, Jr., T.H., and Maley,F. Biochemistry (1972) 11, 1493. 8. LaBadie, J.H. and Aronson, Jr., N.N. Biochim. Biophys. Acta (1973) 321, 603. 9. Marsh, C.A. and Gourlay, G.C. Biochim. Biophys. Acta (1971) 235, 142. 10. Dewald, B. and Touster, O. J. Biol. Chem. (1973) 248, 7223. 11. Herman, R.H. Am. J. Clin. Nutr. (1971) 24, 488. 12. McMaster, W., Desbaillets, L., and Menguy, R. Proc. Soc. Expt. Biol. Med. (1970) J35, 87. 13. Desbaillets, L., McMaster, W.C., and Menguy, R. Proc. Soc. Expt. Biol. Med. (1971) 136, 597. 14. Conchie, J. and Macdonald, D.C. Nature (1959) 184, 1233. 15. Seetharam, B. and Radhakrishnan, A.N. Indian J. Biochem. Biophys. (1972) 9, 59. 16. Snaith, S.M. and Levvy, G.A. Biochem. J. (1968) 106, 53P. 17. Snaith, S.M. and Levvy, G.A. Nature (1968) 218, 91. 18. Jones, G.H. and Ballou, C.E. J. Biol. Chem. (1968) 243, 2442. 19. Eriksson, K.-E. and Winell, M. Acta Chem. Scand. (1968) 22, 1924. 20. Emi, S., Fukumoto, J., and Yamamoto, T. Agr. Biol. Chem. (1972) 36, 991.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

19.

DAVIS

AND

LEWIS

Gatoctomannans

309

21. Tsujisaka, Y., Hiyama, K., Takenishi, S., and Fukumoto, J. Nippon Nogei Kagaku Kaishu (1972) 46, 155. 22. Sugiyama, N., Shimahara, H., Andoh, T., and Takemoto, M. Agr. Biol. Chem. (1973) 37, 9. 23. Krantz, J.C., Carr, C.J. and de Farson, C.B. J. Am. Diet. Assoc. (1948) 24, 212. 24. Wisconsin Alumni Research Foundation, Assay Rept. No. 3110860 and 3110861 (1964). 25. Battelle Columbus Laboratories, NTIS Rept. No. PB-221216 (1972). 26. Bains, G.S. Food Sci. (1963) 12, 344. 27. Grant, W.C. and Fahrenbach, M.J. Proc. Soc. Expt. Biol. Med. (1959) 100, 250. 28. Fahrenbach, M.J., Riccardi, B.A. and Grant, W.C. Proc. Soc. Expt. Biol. Med. (1966) 123, 321. 29. Riccardi, B.A. and Fahrenbach M.J Proc Soc Expt Biol Med. (1967) 124 30. Kiriyama, S., Enishi, Α.,and Yura, K. J. Nutr. (1974) 104, 69. 31. Kodama, T., Nakai, H., Kiriyama, S., and Yoshida, A. J. Jap. Soc. Food Nutr. (1972) 25, 603. 32. Kiriyama, S., Morisaki, H., and Yoshida, A. Agr. Biol. Chem. (1970) 34, 641. 33. Kiriyama, S., Ichihara, Y., Enishi, Α., and Yoshida, A. J. Nutr. (1972) 102, 1689. 34. Bornstein, S., Alumot, E., Mokadi, S., Nachtomi, E., and Nahari, V. Israel J. Agr. Res. (1963) 13, 25. 35. Vohra, P. and Kratzer, F.H. Poultry Sci. (1964) 43, 502. 36. Vohra, P. and Kratzer, F.H. Poultry Sci. (1964) 43, 1164. 37. Ershoff, B.H. and Wells, A.F. Proc. Soc. Expt. Biol. Med. (1962) 110, 580. 38. GRAS Food Ingredients: Carob Bean Gum (Locust Bean Gum), NTIS Rept. No. PB-221203 (1972). 39. Evaliation of the Health Aspects of Carob Bean Gum as a Food Ingredient, NTIS Rept. No. PB-221952 (1972). 40. Sub-chronic Toxicity Study with Guar Gum in Rats, Rept. No. 4095, Centraal Instituut Voor Voedingsonderzoek (1974). 41. Study of Mutagenic Effects of Guar Gum (71-16), NTIS Rept. No. PB-221815 (1972). 42. Teratologic Evaluation of FDA 71-16 (Guar Gum), NTIS Rept. No. PB-221800 (1972). 43. Teratologic Evaluation of FDA 71-16 (Guar Gum), NTIS Rept. No. 223819 (1973). 44. Sub-chronic Toxicity Study with Locust Bean Gum in Rats, Rept. No. 4093, Centraal Instituut Voor Voedingsonderzoek (1974). 45. Study of Mutagenic Effects of Locust Bean Gum (FDA 71-14), NTIS Rept. No. PB-221819 (1972). 46. Teratologic Evaluation of FDA 71-14 (Carob Bean Locust Gum), NTIS Rept. No. PB-221784 (1972).

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

310

PHYSIOLOGICAL EFFECTS OF FOOD CARBOHYDRATES

47. Borecky, L., Lackovic, V., Blaskovic, D., Masler, L., and Sikl, D. Acta Virol. (1967) JJL, 264. 48. Feingold. D.S., Youngner, J.S., and Chen, J. Biochem. Bio­ phys. Res. Comm. (1968) 32, 554. 49. Lackovic, V., Borecky, L., Sikl, D., Masler, L., and Bauer, S. Proc. Soc. Expt. Biol. Med. (1970) 134, 874. 50. Suzuki, M., Chaki, F., and Suzuki, S. Gann (1971) 62, 553. 51. Oka, S., Kumano, N., Sato, K., Tamari, K., Matsuda, K., Hirai, H., Oguma, T., Ogawa, K., Kiyooka, S., and Miyao, K. Gann (1969) 60, 287. 52. Kumano, N., Kurita, K., and Oka, S. Gann (1972) 63, 675. 53. Kumano, N., Kurita, K., and Oka, S. Gann (1973) 64, 529. 54. Oka, S., Kumano, Ν., and Kurita, K. Gann (1972) 63, 365. 55. Deuel, Jr., H.J., Hallman, L.F., Murray, S., and Hilliard, J. J. Biol. Chem. (1938) J25, 79. 56. Barnett, J.E.G., Ralph Α. and Munday K.A Biochem J. (1970) 118, 843 57. Barry, R.J.C., Eggenton, , Smyth, Physiol (London) (1969) 204, 299. 58. Silverman, M., Agánon, M.A., and Chinard, F.P. Am. J. Phy­ siol. (1970) 218, 743. 59. Ghosh, A.K., Mukherji, B., and Sloviter, H.A. J. Neurochem. (1972) 19, 1279. 60. Herman, R.H. Am. J. Clin. Nutr. (1971) 24, 556. 61. Sols, A. and Crane, R.K. J. Biol. Chem. (1954) 210, 581. 62. Hernandez, A. and Sols, A. Biochem. J. (1963) 86, 166. 63. Beutler, E. and Teeple, L. J. Clin. Invest. (1969) 48, 461. 64. Crane, R.K. and Sols, A. J. Biol. Chem. (1954) 210, 597. 65. Arnold, H., Seitz, U., and Löhr, G.W. Hoppe-Seyler's Z. Physiol. Chem. (1974) 355, 266. 66. Slein, M.W. J. Biol. Chem. (1950) 186, 753. 67. Noltmann, E. and Bruns, F.H. Biochem Ζ. (1958) 330, 514. 68. Gracy, R.W. and Noltmann, E.A. J. Biol. Chem. (1968) 243, 3161. 69. Gracy, R.W. and Noltmann, E.A. J. Biol. Chem. (1968) 243, 4109. 70. Gracy, R.W. and Noltmann, E.A. J. Biol. Chem. (1968) 243, 5410. 71. Glaser, L., Kornfeld, S. and Brown, D.H. Biochim. Biophys. Acta (1959) 33, 522. 72. Ginsburg, V. J. Am. Chem. Soc. (1958) 80, 4426. 73. Behrens, N.H. and Cabib, E. J. Biol. Chem. (1968) 243, 502. 74. Letoublon, R.C.P., Comte, J., and Got, R. Eur. J. Biochem. (1973) 40, 95. 75. Levrat, C. and Louisot, P. Can. J. Biochem. (1973) 51, 931. 76. Arnold, D., Hommel, E., and Risse, H.-J. Biochem. Biophys. Res. Comm, (1973) 54, 100. 77. Spiro, R.C. New Eng. J. Med. (1969) 281, 991. 78. Scher, M., Lennarz, W.J., and Sweeley, C.C. Proc. Natl. Acad. Sci., U.S.A. (1968) 59, 1313.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

19.

DAVIS

AND

LEWIS

Galactomannans

311

79. Lahav, Μ., Chiu, T.H., and Lennarz, W.J. J. Biol. Chem. (1969) 244, 5890. 80. Scher, M. and Lennarz, W.J. J. Biol. Chem. (1969) 244, 2777. 81. Baynes, J.W., Hsu, A.-F., and Heath, E.C. J. Biol. Chem. (1973) 248, 5693. 82. Waechter, C.J., Lucas, J.J., and Lennarz, W.J. J. Biol. Chem. (1973) 248, 7570. 83. Behrens, N.H., Carminatti, H., Staneloni, R.J., Leloir, L. F., and Cantarella, A.I. Proc. Natl. Acad. Sci., U.S.A. (1973) 70, 3390. 84. Maestri, N. and DeLuca, L. Biochem. Biophys. Res. Comm. (1973) 53, 1344. 85. Babczinski, P. and Tanner, W. Biochem. Biophys. Res. Comm. (1973) 54, 1119. 86. Lehle, L. and Tanner W Biochim Biophys Acta (1974) 350, 225. 87. Lennarz, W.J. and Scher, M.G. Biochim. Biophys. Acta (1972) 265, 417. 88. Evans, P.J. and Hemming, F.W. Fed. Eur. Biochem. Soc. Lett., (1973) 31, 335. 89. Herscoviçs, Α., Warren, C.D., Jeanloz, R.W., Wedgwood, J.F., Liu, I.Y., and Strominger, J.L. Fed. Eur. Biochem. Soc. Lett. (1974) 45, 312. 90. Tkaez, J.S., Herscovics, Α., Warren, C.D., and Jeanloz, R.W. Biochem. Soc. Trans. (1973) 1, 1174. 91. Warren, C.D., Liu, I.Y., Herscovics, Α., Wedgwood, J.F., and Jeanloz, R.W., Am. Chem. Soc. Abstracts, Atlantic City (1974). 92. Deuel, Jr., H.J., Hallman, L.F., Murray, S., and Hilliard, J. J. Biol. Chem. (1938) 125, 79. 93. Bailey, W.H. and Roe, J.H. J. Biol. Chem. (1944) 152, 135. 94. Wood, Jr., F.C., Leboeuf, B., Renold, A.E., and Cahill, Jr., G.F. J. Biol. Chem. (1961) 236, 18. 95. Kuo, J.F. and Dill, I.K. Biochim. Biophys. Acta (1969) 177, 17. 96. Nijjar, M.S. and Perry, W.F. Diabetes (1970) J9., 155. 97. Grodsky, G.M., Batts, Α.Α., Bennett, L.L., Vcella, C , McWilliams, N.B., and Smith, D.F. Am. J. Physiol. (1963) 205, 638. 98. Lambert, A.E., Junod, Α., Stauffacher, W., Jeanrenaud, B., and Renold, A.E. Biochim. Biophys. Acta (1969) 184, 529. 99. Coore, H.G. and Randle, P.J. Biochem. J. (1964) 9j$, 66. 100. Malaisse, W., Malaisse-Lagae, F., and Mahy, M. Ann. Endo­ crinol. (Paris) (1969) 30, 595. 101. Curry, D.L. Am. J. Physiol. (1974) 226, 1073. 102. Jarrett, R.J. and Keen, H. Metabolism (1968) 17, 155. 103. Goodman, H.M. Endocrinology (1967) 80, 45.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

20 Metabolism and Physiological Effects of Pectins WANDA L. C H E N O W E T H and GILBERT A. L E V E I L L E Food Science and Human Nutrition, Michigan State University, East Lansing, Mich. 48824

Non-digestible dietary carbohydrate y much attention. Although most of this attention has focused on the importance of cereal sources of dietary fiber, poorly digested pectic substances derived primarily from fruit and vegetable sources likewise may be of significance in relation to human health. Pectic substances are complex, colloidal carbohydrate deriv­ atives which occur in or are isolated from plants. They contain a large proportion of anhydrogalacturonic acid units, most likely combined in a chain-like arrangement. The carboxyl groups of the polygalacturonic acids may be partly esterified or may form salts with various cations (1, 2). Pectin is a general term usually employed to designate water-soluble pectinic acids of varying methyl ester content and degree of neutralization capable of forming gels with sugar and acids (1). The amount of pectic substances in several common fruits and vegetables is shown in Table I. Estimation of the total amount of pectic substances in an average diet is not possible because data are available only for a limited number of foods. Comparison of the content of pectic substances in various foods is further com­ plicated by differences in analytical methods and incomplete descriptions of the foods. In addition to pectic substances found naturally in plant foods, pectin may be added to foods during processing. The most common use of pectin is in making jams and jellies; however pectin is an acceptable additive in a number of other foods. Pectins also have found numerous uses in the prepar­ ation of various pharmaceutical products. Much of what i s known about the p h y s i o l o g i c a l e f f e c t of p e c t i n has evolved as a consequence o f i n t e r e s t i n the use of p e c t i n f o r therapeutic purposes. P e c t i n and combinations o f p e c t i n w i t h other c o l l o i d s have been used e x t e n s i v e l y to t r e a t d i a r r h e a l d i s e a s e s , e s p e c i a l l y i n i n f a n t s and c h i l d r e n (3 - 6).

J o u r n a l A r t i c l e No. Station.

7046

, Michigan A g r i c u l t u r a l Experiment 312

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

20.

