Advances in Food and Nutrition Research Volume 37

ADVANCES IN

FOOD AND NUTRITION RESEARCH VOLUME 37

ADVISORY BOARD

DOUGLAS ARCHER Washington. D.C.

JESSE F. GREGORY 111 Gainesville. Florida

SUSAN K. HARLANDER St. Paul, Minnesota

DARYL B. LUND New Brunswick, New Jersey

ROBERT MACRAE Hull, England

BARBARA 0. SCHNEEMAN Davis, Calfornia

STEVE L. TAYLOR Lincoln, Nebraska

ADVANCES IN

FOOD AND NUTRITION RESEARCH VOLUME 37

Edited by

JOHN E. KINSELLA College of Agricultural and Environmental Sciences University of California, Davis Davis, California

ACADEMIC PRESS, INC. A Division of Harcourt Brace & Company San Diego New York Boston London Sydney Tokyo Toronto

This book is printed on acid-free paper. @

Copyright 0 1993 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

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DEDICATION

This book is dedicated to the memory of John Edward Kinsella.

Born in Wexford, Ireland, John E. Kinsella was an internationally distinguished scientist and a dedicated administrator. He received his bachelor’s degree in natural and agricultural sciences from National University in Dublin in 1961, and his master’s degree in biology in 1965 and doctor’s degree in food chemistry in 1967 from Pennsylvania State University. Joining the faculty at Cornell University in 1967, he was named chair of the Department of Food Science in 1977, serving until 1985. He became associate director of Cornell’s Institute of Food Science in 1977, and director from 1980 to 1987. At Cornell, he was designated Liberty Hyde Bailey Professor of Food Chemistry, a named chair, in 1981 and General Foods Distinguished Professor of Food Science, an endowed chair, in 1984. He was a Fulbright Professor of Food Science and Nutrition at University College Cork, Ireland in 1984. In the spring of 1989, he was Campbell-Tyner Eminent Scholar at Florida State University. He came to the University of California, Davis in September 1990 as Dean of the College of Agricultural and Environmental Sciences. Dean Kinsella received numerous academic honors, including the Borden Award (1976) for research in biochemistry of mammary tissue and milk, the Babcock Hart Award (1987) for research in food science and nutrition, the Atwater Award (1988) in recognition of international nutrition and food science research, the Spencer Medal Award (1991) for outstanding research in food and agricultural chemistry, the Chang Award (1991) for research and advancement in lipid and flavor chemistry, and the Tanner Lectureship (1991) for scientific contributions to food chemistry. An authority on the biochemistry of dietary fatty acids, he was also the holder of eight patents. He served as an officer and consultant for many organizations, including the U.S. Department of V

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DEDICATION

Agriculture, World Bank, National Research Council’s Board on Agriculture, National Cancer Institute, and the Food and Nutrition Board of the National Academy of Science’s Institute of Medicine. At the University of California, Davis, Kinsella was instrumental in establishing the University of California FoodSafe Program, a consumer-oriented food safety education program. He was shepherding the College of Agricultural and Environmental Sciences through a bold reorganization aimed at guiding the College’s mission of teaching, research, and public service into the next century. John E. Kinsella died on May 2, 1993. He is survived by his wife Ruth Ann, his sons Sean and Kevin, his daughters Helen and Kathryn, and his granddaughter Hannah.

CONTENTS

CONTRIBUTORS TO VOLUME 37 ............................. PREFACE ................................................

xi xiii

Food. Diet. and Gastrointestinal Immune Function

James J . Pestka I . Introduction ....................................... I1. Overview of the Gastrointestinal Immune System ...... I11. Diseases Involving the Gastrointestinal Immune System ........................................... IV . Impact of Food Constituents and Contaminants on Gastrointestinal Immunity ........................... V . Modification of Gastrointestinal Immunity through Food and Diet .......................................... VI . Research Needs ................................... References ........................................

1 2

22 35 45 53 56

Effect of Consumption of Lactic Cultures on Human Health

Mary Ellen Sanders I. I1. I11. IV .

V.

VI . VII .

Introduction ....................................... General Physiology ................................. Health Targets ..................................... Safety Issues ...................................... Considerations for Strain Selection ................... Research Needs ................................... Conclusions ....................................... References ........................................

67 71 78 114 115 116 121 121 vii

...

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Defining the Role of Milkfat in Balanced Diets

Louise A . Berner

I . Introduction ....................................... I1 . Background Information-Dietary Fat Consumption and Composition ...................................... 111. Dietary Fat. Dairy Products. and Coronary Heart Disease ........................................... IV . Dietary Fat. Dairy Products. and Cancer Risk ......... V . Milkfat as Part of the Total Diet ...................... VI . Conclusions and Research Needs .................... References ........................................

131

133

153 208 224 236 239

Biochemistry of Cardiolipin: Sensitivity to Dietary Fatty Acids

Alvin Berger. J . Bruce German. and M . Eric Gershwin

I . Introduction ....................................... I1 . Discovery of Polyglycerophospholipids ............... 111. Abundance of Polyglycerophospholipids .............. IV . Pathways of PolyglycerophospholipidSynthesis ....... V . Intracellular Location of Polyglycerophospholipid Synthesis ......................................... VI . Conformation of Cardiolipin in Biomembranes ......... VII . Degradation of Polyglycerophospholipids ............. VIII . Oxidation of Cardiolipin ............................ IX . Association of Enzymes with Cardiolipin .............. X . Cardiolipin Acyl Composition ....................... XI . Influence of Diet and Other Factors on Cardiolipin Content ................................ XI1 . Possible Role of Cardiolipin in Resistance to Ethanol-Induced Membrane Disordering .............. XI11. Immunologic Activity of Cardiolipin .................. XIV . Chemical Synthesis of Acyl-Specific Cardiolipin Derivatives ........................................ xv . Chromatographic Separation of Cardiolipin ............ References ........................................

260 261 261 264 267 270 274 275 275 289 300 302 304 314 317 318

CONTENTS

ix

Diseases and Disorders of Muscle

A . M . Pearson and Ronald B . Young 1. I1 . I11. IV . V. VI .

INDEX

Introduction ....................................... Disorders and Diseases of Muscle .................... Disorders of Energy Metabolism ..................... Diseases of the Connective Tissues ................... Research Needs ................................... Summary ......................................... References ........................................

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

340 341 374 387 405 409 410 425

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CONTRIBUTORS TO VOLUME 37

Numbers in parentheses indicate the pages on which the authors' contributions begin.

Alvin Berger, Department of Food Science and Technology, University of California, Davis, Davis, California 95616 (259) Louise A. Berner, Nutrition Consultant, Sun Luis Obispo, California 93401 ( I 3 I ) J . Bruce German, Department of Food Science and Technology, University of California, Davis, Davis, California 95616 (259) M. Eric Gershwin, Division of RheumatologylAllergy and Clinical Immunology, University of California, Davis Medical School, Davis, California 95616 (259) A. M . Pearson, Department of Animal Sciences, Oregon State University, Corvallis, Oregon 97331 (339) James J . Pestka, Department of Food Science and Human Nutrition, Michigan State University, East Lansing, Michigan 48824 (1)

Mary Ellen Sanders, Microbiology Consultant, Littleton, Colorado 80122 (67) Ronald B. Young, Department of Biological Sciences, University of Alabama in Huntsville, Huntsville, Alabama 35899 (339)

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PREFACE

Although the role of foods in providing nutrients (and nonnutrients) to maintain metabolic functions for normal functions (i.e.. energy generation and growth and maintenance of tissue integrity) has been well elucidated, the role of food components in disease prevention, particularly as mediated by the immune system, is finally receiving deserved attention. In this regard, the importance of the gastrointestinal (GI) immune system in health and disease is being systematically clarified. Perturbation or dysfunction of the GI immune system is associated with diseases such as microbial infections, allergies, inflammatory states, autoimmunity, and neoplasms. Ongoing research in cellular and molecular immunology is providing a greater understanding of the interaction of dietary components with the GI immune system. More complete knowledge of dietary factors affecting the GI immune system may provide opportunities to produce foods that ameliorate disturbances and/or enhance GI immune system functions and overall health. In this volume, Pestka reviews the nature of the GI immune system and the complexity and varied functions of the mucosal immune system. Diseases involving the GI immune system, hyperactivity, allergenicity, food-sensitive enteropathies, inflammatory bowel disease, and immunosuppression of the GI system can be affected by dietary components. Nutritional therapies for intestinal diseases, immune-compromised subjects, and autoimmune disorders are now feasible, and the possibility of providing probiotic agents to colonize the gut with beneficial bacteria is of emerging interest. Sanders provides a comprehensive overview of the current information concerning the possible therapeutic benefits of lactic acid bacteria (lactobacilli), particularly those in cultured dairy foods. The author discusses the diversity and complexity of the gastrointestinal ecology and the difficulty of experimentation in this field. The value of food microbes in the treatment of gastric disturbances, ...

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PREFACE

lactose intolerances, diarrhea, and cancer suppression by reducing mutagens, possibly in cholesterol reduction, and by immune stimulation, is reviewed. Probiotics for human food are now being commercialized; however, standards for ensuring functional criteria are needed. The issue of the safety of cultured microbes and the possibility of transfer of genetic material warrants monitoring. The understanding of the role of dietary fat in atherogenesis, atherosclerosis, and thrombosis is still incomplete. Although some useful general guidelines have emerged concerning food fats, the effects of relative amounts and ratios of different fatty acids in different foods is still incomplete. Dietary fat is needed to provide energy, sufficient essential fatty acids (1-3 g/day), and fat-soluble vitamins. In this context it should be relatively easy to prescribe rather accurately the fatty acid needs for various ages and life stages, and the associated energy need patterns, rather than the current approach of advising what fats not to consume. It is time to discuss quantities of specific fatty acids and relate these to the optimum mixture of foods which should be consumed. In this regard, forbidding important foods which deliver essential nutrients (because they contain fats which if eaten in inordinate quantities exacerbate some common chronic diseases) is simplistic at best and may be predisposing certain subjects to the danger of other deficiencies. Thus, the tendency to advise against dairy foods because butterfat is conducive to atherosclerosis, when consumed as the predominant source of fat, may not be generally beneficial to all consumers. Dairy foods are a principal source of calcium, riboflavin, and several other nutrients. The chapter by Berner reviews the literature and addresses the questions of the role of dairy food milkfat in health and disease. The data indicate that dairy foods are desirable in a normal, prudent diet because they ensure a balanced intake of nutrients. Ongoing research to improve the fatty acid composition of dairy foods is outlined. Among the important roles of dietary fatty acids is their influence on composition of cellular and intracellular membranes. This, in turn, can affect several metabolic functions (e.g., receptormediated uptake; mediation of cellular signaling; regulation of membrane-bound enzymes), and in the case of mitochondria, this affects reactions related to energy generation. Cardiolipins are a unique class of acylpolyglycerolphosphatides that are particularly

PREFACE

xv

enriched in the outer membrane of cardiomuscular mitochondria where they may affect enzyme functions. Cardiolipins have an unusual fatty acyl composition that is more resistant to dietary fatty acids than other lipid classes. The role and possible functions of cardiolipins are reviewed; however, little is known about whether modification via diet affects function in animals, and data on humans are not available. Muscle tissue is a significant source of nutrients, and its integrity is important for production efficiency and food quality. The final chapter in this volume catalogs the diseases and disorders of muscle tissue, including sarcoplasmic proteins and connective tissue components. Some of these are related to nutritional disorders and nutrient imbalances. Advances in Food and Nutrition Research fills an important niche in providing a timely, comprehensive review of topics and subject matter that link food, nutrition, health promotion, disease prevention, and amelioration. Scholarly in-depth reviews are welcome. JOHN E. KINSELLA

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ADVANCES I N FOOD AND NUTRITION RESEARCH. VOL. 37

FOOD, DIET, AND GASTROINTESTINAL IMMUNE FUNCTION JAMES J. PESTKA Department of Food Science and Human Nutrition Michigan State University East Lansing. Michigan 48824

1. Introduction 11. Overview of the Gastrointestinal Immune System

111.