CHENOWETH

AND

LEVEILLE

Pectins

313

This use of p e c t i n had i t s o r i g i n i n the treatment of d i a r r h e a w i t h a d i e t of scraped apples, a home-remedy p r a c t i c e d f o r hundreds of years i n Europe and introduced i n t o t h i s country i n 1933 by Birnberg (7). The e f f e c t i v e n e s s of p e c t i n i n t r e a t i n g d i a r r h e a subsequently l e d to i n v e s t i g a t i o n s to determine the mechanism of i t s e f f e c t and i t s f a t e i n the alimentary t r a c t . Experiments i n dogs showed that when 20g p e c t i n was fed i n combination w i t h a mixed d i e t , only 10 percent of the p e c t i n could be recovered i n the f e c e s ; however, i f the same amount was fed during f a s t i n g an average of 50 percent was excreted (Table I I ) . Results obtained w i t h humans fed 50g of p e c t i n d a i l y w i t h a mixed d i e t were s i m i l a r to those i n dogs w i t h approximately 90 percent apparent decomposition of p e c t i n t a k i n g place (8, 9 ) . The degree of decomposition appears to be i n f l u enced by the r e t e n t i o n time i n the i n t e s t i n e , adjustment of the animal to the d i e t , and the degree of e s t e r i f i c a t i o n of the p e c t i n (9, 10). Tests made w i t h huma enzymes i n s a l i v a and g a s t r i c j u i c e which could act on p e c t i n . L i k e w i s e , t r y p s i n , pepsin and rennet had no e f f e c t on p e c t i n i n v i t r o ; however p e c t i n incubated w i t h feces was r a p i d l y decomposed (11). Results of s t u d i e s i n animals and humans w i t h i l e o s t o m i e s i n d i c a t e d that the breakdown of p e c t i n occurs c h i e f l y i n the colon most l i k e l y by the a c t i o n of b a c t e r i a l enzymes ( 9 ) . I s o l a t i o n of the microorganisms which are capable of decomposing p e c t i n r e vealed that the most a c t i v e groups were A e r o b a c i l l u s , L a c t o b a c i l l u s , Micrococcus and Enterococcus (12, 13). The c h i e f products formed during b a c t e r i a l fermentation are carbon d i o x i d e and formic and a c e t i c a c i d . I f g a l a c t u r o n i c a c i d i s produced i t apparently i s broken down r a p i d l y s i n c e only a s m a l l amount i s present i n i n c u b a t i o n mixtures. Although a b a c t e r i c i d a l a c t i o n of p e c t i n has been proposed to e x p l a i n the e f f e c t i v e n e s s of p e c t i n i n t r e a t i n g d i a r r h e a , most experimental r e s u l t s do not support t h i s theory (14, 15). However, recent evidence suggests that under c e r t a i n i n v i t r o c o n d i t i o n s , p e c t i n s may have a s l i g h t a n t i m i c r o b i a l a c t i o n toward E. c o l i (16). Most of the recent i n t e r e s t i n p e c t i n has been i n r e l a t i o n to i t s e f f e c t on l i p i d metabolism. In 1957 L i n and coworkers (17) f i r s t reported that i n r a t s the a d d i t i o n of p e c t i n to a b a s a l d i e t c o n t a i n i n g c h o l e s t e r o l increased the e x c r e t i o n of f e c a l s a p o n i f i able and non-saponifiable l i p i d s and decreased the absorption of exogenous c h o l e s t e r o l . About the same time Keys and coworkers (18) proposed that the lower incidence of a t h e r o s c l e r o s i s associ a t e d w i t h " I t a l i a n - t y p e d i e t s " might be r e l a t e d to the amount of complex carbohydrates such as p e c t i n , h e m i c e l l u l o s e and f i b e r present i n the f r u i t s and vegetables abundant i n these d i e t s . Subsequent i n v e s t i g a t i o n s have thus focused on the e f f e c t of p e c t i n on serum and l i v e r c h o l e s t e r o l concentrations and the f e c a l e x c r e t i o n of l i p i d and s t e r o l s . Numerous experiments i n r a t s (Table I I I ) have shown that

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

314

PHYSIOLOGICAL

EFFECTS

OF FOOD

CARBOHYDRATES

TABLE I PECTIN CONTENT OF VARIOUS FOODS Food

P e c t i c substances %

Apples

fresh basis II II

Bananas

0.5

-

1.6

0.7 - 1.2

Peaches

It

It

0.1 - 0.9

Strawberries

II

tl

0.6 - 0.7

Cherries

II

II

0.2 - 0.5

Green peas

II

Carrots Orange pulp

dry matte It tt

II

12.4 - 28.0

Potato

n

it

it

1.8 - 3.3

Tomato

tt

tt

tl

2.4 - 4.6

Adapted from "The P e c t i c Substances" ( 2 ) .

TABLE I I RECOVERY OF PECTIN FROM FECES BY URONIC ACID ESTIMATION Per Cent Pectin fed with basal d i e t : dogs

8.9

humans

8.7

P e c t i n f e d during f a s t i n g : dogs

51.2

humans

13.8 Adapted from Am. J. Dig. D i s . ( 9 ) .

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975. 0 Ψ Ψ >KNS) 4-(NS) Ψ Ψ + (NS) 0 *(NS)

0 Ψ + (NS)

42 28 28

28 28

28 21 28

28 28

28

18 18

12

5% 2.5% 5%

5% 10%

5% 5% 5%

5% 10%

5%

3% 3%

10%

0 1% 1%

1% 1%

1% 1% 1%

1% 1%

1%

0 1%

0

2

(25) Ψ

Ψ

τ

(23)

0 *(NS)

Ψ Ψ

τ

(33)

(36)

(22)

*(NS) Ψ Ψ

Ψ Ψ Ψ

*

(20)

Ψ Ψ

Ψ Ψ

0

(19)

(17)

0 0 Ψ

f

Ref

0 Ψ Ψ

τ

Bile Acids

NS = no s t a t i s t i c a l l y

significant difference

Results expressed as increase o r decrease compared to r e s u l t s i n r a t s on same d i e t without p e c t i n

50 mg/day

6

Dietary Pectin

500 mg/day

Dietary Cholesterol

E f f e c t of P e c t i n on Plasma and F e c a l L i p i d s i n Rats"^ Fecal Liver Plasma No. Days Total Total on d i e t C h o l e s t e r o l Lipids Sterols Lipids Cholesterol

TABLE I I I

316

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

a d d i t i o n of 3 to 10% p e c t i n to a d i e t c o n t a i n i n g 1% c h o l e s t e r o l counteracts the increase i n l i v e r c h o l e s t e r o l and l i v e r t o t a l l i p i d induced by c h o l e s t e r o l feeding (19-25). A decrease i n serum c h o l e s t e r o l i s u s u a l l y observed as a r e s u l t of p e c t i n supplement a t i o n although i n some experiments the decrease has not been s t a t i s t i c a l l y s i g n i f i c a n t . P r o t o p e c t i n and p e c t i n s w i t h a low methoxy content do not appear t o be e f f e c t i v e i n lowering serum or l i v e r c h o l e s t e r o l . Supplements of tomato p e c t i n have been reported to produce a smaller decrease i n l i v e r c h o l e s t e r o l than c i t r u s p e c t i n (25); however c i t r u s p e c t i n N.F. and apple p e c t i n apparently a r e e q u a l l y e f f e c t i v e (19). An a n t i - h y p e r c h o i e s t e r o l e m i c a c t i o n of p e c t i n has a l s o been reported i n chickens (26-28) and swine (29). I n chickens the a d d i t i o n of 3 to 5% p e c t i n to the d i e t caused a marked increase i n f e c a l e x c r e t i o n of c h o l e s t e r o l and t o t a l l i p i d s i r r e s p e c t i v e of the c h o l e s t e r o l content of the d i e t (26 27) I n guinea p i g s and hamsters p e c t i n apparentl lowering e f f e c t (30). Althoug to a s i g n i f i c a n t decrease i n plasma c h o l e s t e r o l i n male, but not female, r a b b i t s (31), short-term feeding had no e f f e c t (30). In most experiments p e c t i n has been found t o produce an e f f e c t on c h o l e s t e r o l and l i p i d concentrations only i n animals r e c e i v i n g added d i e t a r y c h o l e s t e r o l (19, 29, 32). However, Mokady (33) has r e c e n t l y reported that i n short term feeding experiments i n r a t s , s u b s t i t u t i o n of 10% p e c t i n f o r s t a r c h i n a c h o l e s t e r o l - f r e e d i e t caused a f i v e - t o t e n - f o l d increase i n f e c a l t o t a l l i p i d s and doubled o r t r i p l e d s t e r o l e x c r e t i o n . A s m a l l decrease i n serum c h o l e s t e r o l was observed but r e s u l t s were s i g n i f i c a n t f o r only one of the p e c t i n s t e s t e d , a h i g h molecular weight c i t r u s p e c t i n (Table I V ) . Few i n v e s t i g a t i o n s have been conducted i n humans to evaluate the e f f e c t of p e c t i n supplementation. Keys and coworkers (18) f e d middle-aged men c o n t r o l l e d d i e t s of n a t u r a l foods w i t h and without a d d i t i o n of 15g d a i l y of e i t h e r c e l l u l o s e o r p e c t i n . A three-week p e r i o d of p e c t i n supplementation r e s u l t e d i n a f a l l i n the mean c o n c e n t r a t i o n s of serum c h o l e s t e r o l to l e v e l s approximately 5% below the l e v e l on the same d i e t without p e c t i n (Table V). C e l l u l o s e supplementation, however, f a i l e d t o show any s i g n i f i c a n t e f f e c t on serum c h o l e s t e r o l concentrations. I n a study by Palmer and Dixon (34) an attempt was made to determine the e f f e c t i v e dose of p e c t i n which i s r e q u i r e d to reduce blood c h o l e s t e r o l concentrat i o n s . Sixteen men were f e d v a r y i n g amounts of p e c t i n during s i x 4-week t e s t p e r i o d s . Other d i e t a r y v a r i a b l e s and r i s k f a c t o r s a s s o c i a t e d w i t h a t h e r o s c l e r o s i s were not c o n t r o l l e d . Although not a l l subjects responded e q u a l l y w e l l t o p e c t i n supplementation, d a i l y doses of 8 to 10g p e c t i n caused a s i g n i f i c a n t decrease i n serum c h o l e s t e r o l of these men, a l l of whom had i n i t i a l values which were e i t h e r normal o r only s l i g h t l y elevated. E v a l u a t i o n of the e f f e c t of d i e t a r y p e c t i n on plasma and f e c a l l i p i d s i n humans was made i n an i n v e s t i g a t i o n conducted a t

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

20.

CHENOWETH

AND LEVEILLE

317

PectlHS

TABLE IV EFFECT OF PECTIN ON BLOOD CHOLESTEROL AND FECAL LIPIDS IN RATS FED A CHOLESTEROL-FREE DIET Relative Blood Cholesterol

Control 2 Low MW P e c t i n High MW P e c t i n Low M P e c t i n

1

values f o r

Fecal Lipids

Total Fecal Sterols

%

%

%

100

100

100

91

458

278

76

735

372

86

390

222

83

478

293

Pectin S P e c t i n MR

"Average values f o r 8 rats/group expressed as a percentage of c o n t r o l values ( f o r animals f e d a p e c t i n - f r e e d i e t ) MW = molecular weight; M = methoxy; S = slow s e t t i n g ; MR = medium-rapid s e t t i n g Adapted from Nutr. Metabol. (33).

TABLE V MEAN SERUM CHOLESTEROL CONCENTRATIONS IN 24 MEN FED 15gPECTIN/DAY Major source of d i e t a r y carbohydrate

Serum c h o l e s t e r o l , mg % No P e c t i n

+ Pectin

Legumes

202.4

192.7

Sucrose

221.5

211.3

Δ -9.7 -10.2

Adapted from Proc. Soc. Exper. B i o l . and Med. (18).

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

318

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

the U n i v e r s i t y of Iowa (35). During an i n i t i a l p e r i o d of 4 weeks three healthy male subjects were fed c o n t r o l l e d d i e t s having a composition s i m i l a r to that of the average American d i e t . During a second 5-week p e r i o d the men r e c e i v e d the same d i e t plus 20 to 23g p e c t i n N.F. which was incorporated i n t o the foods i n the d i e t . The f i n a l 5 weeks represented a second c o n t r o l and recovery p e r i o d . R e s u l t s showed that a f t e r p e c t i n feeding there was a s i g n i f i c a n t decrease of 13% i n the mean plasma c h o l e s t e r o l compared to the value f o r the c o n t r o l p e r i o d (Table V I ) . No apprec i a b l e e f f e c t on the plasma t r i g l y c e r i d e l e v e l was observed. During the p e r i o d of p e c t i n i n g e s t i o n the men showed an increase i n t o t a l f e c a l f a t , s t o o l volume, f e c a l s t e r o l and f e c a l d i g i t o nide p r e c i p i t a b l e s t e r o l s . Several mechanisms have been proposed by which d i e t a r y p e c t i n may lower plasma and l i v e r concentrations of c h o l e s t e r o l i n v a r ious s p e c i e s . P o s s i b l e mechanisms would i n c l u d e : 1) r e d u c t i o n i n c h o l e s t e r o l absorption; 3) depression of b i l e a c i mental r e s u l t s reported by L e v e i l l e and S a u b e r l i c h (22) i n d i c a t e that the most important e f f e c t of p e c t i n i n the r a t appears to be i t s i n f l u e n c e on b i l e a c i d a b s o r p t i o n . In c h o l e s t e r o l - f e d r a t s the a d d i t i o n of p e c t i n increased f e c a l b i l e a c i d e x c r e t i o n by 32%, whereas f e c a l n e u t r a l s t e r o l e x c r e t i o n was not a l t e r e d (Table V I I ) . A d d i t i o n a l experiments w i t h i n v e r t e d i n t e s t i n a l sacs demonstrated that p e c t i n decreased i n v i t r o t a u r o c h o l i c a c i d t r a n s p o r t by 50%. Further support f o r the importance of the i n h i b i t i o n of b i l e a c i d absorption was provided by the s i m i l a r i t y i n response of c h o l e s t e r o l - f e d r a t s to p e c t i n and cholestyramine, a known i n h i b i t o r of b i l e a c i d absorption (Table V I I I ) . More r e c e n t l y , however, P h i l l i p s and B r i e n (36) suggested that there may be d i f f e r ent mechanisms f o r the a c t i o n of p e c t i n and cholestyramine s i n c e i n t h e i r experiments p e c t i n d i d not a f f e c t v i t a m i n A absorption whereas cholestyramine i s known to l i m i t absorption of the v i t a min. ^ D i e t a r y p e c t i n somewhat decreases absorption of c h o l e s t e r o l 4- C i n c h o l e s t e r o l - f e d r a t s , as evidenced by decreased deposit i o n of r a d i o a c t i v e c h o l e s t e r o l i n the l i v e r and increased f e c a l e x c r e t i o n of c h o l e s t e r o l - 4 - ^ C (22). However, impaired cholest e r o l absorption induced by d i e t a r y p e c t i n apparently i s only p a r t i a l l y r e s p o n s i b l e f o r the hypochoiesterolemic e f f e c t of p e c t i n . Since p e c t i n e f f e c t i v e l y lowers l i v e r c h o l e s t e r o l even when c h o l e s t e r o l and p e c t i n are fed s e p a r a t e l y on a l t e r n a t e days (19, 22), impaired absorption of the added c h o l e s t e r o l would not appear to be the most c r i t i c a l means by which p e c t i n exerts i t s e f f e c t . In r a t s fed a c h o l e s t e r o l - f r e e d i e t Mokady (37) r e c e n t l y reported that h e p a t i c b i o s y n t h e s i s of c h o l e s t e r o l , as measured by conversion of a c e t a t e - l - ^ ^ C to c h o l e s t e r o l , was s u b s t a n t i a l l y higher i n r a t s fed 10% p e c t i n . In v i t r o i n c o r p o r a t i o n of l a b e l i n t o t r i g l y c e r i d e s , phospholipids and t o t a l l i p i d s i n the l i v e r was a l s o s i g n i f i c a n t l y higher i n the p e c t i n - f e d animals. The

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

20.

CHENOWETH AND

LEVEILLE

319

PectlUS

TABLE VI CHANGES IN PLASMA AND FECAL LIPIDS OF THREE MEN IN RESPONSE TO PECTIN SUPPLEMENTATION Periods

Plasma c h o l e s t e r o l , mg %

Ï

II

Basal Diet

Basal Diet + 20-23 g Pectin

226

±

6.8

84

±

12.8

1.06

±

0.12

1

III Basal Diet

2

196

±

9.9

90

±

10.9

1.23

±

0.12

211

±

8.4

98

±

9.5

0.82

±

0.15

91

±

18.5

Plasma, t r i g l y c e r i d e s , mg % T o t a l f e c a l f a t , g/24 h F e c a l s t e r o l s , g/24 h r Fecal digitonide precip-

69

±

8.3

126

±

3

12.4

2

i t a t e , mg/24 h r Mean

±

standard d e v i a t i o n f o r 3 s u b j e c t s .

D i f f e r s s i g n i f i c a n t l y from mean values f o r Periods I and I I I (p < 0.05). ^ D i f f e r s s i g n i f i c a n t l y from mean value f o r P e r i o d I I I (p < 0.05). Unpublished data adapted from (35).

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

PHYSIOLOGICAL

320

EFFECTS OF

FOOD

CARBOHYDRATES

TABLE V I I EFFECT OF PECTIN SUPPLEMENTATION IN CHOLESTEROL-FED RATS Cholesterol (1%) Plasma c h o l e s t e r o l ,

C h o l e s t e r o l (1%) + p e c t i n (5%)

128

±

3

1

116

±

5

7.4

±

0.3

6.6

±

0.3

10.3

±

0.5

7.5

±

0.3

142

+

5

2

141

±

8

mg/100 ml Liver f a t , % L i v e r c h o l e s t e r o l , mg/g F e c a l s t e r o l s , mg/day F e c a l b i l e a c i d s , mg/da ±

"*"Mean

SE of mean f o r 10 r a t s .

2 Values are means f o r 5 animals. Adapted from J. Nutr. (22).

TABLE V I I I COMPARISON OF THE EFFECT OF PECTIN AND CHOLESTYRAMINE IN CHOLESTEROL-FED RATS Liver Dietary treatment

1% c h o l e s t e r o l

Total Lipid

Cholesterol

Plasma Cholesterol

%

mg/g

mg/100 ml

7.9 ± 0.3

10.3 ± 0.5

128 ± 14

7.1 ± 0.4

7.2 ± 1.1

91 ± 4

5.8 ± 0.2

4.0 ± 0.2

86 ± 4

1% c h o l e s t e r o l + 5% p e c t i n 1% c h o l e s t e r o l + 1% cholestyramine

Mean f o r 5 r a t s * SE of mean Adapted from J. Nutr. (22).

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

20.

CHENOWETH

AND

LEVEILLE

Pectins

321

author suggested that the higher r a t e of h e p a t i c l i p o g e n e s i s observed i n the p e c t i n - f e d r a t s might be due to a r e d u c t i o n i n the absorption of d i e t a r y f a t , since absorbed l i p i d i s known to i n h i b i t hepatic f a t t y acid synthesis. The e f f e c t of d i e t a r y p e c t i n on the number and types of i n t e s t i n a l microorganisms has not been thoroughly i n v e s t i g a t e d ; however, the f a i l u r e of a n t i b i o t i c s to prevent the lowering of blood c h o l e s t e r o l by p e c t i n Ç19, 22) i n d i c a t e s that i n t e s t i n a l m i c r o f l o r a do not c o n t r i b u t e s i g n i f i c a n t l y to i t s e f f e c t . The e f f e c t of p e c t i n on the absorption of n u t r i e n t s other than l i p i d s has received l i t t l e a t t e n t i o n . In r a t s the u t i l i z a t i o n of 3-carotene and absorption of v i t a m i n A was not impaired by p e c t i n supplementation (36). A d d i t i o n of 12 or 24% p e c t i n to d i e t s of r a t s has been found to decrease the d i g e s t i b i l i t y of prot e i n (38). V i o l a and coworkers (39) reported that slow s e t t i n g p e c t i n (55% e s t e r i f i e d ) decreased the apparent d i g e s t i b i l i t y of p r o t e i n but d i d not impai 10% medium-rapid-actin ment of p r o t e i n u t i l i z a t i o n as shown by a decrease i n weight gain per gram of digested p r o t e i n . Both p e c t i n preparations decreased the apparent r e t e n t i o n of calcium by approximately 30%. In summary, experimental r e s u l t s i n v a r i o u s species i n d i c a t e that ingested p e c t i n i s n e a r l y completely broken down i n the colon most l i k e l y by b a c t e r i a l enzymes. The products formed apparently are not e x t e n s i v e l y u t i l i z e d s i n c e p e c t i n makes a n e g l i b l e c o n t r i b u t i o n to the energy value of the d i e t (39). The d i g e s t i b i l i t y and u t i l i z a t i o n of p e c t i n , however, needs to be re-evaluated using more s e n s i t i v e and s p e c i f i c methods. The lowering of plasma and l i v e r c h o l e s t e r o l concentrations by p e c t i n supplementation appears to be r e l a t e d p r i m a t i l y to i t s e f f e c t on b i l e a c i d absorption. R e s u l t s showing an increased e x c r e t i o n of b i l e a c i d s would suggest the p o s s i b i l i t y of an increased h e p a t i c conversion of c h o l e s t e r o l to b i l e a c i d s thus reducing serum and l i v e r c h o l e s t e r o l concent r a t i o n s . By removing the feedback i n h i b i t o r of c h o l e s t e r o l on HMG-CoA reductase these changes would e x p l a i n the increase i n h e p a t i c b i o s y n t h e s i s of c h o l e s t e r o l which has been observed. Further i n v e s t i g a t i o n s are needed to evaluate c h o l e s t e r o l synt h e s i s and turnover as w e l l as the a c t i v i t y of enzymes important i n the r e g u l a t i o n of c h o l e s t e r o l s y n t h e s i s .

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

322

PHYSIOLOGICAL EFFECTS OF FOOD CARBOHYDRATES

Literature Cited

1. Baker, G.L., G.H. Joseph, Z.I. Kertesz, H.H. Mattern and A.G. Olsen. Chem. Eng. News (1944) 22, 105-106. 2. Kertesz, Z.I. "The Pectic Substances", pp. 281-329. Interscience Publishers, Inc., New York, 1951. 3. Howard, P.J. and C.A. Tompkins. J. Amer. Med. Assoc.(1940) 114, 2355-2358. 4. Hunt, J.S. Arch. Ped. (1936) 53, 736-739. 5. Washburn, G. J. Am. Dietet. Assoc. (1938) 14, 34-38. 6. Kutscher, G.W. and A Blumberg Am J. Dig Dis (1939) 6 717-720. 7. Birnberg, T.L. Am. J. Dis. Child. (1933) 45, 18-24. 8. Werch. S.C. and A.C. Ivy. Proc. Soc. Exper. Biol. Med. (1940) 44, 366-368. 9. Werch, S.C. and A.C. Ivy. Am. J. Dig. Dis. (1941) 8, 101-105. 10. Gilmore, N.M. "Effect of 5 percent pectin N.F. or 5 percent pectin L.M. upon growth, excretion, serum proteins and min­ eral contents in liver and kidney tissues of weanling male rats", Ph.D. Dissertation, Michigan State University, 1965. 11. Kertesz, Z.I. J. Nutr. (1940) 20, 289-296. 12. Werch, S.C., A.A. Day, R.W. Jung and A.C. Ivy. Proc. Soc. Exper. Biol. (1941) Med. 46, 569-572. 13. Werch, S.C., R.W. Jung, A.A. Day, T.E. Friedemann and A.C. Ivy. J. Infect. Dis. (1942) 70, 231-242. 14. Erschoff, B.H. and H.B. McWilliams. Am. J. Dig. Dis. (1945) 12, 21-22. 15. Prickett, P.S. and N.J. Miller. Proc. Soc. Exper. Biol. Med. (1939) 40, 27-28. 16. El-Nakeeb, M.A. and R.T. Yousef. Planta Med. (1970) 18, 201209. 17. Lin. T.M., K.S. Kim, E. Karvinen and A.C. Ivy. Am. J. Physiol. (1957) 188, 66-70.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

20.

CHENOWETH

AND

LEVEILLE

Pectins

323

18. Keys, Α., F. Grande and J.T. Anderson. Proc. Soc. Exper. Biol. Med (1960) 106, 555-558. 19. Wells, A.F. and B.H. Ershoff. J. Nutr. (1961) 74, 87-92. 20. Ershoff, B.H. and A.F. Wells. Exper. Med. Surg (1962) 20, 272-276. 21. Ershoff, B.H. and A.F. Wells. Proc. Soc. Exper. Biol. (1962) Med. 110, 580-582. 22. Leveille, G.A. and H.E. Sauberlich. J. Nutr. (1966) 88, 209214. 23. Riccardi, B.A. and M.J. Fahrenbach. Proc. Soc. Exper. Biol. Med. (1967) 124, 749-752 24. Karvinen, E. and M. Miettinen. Acta Physiol. Scand. (1968) 72, 62-64. 25. Anderson, T.A. and R.D. Bowman. Proc. Soc. Exper. Biol. Med. (1969) 130, 665-666. 26. Fisher, H., P. Griminger, H.S. Weiss and W.G. Siller. Science (1964) 146, 1063-1064. 27. Fisher, Η., W.G. Siller and P. Griminger. J. Atheroscler. Res. (1966) 6, 292-298. 28. Griminger, P. and H. Fisher. Proc. Soc. Exper. Biol. Med. (1966) 122, 551-553. 29. Fisher, H., G.W. van der Noot, W.S. McGrath and P. Griminger. J. Atheroscler. Res. (1966) 6, 190-191. 30. Wells, A.F. and B.H. Ershoff. Proc. Soc. Exper. Biol. Med. (1962) 111, 147-149. 31. Fisher, Η., P. Griminger and W.G. Siller. J. Atheroscler. Res. (1967) 7, 381-386. 32. Fisher, H., P. Griminger, E.R. Sostman and M.K. Brush. J. Nutr. (1965) 86, 113-119. 33. Mokady, S. Nutr. Metabol. (1973) 15, 290-294. 34. Palmer, G.H. and D.G. Nixon. Am. J. Clin. Nutr. (1966) 18, 437-442. 35. Hopson, J.J. "Studies on the effect of dietary pectin on plas-

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

324

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

ma and fecal lipids." M.S. Thesis, University of Iowa, 1967. 36. Phillips, W.E.J, and R.L. Brien. J. Nutr. (1970) 100, 289292. 37. Mokady, S. Nutr. Metabol. (1974) 16, 203-207. 38. Gadeken, D. Archiv. Tierenahrung (1960) 19, 409-420. 39. Viola, S., G. Zimmerman and S. Mokady. Nutr. Rep. Internat. (1970) 1, 367-375.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

21 The Physiological Effects of Dietary Fiber JAMES SCALA Thomas J. Lipton, Inc., Englewood Cliffs, N. J.

In the last centur ed more dramatically than either fat or sugar consumption has increased. The9%decline in consumption of cereal fiber compares to a30%increase in fat and a50%increase for sugar. Part of the decline in fiber is probably due to a shift from "crude" sources of carbohydrate such as whole grain cereal, and bread to the "more refined" modern counterparts. Table 1 summarizes the consumption dietary fiber obtained from cereal, potatoes, legumes and fruits and vegetables, as calculated by H. C. Trowell (1) and the author (2). An analysis of the data indicates the change in fiber consumption is largely confined to cereals which have decreased by 75 to 90% fiber from potatoes has decreased by about40%from legumes about20%,while fiber from fruits and vegetables has changed very little. Table 1 Estimates o f D a i l y F i b e r Consumption i n The U.S. i n 1880, 1964, and 1974 F i b e r Source

1880 (1 ) 1964 (1) 1974

Cereals Potatoes Legumes F r u i t s and Vegetables

3.2 1.1 1.0 2.8

0.3 0.5 1.0 3.3

0.8 0.6 0.6 2.0

Total Fiber

8.1

5.1

3.0

(2)

These changes, brought t o the f o r e by B r i t i s h epidemiolog i s t s Drs. Dennis B u r k i t t , Hugh Trowell and the surgeon Dr. N e i l P a i n t e r , r a i s e the question o f what has been s a c r i f i c e d by e l i m i n a t i n g so much c e r e a l f i b e r from our d i e t ( 3 ) . Is f i b e r a f o r gotten n u t r i e n t ? D i e t a r y f i b e r i s defined as p l a n t m a t e r i a l which i s r e s i s t ant t o d i g e s t i o n by the s e c r e t i o n s o f the human g a s t r o i n t e s t i n a l

325 In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

326

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

t r a c t . However, n u t r i t i o n i s t s u s u a l l y speak o f crude f i b e r . "Crude f i b e r " i s the m a t e r i a l l e f t a f t e r treatment with hot a c i d and a l k a l i e , a method o f f i b e r a n a l y s i s developed to t e s t animal feeds f o r undigestable m a t e r i a l . In short, t o p r o t e c t the farmer against poor feed purchases. Consequently, we are l e f t with a value that has l i t t l e , i f any, q u a n t i t a t i v e meaning i n terms of human n u t r i t i o n . Processed g r a i n s c o n t a i n v a r i o u s amounts o f c e l l u l o s e , h e m i c e l l u l o s e , p e c t i n and l i n g i n s , a l l of which are defined as f i b e r ; t h e i r q u a n t i t i e s v a r y with v a r i e t y , p r o c e s s i n g , climate and other f a c t o r s . Consequently, d i e t a r y f i b e r , the und i g e s t i b l e p l a n t carbohydrates, i s from 2 t o 6 times the crude f i b e r content o f food (k). This impreciseness i s a challenge t o food t e c h n o l o g i s t s , and speaks f o r the need t o f i n d a method which w i l l a c c u r a t e l y i d e n t i f y the amount o f " d i e t a r y f i b e r " i n food. I w i l l review the major p h y s i o l o g i c a l e f f e c t s of d i e t a r y fiber. T r a n s i t Time F i b e r increases s t o o l frequency and decreases t r a n s i t time of m a t e r i a l s p a s s i n g through the l a r g e i n t e s t i n e . These two e f f e c t s are i n d i r e c t r e s u l t s of the water b i n d i n g a b i l i t y o f f i b e r and have been demonstrated by comparing s t o o l t r a n s i t times to those on a r e f i n e d , low f i b e r d i e t ; f o r example, an E n g l i s h or U. S. d i e t . Table 2 summarizes data taken from studies by Burk i t t i n which t r a n s i t time was evaluated as a f u n c t i o n o f diet.(5) These comparisons o f p o p u l a t i o n groups have been c l i n i c a l l y e v a l uated and demonstrate that a d i e t h i g h i n f i b e r increases both s t o o l weight and frequency. The increased weight i s due t o the water b i n d i n g c a p a c i t y o f f i b e r which c a r r i e s h t o 6 times i t s weight through the l a r g e i n t e s t i n e . This water i s "bound" i n the s o l i d phase (7) and; consequently, a h i g h f i b e r d i e t w i l l r e s u l t i n l a r g e r and more frequent e l i m i n a t i o n - or i n l a y termsregularity (7). Table 2 T r a n s i t Time as a F u n c t i o n o f D i e t Subject

Diet

T r a n s i t Time

Ugandan V i l l a g e r s and Students

Unrefined

33

35 hours

E n g l i s h Vegetarians, South A f r i c a n P u p i l s and Indian Nurses

Mixed

h2

hours

E n g l i s h Students and Navy Personnel

Refined

69 hours

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

21.