IV.

V.

VI.

A. Anatomy of the Gastrointestinal Tract B. Nonspecific Immune Mechanisms C. Specific Immune Mechanisms Diseases Involving the Gastrointestinal Immune System A. Inadequacy of Normal Function B. Hyperactivity C. Immunosuppression Impact of Food Constituents and Contaminants on Gastrointestinal Immunity A. Microbes and Microbial Products B. Food Allergens C. Chemicals in Food D. Nutritional Composition Modification of Gastrointestinal Immunity through Food and Diet A. Breast-Feeding 8. Detection and Avoidance of Food Antigens and Allergens C. Development of Hypoallergenic Foods D. Control of Microbial Flora E. Autoimmune Therapy by Oral Tolerance Induction F. Nutritional Therapies Research Needs References

1.

INTRODUCTION

The evolution of higher organisms was dependent on the concurrent development of defensive barriers that excluded the external environment from the increasingly complex internal milieu. Although epithelial struc1 Copyrighl 0 1993 by Academic Press. Inc. All rights of reproduction in any form reserved.

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tures such as skin provided for innate defense, more specialized elements were required in the alimentary canal because this was the specific site of absorption from the external environment and because intestinal lumen contents included low and high molecular weight products of digested food, ingested microbes, and the natural commensal microflora. Thus, the intestine of higher organisms evolved as a partially penetrable filter that largely prevents entry of macromolecular material but allows passage of small nutrients. When deleterious components penetrate the barrier, resultant immune reactions must limit this penetration without compromising the function or integrity of the intestine. The importance and complexity of the gastrointestinal (GI) immune system are evidenced readily by serious effects that are manifested in its failure or dysregulation, for example, microbial infections, allergy, neoplasms, inflammatory diseases, and autoimmunity. Advances in cellular and molecular immunology have led to a greater understanding of the GI immune system and its interaction with diet. This information undoubtedly will lead to novel approaches to maintenance of its homeostasis through diet as well as through therapies for immunologically mediated diseases. In addition, regulatory concerns may arise over the foods eaten by certain sensitive individuals. Ultimately, this new information could afford opportunities for the development of novel foods that benefit general or select populations. The purposes of this chapter are (1) to provide an overview of the GI immune system in health and disease, (2) to relate its function to food composition and nutritional value, and (3) to discuss the current status of and future prospects for beneficial modification of this system through food and diet. Although emphasis is placed on the human GI immune system, generalities are drawn from well-developed animal models. II. OVERVIEW OF THE GASTROINTESTINAL IMMUNE SYSTEM

A. ANATOMY OF THE GASTROINTESTINAL TRACT Discussion of the GI immune system requires a general understanding of the anatomy of the digestive tract. The GI tract (Fig. I) is approximately 30 feet long and is composed of four layers including a mucus membrane lining, a highly vascular submucus coat, longitudinal muscle layers, and an additional oblique layer of muscle fibers (Burke, 1985). Food entering the mouth is disrupted mechanically by the teeth and mixed with lubricating salivary secretions. This material then travels via the pharynx and esophagus to the stomach. On entering the stomach, food is digested by gastric

FOOD, DIET, AND GASTROINTESTINAL IMMUNE FUNCTION

3

FIG. I . Anatomy of the gastrointestinal tract.

juices that contain pepsin, lipases, and acids. The stomach absorbs little food because the food is not yet in diffusible form. Opening the pyloric valve allows stomach contents to enter the small intestine (Burke, 1985). This organ is approximately 20 feet long and has been subdivided into sections called the duodenum, jejunum, and ileum (Fig. 1). The inner mucosal surface has numerous circular folds that con-

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FIG. 2. Sectional views of the small intestinal lining. (A) Circular folds containing villi.

(B)Expanded view of multiple villi found on folds. (C) Expanded view of villi containing absorptive lymphatic and blood capillaries.

tain small protuberances called villi (Fig. 2). Villi effectively increase the total surface area almost 600-fold, thus enhancing the absorptive capacity of the small intestine. Villi contain blood capillaries that permit absorption of monosaccharides and amino acids as well as lymph capillaries that facilitate absorption of fatty acids and glycerol. Enzymes and hormones secreted by the liver and pancreas provide numerous accessory functions to the small intestine. Because of its length, surface area (equivalent to a singles tennis court), and digestive capacity, most absorption (>80%) occurs in the small intestine. Undigested food material enters the large intestine, travels to the colon, and finally exits the anus. Clearly, the large surface area, high nutrient content, and absorptive capacity of the alimentary canal make it particularly prone to penetration by microbial agents and food macromolecules. This challenge is met by an extraordinary array of nonspecific and specific immume mechanisms.

B. NONSPECIFIC IMMUNE MECHANISMS Nonspecific or “innate” immunity is the front line of host defense against microorganisms in the gut and other sites (reviewed by Walker,

FOOD, DIET, AND GASTROINTESTINAL IMMUNE FUNCTION

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TABLE I MECHANISMS OF NONSPECIFIC IMMUNITY IN THE GASTROINTESTINAL TRACT

Mechanism Physical Secretory Microfloral Cellular

Examples Epithelial membranes, intestinal mobility Gastric acidity, bile acids, proteolytic enzymes, mucus Site occupation, bacteriocins, volatile fatty acids Macrophages, pol ymorphonuclear phagocytes

1983; Newby, 1984). These mechanisms might be considered constitutive properties of the normal host. The general types of nonspecific immunity in the gut are summarized in Table I. A fundamental mechanism used by a host to avoid microbial infection is performed by the epithelial barriers, which exclude most (299%)proteins in the intestinal tract (Newby, 1984). Constant renewal of epithelial cells as they move from the crypt insures that damaged villi do not remain in the intestine as foci for infection. Motility of the gut contents also keeps the small intestinal microfloraat low levels relative to the large intestinal microflora, which exist in a more static environment. Secretions provide another major form of nonspecific immunity in the GI tract. For example, the low pH of the stomach (c4.0) along with popsin facilitates destruction of pathogens, as well as of their toxins and immunogenic macromolecules. Bile acids and pancreatic secretions that contain proteases such as trypsin and carboxypeptidase also can function as protectants against microbial pathogens. Also, gastric and intestinal epithelia are covered by a moving layer of mucus that is shed continuously into the lumen. Mucus is a glycoprotein that consists of a long polypeptide chain surrounded by oligosaccharide units that protect it against proteolytic attack. In addition to functioning as a lubricant and as protection for the stomach and intestine from acidic pH, mucus provides a vehicle for antibacterial substances (secretory immunoglobulin A, lysozyme, lactoferrin) and prevents passage of large molecular weight materials into enterocytes. The intestinal microflora represent a stable ecosystem that diminish opportunities for microbial infection. By occupying binding sites on the enterocytes, decreasing gut pH, producing volatile fatty acids, elaborating

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bacteriocins, and increasing motility, these commensal microbes provide an important element of nonspecific defense (Newby, 1984). Microbial agents or antigens that do penetrate the epithelial barrier first may encounter mononuclear phagocytes (blood monocytes or tissue macrophages) and polymorphonuclear phagocytes (PMNs or granulocytes) which nonspecifically defend the systemic compartment. PMNs can cross over blood vessels and are a primary defense against infectious agents, whereas macrophages can be recruited to an inflamed site and can be activated subsequently to an enhanced killing state. Certain proteins in blood also can serve as reinforcing nonspecific defense mechanisms (Pestka and Witt, 1985). Interferon, formed by virus-infected cells, can inhibit replication of other unrelated viruses. Kinins constitute a group of peptides that, when activated, are involved in inflammation and blood clotting. Finally, the complement system, a series of proteins and enzymatic reactions, can bring about the lysis of an invading cell. The complement cascade is activated either nonspecifically by bacterial components (endotoxin, protein A) in the “properdin pathway” or specifically by attachment of an antibody to an invading cell in the “classical pathway.” The nonspecific mechanisms described here act synergistically to prevent entry of intestinal macromolecules or establishment and infection by enteric microorganisms. Thus, under normal conditions, relatively large numbers of a microorganism would be required to initiate infection. However, abilities such as attachment and motility can enhance the virulence of a microbe and contribute to the failure of innate defense. Nonspecific immunity also might be depressed by a variety of factors including decreased gastric acidity via antacid ingestion, diminished commensal microflora as a result of antibiotic administration, or damage to epithelial barriers. In such cases, the numbers of a pathogen such as Salmonella that are required for establishment of an infection would be diminished greatly. Intricate specific defense mechanisms therefore must have evolved to protect higher organisms under these circumstances. C. SPECIFIC IMMUNE MECHANISMS When a microbial invader overcomes nonspecific immune defenses, a mammalian host can activate a system that recognizes and then inactivates a foreign material or “antigen” that is then removed or destroyed (Pestka and Witt, 1985). Fundamental to this specific or “acquired” immune system are capacities (1) to recognize minute differences in the chemical structure of an antigen and (2) to “remember” these structures for long periods of time. Antigens are typically high molecular weight (>10,000) proteins or polysaccharides. Components of bacteria, fungi, and viruses

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such as cell wall, flagella, capsule, and toxins are excellent antigens and are multivalent, that is, have more than one antigenic determinant or recognition site. Abbas et af. (1991) suggested that specific responses could be divided into (1) a cognitive phase, (2) an activation phase, and (3) an effector phase (Fig. 3). The cognitive phase refers to the binding of foreign antigens to specific receptors on lymphocytes that are present prior to antigen stimulation. The activation phase refers to the series of events that is induced in lymphocytes as a consequence of specific antigen recognition. Activation events include proliferation, which leads to expansion of antigen-specific lymphocytes and differentiation from cognitive to effector functions. Activation requires antigen as well as “helper” or “accessory” signals from another cell for a complete signal. Finally, the effector phase represents the active functional manifestations of antigen recognition and activation. Several effector responses to an antigenic stimulus exist. First, one or more components of the specific immune system can be induced to bring about the removal of the antigen. Second, cooperative interactions can occur between specific and nonspecific immune mechanisms, thereby enhancing overall host defense. Third, an antigenic stimulus can induce

FIG. 3. Phases of the specific immune response. Based on Abbas et al. (1991).

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“tolerance,” a “specific” type of unresponsiveness. Thus, a host can recognize and tolerate self-antigens. Therefore, the ability of the immune system to develop a memory allows the host to prevent future reinfection by an invading organism as well as to avoid mounting a self-destructive immune response. I . lmmunocompetent Cell Types Many highly specialized cell types that facilitate varied and intricate specific humoral and cell-mediated immune reactions have been described. Leukocytes are the cells within the immune system that carry out these critical functions, which include resistance to infection, homeostasis of cell maturation, antibody production, and immune surveillance against nascent neoplastic cells (Dean and Murray, 1991). These specialized cells are derived from stem cells in the bone marrow that proliferate, differentiate, and mature into lymphocytes, granulocytes, macrophages, and other specialized cells during a process called hematopoiesis (Fig. 4). Many aspects of leukocyte development are regulated by cell-to-cell interactions and by cytokines. Cytokines, including interleukins, lymphokines, and monokines, are soluble protein factors that have specific effects on cell growth, differentiation, and maturation. Leukocyte subsets can be identified by cell differentiation (CD)antigens present on the cell surface using techniques such as immunofluorescenceand flow cytometry or using functional assays in cell culture. In most cases, the cell types involved in generalized systemic immunity (Table 11) also play key roles in gastrointestinal immunity. Lymphocytes carry out critical regulatory and effector activities in specific immunity. Lymphocytes that mature in the tissue equivalent of the avian bursa are termed B cells. B cells are responsible for humoral (antibody-mediated)immunity and carry immunoglobulins on their surface. Lymphocytes that mature in the thymus are known as T cells, which can have both effector and regulatory functions (Table 11). Effector T cells are involved in cell-mediated immune sequelae such as cytotoxicity and delayed-type hypersensitivity. Regulator T cells control the maturation of effector T and B cells via cognate (cell-to-cell)interactions and cytokines. The immune response thus can be enhanced or depressed through T helper (Th) or T suppressor (T,) cells, respectively. Regulator T cells also contribute to the development and maintenance of tolerance. Committed B and T cells undergo a second stage of differentiation on encountering antigen in “secondary” lymphoid organs such as spleen and gut-associated lymphoid tissue. Here, B and T cells segregate into specific areas where they are exposed to filtering blood and lymph, thus facilitating contact between lymphocytes and circulating antigens. This contact ulti-

FOOD, DIET, AND GASTROINTESTINAL IMMUNE FUNCTION ERY'THROCYTES 1

GRANULOCYTES: Neutrophlls Eosinophlls Basophlls

MEGAKARYOCYTES

1

MONOCYTES LYMPHOCYTE PROGENITORS

I

PLATELETS

\( MACROPHAGES

!

B ELL

THYMUS T CELL (Thymocyte)

PLA'SMA CELLS

T HELPER

T EFFECTOR

FIG. 4. Hematopoiesis and differentiation of lymphocyte progenitors.

TABLE I1 GENERAL FUNCTIONS OF LEUKOCYTES I N SPECIFIC IMMUNITY

Cell type Lymphocytes B cells T cells Accessory cells Macrophages Monocytes Killer cells Natural killer cells Mast cells

Functions Antigen presentation, antigen recognition, antibody secretion Antigen recognition, control maturation of R cells and T effector cells, cytotoxicity Phagocytosis, antigen presentation Phagocytosis, antigen presentation Antibody-mediated cellular cytotoxicity Tumor cell lysis IgE-mediated hypersensitivity

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mately results in the observed humoral and cell-mediated effector functions of specific immunity. The mechanisms of antigen uptake in the gut and subsequent immunologic events are among the most remarkable aspects of immunity in higher organisms. In addition to B and T cells, macrophages and monocytes derived from hematopoiesis can phagocytose infectious particles and function in antigen presentation. Mast cells can respond to various antigens and generate a hypersensitivity response. Additionally, mononuclear cells known as “killer” cells can bind to antibodies and facilitate tumor cell lysis. Other cell types with spontaneous cytolytic activity for neoplastic cells have been called natural killer (NK) cells.

2 . Gut-Associated Lymphoid Tissue Differentiating generalized systemic immunity from mucosal immunity is useful (Fig. 5 ) . The systemic immune system includes all tissue involved in protecting the internal milieu from invading microorganisms. The mucosal immune system encompasses lymphoid tissue that borders the external environment of the gut lumen or other sites such as the bronchus and nasal regions. Although this classification is useful when analyzing diverse functions, many of the specific activities of lymphoid tissue in the systemic and mucosal compartments overlap; one compartment can impact the function of the other. Leukocytes are found at prominent locations in the gut-associated lymphoid tissue (GALT). GALT consists of extrinsic and intrinsic components (Mayrhofer, 1984). The extrinsic lymphoid tissue comprises the single mesenteric lymph node in rodents or multiple (>loo) lymph nodes found in the mesentery of humans and other animals. This extrinsic tissue includes sites that collect intestinal lymph and therefore interface between the gut mucosal and systemic immune compartments. The intrinsic component includes ( 1) organized follicles and groups of follicles (Peyer’s patches) in the small intestine, appendix, and large intestinal submucosa, (2) more diffuse lymphoid tissue, namely, lymphocytes, macrophages, and mast cells in the lamina propria, and (3) intraepithelial lymphocytes in the gut wall (Fig. 6). The intrinsic GALT components contain numerous subsets of immunocompetent cells that play a variety of roles. In humans, the pharynx is surrounded partially by tonsillar lymphoid aggregates that likely participate in immunity. Since the pharynx is shared by the alimentary tract and the respiratory system, inhaled particulate matter can be propelled into this area by ciliary action and swallowed, thus exposing the gut to inhaled antigens (Mayrhofer, 1984). Peyer’s patches have been studied thoroughly and found to contain a full complement of immune cells necessary for

FIG. 6. Gut-associated lymphoid tissue (GALT). Key elements of the intrinsic GALT include the Peyer's patches and lamina propria. Locations of B and T lymphocytes, macrophages (Ma), mast cells (MC), and intraepithelial lymphocytes are shown.

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induction of an immune response, that is, B, T, macrophage, and accessory cells. Lamina propria and intraepithelial lymphocytes apparently function primarily as effector cells. The intestinal lamina propria contains both immunoglobulin(1g)-secretingB cells (plasma cells) and highly specialized T cells that have the phenotype of memory T cells. When activated, these T cells can be characterized functionally as differentiated effector lymphocytes (Zeitz et al., 1990). The pattern of cytokines produced by lamina propria T cells and the responsiveness to certain cytokines also differ from those of other lymphocyte populations (James et al., 1990). Since T-cellderived cytokines are critical regulators for epithelial growth and differentiation as well as for connective tissue metabolism, lamina propria T cells might have additional significance in mucosal growth and transformation. The intraepithelial lymphocyte population has been subject to intense investigation and classified into several types based on differentiation markers and cytokine patterns. These lymphocytes have been suggested to contain regulatory and cytotoxic T cells (Kiyono et a / . , 1991; Lefrancois, 1991; Taunk et al., 1992). 3. Antigen Uptake in the Gut

The movement of high molecular weight antigens from the gut lumen to the blood circulation has been demonstrated experimentally in humans and animals on numerous occasions. Prior to uptake, the antigens must resist proteolytic activity in the lumen and penetrate the mucus/IgA layer to interact with the various absorptive cell types. Factors that disrupt mucosal barrier function and cause extensive uptake include immature gastrointestinalfunction, malnutrition,inflammation,and immunoglobulin deficiencies (Walker, 1987). Macromolecules may be taken up by the gut by at least two distinct mechanisms (Stokes, 1984). In the first, the intestinal epithelial cell can endocytose macromolecular aggregates and deliver these to the subepithelial space. In the second, antigens can be deliberately “sampled” by the Peyer’s patches. a . Uptake via the Intestinal Cell. Normally, epithelial cells are constantly proliferating in the crypt and migrating up the villus surface to the villus tip. Thus, cells of the crypt are undifferentiated whereas cells at the villus tip are differentiated with the capacity to absorb nutrients and provide a mucosal barrier. The microvillus and glycocalyx on the luminal surface are the key elements that facilitate this function. Walker (1985,1987) has reviewed extensively all aspects of antigen uptake by the small intestinal cell. Briefly, when luminal macromolecules are at suffi-

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cient concentrations to evade proteolysis or antibody neutralization and to penetrate the active glycocalyx compartment, they adsorb to the microvillus membrane (Fig. 7). Following evagination of the membrane, phagosomes arise from coalesced vacuoles and fuse with lysosomes to form the phagolysosomes, in which intracellular digestion occurs. Intracellular antigens that escape digestion migrate to the basal surface and are released into the interstitial space. Here, the macrophage functions as nonspecific defense to prevent movement of the antigens into the systemic compartment. Nevertheless, when present at high levels, macromolecular antigens may escape into the circulation and induce a series of immunologic sequelae. Thus, the fact that a small percentage of luminal antigens

Luminal Antigens

II

Phagosome formation I I

J!

0 t

Digestion

@

Exocytosis

L

Antigen and antigen fragments in ints titial space

FIG. 7. Antigen uptake by an epithelial cell. Luminal antigens at sufficient concentrations evade mucus and IgA barrier and interact with microvillous membrane of intestinal cell. Following adsorption/invagination,phagosomes are formed that fuse with lysosomes. Undigested antigen is released into the interstitial space following exocytosis. In some cases, these antigens can enter the systemic compartment and induce immunologic sequelae. Based on Walker (1987).

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JAMES J. PESTKA

can be detected in mesenteric lymph and portal blood is not surprising (Stokes, 1984). Husby (1988) has indicated that dietary antigens are taken up in amounts that are nutritionally insignificant but may be of immunologic importance. Local or systemic antibodies may retard the uptake but, in addition, actually may increase the uptake of unrelated antigens. In humans, the uptake of intact dietary antigen, free or in immune complexes, was reported in studies of healthy subjects and of patients with immune deficiency or allergy. For example, when the uptake of dietary antigen in healthy persons was investigated after a test meal using enzyme immunoassay and high performance liquid chromatography (HPLC) of serum samples, ovalbumin was taken up as intact antigen or as a high molecular weight immune complex constituent in all subjects. The antigen was measurable in serum up to 2 days after the meal. b. Uptake via the Peyer’s Patch. As an alternative to the process just described, antigens can be sampled by lymphoid nodules and groups of nodules known as Peyer’s patches (Fig. 6). These patches are the primary inductive sites for the gut mucosal immune response (Fig. 8). Size and

FIG. 8. Antigen uptake by the Peyer’s patch and lymphocyte homing to mucosal sites. Antigen (Ag) is sampled by M cells in dome epithelium and passed on to underlying lymphocytes. After Ag presentation, activated B and T cells proliferate, differentiate, and migrate via the mesenteric lymph node (MLN)and thoracic duct (TD)to general circulation. From here, they populate the lamina propria and other mucosal sites. Subsequent encounters with Ag result in differentiation of B cells to plasma cells that secrete immunoglobulin (Y), primarily IgA.