SCALA

327

Dietary Fiber

T r a n s i t times reported i n Table 2, were evaluated by the method o f Hinton (6) i n which radiopaque p e l l e t s ( r i c e g r a i n i n s i z e ) are f e d to volunteers and are observed by X-ray o f the passed, c o l l e c t e d , s t o o l s . Hinton defines t r a n s i t time as the time r e q u i r e d t o pass 80& o f the p e l l e t s . D i v e r t i c u l a r Disease D i v e r t i c u l a r disease a f f e c t s about 20& of a d u l t s i n the United States - and with increased l o n g e v i t y , i t s numbers i n crease every year. Estimates by B u r k i t t and Trowell i n d i c a t e that i t i s growing at a r a t e of l6& per year ( l ) . T h i s r a t e can be questioned because i t i s not age and l o n g e v i t y compensated. Table 3 summarizes h e a l t h s t a t i s t i c s i n the U. S. which demons t r a t e the age r e l a t i o n s h i p o f d i v e r t i c u l a r disease ( 8 ) . Other e p i d e m i o l o g i c a l surveys have shown c o r r e l a t i o n s of d i v e r t i c u l o s i s with a p p e n d i c i t i s , (9) v a r i c o s Table 3 D i v e r t i c u l a r Disease as a Function of

Age

Incidence

Age Under 35

^5 - 5^ 60 - 70 over

70

10& 25&

D i v e r t i c u l o s i s i s c h a r a c t e r i z e d by small defects which develop as bulges i n the w a l l of the colon; they are s i m i l a r i n appearance to the bubble which appears at a weak p o i n t on an i n f l a t e d rubber tube. C l i n i c a l l y , they are s a c c u l a r outpouchings o f the mucosa and submocosa which extrude through defects i n the muscularis. As these evaginations f i l l with i n t e s t i n a l contents or gas, they become i n f e c t e d and i n extreme cases, gangrenous. In a l l cases, they are p a i n f u l and u s u a l l y r e q u i r e surgery, i n which the diseased p o r t i o n of the i n t e s t i n e i s removed - a process which o f t e n i n c l u d e s a temporary colestomy. In 1967 Dr. N e i l P a i n t e r found that a high f i b e r d i e t would r e l i e v e the symptoms of d i v e r t i c u l o s i s and could, i n some cases, obviate the need f o r surgery ( l l ) . Of the 70 cases he t r e a t e d with a high f i b e r d i e t , 62 could be spared from surgery. Data from Dr. P a i n t e r s o r i g i n a l 62 p a t i e n t s ( t a b l e k) show that previous t o the high f i b e r regimen the d i v e r t i c u l a r p a t i e n t s had i r r e g u l a r , infrequent, hard s t o o l s , and the simple procedure o f t a k i n g about 15 grams of bran d a i l y i n three servings of two tablespoons each, produced r e g u l a r i t y and eliminated the symptoms. The means by which f i b e r r e l i e v e s , the symptoms of d i v e r t i c u l o s i s has been more c l e a r l y shown by Eastwood who s t u d i e d d i v e r t i c u l a r p a t i e n t s on a high f i b e r d i e t and compared them t o a T

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

328

PHYSIOLOGICAL

group o f normal h e a l t h y volunteers

EFFECTS

OF

FOOD

CARBOHYDRATES

(7).

Table k Bowel Habits of 62 P a t i e n t s Before and A f t e r Taking B r a n ( l 2 ) Bowel Habit (Frequency of E l i m i n a t i o n s ) Irregular Every 3 days Every 2 days Once d a i l y Twice d a i l y Three times d a i l y Frequent s t o o l s

Before Bran

13 7 8 28 3 none

3

After Bran

31 25 6 none

The d i v e r t i c u l a r p a t i e n t firmed P a i n t e r ' s f i n d i n g s and e x h i b i t e d : l ) more normal waste t r a n s i t times when put on a bran d i e t v e r i f y i n g P a i n t e r s observ a t i o n s , 2) t h e i r i n t r a c o l o n i c pressure was reduced to more normal values s i m i l a r t o the observations of Hodgson, (13) and 3) the r a t i o of bound water to l i q u i d water i n the feces becomes more s i m i l a r to the normal p a t i e n t s . These observations suggest that f i b e r i s e f f i c a c i o u s by t r a n s p o r t i n g a l a r g e r volume o f water s t o o l s i n t o the l a r g e i n t e s t i n e . These s o f t e r , b u l k i e r s t o o l s reduce the i n t r a c o l o n i c pressure p r e v e n t i n g d i s t e n t i o n o f the d i v e r t i c u l a . However, i t i s more important t h a t f i b e r probably prevents the development o f high i n t r a c o l o n i c pressures which l e a d t o the formation of d i v e r t i c u l a . In a d d i t i o n , a high f i b e r d i e t may induce a much stronger i n t e s t i n a l musculature c o n t a i n i n g fewer weak spots which are p o t e n t i a l s i t e s o f d i v e r t i c u l a . This prev e n t i v e p o t e n t i a l f o r f i b e r could be t e s t e d by animal s t u d i e s . 1

Cardiovascular Disease The low incidence of ischaemic heart disease and low serum c h o l e s t e r o l among p o p u l a t i o n groups on an u n r e f i n e d , high residue d i e t has been a t t r i b u t e d , i n p a r t , to the hypocholesterolemic e f f e c t o f n o n d i g e s t i b l e carbohydrate ( l U ) . The d i e t o f these people i s o f t e n , but not always, low i n f a t ; long term studies with South A f r i c a n White and Bantu p r i s o n e r s on c o n t r o l l e d d i e t s has confirmed the hypocholesterolemic e f f e c t o f f i b e r (15). Comp a r a t i v e s t u d i e s of p o p u l a t i o n groups are summarized on Table 5·

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

21. se A L A

329

Dietary Fiber Table 5 Serum C h o l e s t e r o l i n Population Groups Where D i e t D i f f e r s i n F i b e r Content (ik) (15) (l6)

Group

Blood Cholesterol

Diet

New Guinea (Male) New Guinea (Female) New Guinea (Male) New Guinea (Female) Non Vegetarians Lacto Vegetarians Vegan Vegetarians T r a p p i s t Monks Benedictine Monks

Unrefined Native Unrefined Native Western Refined Western Refined Western Vegetarians (Dairy) S t r i c t Vegetarian Lacto Vegetarian Mixed

lOÇjmg^ 13*+ 183 187 291 256 206 l80 225

Other s t u d i e s hav hypocholesterolemic o f d i e t a r y f i b e r . In general, these s t u d i e s i n d i c a t e that a d d i t i o n o f f i b e r t o the d i e t reduces serum c h o l e s t e r o l by preventing absorption o f d i e t a r y c h o l e s t e r o l and by i n c r e a s i n g the e l i m i n a t i o n o f b i l e a c i d s ; thereby, the removal o f h e p a t i c synthesized c h o l e s t e r o l . These f i n d i n g s have been confirmed by extensive animal s t u d i e s (33), (3J0. These s t u d i e s are summarized i n Table 6. In general, these s t u d i e s have been done over short periods and have u t i l i z e d f i b e r as r e f i n e d c e l l u l o s e , or from n a t u r a l sources. Table 6 The E f f e c t o f D i e t a r y F i b e r on Serum C h o l e s t e r o l on Man Control Diet

Subjects Young G i r l s

(19)

(Cellulose)

226 Male Volunteers (20)

(21)

170 (Oats)

251 Male Volunteers

Experimental Diet

206

223 (Bengal Gram) l60

Although the hypocholesterolemic aspects o f f i b e r has not been q u a n t i f i e d , these s t u d i e s demonstrate i t s e f f e c t i v e n e s s as i n f e r r e d from serum c h o l e s t e r o l . F i b e r helps i n e l i m i n a t i n g both d i e t a r y and h e p a t i c c h o l e s t e r o l thereby reducing, t o some extent, one major r i s k f a c t o r o f c a r d i o v a s c u l a r disease - serum c h o l e s terol.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

330

PHYSIOLOGICAL

Colonic

EFFECTS

O F FOOD

CARBOHYDRATES

Cancer

Since f i b e r i n c r e a s e s the speed o f m a t e r i a l through the gut and the volume which i s passed, i t would be expected t o reduce the exposure time o f the gut t i s s u e s t o any non d i g e s t i b l e com­ ponent. Therefore, duductive reasoning leads us t o expect i t t o reduce the likelihood o f c o l o n i c t o x i c i t y d e r i v e d from any i n ­ gested o r p h y s i o l o g i c a l l y produced t o x i c agent. Toxic agents o f greatest concern i n the gut are carcinogens. Although s t u d i e s t o r e l a t e f i b e r , i n g e n e r a l , t o the reduc­ t i o n o f cancer have been u n s u c c e s s f u l , the recent e p i d e m i o l o g i c a l e v a l u a t i o n o f Drs. I r v i n g and Drasar have shown a s i g n i f i c a n t a l b i e t small, negative c o r r e l a t i o n with c o l o n i c cancer and c e r e a l consumption ( 2 2 ) . Although the c o r r e l a t i o n s account f o r a small p o r t i o n o f c o l o n i c cancer, i n t u i t i o n and epidemiology are i n such obvious agreement that the t r e n d i s s i g n i f i c a n t and a milestone i n epidemiology. Thes Table 7 C o r e l a t i o n o f Cancer o f the Colon With Consumption of Various F i b e r Containing Foods (22) Correlation Coefficient

Source o f F i b e r Cereals Potatoes and Starches Pulses Nuts and Seeds Vegetables Fruit

-0.30 -0.07 +0.07 +0.05 +0.22

Statistical Significance 0.10^P^0.05 N.S. N.S. N.S. N.S.

C o l o n i c cancer i s second only t o lung cancer as a k i l l e r among cancers. I t occurs l e a s t f r e q u e n t l y i n populations with a h i g h residue u n r e f i n e d d i e t such as i n C e n t r a l A f r i c a ( 2 3 ) . This has been observed by e p i d e m i o l o g i s t s who have evaluated b l a c k s l i v i n g i n A f r i c a against b l a c k s i n the U. S. o r Japanese i n Japan t o those i n the U. S. These data presented i n Table 8 are taken from evaluations by Dr. Robert D o l l at Oxford, and have been con­ firmed by other e p i d e m i o l o g i c a l surveys (2h). Colon-Cancer

Table 8 Incidence i n Males by Race and Country

Country U. S. ( C a l i f o r n i a ) U. S. (Hawaii) U. S. (Hawaii) Rhodesia Japan (Rural) South A f r i c a Nigeria

Race Black Caucasian Japanese Black Japanese Black Black

Incidence 69.8 68.0 66 Λ 18.2 11.8 10.8 5·8

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

21.

se A L A

331

Dietary Fiber

Colonic cancer i s highest i n c o u n t r i e s which e x h i b i t a high incidence o f c a r d i o v a s c u l a r disease and the two are h i g h l y c o r r e ­ l a t e d (25). Therefore, since c h o l e s t e r o l i s the most widely accepted r i s k f a c t o r i n c a r d i o v a s c u l a r disease, i t i s a common b a s i s on which to compare the two d i s e a s e s . Rose observed 5 groups o f people with high r i s k f o r c a r d i o v a s c u l a r disease by t h e i r h i g h serum c h o l e s t e r o l ; then searched f o r the d i f f e r e n c e s among those who had c o l o n i c cancer (26). As Table 9 i n d i c a t e s , the c o l o n i c cancer p a t i e n t s e x h i b i t e d lower than expected serum c h o l e s t e r o l l e v e l s f o r a group at r i s k , as compared to other cancers o f the alimentary system. Table 9 Serum C h o l e s t e r o l D e v i a t i o n s i n Men Who Died o f Alimentary Carcinoma* Site

Mea +0.20 -0.13 -O.512

A l l cancer except colon Colon Colon

^-Adapted from Rose

( W h i t e h a l l Study) (Whitehall Study) (General E l e c t r i c Study)

(26).

Dr. E r i k B j e l k e had conducted a r e t r o s p e c t i v e and prospec­ t i v e study on the i n t e r r e l a t i o n s h i p between d i e t and c h o l e s t e r ­ o l (27). The group e x h i b i t e d an elevated c h o l e s t e r o l ; however, those with lower c h o l e s t e r o l e x h i b i t e d the higher incidences o f c o l o n i c cancer. As Table 10 i n d i c a t e s , t h i s r e l a t i o n s h i p t e s t e d out c o n s i s t e n t l y over the f i v e year p e r i o d during which the group was followed confirming the f i n d i n g s o f Rose and adding the v a l ­ uable dimension o f p r o s p e c t i v e epidemiology. Table 10 R e l a t i v e Risk f o r C o l o n i c Cancer As a Function o f Serum C h o l e s t e r o l *

Mean C h o l e s t e r o l

Retrospective Relative Risk

291 287 271

Prospective Relative Risk

1.0 1Λ 2.1

^Adapted

from B j e l k e

1.0 2.3 2.0 (27).