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15

numbers of follicles within Peyer’s patches and small intestinal distribution are both age and species dependent (reviewed by Owen and Ermak, 1990). For example, in rodents Peyer’s patches contain 2-1 1 follicles of uniform size and are found throughout the small intestine. In contrast, human Peyer’s patches within the duodenum are small and consist of few follicles but become larger more distally in the ileum; thus, they correspond to the presence of endogenous microflora. Up to 1000 follicles have been noted in the large terminal ileal Peyer’s patch. These large aggregates slow the movement of gut luminal contents, thereby increasing the potential for sampling of antigens. Peyer’s patches are dome shaped and have epithelium absent of villi that contains M cells and M-cell-associated lymphocytes (Owen and Ermak, 1990). M cells exhibit short microvilli and do not digest antigen. Thus, they facilitate delivery of intact antigen into the underlying lymphoid tissue. Since antigenically stimulated lymphoid follicles in the Peyer’s patches protrude into the gut lumen, they have an enhanced capacity for antigen sampling and thus act as sentinels. Macromolecular and particulate antigens-including viruses, bacteria, and small parasites-can be phagocytosed by M cells and passed into the Peyer’s patches. B cell zones that contain germinal centers exist beneath the Peyer’s patch dome. These locales are the sites of B cell proliferation, switching to an IgA+ phenotype, and maturation that is based on affinity for antigen. Near the B cell zones are T cell areas that contain regulatory and effector subsets. In addition to B and T cells, Peyer’s patches contain “accessory cells” (macrophages and dendritic cells) that are capable of presenting antigens. Both B and T cells from the Peyer’s patch are capable of “homing” to distal sites in the body and, thus, can mediate a variety of wide-ranging effects (Fig. 8). This full complement of immunocompetent cells in the Peyer’s patches specifically facilitates ( 1 ) humoral and (2) cell-mediated responses that are focused primarily in the gut, as well as (3) concurrent regulatory effects in the gut and systemic compartment that include oral tolerance. 4. Specific Humoral Responses in the Gut

Humoral immunity is mediated by highly specific proteins known as antibodies, which are secreted by plasma cells derived from B cells in response to antigens. Antibodies belong to the globulin class of serum glycoproteins and thus have been termed immunoglobulins (Igs). The general structure of an Ig consists of two heavy (H)and two light (L) polypeptide chains (Fig. 9) resulting in a total molecular weight in the range of 150,000 to 190,000 daltons for a basic monomer. Although interchain disulfide bonds exist, hydrophobic, electrostatic, and hydrogen bonding

16

JAMES J. PESTKA

r

ANTIGEN

‘ITES

1

FIG. 9. General structure of an immunoglobulin. An immunoglobulin (Ig) consists of heavy

(H)and light (L)chains. An Ig has two specific binding sites, each composed of the variable regions of the H and L chains. The F, region contains determinants for various functions such as placental transfer, complement fixation, and mast cell binding.

interactions are primarily responsible for maintaining the basic Ig structure. Amino terminal ends of both heavy and light chains form the variable regions of the Ig and specifically bind antigen. These variable regions form a binding site that can recognize an antigenic determinant (epitope) approximately 6-7 amino acids in size. Since two pairs of heavy and light chains form identical binding sites, an Ig is said to be bivalent. This ability to bind two antigens simultaneously is used in macroscopic immunoassays such as the precipitation and agglutination reactions. The carboxyl end of the Ig is called the F, region. This region determines the biological functions of the antibody. The five major classes or “isotypes” of Igs are based on heavy chain structure (Table 111). Of these isotypes, IgA is of predominant importance in local immunity in the gut. a. IgA. IgA is critical in immunity to antigens in the gut and other mucosal sites, and accounts for 60% of total daily antibody production in humans (Mestecky, 1988; McGhee et al., 1989,1990). IgA is found both in mucus secretions (secretory IgA) of the gut and as a circulating Ig. Multiple IgA subclasses exist in humans, apes, and rabbits, whereas other species, such as mice, exhibit a single class (Ken-, 1990; Russell et al., 1991). The two human subclasses, IgAl and IgAz , have different distributions in the mucosal (secretory IgA) and systemic (circulatory IgA) compartments. The molecular form (polymeric or monomeric) and subclass are dependent on ( 1 ) type of antigen, (2) duration of immune response, and (3) route of exposure. The molecular properties, cellular origin, and possi-

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TABLE 111 PROPERTIES OF MAJOR IMMUNOGLOBULIN CLASSES

Immunoglobulin class

Percentage of total circulating Ig

Structure

Characteristics

IgA

15

Monomer, polymers

sIgA

-

Dimer

IBE

0.05

Monomer

IgG

80

Monomer

IOM

5-10

Pen tamer

IgD

0.05

Monomer

Found in blood and lymph Found in secretions such as saliva. milk, mucus; protects mucous membranes Found in blood and lymph; attaches to mast cells and basophils; associated with allergies and chronic parasitic infections Found in blood and lymph; transfers across placenta; fixes complement; attaches to phagocytes Found in blood, lymph; fixes complement; attaches to phagocytes Found mainly on surface of B cells

ble physiologic role of the IgA immune system have been the subject of several reviews (Bogers et al., 1991; Russell el al., 1991). Secretory IgA (sIgA) occurs as a dimer and includes two additional peptides: a J (joining) chain and a secretory component that enhances its transfer and enteric survivability (Mestecky et al., 1991). Antigens in the gut are most likely to encounter sIgA before any other Ig. Peyer’s patches usually are considered inductive sites for the IgA response. Antigens sampled by the Peyer’s patches encounter antigen-presenting cells, regulatory T cells, and B cells. From this location, committed IgA plasma cell precursors migrate via the mesenteric lymph node and thoracic duct to blood, spleen, and liver, and return to the gut lamina propria or are localized at distant mucosal sites (Fig. 8) (Czerkinsky et al., 1987). On differentiation at the mucosal level,. the resultant plasma cells produce dimeric IgA that, by noncovalent association, become complexed with secretory component, a protein that is synthesized by glandular cells.

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JAMES J. PESTKA

Secretory component facilitates transfer of sIgA across the epithelial layer into the lumen. Primary roles that have been suggested for sIgA are antigen exclusion, inhibition of adherence of microorganisms, intracellular virus neutralization, and excretion of IgA immune complexes. A key feature of sIgA is its inability to induce mechanisms such as opsinization or complement activation, which potentially could be damaging to the intestine. Rather, IgA induces antigen clearance by taking advantage of the normal clearing activities of the gut (Newby, 1984). Interaction with nonspecific immune parameters enables sIgA to inhibit entry of soluble antigens and restrict epithelial colonization of bacteria and viruses. Although the function of serum IgA is less defined, one possible role is the noninflammatory neutralization of toxins, viruses, and enzymes (Mestecky, 1988; Kerr, 1990). B cell activation, switching, proliferation, and differentiation to IgA synthesis are regulated tightly in the mucosal and systemic immune compartments by several cell types and cytokines (McGhee et al., 1989). A key element in differentiation of B cells to IgA production is the type 2 T helper cell, which can secrete the cytokines IL4, ILS, and IL6 (McGhee et al., 1989,1990;Taguchi et al., 1990). IL5 and IL6 stimulate IgA production in mitogen-stimulated B cells, a process that is enhanced by the presence of IL4 (Bond et al., 1987; Coffman et al., 1987; Murray et al., 1987). Another cytokine, TGF-P 1 , is a product of several cell types including T cells that is capable of inducing switching of IgM' cells to IgA+, as well as having a number of other pleiotropic effects (Coffman et al., 1989; Chen and Li, 1990; Kim and Kagnoff, 1990a,b). Finally, orally administered antigens can induce proliferation of Peyer's patch T cells that bear an F, a receptor. These cells may collaborate selectively with IgA-committed B cells via a putative immunoglobulin binding factor, but this factor has not been characterized molecularly (McGhee et d., 1989,1990). 6 . IgE. IgE is another key immunoglobulin that is thought to be involved in responses to parasitic infections. Present only in small amounts in serum, IgE exists primarily in association with mast cells and basophils. Binding of antigen to mast-cell-associated IgE and subsequent cross-linking results in the release of histamine and other inflammatory mediators (Fig. 10). Barrett and Metcalfe (1988) have reviewed the relationship between IgE and mucosal mast cells extensively. Numerous mast cells can be found within and beneath the gut mucosa (Fig. 6). For example, the human intestine contains 20,000 mast cells per mm3 (Norris et al., 1963). Thus IgE may be carried into the gut mucosa by mast cells and facilitate degranulation with histamine release on interaction with a food or microbial antigen. When this occurs in association with epithelia, the

FOOD. DIET. AND GASTROINTESTINAL IMMUNE FUNCTION

19

A

,;y-y,, .*"

MASTCELLS

".

B

FIG. 10. Activation of mast cells via IgE crosslinking. (A) Multivalent antigen (Ag) specifically binds to IgE attached to mast cells via F, receptors. (B) Crosslinking by Ag initiates degranulation of mast cells with concurrent release of vasoactive amines.

volume and flow of secretion can increase as can flushing action that removes the offending agent from the intestine. This response also contributes to certain food allergies (discussed in Section IILB). c. Other lgs. Protective function in the gut analogous to that served by IgA also may be attributed to secretory IgM, because on occasion its mucosal synthesis is elevated, particularly in selective IgA deficiency. IgG is not considered a secretory immunoglobulin since its external translocation requires passive intercellular diffusion. By binding complement, IgG actually can cause increased mucosal permeability and tissue damage, and thus contribute to long-term immunopathology in gut mucosal lesions.

5.

Specific Cell-Mediated Responses in the Gut

Understanding mechanisms for cell-mediated immune responses in the gut has just begun. These processes are likely to involve the two major types of cell-mediated immune response found in systemic immunity, namely, cytotoxicity and delayed-type hypersensitivity (DTH) reactions. Cytotoxic T cells defend a host against living antigens such as virusinfected cells or intracellular pathogens (Fig. 1 IA). In such a response, a target cell bearing a surface antigen interacts directly with a cytotoxic T

JAMES J. PESTKA

20 A

- -

0 . target cell

CTL

cJ+

lysed target cell lysis of more target cells

CTL

B

nonspecific cell killing lymphokine release

t

TDTHCBIl macrophage attraction, immobilization, and activation

FIG. 11. Major types of cell-mediated specific immune responses. (A) Cytotoxicity: cytotoxic T cell specifically recognizes and lyses target cell. (B) Delayed hypersensitivity: T cell releases cytokines that facilitate killing of target.

cell, resulting ultimately in the lysis of the target cell. The killing is unidirectional, so a cytotoxic T cells can kill target cells repeatedly. In allergic contact hypersensitivity, low molecular weight chemicals (haptens) can conjugate to proteins in the host and elicit a reaction involving cytotoxic T cells. Intestinal intraepithelial lymphocytes may have a novel and important role in recognizing and destroying transformed epithelial cells and colon cancers (Taunk et al., 1992). Certain other cell-mediated hypersensitivity reactions are referred to as "delayed-type'' since the time required for onset of effects is long compared with the time required for immediate-type reactions (Fig. 11B). DTH reactions can occur systemically for any type of antigen, living or nonliving; the sole requirement is that the antigen be bound to the cell surface. Recognition of the antigen by a specific T cell receptor induces activation of a T cell and subsequent cytokine secretion. These cells may cause nonspecific cell killing; others act on macrophages by drawing them to the

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21

site of inflammation where they are immobilized and activated. Such macrophages have enhanced ability to kill and destroy pathogens that normally would survive macrophage ingestion. Once formed, these macrophages kill nonspecifically, but their activation occurs through antigenspecific cytotoxic T cells. Rejection of allografts is the rejection of tissue grafts between members of the same species that differ genetically. Certain genetically determined cell surface antigens, called major histocompatibility antigens, are responsible for initiating this response. In this case, a local DTH reaction involving cytotoxic T cells occurs that produces an intense inflammation at the graft site. 6 . Common Mucosal Immune System

Antigen stimulation in the gut has been suggested to result in IgA secretion at other mucosal sites such as salivary glands and genitourinary sites. As described earlier, on antigen presentation in the Peyer’s patches, B and T cells migrate via the mesenteric lymph node into the systemic circulation. In addition to entering the lamina propria of the intestine, these cells can migrate to the salivary glands, to the eye via lacrimal glands, and into milk via the mammary glands (Fig. 8). The capacity of antigen-activated IgA B cell blasts from the Peyer’s patches to migrate to multiple mucosal sites has led to the concept of a common mucosal immune system (McDermott and Bienenstock, 1979). Although largely demonstrated in experimental animals, evidence exists for a common mucosal immune system in humans, based on detection of gut antigen-specific IgA at anatomically remote sites. Further, antigenspecific IgA-producing cells can be found in blood after oral immunization and before their appearance in saliva and tears (Russell et al., 1991). The advantage of a common mucosal response probably relates to the mobilization of humoral and cellular immune elements to various sentinel sites (e.g., mouth, eye, genitourinary tract) that can prevent infection on subsequent reexposure to a mucosal pathogen. 7. Stimulation of Specific Immunity

Stimulating the specific immune response within the gut to protect against various microbial illnesses clearly, is desirable. However, achieving long-term memory when immunizing orally is difficult because these antigens to be degraded by acidic pH and proteolysis in the gut (Stokes, 1984). Oral immunization with live organisms rather than nonreplicating ones is generally more effective for induction of IgA responses, implying that colonization and/or replication in the GI tract is

22

JAMES J. PESTKA

required (McGhee et al., 1992). Further, particulate antigens function much more effectively than soluble ones. Thus, close contact with key components of the gut is required to induce a GI immune response. The immunogenic character of an orally presented antigen can be preserved by administration with large amounts of sodium bicarbonate or in protective capsules (McGhee er al., 1992). Several adjuvants also have been used to induce gut IgA responses, including ox bile, muramyl dipeptide, concanavalin A, peptidoglycan and vitamin A, streptomycin, lysozyme, polyvalent cations, and DEAE dextran (Ernst et al., 1988). Other oral antigen administration systems have employed cholera toxin, biodegradable microspheres, and selective delivery of antigens by recombinant bacteria (McGhee et al., 1992). 8. Oral Tolerance as a Specific Response Effects on systemic immunity from encountering antigen by the oral route can range from the active sensitization described earlier to oral tolerance (immunological unresponsiveness) (Strobel and Ferguson, 1985). This effect is dependent on timing, dose, and frequency of antigen administration. Systemic unresponsiveness often can be observed concurrently with an intestinal immune response (Challacombe and Tomasi, 1980; Richman et al., 1981; Mattingly, 1983). The coexistence of a diminished systemic response with an enhanced mucosal response is thought to be favorable, since it reduces the chances of unfavorable inflammatory events while still protecting the host against agents in the gut environment. Oral tolerance in mice apparently becomes impaired with age (Kawanashi et al., 1990). Stokes (1984) and Ernst er al. (1988) have summarized possible mechanisms and the potential significance of oral tolerance. Ill.