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

332

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

Other forms o f suspected d i e t r e l a t e d cancer d i d not e x h i b i t t h i s same p a t t e r n - adding more confirmation t o the f i n d i n g ; t h a t i s , c o l o n i c cancer among high serum c h o l e s t e r o l c a r d i o v a s c u l a r disease r i s k p a t i e n t s i s i n v e r s e with serum c h o l e s t e r o l . These s t u d i e s r a i s e an obvious question, "What do these people pass i n t o t h e i r l a r g e i n t e s t i n e s which w i l l reduce serum c h o l e s t e r o l below i t s expected value?" One component i s obviousb i l e a c i d s , d i e t a r y s t e r o l s and f a t . Since 19^0, i t has been known t h a t deoxydiolate, a b i l e a c i d by-product i s a weak carcinogen (28). High f i b e r u n r e f i n e d d i e t s produce a spectrum o f b i l e s a l t s which are s u b s t a n t i a l l y d i f f e r ­ ent from those observed on a r e f i n e d low residue d i e t . This i s i l l u s t r a t e d by comparing the b i l e a c i d by-products i n r u r a l A f r i c a n s t o Europeans, as done by Dr. James F a l a i y e o f N i g e r i a (29). His data (Table 11) shows t h a t there i s more deoxycholate i n the feces o f people on low residue than on high residue d i e t s

B i l e S a l t Ratios and D i e t * Population Group

Diet

N i g e r i a n Adults E n g l i s h Adults

Unrefined Refined

Cholate 1.5 l.h

Data adapted from F a l a i y e

ChenοDeoxycholate

DeoxyCholate

1.0 1.0

O.k 0.9

(29).

These b i l e a c i d by-products are produced by the anaerobic m i c r o f l o r a . People on a r e f i n e d d i e t have a much greater popu­ l a t i o n o f anaerobes i n t h e i r l a r g e i n t e s t i n e (29). This i s apparent by c o n t r a s t i n g the r a t i o o f i n t e s t i n a l aerobes of Black Ugandins to Blacks i n the U. S. (30), (31) Along with the b i l e a c i d hypothesis, components of the food i t s e l f should be considered. Nitrogen metabolites ranging from ammonia, n i t r o s o compounds and other amines have been i m p l i c a t e d (32). T h e i r production s i m i l a r l y depends upon transformations by the i n t e s t i n a l f l o r a and the l o g i c o f development f o l l o w s a p a t t e r n s i m i l a r to the one considered f o r b i l e a c i d s . A v i r u s hypothesis i s s i m i l a r except t h a t t r a n s i t time and volume are the cogent f a c t o r s . In each hypothesis, f i b e r emerges as a preven­ t i v e agent. Conclusions The r o l e o f f i b e r i n the d i e t i s emerging as a preventive agent f o r problems o f the alimentary and v a s c u l a r system. This c o n c l u s i o n i s supported by epidemiology, d i r e c t experimentation, and i n f e r e n c e s from d i e t a r y p a t t e r n s i n ethnic and socio-economic

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

21.

SCALA

Dietary Fiber

333

groups. More research i s r e q u i r e d by food s c i e n t i s t s on f i b e r as a food component and by b i o m e d i c a l s c i e n t i s t s on the l o n g term e f f e c t s o f f i b e r i n the d i e t . I n view o f t h e complexity o f t h e d i e t a r y relevance such s t u d i e s should be l o n g term and cover s e v e r a l age groups. Much work remains t o be done on animals; e s p e c i a l l y f e e d i n g s t u d i e s on primates.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

334

PHYSIOLOGICAL

Literature

EFFECTS

O F FOOD

CARBOHYDRATES

Cited

1Burkitt, D.P., Walker, A.R.P., Painter, N.S., Effect of Dietary Fiber on Stools and Transit Times and Its Role in the Cau­ sation of Disease. Lancet, (1972),1408,ii. 2Revelle, R., Food and Population. Scientific American, (1974), 160, 231. Increases in Food Can Help to Create Conditions That Stabilize Population Levels. 3Painter, N.S. and Burkitt, D.P., Diverticular Disease of the Colon: A Deficiency Disease of Western Civilization. British Medical Journal (1971), 2, 450-454. 4Williams, R.D. and Olmsted, W.H., A Biochemical Method for Det­ ermining Indigestible Residue in Feces. Lingin, cellulose, and non-water-soluble hemicelluloses. J. Biol. Chem. (1936) 108, 653-666. 5Burkitt, D.P., Walker A.R.P and Painter N.S. Dietary Fiber and Disease. J.A.M.A 6Hinton, J.M., Lennard-Jones, Young, , Studying Gut Transit Times Using Radio-Opaque Markers. J. Br. Soc. Gastroentrol, (1969), 10, 842-847. 7Eastwood, M.A., Findlay, J.M., Mitchell, W.D., Smith, A.N. and Anderson, A.J.B., Effects of Unprocessed Bran on Colon Fun­ ction in Norman Subjects and in Diverticular Disease. Lancet (1974),146-149,i. 8Brown, J. W., Colonic Diverticulosis in the Aged. J. Am. Geri­ atrics Soc. (1969), 17, 366-370. 9Burkitt, D.P. The Aetology of Appendicitis. Br. J. Surg. (1971) 58, 695-699, i. 10Latto, C., Gilmore, O.J.A. and Wilkinson, R.W., Diverticular Disease and Varicose Veins. Lancet, (1973) 1089. 11Burkitt, D.P., James, P.Α., Low-Residue Diets and Hiatus Hernia. Lancet (1973), 128-130, 2. 12Painter, N.S., Almeida, A.Z., and Colebourne, K.W., Unprocessed Bran in Treatment of Diverticular Disease of the Colon. British Medical Journal, (1972), 729-731, 3. 13Hodgson, J., Effect of Methylcellulose on Rectal and Colonic Pressures in Treatment of Diverticular Disease. British Medical Journal, (1972),729-731,3. 14Lyken, R. and Janse, A.A.J., The Cholesterol Level in Blood Serium of Some Population Groups in New-Guinea. Trop. Geogr. Med., (1960), 145-148, 12. 15Hardinge, M.G., Chambers, A.C., Crooks, H. and Stare, F.J., Nutritional Studies of Vegetarians III Dietary Levels of Fiber, Am. J. Clin. Nutr., (1958), 523-525, 6. 16Groen, J.J., Tijong, K.B., Koster, M., Willebrands, A.F., Verdonck, G., and Pierloot, Μ., The Influence of Nutrition and Ways of Life on Blood Cholesterol and the Prevalence of Hypertension and Cornary Heart Disease Among Trappist and Behedictine Monks. Amer. J. Clin., (1962), 456-47O, 10.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

21.

SCALA

Dietary

Fiber

335

17Keys, Α., Anderson,J.T.,and Grande, F., Diet-Type (fats constant) and Blood Lipids in Man. J. Nutr., (1970), 257266, 70. 18Antonis, A. and Bersohn, I,, The Influence of Diet on Fecal Lipids in S. Afr. white and Bantu Prisoners. Am.J.Clin. Nutr., (1962), 142-155 ,11. 19Shurpaleakar, K.S., et. al., Effect of Cellulose in an Athero­ genic Diet on the Blood Lipids of Children. Nature, (1971), 554-555, 232. 20de Groot, A.P., Luyken, R., and Pikaar, N.A., CholesterolLowering Effect of Rolled Oats. Lancet, (1963), 303-304, 2. 21Mathur, K.S., Khan, M.A. and Sharma, R.D., Hypocholesterolaemic Effect of Bengal Gram; a Long Term Study in Man. Brit. Med. J., (1968),30-31,1. 22Irving, D., Drasar B.S. Cancer Br J., (1973) 462, 28 23Burkitt, D.P., Epidemiolog Cancer, (1971) 3-13, 28. 24Doll, R., Cancer. Br. J., (1969), 1, 23. 25World Health Organization Statistics. Vol. 1, (1969). 26Rose, G. et. al., Lancet, (1974, 7850, i . 27Bjelke, Ε., Lancet, (1974, 1116, i . 28Cook. J. W., Kennaway, N.M., Production of Tumors by Deoxycholic Acid. Nature, (1940), 627, 145. 29Hill, M.J. et. al., Bacteria and the Etiology of Cancer of the Large Bowel. Lancet, (1971), 95-100, 1. 30Aires, V., et. al., Bacteria and Etiology of Cancer of the Large Bowel. Gat, (1969), 334-335, 10. 3lHill, M.J. et. al., Bacteria and Etiology of Cancer of the Large Bowel. Lancet, (1971), 95-100, i . 32Visek, W.J., Some Biochemical Considerations in Utilization of Non-specific Nitrogen. Agr. Food Chem., (1974), 174-184, 22. 33Trowell, Η., Ischemic Heart Disease and Dietary Fiber. The Am. J. of Clin. Nutr., (1970), 926-932, 25. 34KBruns, P., Nutritional, Microbiological and Physicochemical Studies on Chemically Modified Tapioca Starch Thesis, (1974, Cornell University.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

22 Digestibility of Food Polysaccharides by Man: A Review A L L E N E JEANES Northern Regional Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Ill. 61604

Introduction Polysaccharides that are commonly ingested by man but are not digested have been indicated in this symposium to be s i g n i f ­ icant i n the human d i e t . Nondigestible polysaccharide constitu­ ents of foods are beneficial in providing bulk, binding water and certain toxic substances (1), and i n exerting hypocholester­ olemic action (1, 2, 3). Polysaccharide food additives such as carrageenan (4) and the alginates and xanthan (5) serve advan­ tageous functions i n food products through t h e i r specific physical and c o l l o i d a l properties without regard to digestibility. In this review, further consideration i s given to the digest­ ibility of the great variety of other polysaccharide food addi­ tives that serve to impart aesthetic appeal and processing and marketing advantages to foods. Since these substances usually suffice i n low concentrations, the question of t h e i r digestibility has more import physiologically than n u t r i t i o n a l l y . Included i n t h i s consideration are polysaccharides that have been proposed for use i n foods but do not now have significant application as w e l l as several types that are present naturally in foods but are poorly utilized. A t t e n t i o n i s g i v e n f i r s t , however, to the food p o l y s a c c h a r i d e s t h a t are d i g e s t e d and to the b a s i s f o r t h e i r d i f f e r e n t i a t i o n from those t h a t are n o n d i g e s t i b l e . Review o f these f a c t s d i s c l o s e s areas where i n f o r m a t i o n i s l a c k i n g and f u r t h e r r e s e a r c h i s needed. Before d i s c u s s i n g the i n d i v i d u a l p o l y s a c c h a r i d e s i t i s necessary to define d i g e s t i o n and to e v a l u ­ ate the p o s t u l a t e d r o l e o f m i c r o f l o r a i n the s m a l l i n t e s t i n e .

Digestion:

Origin of Depolymerases

Digestion consists i n depolymerization of large molecules into t h e i r monomeric units which may be absorbed into the c i r c u ­ latory system for d i s t r i b u t i o n to the body tissues. Several aspects of the digestive process have been considered already i n t h i s symposium. Crane (6) depicted the generally recognized 336 In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

22.

J E A N ES

Digestibility

of Polysaccharides

337

p r i n c i p l e that, i n the small intestine, digestion and absorption of carbohydrates occur, whereas i n the large intestine (colon) sugars are not absorbed but undergo fermentation by the indigenous microbial f l o r a . Hehre (7) has pointed out that microflora may exist also i n the small intestine and would be a potential source o f digestive depolymerases, especially for nonamylaceous polysaccharides. Rackis (8) has correlated data on anaerobic fermentation o f indigestible soybean oligosaccharides (raffinose, stachyose and verbascose) i n the human i n t e s t i n a l tract with the findings of other investigators that established the identity, d i s t r i b u t i o n and action of the effective microflora i n test animals. Thus, i n the small intestine o f the dog, the greatest bacterial metabolic a c t i v i t y occurred i n the ileum and much less i n the duodenum and jejunum (8). The microbial f l o r a indigenous to the alimentary canal may vary from species to species and to some degree within a species (9). Conventional laborator microflora throughout thei several types are localized, often i n thick layers, on s p e c i f i c mucosal surfaces o f the stomach, caecum and colon, and long chains o f gram-negative rod- or coccal- shaped bacteria have been found on the v i l l o u s epithelium of the ileum (10). In the normal healthy human, biopsied tissue or fluioTTrom the lumen of the small intestine was found to contain only small numbers of microorganisms such as nonpathogenic streptococci or l a c t o b a c i l l i (11) which apparently were not growing (12). Comparisons o f germ-free and conventional rats support the conclusions that the maltase, invertase (sucrase), lactase, c e l lobiase and trehalase a c t i v i t i e s of small i n t e s t i n a l tissue homogenates come from the animal i t s e l f and that the i n t e s t i n a l microorganisms do not contribute to any major extent to their production (13, 1£, 15). Isomaltase, which was not measured spec i f i c a l l y , c h a r a c t e r i s t i c a l l y accompanies sucrase. The same conclusions resulted from comparable studies on oligo-1,6glucosidase a c t i v i t y from i n t e s t i n a l homogenates of the rat (13) and hog (13, 16). From~"Kis experimentation on the conventional p i g , Dahlqvist concluded that the dextran-degrading a c t i v i t y (dextranase ) found i n homogenates o f i l e a l mucosa did not originate i n contaminating, adhering microflora. When mucosa from the colon was tested i n the same way, dextranase a c t i v i t y from the colon as compared with that from the ileum was i n the r a t i o of 1 to 5.5 (17). As i s detailed l a t e r i n t h i s review, dextranase a c t i v i t y i n homogenates of small i n t e s t i n a l mucosa has been found consistently i n close association with sucrase and isomaltase. Extensive 1

In t h i s review dextranase designates dextran-degrading a c t i v i t y rather than a s p e c i f i c enzyme since the identity of the enzyme has not been established.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

338

PHYSIOLOGICAL

EFFECTS OF

FOOD

CARBOHYDRATES

research by numerous investigators has established the location, o r i g i n and development of the c e l l s of the small i n t e s t i n a l brushborder membrane i n which the disaccharidases sucrase-isomaltase, maltase, lactase and several others occur. The sucrase-isomaltase complex has been v i s u a l i z e d i n s i t u i n biopsy specimen of human small i n t e s t i n a l mucosa by immunofluorescence staining (18). Among reviews on the disaccharidases of the brush border are those of Gray (19) i n t h i s symposium and of Prader and Auricchio (20). Digestion of polysaccharides i s dependent upon the presence of the requisite s p e c i f i c hydrolytic enzymes (s) and the absence of s p e c i f i c inhibitors or chemical or physical aspects of the polysaccharide that could interfere with enzyme action. The only digestive polysaccharidases that have been established i n man are the α-amylases of s a l i v a and pancreatic juice and a particle-bound (21) glucoamylase (£, 19) of small i n t e s t i n a l mucosa which i s designated γ-amylase by some investigators (21, 22) Polysaccharides Known t Amylaceous Polysaccharides. Salivary and pancreatic aamylases hydrolyze 1,4-a-D-glucopyranosidic linkages i n amylaceous polymers with formation οΐ maltose and α-limit dextrins. These fragments are hydrolyzed to glucose by saccharidases of the intes­ t i n a l brush-border membrane (19) for transport through the absorp­ t i v e c e l l s of the i n t e s t i n a l mucosa (6). α-Amylase acts on starch when both are free i n the i n t e s t i n a l cavity (23). As to i t s s p e c i f i c i t y , Whelan (24) has stated that 'There i s no evidence contrary to the belielTthat α-amylase i s e n t i r e l y s p e c i f i c for the hydrolysis o f 1,4-a-D-glucopyranosidic bonds". The report of α-amylase action on salep mannan (a water-soluble 1,4-3-D-mannan) was shown by A l l e n and Whelan (25) to be erroneous. Action of salivary and pancreatic α-amylases i s blocked by the 1,6-a-Dglucopyranosidic branch points or by substituent groups sucR as the phosphate of potato starch. Thus, the smallest fragment from α-amylolysis that contains a branch point or phosphate group i s a tetrasaccharide (24). Experimental animals usually are fed raw starches either as the granules or ground whole grain. Raw starches from various plant sources d i f f e r i n their s u s c e p t i b i l i t y to i n v i t r o action of salivary and pancreatic α-amylases (26, 27) ancT to i n vivo d i g e s t i b l i t y (28). In general, root or tuber starches are much more resistant~than are the cereal starches. Two types of nontuberous starches are, however, also unusually resistant to aamylolysis. These starches have amylose contents i n the range of 60-751 (as compared with about 25% i n most ordinary starches) and gelation temperatures i n the range o f about 130-150° C (as com­ pared with about 75-80° C for most ordinary starches). These resistant starches are from (a) a wrinkle-seeded variant of the common garden pea, Pisum sativum, and (b) hybrids from geneti­ c a l l y selected lines o f Zea mays which produce good yields of an

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

22.