DISEASES INVOLVING THE GASTROINTESTINAL IMMUNE SYSTEM

The relationship between disease and the GI immune system is exceedingly complex (Fig. 12). For example, normal function may be overridden by a highly virulent microorganism or by a series of mutation events leading to a neoplasm. Under these circumstances, inflammatory responses in the gut actually might contribute to the overall symptoms of the disease. Also, hyperactivity might lead to inflammatory sequelae such as food allergies or enteropathies and, on a more speculative level, to inflammatory bowel diseases and various immune complex and auto-

FOOD. DIET, AND GASTROINTESTINAL IMMUNE FUNCTION

23

NORMAL FUNCTION ADEQUATE

No disease

Increased sensitivity to microbial infections, allergies,

Specific and nonspecific gastrointestinal immune mechanisms

Microbial infection, neoplasm

inflammatory bowel disease, autoimmune disorders HYPERACTIVITY

FIG. 12. Complex relationships between disease and the GI immune system.

immune disorders. A third possibility is that generalized immunosuppression can bring about increased sensitivity to microbial infections, allergies, or neoplasms. These options are complicated further by the fact that, in all these cases, mutually excluding GI immunity and general systemic immunity is difficult.

A. INADEQUACY OF NORMAL FUNCTION 1 . Microbial Infection A multitude of bacteria, parasites, and viral agents cause gastroenteritis or penetrate the gut prior to eliciting systemic infection. The capacity to override the GI immune system is dependent on the numbers of ingested pathogen and on virulence factors such as toxigenicity, adherence, and invasiveness (Walker and Owen, 1990). Thorne (1986) outlined five levels of pathogenesis for bacterial diarrheal diseases: (1) bacteria produce toxin but do not adhere and multiply (e.g., Bacillus cereus, Staphylococcus aureus, Clostridium perfringens, Clostridium botulinum); ( 2 ) bacteria adhere and produce toxin (e.g., enterotoxigenic Escherichia coli, Vibrio cholerae); ( 3 ) bacteria adhere to the mucosa and destroy the brush border (e.g., enteropathogenic E. coli); (4) bacteria invade the mucosa and multiply intracellularly (e.g., Shigella spp.); and

24

JAMES J. PESTKA

(5) bacteria penetrate mucosa and spread to lamina propria and lymph nodes (e.g., Campylobacter, Yersinia). With each increasing level of action, the pathogen focus moves from the mucosal to the systemic compartment. Hence, the specific immune response must be escalated. For example, some types of Salmonella can move into the systemic compartment causing generalized illnesses such as enteric and typhoid fevers. This escalation concurrently can unleash destructive potential that will damage the host seriously. In addition to the aforementioned mechanisms of virulence, the antigen sampling process itself may become a major portal of entry for pathogens (Owen and Ermak, 1990). Wells et al. (1988) hypothesized that, in some instances, a motile phagocyte may ingest an intestinal bacterium, transport it to an extraintestinal site, fail to accomplish intracellular killing, and liberate the bacterium at the extraintestinal site. This hypothesis was based on the observation that intestinal bacteria that most readily translocate out of the intestinal tract are categorized as facultative intracellular pathogens. Also, intestinal particles without inherent motility (e.g., yeast, ferritin, starch) move out of the intestinal lumen within hours of their ingestion. Finally, the rate of translocation of intestinal bacteria can be altered with agents that modulate immune functions such as phagocytosis. Thus, systemic infection by translocating intestinal bacteria could be a result of the antigen-sampling process that evolved to regulate the immune response to intestinal antigens. A normal mucosal immune response occasionally might enhance viral infection. Sixbey and Yao (1992) have shown that, when bound to dimeric IgA, Epstein-Barr virus (EBV) can enter epithelial cells through secretory component-mediated IgA transport. This IgA-dependent infection of mucosal tissue may be a mechanism for the involvement of EBV in cases of human nasopharyngeal carcinoma. 2 . Intestinal Cancer Lower intestinal tract cancer is the second most frequent cause of cancer death in the United States, typically affecting persons over age 50. Diet is thought to be a major factor in the prevalence of this disease. Successful establishment of intestinal neoplasms suggests a failure in normal immune surveillance. Immune surveillance refers to the capacity of cell-mediated immune-effector cells (1) to recognize unique cell surface antigens that distinguish spontaneously arising tumors from normal cells and (2) to destroy the neoplasm (Dean and Murray, 1991). This concept is supported by the observation of increased neoplasia associated with primary immunodeficiency diseases and immunosuppressive

FOOD, DIET, AND GASTROINTESTINAL IMMUNE FUNCTION

25

therapy. Immune surveillance may involve direct T cell killing, antibodydependent cellular cytotoxicity, or natural killer cell cytolysis. Some investigators question the validity of the concept of immune surveillance, since it largely has been demonstrated with a limited subset of cancers caused by oncogenic viruses (Abbas et al., 1991). Immunologic mechanisms in intestinal malignancy are reviewed in depth by Itzkowitz and Kim (1988). B. HYPERACTIVITY 1 . Adverse Reactions: Intolerance or Allergy

Adverse food reactions can be defined as clinically abnormal responses to a food or food additive. The concepts of “food allergy” and “food intolerance” frequently are misused and interchanged when discussing adverse reactions. Based on the criteria of the Committee on Adverse Reactions of the American Academy of Allergy and Immunology (Atkins and Metcalfe, 1984), a food intolerance is defined as an idiosyncratic, pharmacologic, metabolic, or toxic reaction in which the immune system does not participate. Known and alleged food intolerances are summarized in Table IV. These nonimmunologic effects have been discussed extensively by Anderson ( 1984); further discussion is beyond the scope of this chapter. Other poorly defined symptoms that have been attributed to foods include allergic tension fatigue syndrome, hyperactiv-

TABLE IV GENERAL CLASSES OF FOOD INTOLERANCE‘

Type of reaction Anaphylactoid Metabolic Pharmacologic Food intoxication Food additive

Examples Food-induced histamine release, histamine poisoning Lactose intolerance, hypoglycemia Caffeine Natural food toxins, bacterial and fungal toxins Salicylates, tartrazine, monosodium glutamate, sulfites

Based on Anderson (1984).

26

JAMES J . PESTKA

ity, schizophrenia, cerebral allergy, and environmental illness (Kettelhut and Metcalfe, 1987). A variety of controversial diagnostic tests has been used to verify these other syndromes. Metcalfe (1992) evaluated these conditions and concluded that (1) in most instances no clear relationship exists between these diagnoses and food ingestion, (2) the lack of association with food suggests other etiologies, and (3) well-designed clinical trials rather than controversial tests such as sublingual provocation, subcutaneous/intracutaneous provocation, and in vitro leukocytotoxic tests should be used to evaluate these syndromes. In contrast to food intolerances, food allergies or hypersensitivities are indicative of adverse reactions in which the gut and systemic immune systems play distinct roles (reviewed by Taylor, 1980; Atkins and Metcalfe, 1984; Ferguson, 1984; Stern and Walker 1985). Sampson and Metcalfe (1991) estimated that 1-2% of the general population may exhibit food allergies. Allergies can be classified pathogenically into reaginic (involving IgE) and nonreaginic classifications (Gel1 et al., 1975) (Table V). Sometimes these reactions are categorized as immediate or delayed-onset type. In such food-related hypersensitivities, the gut immune system may be an initial target, but hypersensitivity can be manifested further at the systemic level. Absolute separation into immediate and delayed hypersensitivities must be done with caution because the interval between ingestion and symptoms is dependent on quantity of food ingested, degree of hypersensitivity, threshold for complaints, and other factors that are not always related directly to the immune event taking place (Taylor, 1980). TABLE V CLASSIFICATION OF HYPERSENSITIVITIES

Classification Reaginic Type I: Immediate hypersensitivity Nonreaginic Type 11: Antibody mediated Type 111: Immune-complex mediated Type IV: T-cell mediated

Mechanisms Involves IgE-mediated release of vasoactive amines, arachidonic acid metabolites, and cytokines from mast cells IgM and IgG specific for tissue or cell surface antigens induces complement activation. leukocyte recruitment and activation Circulating immune complexes and IgM/lgG induce complement activation, leukocyte recruitment and activation Delayed hypersensitivity caused by T-cell activation; direct target cell lysis by cytotoxic T cells

FOOD. DIET. AND GASTROINTESTINAL IMMUNE FUNCTION

27

2 . Reaginic (Type I ) Hypersensitivities Key components of a Type I reaction to a food are a sensitizing antigen, specific IgE response, and IgE-binding mast cells or basophils. Examples of antigenic substances that have been identified in foods include cow's milk protein, egg, codfish, shrimp, peanut, and soybean. Although serum antibodies to ingested proteins can be detected early in life (Gunther et al., 19601, the capacity to produce problematic antigen-specific IgE responses apparently is determined genetically. IgE-secreting cells can be found in the gut lining and other mucosal sites as well as in the systemic compartment. Mast cells and basophils have receptors that bind the F, region of IgE (Barrett and Metcalfe. 1988). On cross-linking of these receptors through binding of multivalent antigen (allergen), the mast cell releases various inflammatory mediators including histamine (Fig. 10). Mast cell degranulation with subsequent exposure of adjacent tissues to mast cell mediators can cause local changes in vasopermeability, enhanced mucus production, muscle contraction, pain, and inflammatory cell recruitment. This local anaphylaxis also can bring about translocation of macromolecules across the intestinal barrier. Mast cells bound to IgE molecules that react with food antigens can be identified throughout the body, even in skin. Thus, food antigens entering the systemic compartment by mechanisms described earlier can initiate degranulation of mast cells at other sites in the body. Clinical manifestations of reaginic hypersensitivities may range from minor to more ominous effects that may be localized to the GI tract, to more distant sites, or to both places. The oral cavity often is the initial site of symptoms such as swelling and burning of the lips, mouth, and throat. Responses in the gut may result in abdominal swelling, nausea, cramping, vomiting, and diarrhea. Other sites of clinical signs include skin where itching, fluid accumulation, and eczema might be seen. Asthma and allergic rhinitis also can occur, particularly in children. Systemic anaphylaxis describes the simultaneous onset of a reaginic response at multiple organ sites that includes the effects just described coupled with sometimes fatal hypotension with shock (Sampson et al., 1991b). Systemic anaphylaxis can occur after minor symptoms have been observed in previous exposures to the offending food or may be manifested unexpectedly. Unequivocal verification of reaginic food allergy requires antigen identification, demonstration of a relationship between antigen exposure and adverse reaction, and determination of the immunologic mechanism involved (Atkins and Metcalfe, 1984). Hence, no single test can verify a

28

JAMES J. PESTKA

TABLE VI METHODS FOR DIAGNOSIS O F TYPE I FOOD ALLERGIES

Preparation of medical history In vivo skin test In vivo tests RAST ELISA Basophil histamine release Oral food challenge Elimination diets

food allergy. Various methods that can be used to diagnose reaginic food allergies are listed in Table VI. Initially, a careful medical and dietary history must be taken to exclude reactions that might be misinterpreted as food allergies. Such reactions include enzyme deficiencies, gastrointestinal disease, anatomical defects, chemical reactions, collagen vascular diseases, endocrine disorders, and psychological factors (Metcalfe, 1992). Initial evaluation can be followed up by immunologic testing and food challenge elimination trials. Immunologic testing consists of in uiuo and in uitro procedures that employ food extracts. For example, skin testing can be performed by applying aqueous food extracts to a scratch or puncture. A demonstrable weal compared with a control site can be used to support the possible involvement of suspect food antigens. In uitro tests such as the radioallergosorbent test (RAST) and enzyme-linked immunosorbent assay (ELISA) are useful in identifying antigen-specific IgE in patient serum (Atkins and Metcalfe, 1984). Basophil histamine release assays have been found to correlate with skin tests but are difficult and expensive to perform. Although other immunologic tests such as cytotoxic testing, provocative subcutaneous testing, and provocative sublingual testing employ the use of extracts, these tests are unproven (Metcalfe, 1992). Final verification that a particular dietary antigen causes an allergic response is dependent on observing the response to oral challenge, particularly when the association between certain foods and a clinical sign remains uncertain. Because of the threat of systemic anaphylaxis, oral food challenge always should be conducted under supervision of a physician. 3. Nonreaginic (Types 11, III, I V ) Hypersensitivities

Strong evidence is not available to suggest the potential for Type I1 food-mediated allergies. However, food-related Type I11 reactions have been noted that involve deposition of antigen-antibody complexes at a

FOOD, DIET, AND GASTROINTESTINAL IMMUNE FUNCTION

29

reaction site, followed by complement fixation and cell-mediated tissue damage. Circulating immune complexes that contain food antigens have been noted (Cunningham-Rundles et al., 1979a,b; Pagnelli et al., 1979, 1980; Frick, 1982). Other experimental evidence includes translocation of cow’s milk antigen (Bock et al., 1983), elevated Ig-producing cells in cow’s milk allergy (Savilahti, 1973; Stern et al., 1982), and deposition of IgG, IgM, and complement (Matthews and Soothill, 1970; Shiner et al., 1975). The precise mechanisms of Type I11 hypersensitivities remain elusive. Based on transplantation studies, Strobel (1990), postulated that Type 111 reactions in gut mucosa lead to aberrant expression of histocompatibility antigens, infiltration of inflammatory cells, and an increase in intraepithelial lymphocytes. These changes are followed by an increase in crypt cell turnover and elongation of crypt cells. Type IV hypersensitivities are referred to as delayed-type hypersensitivities because of the time required for onset of effects. These reactions involve recognition of a cell-bound antigen by a T cell and subsequent cytotoxic action; reactions can occur between 6 and 25 hr after ingestion of food. Clinical manifestations of Type IV hypersensitivity involve inflammation of the GI tract with attendant symptoms (Atkins and Metcalfe, 1984). Elevation of intraepithelial lymphocytes, a group of cells including T cells or cells that share many phenotypic markers and functional activities with T cells, appear to be a common element in foodmediated Type IV reactions such as cow’s milk allergy (Asquith, 1970; Ashkenazi et al., 1980; Stern, 1982). Intraepithelial lymphocytes may function locally in Type IV responses by a mechanism as yet unknown. 4 . Food-Sensitive Enteropathies

Several diseases exist in which small intestinal pathology appears to be associated with a local immune response in the gut (Mowat, 1984). Clinical features of food-sensitive enteropathies in infants include diarrhea, malabsorption, and failure to thrive, all of which occur soon after the introduction of antigen in diet. Although sometimes defined in a descriptive fashion, these diseases very likely involve reaginic or nonreaginic mechanisms. Examples include celiac disease, cow’s milk protein intolerance, and soy enteropathy. Strobe1 (1990) has suggested that foodsensitive enteropathies result from a breakdown of oral tolerance, which allows a cell-mediated and/or IgE-mediated immune response to develop. Underlying reasons are multifactorial and encompass genetics, environment, maturity, infection, and autoimmune responses. a . Celiac Disease. Celiac disease is defined as a permanent condition of gluten intolerance that is associated with characteristic gluten-

30

JAMES J. PESTKA

sensitive changes in the small intestinal mucosa (Ferguson, 1984). This tissue characteristically returns to normal with a gluten-free diet but relapses on further challenge with gluten. Celiac disease involves interactions between genetic and immunologic factors and diet (Cole and Kagnoff, 1985). The condition typically begins in the first 2 years of life following exposure to gluten and is related to initial exposure to an offending food antigen (Mowat, 1984). Major histocompatibility complex genes apparently represent a major component contributing to disease susceptibility (Kagnoff, 1989). A viral protein also may play a role in the pathogenesis of celiac disease, perhaps via immunologic cross-reactivity between an antigenic determinant shared by the viral protein and (Y gliadins. When Husby (1988) studied uptake of ovalbumin and p-lactoglobulin in children with celiac disease, either on a gluten-free diet or after gluten challenge, and in controls with normal gut mucosa, both antigens were present in high molecular weight fractions of the sera, presumably as immune complexes. Levels of the antigens in serum did not differ among the children with celiac disease and the controls eating the gluten-free diet. However, in 4 of 5 celiac children, the uptake of both antigens was increased after gluten challenge, indicating the potential for increased antigen uptake in celiac disease. Several studies suggest that cellmediated reactions, particularly of intraepithelial lymphocytes, are of critical importance in celiac disease (Kagnoff, 1989; Russell et al., 1991; Troncone and Ferguson, 1991). Intestinal T cell-mediated reactions in experimental animals have shown pathologic features similar to those of celiac disease. This pathology includes changes in villus and crypt architecture, crypt hyperplasia, elevated numbers of intraepithelial lymphocytes, and increased intraepithelial lymphocyte mitosis. Since pathogenesis in celiac disease might be viewed as failure of the normal inhibition of immune responses to gluten in the gut, therapeutic control of these immunoregulatory mechanisms might provide an approach to treating this disease and other food protein-sensitive enteropathies.

b. Cow’sMilk Protein Intolerance. Cow’s milk protein intolerance (CMPI) is an enteropathy that includes syndromes resulting from intolerance of one or more cow’s milk proteins. The condition is estimated to affect between 1 and 3% of children (Mike and Asquith, 1987). CMPI occurs during the first 6 months of life but, unlike celiac disease, subsides in subsequent years. Symptoms include diarrhea, vomiting, weight loss, urticaria, wheezing, and eczema. Interestingly, breast-fed infants can be sensitized to cow’s milk that has been ingested by the mother (Gerrard and Shenassa, 1983). Mike and Asquith (1987) suggested that Type I, 111, and IV responses may be active in CMPI to varying extents

FOOD, DIET, AND GASTROINTESTINAL IMMUNE FUNCTION

31

among children. In a model proposed by Jackson et a f . (1983), CMPI was related to increased permeability during the course of acute gastrointestinal infections. Type I reactions may occur on entry of unprocessed antigens, thereby enhancing entry of more antigens into the systemic compartment. Ensuing local and systemic reaginic and nonreaginic effects then might determine the extent of the illness. c . Soy Protein Enteropathy. Mucosal and systemic reactions to soy proteins have been identified in children and adults. Mechanistically, this response appears to involve a Type 111 reaction mediated by IgG antibodies (Mike and Asquith, 1987). Young animals fed soy protein develop very high titers of the IgG, a subclass that could form immune complexes, activate complement, and elicit a Type I11 hypersensitivity response (Barrett et al., 1978). Morphologic effects are consistent with this possibility and include villous atrophy and infiltration of the villi and lamina propria with mononuclear cells. Nevertheless, the immunologic mechanisms of soy protein enteropathy are still vague.

5 . Inflammatory Bowel Disease Inflammatory bowel disease is a classification that includes two similar clinical entities, ulcerative colitis and Crohn’s disease (regional enteritis). Ulcerative colitis is an inflammatory disease that begins in the rectum and proceeds upward (Burke, 1985). The condition usually affects young adults under age 30. Extensive sloughing of mucosal cells occurs in this disease, and involves ulcerations that can coalesce into the deep muscular layer of the colon. Crohn’s disease patients exhibit similar symptoms; the terminal ileum is affected particularly but the disease can be found in any segment of the intestinal tract. The pathogenic mechanisms of inflammatory bowel disease remain enigmatic and have been proposed to include autoimmunity, toxic environmental factors, and diet (Mike and Asquith, 1987). Immune sensitization to intestinal epithelial antigens is common in families with chronic inflammatory bowel disease. The high frequency of sensitization among asymptomatic relatives suggests that it predisposes individuals to gut tissue injury (Fiocchi et al., 1989). Although heightened humoral and cell-mediated immunologic manifestations clearly occur in the gut, whether these conditions actually cause the disease or are serious secondary effects that perpetuate the inflammatory process is not clear. With respect to humoral immune function, both numbers and functional characteristics of B and T cells vary with inflammatory bowel disease, but a definitive correlation has not been proven (Fiocchi, 1990).