JEANES

Digestibility

of Polysaccharides

339

i n d u s t r i a l l y useful high-amylose starch (29). This high-amylose corn starch i s , however, finding increasing application i n convenience foods because of i t s different physical properties (30). Starch (63% amylose) and the corresponding ground whole grain from high-amylose corn, when tested by pancreatin i n v i t r o , were 42% and 37% as d i g e s t i b l e , respectively, as normal starch cont r o l s . Under the same conditions, wrinkle-seeded pea starch (65% amylose) was about 85% as "digestible" as the normal corn starch control (26). When tested i n the r a t , the ground high-amylose corn was Tôund to be 77% as digestible as normal corn (31). Resistance of these novel starches to gelation and α-amylase action appears to r e s u l t from a combination of factors which includes unusual granule morphology, strong micellar structures, and abnor­ mally low molecular weight of both the amylose and amylopectin fractions (29). The efïêcts of granule disruption and gelatinization on the s u s c e p t i b i l i t y of high-amylos starches to α-amylolysi might be expected to be favorable. Walker and Hope (27) showed that the action of α-amylases i s much more rapid and extensive on starch pastes than on the raw granules. P a r t i a l g e l a t i n i z a t i o n increases the n u t r i t i o n a l a v a i l a b i l i t y o f starch and wheat bran for chickens (32) and f l a k i n g and steaming improves the u t i l i ­ zation of sorgnûm by c a t t l e (33). Cooking under suitable conditions i s necessary to r a i s e tEë d i g e s t i b i l i t y of dry-bean starches up to about 87% (34). Cooking provTcfes the added benefit o f diminishing or destroying the a c t i v i t y o f glycoprotein-type i n h i b i t o r s of mammalian α-amylases which occur i n certain wheats, sorghum, legumes and other plant products (32, 35). Heat-stable polyphenolic i n h i b i ­ tors of mammalian α-amylases also occur i n numerous plant products (35). In contrast to the action o f these i n h i b i t o r s , the neutral α-β-glucan dextran increases the rate of reaction i n v i t r o of aamylase on an insoluble starch substrate by enhancing the inter action between the enzyme and i t s substrate (36). Modified food starches (37, 38), such as~~the acid-converted or dextrinized, are u t i l i z e d less completely than most natural starches because non-l,4-glucosidic linkages are introduced by the processing (39). Chemically derivatized starches, such as the phosphorylatêcT or hydroxyalkylated, are attacked by a-amylases i n inverse r e l a t i o n to t h e i r degree of substitution (39, 40). An apparent anomaly to this p r i n c i p l e reported for hydroxyetnyl starch was resolved by Banks et a l . (41). Dextran. A dextran having 95% 1,6-a-D-glucopyranosidic linkages (42) produces a rapid increase i n blood sugar and l i v e r glycogen wïïëh ingested by man (43) or the rat (45, 44). Digestib i l i t y and c a l o r i c a v a i l a b i l i t y assays i n the r a t incTicated that t h i s dextran was highly digestible (90%) and was u t i l i z e d for

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

340

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

growth (45). The 5% a-l,3-linkages at branch points are believed to prevent complete digestion (42). Dextran-hydrolyzing a c t i v i t y has been demonstrated i n small i n t e s t i n a l mucosa of man (46, £7), the rat (22, 48) and the p i g (17). This a c t i v i t y was d i s t i n c t from that oT α-amylase but closely associated with invertase (46) and with isomaltase (48). R u t t l o f f et a l . (22) differentiated dextranase and isomaltase and consi3è~rècT botïï to be particle-bound. The dextran used by Bloom, Wilhelmi and Adrouny (43, 46), Booth et a l . (45) and Dahlqvist (17, 47) was the same s t r u c t u r a l l y , ancT haîTbeen described by Jeanes (42). Dahlqvist (17), using homogenates of pig tissues, showed that dextranase a c t i v i t y was absent i n the stomach and pancreas and present to a low degree i n the colon. Although of no import for digestion, dextran-degrading enzymes do occur i n lysosomes, l i v e r , spleen and kidney tissues of man and other mammals (49,50) L i t t l e further attentio dextran by i n t e s t i n a l enzymes of the substrates f o r , the intimately occurring mixture of aglucosidases of the brush-border membrane that shows isomaltase, sucrase and maltase a c t i v i t i e s , however, constitutes progress towards eventual c l a r i f i c a t i o n of how dextran i s depolymerized i n the small intestine. Isomaltose, the major disaccharide repeating unit of the dextran used i n research reviewed here, i s s p l i t by the isomaltase-sucrase complex of the brush-border membrane (19, 51) and also by the isomaltase subunit after separation as an independent entity (52, 53). The isomaltase-sucrase complex shows weak a c t i v i t y on higFmolecular weight dextran (51). Gray (19, 54) indicates that i n i n t e s t i n a l digestion α-amylase l i m i t dext r i n s are the natural substrate f o r the isomaltase (α-dextrinase)sucrase complex. He equates t h i s complex (54) with the oligo-1,6glucosidase isolated from hog small i n t e s t i n a l mucosa by Larner and McNickle (16). This oligo-l,6-glucosidase hydrolyzed the a1,6-linkages of isomaltose, panose and α-limit dextrins but, under the test conditions used, did not degrade a dextran (95% a-1,6linkages) of molecular weight 75,000 (16). Hehre and Sery (55) showed that large numbers of dextrans p l i t t i n g anaerobic bacteria belonging to the Bacteroides genus occur i n the human colon. Pure cultures of these enteric bac­ t e r i a growing anaerobically i n the presence of dextran appear to depolymerize the dextran and then metabolize the liberated glu­ cose. The soluble dextranase system produced when a s t r a i n isolated from human feces was grown anaerobically i n dextran broth was precipitated by half-saturated ammonium sulfate at 4° and found to show two major, pH-dependent a c t i v i t i e s (56). At pH 7.0-7.5 exolytic-type action occurred with l i b e r a t i o n of glu­ cose from a variety of s t r u c t u r a l l y different dextrans and also from the long outer chains of amylopectins; at pH 5.0-5.5, endolytic-type action predominated with rapid lowering of dextran viscosity. ff

M

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

22.

JEANES

Digestibility

of

Polysaccharides

341

In contrast to these properties of the bacteroides dextranases, the dextran-degrading system isolated by Bloom and coworkers (46) from duodenal and jejunal i n t e s t i n a l mucosa of the rat showed a pH optimum of 6.0-6.2 and s o l u b i l i t y i n halfsaturated ammonium sulfate. Other strains of Bacteroides, growing i n other environments, would not necessarily produce dextranases with the same pH dependencies found by Sery and Hehre (56). Bacteroides, however, are reported to colonize only theTarge bowel of mice (9). In the small intestine of the normal human, Bornside et a l . (12) found Bacteroides i n very low numbers and Gorbach et a l . (577 found them i n the ileum of only 4 out of 13 normal humans. Bacteria of other genera, however, cannot be excluded a r b i t r a r i l y as the possible dextranase producers. Nonpathogenic l a c t o b a c i l l i and anaerobic streptococci occur sparsely i n the human small intestine (11) and i n the stomach and small intestine of the mouse (10) Mthough many members of these genera produce extracellula to produce extracellula from bovine rumen and from feces of an infant (58). Other Polysaccharides Plant Polysaccharides. Many variously constituted polysaccharides are u t i l i z e d i n food preparations, usually as additives (37, 59). Some are simply constituted, having only one or two types of constituent sugar such as cellulose and i t s hydrophilic derivatives, and the galacto- and glucomannans (2). The pectins are more complexly constituted (3). Numerous otKer polysaccharides such as the plant gums and mucilages and the hemicelluloses are heterogeneous i n both composition and linkages. From among a l l these heteropolysaccharides, experimental data on feeding tests have been found only for gum arabic. This plant exudate gum, which i s extensively used i n food preparations (37) under the GRAS c l a s s i f i c a t i o n (60), has been shown to be 7lT~digested by the rat and to be u t i l i z e d for growth (45). Several other studies, but not a l l , support these observations (60). Guinea pigs (60, 61) and rabbits (60) also appear to u t i l i z e gum arabic for energy. As part of a comprehensive review of information on the s u i t a b i l i t y of gum arabic for use i n human food, the Food and Drug Administration has concluded that "gum arabic i s capable of being digested to simple sugars i n herbivores, and to some extent i n omnivores, such as man" (60). The main structural feature of gum arabic i s a backbone chain of 1,3-3-g-galactopyranose units, some of which are substituted at the C-6 p o s i t i o n with various side chains. These side chains are composed mainly of L-arabinofuranose and L-rhamnopyranose u n i t s , the glycosidic linkages of which are acid l a b i l e (62). Current concepts of depolymerization of such a heterogeneous macromolecule indicate that a combination of s p e c i f i c enzymes would be required, none of which are known to occur i n the rat or man.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

342

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

The only related i n t e s t i n a l enzyme known i s the 3-galactosidase, lactase, which appears to have cellobiase and gentiobiase a c t i v i t y also (47, 63) or to be closely associated with these other enzymes. Enzymic depolymerization of gum arabic would be simplif i e d , however, i f the a c i d - l a b i l e groups on the periphery of the molecule had already been removed by gastric a c i d i t y . I f microf l o r a of the rat small intestine are responsible for the depolyme r i z a t i o n of gum arabic, s i m i l a r a c t i v i t y might be expected also on other heteropolysaccharides such as hemicelluloses or, even more l i k e l y , on the more simply constituted galactomannans (2). These expectations, however, are not confirmed by observations. Obviously, d e f i n i t i v e experimentation i s necessary to resolve the enigma of gum arabic d i g e s t i b i l i t y . Inhabitants of coastal regions have always used seaweeds or t h e i r polysaccharide extracts f o r food purposes (59, 64). Many of these polysaccharides resemble agar or carrageenan i n compos i t i o n and, apparently to be indigestible (4) ; i n the rat (45) and to provide some growth i n the guinea p i g (65). The exceptional seaweed, Rhodymenia palmata, however, i s reported (without documentation) to be completely digestible by man (64, 66, 67) and to have been an important food i n coastal regions, especially i n northern latitudes. This alga, commonly c a l l e d dulse or red kale, contains about 22% protein, 3-4% f a t , and 45% polysaccharide. The polysaccharide consists mainly of a 1,3and 1,4-linked 3-D-xylan and, to lesser extent, of starch- and c e l l u l o s e - l i k e materials. The claim of d i g e s t i b i l i t y of this xylan, which i s not supported by controlled experimentation, i s included i n order to complete the coverage of this review. Glycoproteins. Mucopolysaccharides and other types of glycoproteins enter man's diet from animal products such as milk, eggs, connective tissue and tendons of meat, and organs such as l i v e r . Glycoproteins occur also i n plant seeds (68). The carbohydrate components of glycoproteins, which are covalently bound to the peptide moieties, may consist of amino sugars and one to s i x other sugar types and constitute from 1% up to 80% of the weight of the molecule (69). Glycosidic digestion of glycoproteins i s very d i f f i c u l t to effect. The protein moiety, however, i s acted upon by gastrointestinal proteases, some of which are s p e c i f i c f o r certain glycoproteins, u n t i l obstructed by the attached carbohydrate (68) as has been demonstrated f o r a soybean hemagglutinin (70). Trie peptide-bound oligo- or higher saccharides l e f t wouIcT not be expected to be attacked by intest i n a l carbohydrases since they probably would be heterogeneous i n composition as w e l l as linkages. Fructans. Inulin, a r e l a t i v e l y low-molecular-weight polymer of (2+1) -linked 3-D-fructofuranose, occurs as a reserve material i n the tubers of certain plants such as the sunflower called

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

22.