32

JAMES J. PESTKA

Further, anticolon antibodies are detectable in ulcerative colitis, but similarly are found in other conditions. Altered immune regulation sometimes results in a disproportionately increased number of IgG-producing cells in the mucosa; this immune dysregulation may contribute to persistence of inflammatory bowel diseases (Brandztaeg et al., 1985). Vascular complement activation. also might be a process in active inflammatory bowel disease lesions, and presumably relates to the degree of inflammation and immune complex formation (Halstensen e? al., 1989). Circulating antigen-nonspecific suppressor T cells are found in the early stages of Crohn’s disease (James et al., 1987). These and other data suggest that the suppressor T cells are markers of an underlying and persistent antigen-specific immune response to an as yet unidentified antigen or set of antigens. James et al. (1987) have postulated that this underlying antigen-specific response is the result of a primary immunoregulatory abnormality involving an imbalance between the effects of antigen-specific helper and T suppressor cells that recognize a common antigen or antigens present in the mucosal environment. Critical areas of study in intestinal inflammatory diseases are identification of unique and nonunique lymphoid cell sets in the intestine and clarification of how these sets operate in the intestinal microenvironment (Elson et a/., 1986). The latter goal will require understanding the mechanisms by which these sets communicate with and regulate one another via cell surface molecules and soluble mediators. Cytokines that have been investigated in some detail or in a preliminary fashion include IL1, IL2, IL4, interferon y (IFNy), and colony-stimulatory factors (Fiocchi, 1989). For example, Kusugami et al. (1989) demonstrated that reactivity to IL2 distinguishes intestinal mononuclear cells from inflammatory bowel disease patients from those from controls. How intestinal immune homeostasis is maintained in normal individuals, so they do not have inflammatory disease despite the presence of endogenous stimulatory factors such as endotoxin that are inflammatory elsewhere in the body, is particularly unclear. The role of food and diet in inflammatory bowel diseases is also complicated (Frieri et al., 1990). Clarification of these processes perhaps will provide new perspectives on mechanisms involved in chronic intestinal inflammatory diseases and clarify the role of food in initiating or exacerbating these diseases. 6 . Other Immunologic Diseases: Immune Complex and Autoimmune Types

The gut immune system may play a pivotal regulatory role in other immune diseases that are mediated by the nonreaginic hypersensitivity

FOOD, DIET, AND GASTROINTESTINAL IMMUNE FUNCTION

33

reactions described in Table V. These diseases result from aberrant, excessive, or uncontrolled immune reactions or via autoimmunity resulting from immune responses to self-antigens (Abbas et al., 1991). Some immunologic diseases involve antibodies that can (1) form immune complexes and deposit at various tissue sites (e.g., poststreptococcal glomerulonephritis, systemic lupus erythematosus) or (2) attach to self-antigens that are circulating or fixed, thus inducing an autoimmune response (e.g., Goodpastures’s syndrome, Grave’s disease). Others involve T-cellmediated injury (e.g., myasthenia gravis, viral myocarditis). Jonsson, Mountz, and Koopman (1990) have evaluated pathogenesis of autoimmunity relative to oral mucosal diseases. Although the role the gut immune system plays in most autoimmune and immune-complex diseases is vague, evidently this system is involved in a syndrome known as IgA nephropathy. IgA nephropathy is the most common form of glomerulonephritis that, in some cases, can lead to kidney failure (Schena, 1990). Apparently, this disorder is caused by mesangial deposition of IgA-containing immune complexes formed by polymeric IgA (pIgA) that is overproduced in response to antigens presented at mucosal surfaces (Emancipator and Lamm, 1989). To test whether antibodies to dietary antigens might be involved in the pathogenesis of IgA nephropathy, Nagy et al. (1988) measured IgG and IgA serum antibody activities to gluten, a gluten fraction called glyc-gli, a-lactalbumin, p-lactoglobulin, casein, and ovalbumin in patients with IgA nephropathy. The IgA activities to gluten antigens and a-lactalbumin were increased significantly in IgA nephropathy over levels seen in age-matched healthy controls. These researchers suggest that their results showed a relationship between the intestinal humoral immune system and IgA nephropathy, and indicated that antibodies to dietary antigens in some patients may be involved directly in the pathogenesis of IgA nephropathy.

C. IMMUNOSUPPRESSION The GI immune system can be depressed for a variety of reasons, which can impact a host’s capacity to defend against microbial pathogens in the gut and to control intake of protein antigens. The association between various immunodeficiency disorders and heightened incidence of gastrointestinal disease has been reviewed extensively by Ament ( 1984). For example, selective IgA deficiency is a type of congenital immunodeficiency that occurs in 1 of 400 persons. Absence of sIgA in the intestine allows uptake of protein antigens and creates a propensity for increased

34

JAMES J. PESTKA

Type I and 111 allergies (Cunningham-Rundles, 1991). Also, radiation and chemotherapy used in transplantation and cancer treatment also can contribute to depressed GI immune function (Cunningham-Rundles and O’Reilley, 1986). Acquired immunodeficiency syndrome (AIDS) affects all aspects of immunity because of a depressed T helper population. Because it is the largest lymphoid organ in the body, the GI tract is considered a potential reservoir for human immunodeficiency virus (HIV), the agent that causes AIDS. Indeed, most AIDS patients exhibit a variety of gastrointestinal symptoms including diarrhea. Initial depletion of CD4+ (T helper cells) has been postulated to result in impaired IgA+ B cell development, which reduces IgA secretion in the lamina propria (Smith et al., 1992). This change, coupled with impaired gastric acid secretion, results in increased bacterial colonization leading to recruitment of activated monocytes and macrophages that promote chronic inflammation, villus atrophy, and malabsorption. Progression of the HIV disease results in reduced function among cytotoxic T cells, mucosal macrophages, and monocytes. Immunosuppression and weight loss brought about by these events makes the AIDS patient increasingly susceptible to a variety of enteric pathogens that are considered rare in normal populations (Table VII).

TABLE VII ENTERIC PATHOGENS ASSOCIATED WITH

HIV INFECTION^ Group Viruses Bacteria

Fungi Protozoa

GenudSpecies Cytomegalovirus Herpes simplex virus Salmonella spp. Shigella flexneri Campylobacterjejuni Clostridium dificile Candida spp. Giardia lamblia Entamoeba histolytica Isospora belli Cryptosporidium spp. Microsporidium spp.

Based on Smith et al. (1992).

FOOD. DIET, AND GASTROINTESTINAL IMMUNE FUNCTION

IV.

35

IMPACT OF FOOD CONSTITUENTS AND CONTAMINANTS ON GASTROINTESTINAL IMMUNITY

A. MICROBES AND MICROBIAL PRODUCTS Many food- and waterborne microbial agents can colonize the GI tract and induce gastroenteritis (Centers for Disease Control, 1990). These agents can be bacterial (Table VIII), viral (Table IX), or parasitic (Table XI. As discussed earlier, other foodborne microbial agents can evade the GI immune system and cause systemic disease. Endogenous components or secreted products of bacteria can cause a variety of toxic effects. Clearly, staphylococcal enterotoxin and botulinum toxin must survive ingestion and evade GI immunity in the gut prior to inducing their specific toxic effects. Bacterial products also can affect immune function directly. For example, lipopolysaccharide (LPS or endotoxin), a component of gram-negative bacteria, is a potent polyclonal activator of B cells and can induce cytokine release by monocytes. McGhee et al. (1989) proposed that gram-negative bacterial antigens and LPS actually contribute to the normal differentiation and development of Peyer’s patches. Staphylococcal enterotoxin has been deemed a “superantigen” because of its capacity to activate a large number of T cell clones (Marrack and Kappler, 1990). What the long-term effects on GI immunity of such T cell activation in the intestinal tract would be is not clear. Finally, cholera toxin is the most potent mucosal immunogen known (McGhee et al., 1992), and can act as an adjuvant when ingested simultaneously with other proteins to induce both mucosal and systemic Ig responses. Mycotoxins are low molecular weight secondary metabolites that are produced by various fungi and frequently are found in foods (Pestka and Casale, 1990). Because they resist digestion and processing, mycotoxins can enter into the intestinal tract; because of their size, they evade normal GI immune mechanisms of protection. Their toxic effects are variable and can include cancer, impaired reproduction, and gastroenteritis. The capacity of mycotoxins to impair a variety of normal immune functions has been reviewed by Pestka and Bondy (1990). In at least one case, the GI immune system is known to be affected specifically. In the mouse, vomitoxin (deoxynivalenol) can stimulate B cells polyclonally to produce hyperelevated levels of serum IgA, probably via a T-cell-related mechanism (Bondy and Pestka, 1991). The quality and quantity of this IgA results in increased IgA immune complex formation and accumulation in the kidney, with current hematuria (Dong et al., 1991). This experimental model bears many similarities to human IgA neDhroDathv.

TABLE VIII INFORMATION RELEVANT TO OUTBREAKS OF BACTERIAL GASTROENTERITIS"

Selected symptoms Causative agent

Patient age

Vomiting

Fever

Diarrhea

Incubation period

Duration of illness

Mode of transmission

All

Common

Rare

Usually not prominent

1-6 hours

<24 hours

Food

All groups, especially < I year old children and young adults

Variable

Variable

May be dysenteric

3-5 days (1-7 days)

1-4 days, occasionally > 10 days

Food, water, pets, fecal-oral

Occasional

Variable

12-72 hours 2-6 days

3-5 days

Enteropathogenic

Adults, infants, and children Infants

Enteroinvasive

Adults

Occasional

2-3 days

1-2 weeks

Food. water, PTP," fecal-oral Food, water, PTP, fecal-oral Food, water, FTP, fecal-oral

Bacillus cereus and Staphylococcus aureus Carnpylobacterjejuni

Escherichia coli Enterotoxigenic

Variable

Watery to profuse watery Variable Watery to profuse watery Common May be dysenteric

1-3 weeks

Enterohemorrhagic

'

Salmonella spp.

Shigella spp.

Yersinia enterocolitica

Vibrio cholerae

(10 years of age (So%), 15 months-75 years All groups, especially infants and young children All groups, especially 6 months-10 years All groups, especially older children and young adults All groups

Common

Rare or mild

Occasional

First watery. then grossly bloody

3-5days

7-10days (1-12 days)

Food, PTP, fecaloral

Common Loose, watery, occasionally bloody

8-48 hours

3-5 days

Food, water, fecaloral

Occasional

Common May be dysenteric

1-7 days

4-7 days

Food. watar, PTP, fecal-ord

Occasional

Common Mucoid, occasionally bloody

2-7 days

I day-3 weeks (average 9 days)

Food. water, PTP. pets, fecal-oral

Common

Variable

9-12 hours

3-4 days

Fecal-oral, food, water

From the Centers for Disease Control (1990). PTP, Person-to-person.

May be profuse and watery

00 W

TABLE IX INFORMATION RELEVANT TO OUTBREAKS OF VIRAL GASTROENTERITIS"

Selected symptoms" Causative agent

Patient age

Vomiting

Fever

Incubation period

Duration of illness

Mode of transmission

Young children and elderly people Infants, young children, and adults

Occasional

Occasional

1-4 days

Common for infants. variable for adults

Occasional

1-3 days

2-3 days. occasionally 1-14 days 1-3 days

Young children

Common

Common

7-8 days

8- 12 days

Older children and adults

Common

Rare or mild

18-48 hours

12-48 hours

Food. water, PTP,' air,(/fecal-oral

Rotavirus Group A

Infants and toddlers

Common

Common

1-3 days

5-7 days

Group B

Children and adults

Variable

Rare

3-7 days

Group C

Infants, children, and adults

Unknown

Unknown

56 hours (average) 24-48 hours

Water, PTP, food,'/ air." nosocomial. fecal-oral Water. PTP. fecal-oral

3-7 days

Fecal-oral

Astrovirus Calicivirus

Enteric adenovirus Nonvalk virus

From the Centers for Disease Control (1990).

" Diarrhea is common and is usually loose, watery, and nonbloody when associated with gastroenteritis. '-PTP, Person-to-person. Not confirmed.

Food. water. fecal-oral Food. water. nosocomial. fecaloral Nosocomial. fecal-oral

TABLE X INFORMATION RELEVANT TO OUTBREAKS OF PARASITIC GASTROENTERITIS"

Selected symptoms Causative agent

Patient age

Fever

Balantidium coli

Unknown

Cryprosporidiurn spp.

Children, adults with AIDS

Enrarnoeba histolytica

All groups, adults

Variable

Giardia lamblia

All groups, children

Rare

lsospora bclli

Adults with AIDS

Unknown

" From the Centers for Disease Control (1990).

' PTP, Person-to-person.