JEANES

Digestibility

of Polysaccharides

343

Jerusalem artichoke. The tuber apparently i s not metabolized by c a t t l e . Man does not metabolize i n u l i n (71) either from the artichoke tuber when i t i s used i n pickles and relishes (72) or when the extracted fructan i s used i n bread f o r diabetics~T73). Levans, i n which 3-D-fructofuranose units are linked predominantly by (2-*6)-bon3s, occur i n many plant tissues and are produced e x t r a c e l l u l a r l y by many types of microorganisms. Levan from Aerobacter levanicum, when reduced to suitable molecular s i z e , serves e f f e c t i v e l y as a nontoxic, nonantigenic blood volume expander which i s slowly eliminated from the body (74). Data on d i g e s t i b i l i t y have not been found. 3- (1+3) -Glucans. Two s t r u c t u r a l l y different 1,3-3-D-glucans have been proposed as food additives. The polysaccharide class of which these glucans are members i s widely distributed i n nature and includes th d produc laminari d th i n t r a and extracellular product (75) . These two microbial glucans have a backbone structure of 1,3-3-D-glucopyranosldic residues; the curdlan-type i s unbranched (76) . The PolytraiP- (or scleroglucan-) type has a glucose sidecEâin attached by a 3-1,6-bond to every t h i r d l i n e a r chain r e s i due (77). These glucans are essentially insoluble i n cold water. Mien Heated, the unbranched type forms a gel of unusual propert i e s (78). The branched type swells greatly i n water and forms t a c t i l y d i s t i n c t gels. In the r a t , t h i s branched 3-1,3 glucan has c a l o r i c value equivalent to starch and, after a period of apparent adaptation, dogs u t i l i z e d t h i s glucan well (77). These results are unexpected on the basis of currently available information on the d i g e s t i b i l i t y of polysaccharides. Tests o f human d i g e s t i b i l i t y have not been reported. Conclusions From among the many and varied plant polysaccharides, man's u t i l i z a t i o n f o r n u t r i t i o n i s r e s t r i c t e d almost exclusively to the amylaceous substances. Depolymerization of these substances to glucose i n the small intestine i s accomplished by man's own enzymes. Man and the r a t derive high c a l o r i c value also from the predominantly a-(l->6)-linked bacterial glucan dextran. A dextrandegrading enzyme, which has been demonstrated frequently i n homogenates of the small i n t e s t i n a l mucosa, has not yet been i d e n t i f i e d and isolated. The r a t and the dog derive high c a l o r i c value from a predominantly 3- (1+3)-linked bacterial glucan. D e f i n i t i v e experimentation i s needed to establish the o r i gin, either mammalian or microfloral or both, of the small intest i n a l enzymes that make possible the high n u t r i t i v e u t i l i z a t i o n of dextran and 3- (1+3) -glucan.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

344

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

The reported high c a l o r i c u t i l i z a t i o n of the heteropolysac­ charide gum arabic i n some rat-feeding tests and not i n others, indicates need for further experimentation. Dextran, 3-(l+3)-glucan and gum arabic are not important n u t r i t i o n a l l y to man. I t i s important, however, to obtain through use of these polysaccharides information now unknown on digestive processes that occur i n the small intestine of man, the rat and other laboratory test animals.

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

Scala, J. This Symposium (1975), Part C, Chapter 21. Lewis, Betty A. This Symposium (1975), Part C, Chapter 19. Chenoweth, Wanda and Leveille, G. A. This Symposium (1975), Part C, Chapter 20. Stancioff, D. J. and Renn D W This Symposium (1975) Part C, Chapter 18 McNeely, W. H. an , Symposiu (1975), C, Chapter 17. Crane, R. K. This Symposium (1975), Part A, Chapter 1. Hehre, E. J. This Symposium (1975), Part B, Introductory Remarks. Rackis, J. J. This Symposium (1975), Part B, Chapter 13. Savage, D. C. Amer. J. Clin. Nutr. (1970) 23, 1495. Savage, D. C. J. Bacteriol. (1969) 97, 15057 Plaut, A. G., Gorbach, S. L., Nahas, L . , Weinstein, G., Spanknebel, G. and Levitan, R. Gastroenterology (1967) 53, 868. Bornside, C. Η., Welsh, J. S. and Cohn Jr., I. J. Amer. Med. Ass. (1966) 196, 1125. Larner, J. and Gillespie, R. E. J. Biol. Chem. (1957) 225, 279. Dahlqvist, Α., Bull, B. and Gustafsson, Β. E. Arch. Biochem. Biophys. (1965) 109, 150. Reddy, B. S. and"Wstmann, B. S. Arch. Biochem. Biophys. (1966) 113, 609. Larner, J. and McNickle, C. M. J. Biol. Chem. (1955) 215, 723. Dahlqvist, A. Biochem. J. (1961) 78, 282. Dubs, R., Steinmann, B. and Gitzelmann, R. Helv. Paediat. Acta (1973) 28, 187. Gray, G. M. "This Symposium (1975), Part B, Chapter 11. Prader, A. and Auricchio, S. Ann. Rev. Med. (1965) 16, 345. Dahlqvist, A. and Thomson, D. L. Biochem. J. (1963) 89, 272. Ruttloff, R., Noack, R., Friese, R., Schenk, G. and P r o l l , J. Acta Biol. Med. Ger. (1967) 19, 331. Chem. Abstr. (1968) 68, 111, 599a. Gray, G. M. Gastroenterology (1970) 58, 96.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

22.

JEANES

Digestibility

of

Polysaccharides

345

24. Whelan, W. J., in "Methods in Carbohydrate Chemistry," Vol. 4, Whistler, R. L., Ed., Academic Press, New York, N.Y., 1964, p. 252. 25. Allen, P. Z. and Whelan, W. J. Biochem. J. (1963) 88, 69. 26. Sandstedt, R. M . , Strahan, D., Ueda, S. and Abbott, R. C. Cereal Chem. (1962) 39, 123. 27. Walker, Gwenn J. and Hope, Pamela. Biochem J. (1963) 86, 452. 28. Booher, Lela E., Behan, Ida, McMeans, Evelyn and Boyd, H. M. J. Nutr. (1951) 45, 75. 29. Senti, F. R. and Dimler, R. J. Food Technol. (1959) 13, 663. 30. Hullinger, C. H . , Van Patten, E. and Freck, J. A. Food Technol. (1973) 27, 22. 31. Borchers, R. Cereal Chem. (1962) 39, 145. 32. Saunders, R. M. Cereal Science Toctay (1975), in press. 33. Osman, H. F . , Theurer Β. Hale W H and Mehen S M J. Nutr. (1970) 100, 1133 34. Hellendoorn, E. W (1969) 35. Marshall, J. J., Lauda, Carmen A. and Whelan, W. J. This Symposium (1975), Part B, Chapter 16. 36. Ceska, M. Experientia (1971) 27, 767. 37. Glicksman, M. "Gum Technology in the Food Industry," Academic Press, New York, N.Y., 1969, pp. 590. 38. Wurzburg, O. B . , in "Handbook of Food Additives," Furia, T. E . , Ed., The Chemical Rubber Co., Cleveland, Ohio, 1968, p. 377. 39. Reussner, Jr., G., Andros, J. and Thiessen, J r . , R. J. Nutr. (1963) 80, 291. 40. Parrish, F. W. and Whelan, W. J. Staerke (1961) 13, 231. 41. Banks, W., Greenwood, C. T. and Muir, D. D. StaerTce (1972) 24, 181. 42. Jeanes, Allene. Food Technol. (1974) 28, 34. 43. Bloom, W. L. and Wilhelmi, A. E. Proc. Soc. Exp. Biol. Med. (1952) 81, 501. 44. Parkinson, T. M. Nature (London) (1967) 215, 415. 45. Booth, Α. Ν., Hendrickson, A. P. and DeEds, F. Toxicol. Appl. Pharmacol. (1963) 5, 478. 46. Adrouny, G. Α., Bloom, W. A. and Wilhelmi, A. E. Fed. Proc. (1957) 16, Abstr. 618. 47. Dahlqvist, A. J. Clin. Invest. (1962) 41, 463. 48. Dahlqvist, A. Biochem. J. (1963) 86, 72". 49. Fischer, Ε. H. and Stein, Ε. Α., in The Enzyme Vol. 4, Part A , " 2nd ed., Boyer, P. D., Lardy, H . , Myrback, K., Ed., Academic Press, New York, N.Y., 1960, p. 306. 50. Aimion, R. Enzymologia (1963) 25, 245. 51. Kolinska, J. and Semenza, G. Biochim. Biophys. Acta (1967) 146, 181. 52. Cogoli, Α., Eberle, Α., Sigrist, Η., Joss, Christine, Robinson, Etheria, Mosimann, H. and Semenza, G. Eur. J. Biochem. (1973) 33, 40.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

346

PHYSIOLOGICAL EFFECTS OF FOOD CARBOHYDRATES

53. Conklin, Κ. Α., Yamashiro, Κ. M. and Gray, G. M. J. Biol. Chem. (1975), in press. 54. Gray, G. M. Fed. Proc. (1967) 26, 1415. 55. Hehre, E. J. and Sery, T. W. J. Bacteriol. (1952) 63, 424. 56. Sery, T. W. and Hehre, E. J. J. Bacteriol. (1956) 71, 373. 57. Gorbach, S. L . , Banwell, J. G., Jacobs, B., Chatterjee, B. D., Mitra, R., Sen, Ν. N. and Mazumder, D. N. G. Amer. J. Clin. Nutr. (1970) 23, 1545.

58. Bailey, R. W. and Clarice, R. T. J. Biochem. J. (1959) 72, 49. 59.

Whistler, R. L., BeMiller, J. Ν., "Industrial Gums: Polysac­ charides and Their Derivatives," 2nd ed., Academic Press,

New York, N.Y., 1973, numerous contributed chapters. 60. Food and Drug Administration, "Food Additives and GRAS Sub­ stances," Federal Register (1974) 39 (185, Part II), 34203. 61. Hove, E. L. and Herndon J. F J. Nutr (1957) 63 193 62. Glicksman, M. and charides and Thei Derivatives, , , and BeMiller, J. Ν., Ed., Academic Press, New York, N.Y., 1973, p. 197. 63. Semenza, G., Auricchio, S. and Rubino, A. Biochim. Biophys. Acta (1965) 96, 487. 64. Chapman, V. J., "Seaweeds and Their Uses," Methuen and Co. Ltd., London, 1950, p. 186; 2nd ed., 1970, p. 118. 65. Booth, A. N . , Elvehjem, C. A. and Hart, Ε. B. J. Nutr. (1949) 37, 263. 66. Swartz, M. D. Trans. Conn. Acad. Sci. (1911) 16, 000. 67. Sand, R. E. and Glicksman, Μ., in "Industrial Gums: Polysac­ charides and Their Derivatives," 2nd ed., Whistler, R. L. and BeMiller, J. N., Ed., Academic Press, New York, N.Y., 1973, p. 185. 68. Yamashina, I., in "The Amino Sugars," Vol. II B, Balazs, E. A. and Jeanloz, R. W., Ed., Academic Press, New York, N.Y., 1966, p. 81. 69. Spiro, R. G. Ann. Rev. Biochem. (1970) 39, 599. 70. Lis, Halina, Sharon, N. and Katchalski, E. Biochim. Biophys. Acta (1969) 192, 364. 71. Schanker, L. S. and Johnson, J. M. Biochem. Pharmacol. (1961) 8, 421. 72. Encyclopedia Britannica, Vol. 2, Wm. Benton, Publisher, Chicago, I11., 1964, p. 525. 73. The Merck Index of Chemicals and Drugs, 7th Edition. Merck & Co., Inc., Rahway, N.J., 1960, p. 557. 74. Avigad, G., in "Encyclopedia of Polymer Science and Technol­ ogy," Vol. 8, Bikales, Ν., Ed., 1968, p. 711. 75. Bull, A. T. and Chesters, C. G. C. Advan. Enzymol. (1966) 28, 325. 76. Harada, T., Misaki, A. and Saito, H. Arch. Biochem. Biophys. (1968) 124, 292.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

22.

JEANES

Digestibility

of Polysaccharides

77. Rodgers, Ν. E . , in "Industrial Gums: Polysaccharides and Their Derivatives," 2nd ed., Whistler, R. L. and BeMiller, J. Ν., Ed., Academic Press, New York, N.Y., 1973, p. 499.

78. Harada, T. Proc. Biochem. (January 1974) page 21.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

347

INDEX A Absorption bile acid 318 of calcium 100 of carrageenan 286 and metabolism, mannose 303 of nutrients, effect of pectin on the 321 radioactive strontium 275 of sugars, physiology of the intestinal 1 and toxicity of carrageenan Absorptive surface, digestive 8 Acid absorption, bile 318 ACTH 56 Activity of pepsin, proteolytic 287 Adipose tissue fatty acid synthesis 46 lipid metabolism 51 lipogenesis 49 response to food carbohydrates 46 Adrenal cortex, effects of steroid hormones of the 56 Adrenalin 56,60 Agar 342 Alactasia 102 Alcoholism 58 Alditols, metabolism and physiological effects of 123 Aldose reductase 130 Alginate, propylene glycol 272 Alginate, sodium 273 Alginates, toxicity studies of 269 Alimentary carcinoma 331 Amylaceous substances 338, 343 α-Amylase inhibitors 244 assay of 246 detection of 247 from phaseolus vulgaris 257 from plants 244 physiological effects of 259 properties of 249 from wheat 253 α-Amylases of saliva and pancreatic juice 338 Amylose starch, high339 Anaerobic fermentation of soybean products 213 Analysis, Bio-Rad 60 Anti-hypercholesterolemic action of pectin 316 Antihyperglycemic agent 263 Arabitol 123

L-Ascorbic acid from D-glucose Artichoke, Jerusalem Atherogenic diet ATP level, hepatic Atypical mucopolysaccharidosis

140 343 26 50 147

Β Bile acid absorption Bile acids

318 329

Biosynthesis of galactose-1phosphate 113, 116 Biosynthesis, lactose 102 Biosynthesis of uridine diphosphate glucose and uridine 109 Blood lipids in rats 26 Blood sugar 202 effect of mannose on 307 regulation 59 Blood triglycerides in man 25 Bone collagen 100 Bowel, water content of the 289 Breakdown of carrageenan 286 Brewers yeast extract 168 Brush-border enzyme activities 7 Brush-border membrane 3, 338 Brush-border oligosaccharidases, human 181,183 Brush-border saccharidases 7 Butanediol 47 C Calcium, absorption of 100 Calorie sources for intravenous feeding 74 Cancer, colonic 330 Carbohydrate in adipose tissue 46 diet, high-protein, low 60 digestion 7,181 substrate in intravenous feeding, maltose as a 74 Carbohydrates adipose tissue response to food 46 advantages of substituting fructose for other 69 in dental caries, role of 150 insulin-inducing 56 metabolic effects of dietary 20 Carbon dioxide 214

349

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

350

PHYSIOLOGICAL

Carcinoma, alimentary 331 Cardiovascular disease 328 Cariogenicity of sorbitol 153 Carrageenan 282, 299, 342 absorption of 286 breakdown of 284, 286 physiological effects of 287, 289 toxicity of 282 Carrier, Na -dependent 12 Catecholamine products in the urine 58 Cellobiase activity 337 Ceramine-lactoside lipidosis 102 Cereals 325 Cerebral hemorrhage 69 Cholesterol 59, 318, 328 levels 170 plasma 299 Chromatography 223 Chromium 157, 162 deficiency in man 16 in food refining, losses of 17 in nutrition, role of 166 trivalent 166 Chstridium perfringens 214 Cobalt 157,163 Collagen, bone 100 Colonic cancer 330 Copper 157,163 COMPT 56 Coronary damage 69 Crave-eater 58 Crigler-Najjar disease 148 Cyanogen bromide 255 Cyclic AMP 56, 238 +

D Deactivation of the catecholamine hormones, enzymatic Deficiency of intestinal oligosaccharidase Degraded carrageenan physiological effects of Dental caries role of carbohydrates in Dental plaque Deoxycholate D-2-Deoxyribose Depolymerases, origin of Detection of α-amylase inhibitors Dextran Dextranase activity Diabetes mellitus Diabetic humans fructose use by Diarrheal diseases Diet and cholesterol Diet, heart disease and Diet, high-protein, low carbohydrate Dietary fiber, physiological effects of

57 186 284 289 128 150 151 332 135 336 247 339 337 75 126 55 312 331 23 60 325

EFFECTS

O F FOOD

CARBOHYDRATES

Dietary fructose in alleviating the human stress response Dietetic agents, inhibitors as Digestibility of food polysaccharides by man Digestion of carbohydrate of mannans of polysaccharide, intraluminal Digestive-absorptive surface Disaccharidase déficiences, treatment of Disaccharides as a source of calories, intravenous Diverticular disease Dulcitol

54 263 336 336 181 298 181 8 186 77 327 123

Ε Eater crave

58

Effects of dietary carbohydrates, metabolic Effects of polyols on plaque formation Effects of steroid hormones of the adrenal cortex Electrophoresis Elements, micro-nutrient Elimination of flatulence Enzymatic deactivation of the catecholamine hormones Enzymatic reaction of polyols Enzyme activities Enzyme, human liver Enzyme inhibitors, hydrolytic Enzymes of glycogen metabolism Enzymology of the polyols Erythritol Essential trace elements lost during food refining Extracellular polysaccharide

20 153 56 224 158 220 57 124 7 223 244 235 123 123 174 151

F Fabry's disease 102 Facilitated diffusion carrier 12 Fatty acid synthesis, adipose tissue 46 role of carbohydrate in 46 Fermentation of soybean products, anaerobic 213 Fiber, physiological effects of dietary 325 Fibroblast sonicates, glycogen hydrolysis by 229 Fibroblasts 228 Film surface, molecular orientation of copolymer 324 Flatus problem, a-galactosidase activity and 207 Flatulence 211, 212, 220 Fluoridation, water 153 Fluoride 152,157,165

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

351

INDEX

Food carbohydrates, adipose tissue response to 46 Food legumes, oligosaccharides of 207 Food polysaccharides by man, digestibility of 336 Food refining, losses of essential trace elements during 174 Fructans 342 Fructose 3, 75, 153, 236 in alleviating the human stress response 54 infusion 75 intolerance 70, 153 metabolism 49, 52, 55 use of diabetics 55 utilization of sorbitol and 130 Fruits 325 Function of the stomach 11

Galactomannans 29 Galactose 5,236 biosynthesis in the mammary gland 104 metabolism 100,104 pyrophosphorylase pathway 109 -1-phosphate from glucose, uncon­ trolled biosynthesis of 116 -1-phosphate from UDP-galactose, biosynthesis of 113 -1-phosphate toxicity 112 Galactosemia 110, 131 α-Galactosidase activity and flatus 207 Galactosidases, lysosomal 102 Gangliosidosis, generalized 102 Gargoylism 147 Gastric secretion 288 Gels, isoelectric focusing 224 Genetics of transferase deficiency 111 Gliadins, wheat 256 £-(1^3)-Glucans 343 Glucoamylase 338 Glucocorticoids 240 Glucogen 240 Glucomannans 296 Glucose 5, 54, 74, 140, 153, 195, 235,238, 240 infusion 74 metabolism of 86 in parenteral nutrition, xylitol as a substitute for 128 -1-phosphate 236 tolerance factor 166,168 uncontrolled biosynthesis of galac­ tose-1-phosphate from 116 α-Glucosidases of mammalian tissues, lysosomal 223 Glucuronate-xylulose cycle 126,140 D-Glucuronic acid 144,147 as a metabolic intermediate 138 utilization and production of 146

Glycogen formation, effect of mannose on hydrolysis byfibroblastsonicates . in the liver metabolism, enzymes of storage disease (Pompe's disease) synthetase 236, Glycoproteins GTF supplementation of diet

235 307 229 55 235 223 242 342 171

H

Heart disease and diet Hepatic ATP level Hexitols Hexoses High-protein, low-carbohydrate diet Hormones of the adrenal cortex, effects of steroid Hormones enzymati deactivatio f Human brush border oligo­ saccharidases Humans, flatulence in Hurler's disease Hydrogen Hydrolysis by fibroblast sonicates, glycogen Hydrolytic enzyme inhibitors Hydrolyzed milk, lactoseHyperactive, human Hypocholesterolemic activity 288, Hypoglycemia Hypomanic activity Hypothermia Human liver enzyme

23 50 130 3 60 56

183 211 147 214 229 244 201 58 298 54 58 75 223

I L-Iduronic acid 145, 147 Impaired glucose tolerance 168 Indications for intravenous feeding 74 Infusion 75 fructose 75 glucose 74 maltose 73,77 sorbitol 76 xylitol 76 Inhibition, tumor 302 Inhibitor from phaseolus vulgaris, α-amylase 257 Inhibitors 250 of α-amylase 246, 247, 250 physiological effects of 259 from plants 244 from wheat 253 as dietetic agents 263 invertase 246 Injected maltose, metabolism of 77 Inorganic pyrophosphate 115 Inositol 142

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

352

PHYSIOLOGICAL

Insulin 52, 54, 240, 342 carbohydrates which induce 56 levels 170 secretion, effect of mannose on 307 Interferon stimulation 302 Intermediates, pentoses and D-glucuronic acid as metabolic 138 Intestinal absorption of sugars 1 brush border, oligosaccharidases of the small 181 mucosa, metabolism of oligosaccha­ rides in the 208 oligosaccharidase 182, 186 Intestine, role of microflora in the small 336 Intolerance, lactose 191 Intraluminal digestion of polysaccharide 181 Intracellular polysaccharide 15 Intravenous disaccharides as a of calories 77 Intravenous feeding 74, 80, 83 caloric sources for 74, 80, 83 indications for 74 maltose as a carbohydrate substrate in 74 Invertase inhibitors 246 Iodine 157,160 Iron 157,160 Isoelectric focusing gels 224 Isomaltitol 131

J Jerusalem artichoke

343 Κ

Kale, red Konjac mannan Krabbe's disease

342 299 102

L Lactase activity, low deficiencies Lactic acid in plaque Lactose biosynthesis catabolism hydrolyzed milk intolerance synthetase Legumes oligosaccharides of food Leloir pathway Lipid metabolism in adipose tissue Lipidosis, ceramide-lactoside

337 203 101,186 152 100 102 100 191, 201 101,191 103 325 207 107 49, 313 51 102

EFFECTS

O F FOOD

CARBOHYDRATES

Lipids 318 in rats, blood, serum, and liver 26, 28 Lipogenesis, adipose tissue 49 Liver enzyme, human 223 Liver, glycogen in the 55 Losses of essential trace elements during food refining 173, 174 Lysosomal α-glucosidases of mammalian tissues 223 Lysosomal galactosidases 102 Lysosomes 223

M Macrocystis pyrifera Maillard reaction Maltase Maltitol Maltose carbohydrat substrat

269 75, 128 337 131 224 i

infused 73,77 oxidation of 83 Maltosyloligosaccharides 80 Mammalian tissues, lysosomal α-glucosidases of 223 Man and animals, trace elements with beneficial effects in 161 Manganese 157, 163 Mannan, konjac 299 Mannans 296 digestion of 298 hypocholesterolemic activity of 298 D-Mannitol 123 Mannose, absorption and metabolism 303 Membrane, brush-border 3, 338 Membrane transport, types of 15 Metabolic effects of dietary carbohydrates 20 Metabolic intermediates, pentoses and D-glucuronic acid as 138 Metabolism in adipose tissue, lipid 51 enzymes of glycogen 235 fructose 49, 52 galactose 100,104 of infused maltose and other sugars 73 of injected maltose 77 of lactose and galactose 100 lipid 49,313 of maltose 86 mannose absorption and 303 and nutrition, xylitol in 125 of oligosaccharides in the intestinal mucosa 208 and physiological effects of pectins 312 and physiological effects of the pentoses and uronic acids 135 and physiological effects of the polyols (alditols) 123

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

353

INDEX

Metabolism (continued) pyrophosphorylase pathway of galactose 109 trace elements in human nutrition and 156 Methane 214 Microflora in the small intestine 336 Micro-nutrient elements 158 Microorganisms 100 Milk, lactose-hydrolyzed 191,201 Milk rejection 198 Molybdenum 157,163 Mucopolysaccharidosis, atypical 147 Mucosa, metabolism of oligosaccha­ rides in the intestinal 208 Ν Na -dependent carrier Navy beans Nickel 157 Nitrogen Noradrenalin Normetadrenalin Nutrients, effect of pectin on the absorption of Nutrition and metabolism, trace elements in human parenteral role of chromium in xylitol in 125,

12 24 21 56 60 321 156 73 166 128

Ο Oats 251 Oligosaccharides of food legumes 207 Oligosaccharides in the intestinal mucosa, metabolism of 208 Oligosaccharidase, deficiency of intestinal 186 Oligosaccharidases, human brush border 181, 183 Origin of depolymerases 336 Osmoreceptors, sugar-specific 12 Oxidation of intravenously administered maltose 83

Ρ Pancreatectomy Pancreatic amylase Pancreatic juice, α-amylases of Pancreatitis Parenteral nutrition xylitol as a substitute for glucose in Pathological aspects of the pentoses Pectin on the absorption of nutrients, effect of anti-hypercholesterolemic action of

75 7 338 75 73 128 143 299 321 316

Pectin (continued) metabolism and physiological effects of 312 Pentitols, utilization of free 129 Pentoses, pathological aspects of the 135,136,143 Pentosuria 126,143 Pepsin, proteolytic activity of 287 Peptic ulcers 282 Phaseolus vulgaris 247 α-amylase inhibitor from 257 Phosphatase system, regulation of the 240 Phospholipids 318 Phosphorylase 236 kinase 236 reaction, regulation of the 238 pH on the tooth surface 152 Physiological effects of α-amylase inhibitors 259 of dietary fiber 325 of pectins, metabolism and 312 of the pentoses and uronic acids, metabolism and 135 of the polyols (alditols), metabolism and 123 Physiologically important uronic acids 144 Physiology of the intestinal absorption of sugars 1 Plant polysaccharides 341 Plants, α-amylase inhibitors from 244 Plaque formation 152 effects of polyols on 153 Plaque, lactic acid in 152 Plasma cholesterol 299 Polyfructan 151 Polyglucan 151 Polyols (alditols) 123 in animal metabolism 123 enzymatic reactions of 124 on plaque formation, effects of 153 Polysaccharide extracellular 151 intracellular 151 intraluminal digestion of 181 Polysaccharides amylaceous 338 digestibility of food 336 plant 341 Potatoes 325 Potato tubers 249 Propylene glycol alginate 272 Protein, low-carbohydrate diet, high 60 Proteolysis 259 Proteolytic activity of pepsin 287 Pyrophosphate, inorganic 115 Pyrophosphorylase pathway of galactose metabolism 109

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

354

PHYSIOLOGICAL

Q Quar gum

299

R Radioactive strontium absorption 275 Raffinose 207 Rats, lipids in 26, 28 Reactions of UDP-D-glucuronic acid 147 Red kale 342 Refined white sugar 174 Refining, losses of chromium in food 173 Refining, losses of essential trace elements during food 174 Refining of wheat 174 Regulation of the phosphatase system 240 Regulation of the phosphorylase reaction 238 Ribitol 123 D-Ribose 13 D-Ribulose 13 Role of carbohydrates in dental caries 150 Role of chromium in nutrition 166 Role of dietary carbohydrate in adi­ pose tissue fatty acid synthesis 46 Role of microflora in the small intestine 336 Rye 251

S Saccharidases 7 Saliva and pancreatic juice 338 Seaweed 342 Secretion, effect of mannose on insulin 307 Secretion, gastric 288 Selenium 157,164 Silicon 157,165 Small intestine, function of 11 Small intestine, role of microflora in the 336 Sodium alginate 273 Sonicates, glycogen hydrolysis by fibroblast 229 Sorbitol 123, 124, 130 dehydrogenase 125 infusion 76 Sorghum 249 Source, properties, and uses of carrageenan 283 Soybean products 213 Soy protein products 212 Stachyose 208 Starch 249 digestion 259 high-amylose 339 Steroid hormones of the adrenal cortex 56 Stimulation, interferon 302 Stomach, function of 11 Streptococcus mutatis 151

EFFECTS

O F FOOD

CARBOHYDRATES

Stress response, benefits of dietary fructose in alleviating the human Strontium absorption, radioactive Sucrase Sugar, blood 59, Sugar, refined white Sugar-specific osmoreceptors Sugars, metabolism of infused maltose and other Sugars, physiology of the intestinal absorption of Sympathetic hormones Synthesis, adipose tissue fatty acid

54 275 337 202 174 12 73 1 56 46

Τ Tin Tooth surface, pH on the Toxicity f

157,165 152 301 282

Trace element analysis Trace elements with beneficial effects in man and animals Trace elements in human nutrition and metabolism Transferase deficiency, genetics of Transport, types of membrane Trehalase Trehalose, intravenously administered Triglycerides 25, 47, 59, Trivalent chromium Tryptophan Tumor inhibition

158 161 156 111 15 337 80 318 166 254 302

U Uncontrolled biosynthesis of galac­ tose-1-phosphate from glucose Uricemia Uridine diphosphate galactose C-4-epimerase Uridine diphosphate glucose and uridine, biosynthesis of Uremia Urine, catecholamine products in the Uronic acids, physiologically important 135, Utilization of free pentitols Utilization of pentoses Utilization and production of glucuronic acid Utilization of sorbitol and fructose ....

116 128 110 109 75 58 144 129 136 146 130

V Vanadium Vegetables Verbascose

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

157,165 325 208

355

INDEX

W Water content of the bowel Water fluoridation Wheat, α-amylase inhibitors from Wheat gliadins Wheat, refining of

289 153 253 256 174

Xylitol (continued) as a substitute for glucose in parenteral nutrition utilization Xylose Xylulose cycle, glucuronate

X Xanthan gum toxicity studies with Xylitol infusion in metabolism and nutrition

128 128 135,143 135 126,140

Y

269, 275 277 123 76 125

Yeast extract, brewers

168

Ζ Zinc

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

157,160