Diarrhea

Rare

Occasional mucus or blood Occasional Profuse, watery

Abdominal

Incubation period

Duration of illness

Mild to severe pain

Unknown

Unknown

Occasional cramping

Occasional mucus or blood Loose, pale, greasy stools

Colicky

Loose stools

Unknown

Cramps, bloating, flatulence

Mode of transmission

Food, water. fecal-oral 1-2 weeks 4 days-3 Food. water, weeks FTP," pets. fecal-oral 2-4 weeks Weeks to Food. water, months fecal-oral 5-25 days 1-2 weeks Food. water, to months fecal-oral and years 9-15 days 2-3 weeks Fecal-oral

40

JAMES J. PESTKA

TABLE XI SIMILARITIES BETWEEN VOMITOXIN-

INDUCED

IgA NEPHROPATHY A N D H U M A N IgA NEPHROPATHY

Increased serum IgA Increased po1ymeric:monomeric IgA Increased in uirro IgA production Increased IgA specific for dietary antigens Increased CD4:CD8 T-cell ratio Increased IgA-bearing cells Increased mesangial IgA Increased serum IgA complexes Hematuria Predilection for males Long-term persistence

the most common form of human glomerulonephritis (Table XI). Males particularly are prone to disease in the experimental model and in human disease and can be affected by as little as 2 ppm of vomitoxin in the diet (Greene et al., 1992). Since this level can be found in cereal-based food products (Abouzied et al., 1991),vomitoxin might be speculated to be an etiologic agent in human IgA nephropathy.

B. FOODALLERGENS Although foods contain a multitude of proteins, very few of these can trigger IgE-mediated food allergies. Typical food allergens are naturally occurring water-soluble proteins that are heat and acid stable and resist digestion. Taylor (1992) has noted that, with the exception of cow’s milk proteins and egg proteins, most allergenic proteins in foods are of plant or marine origin. Some common allergenic foods are listed in Table XII, TABLE XI1 C O M M ~ NALLERGENIC

FOODS^

Legumes, especially peanuts and soybeans Crustacea: shrimp, crab, lobster, crayfish Milk, including cows’ milk and goats’ milk Eggs from all avian species Tree nuts: almonds, walnuts, Brazil nuts, hazelnuts, etc. Fish: cod, haddock, salmon, trout, etc. Mollusks: clams, oysters, scallops, etc. Wheat Reprinted with permission from Taylor (1992).

FOOD, DIET, AND GASTROINTESTINAL IMMUNE FUNCTION

41

Many food proteins can be perceived as “foreign” by a host and can stimulate both humoral and cellular immune responses. However, most immunogenic food proteins stimulate IgG rather IgE (Taylor, 1992). Thus, allergenicity does not correlate with immunogenicity. Production of IgE antibodies in food allergies apparently is associated with a heritable genetic predisposition (Aas, 1978). A s discussed earlier, binding of an allergen by specific IgE present on the surface of mast cells and basophils results in degranulation with release of histamine and other allergy mediators (Fig. 10). To induce this response, the allergen must be of sufficient size to “bridge” between two IgE molecules. The optimal molecular weight for an allergen is in the range of 10,000 to 70,000 daltons (Taylor et al., 1987). These size estimates are based on (1) capacity to be immunogenic, (2) intestinal permeability of the protein, and (3) the bridging requirement (Taylor, 1992). A second key characteristic of an allergen is the presence of multiple immunogenic sites (or epitopes) that facilitate bridging between two IgE molecules. These epitopes must be spaced at an appropriate distance on the protein molecule. Finally, a common feature of allergenic proteins is the capacity to resist the digestive process as well as heat and acid treatments (Taylor, 1992). Taylor (1992) has reviewed the chemistry and detection of specific food allergens extensively. He noted that progress in food allergen identification has been facilitated by protein separation techniques such as SDS-polyacrylamide gel electrophoresis and immunoblotting with serum from food-allergic individuals. Allergenic food proteins identified by such techniques are summarized in Table XIII. C. CHEMICALS IN FOOD When exogenous chemicals interact with lymphoid tissue, immune homeostasis might be disrupted and induce undesirable “immunotoxic” effects such as ( 1 ) immunosuppression, (2) uncontrolled proliferation, (3) impaired host resistance, (4) allergy, and (5) autoimmunity (Dean and Murray, 1991). Therefore, foodborne chemicals (not including macromolecules) have the potential to interact with the gastrointestinal immune system. Chemicals that are potentially immunotoxic in the gut might be found among natural components, additives, growth promoters, animal drugs, and various contaminants (Table XIV). However, most information relative to these chemicals often is based on systemic immune effects that follow injection at relatively high doses. Miller (1987) reviewed intolerance to food additives such as tartrazine, benzoates, food flavors, and colors and concluded that the mechanisms for production of adverse reactions have not been demonstrated to have

42

JAMES J. PESTKA

TABLE XI11 K N O W N FOOD ALLERGENSU

Source

Allergen

Cows' milk Egg white Egg yolk Peanut Soybean

P-Lactoglobulin. a-lactalbumin. caseins Ovomucoid ( g a l dI), ovalbumin (gal dII). conalbumin (gal dIII) Lipoprotein, livetin, apovitellenin I, apovitellenin V1 Peanut I. concanavalin A-reactive glycoprotein Kunitz trypsin inhibitor, P-conglycinin. glycinin, unidentified protein (20.000 daltons) Albumin protein (1800 daltons) Unidentified proteins ( 16.000-30.000 daltons) Unidentified protein (30,000 daltons) Papain Glutelin fraction, albumin proteins (14,000-16.000 daltons) Trypsin inhibitor Albumins and globulins Allergen M (gad c l ) , a-parvalbumin Antigen 1 (9000-20,000 daltons), antigen I1 (31.000-34.000 daltons), transfer ribonucleic acid

Green pea Potato Peach Papaya Rice Buckwheat Wheat Codfish Shrimp

" Reprinted with permission from Taylor (1992).

immunologic components that usually are associated with hypersensitivity reactions. Animal growth regulators that exist as residues in food theoretically could alter immune function. For example, estrogenic compounds such as diethylstilbestrol (DES) can depress T-cell-related functions including aspects of cell-mediated immunity and helper activity for antibody responses (Kalland er al., 1979; Luster er al., 1979,1980; Kalland, 1980).

TABLE XIV EXAMPLES OF POTENTIALLY IMMUNOTOXIC

INGESTED CHEMICALS ~~

~~

Type

Examples

Growth promoters Drugs Pesticides

Diethylstilbestrol Penicillin, ethanol Carbamates, organochlorines, organophosphates, organotins Polychlorinated biphenyls, polybrominated biphenyls

Contaminants

FOOD, DIET, AND GASTROINTESTINAL IMMUNE FUNCTION

43

Host resistance to Lesreria, Trichinella, and transplantable tumors also is depressed in DES-exposed mice (Dean et al., 1980). Drugs entering foods as residues have the potential to induce an immunologic effect. For example, p-lactam antibiotics including penicillin are capable of inducing allergic responses (Ahlstedt et al., 1980). Although penicillin alone cannot elicit an immune response, biotransformation products that react with self proteins can induce antibody responses (Parker, 1982) or be carried by gastrointestinal contents, bacteria, or bacterial products (Dean and Murray, 1991). Notably, a higher frequency of allergy occurs after intramuscular penicillin administration than after oral administration (Ahlstedt et al., 1980; Van Arsdel, 1981). Ingestion of drugs of abuse also can alter immunologic function. Alcohol abuse in humans results in greater numbers and severity of infections (Tapper, 1980), impaired humoral immunity (Gluckman et al., 1977). and dysregulation of T cell function (Berenyi et al., 1975). Pesticide residues also might modulate immune function. Dean and Murray (1991) reviewed evidence that suggests certain pesticides or formulation contaminants can impair immunity in rodent models. These chemicals include four general classes: ( I ) carbamates, (2) organochlorines, (3) organophosphates, and (4) organotin compounds. Greatest risk was suggested to be posed by pesticides such as organochlorine that are stable in the environment. Pesticide contamination also presents a greater risk to the developing immune system than to the mature adult. Finally, inadvertent food contaminants can pose an immunotoxic risk. For example, contamination of rice with polychlorinated biphenyls (PCBs) in Japan increased susceptibility of exposed persons to respiratory infections (Shigamatsu et al., 1978). People exposed to PCBs in rice in China exhibited depressed serum IgM and IgA and altered T cell number and function (Chang et al., 1980; Lee and Chang, 1985). The accidental substitution of the flame retardant Firemaster BP-6 for a magnesium oxide food supplement in livestock feed resulted in widespread contamination of milk and dairy products with polybrominated biphenyls (PBBs) in Michigan during 1973. High levels of PBBs were found in serum and adipose tissue of Michigan residents (Bekeisi et al., 1978). Exposed persons exhibited a variety of immunologic abnormalities including depressed T cell numbers, depressed lymphocyte function, and increased Ig levels (NIEHS, 1983; Bekesi et al., 1987). Although substantial information is available that certain ingested chemicals have the potential to alter systemic immunity, a definite need exists to expand these findings to the GI immune system using models of oral exposure at realistic doses.

44

JAMES J. PESTKA

D. NUTRITIONAL COMPOSITION That the frequency and severity of GI infections increase in undernourished individuals is well known (Chandra and Wadhwa, 1989). Protein-energy malnutrition is a primary cause of immunodeficiency (Christou, 1990). Fatal outcomes in children with diseases such as mumps and measles can occur because of such generalized immunosuppression. Specific immunologic effects include thymic atrophy, decreased spleen weight, decreased T cell counts, and impaired production of cytokines, thymic hormones, and antibodies. Thus, both cellular and humoral immunity are affected. With respect to GI immunity, McMurray et al. (1977) showed that moderate malnutrition depressed secretory IgA levels. Chandra and Wadwha (1989) reviewed specific effects of malnutrition on gut immunity (Table XV), including depression of IgA-producing cells and fewer intraepithelial lymphocytes. Antibody responses after viral vaccine administration are reduced and natural killer cell activity decreases. Additionally, the number of bacteria that bind to epithelial cells is increased. Osogoe et al. (1991) examined the effects of refeeding subsequent to starvation on the plasma cell population in the lamina propria of the small intestinal villi in adult rats, using an immunohistochemical method to detect IgA, IgM, and IgG. Extensive hyperplasia of intestinal plasma cells could be induced by refeeding after starvation. A large majority of the proliferating intestinal plasma cells expressed IgA, whereas cells bearing IgM or IgG occurred in extremely small numbers in the lamina propria. The mechanism of this extensive plasma cell hyperplasia might involve enhanced transmission of antigenic macromolecules across the mucosal barrier. Also, the origin of the expanded plasma cell population was sugTABLE XV EFFECTS OF MALNUTRITION ON GASTROINTESTINAL IMMUNITY

Secretory component synthesis depressed Reduced numbers of IgA-producingcells Decreased total secretory IgA Decreased specific secretory IgA Reduced numbers of intraepithelial lymphocytes Impaired lymphocyte migration Increased bacterial adherence to epithelial cells L1

Based on Chandra and Wadhwa (1989).

FOOD, DIET, AND GASTROINTESTINAL IMMUNE FUNCTION

45

gested to be correlated with B cell precursors in the germinal centers of the Peyer’s patches, which apparently were more resistant to starvation than other lymphoid cells. Single nutrient effects on immunity also have been characterized and reviewed extensively (Chandra and Chandra, 1986; Delafuente, 1991 ;Latshaw, 1991) with respect to systemic immunity. These effects are likely to be equally important in gut immune function.

V.

MODIFICATION OF GASTROINTESTINAL IMMUNITY THROUGH FOOD AND DIET

A.

BREAST-FEEDING

Innate and specific defense mechanisms must be well developed for the epithelium to function as an impermeable barrier to proteins and their fragments. However, such defense mechanisms are not fully developed in the infant during the postpartum period, particularly when born prematurely (Walker, 1987). Newborn infants are especially susceptible to pathologic penetration by deleterious intestinal contents because of this delayed maturation of the mucosal barrier. Repercussions of this immaturity include enhanced susceptibility to infection, potential for hypersensitivity reactions, and formation of immune complexes. In some situations, these conditions can be fatal. Clinical conditions that have been associated with an immature mucosal barrier include allergy, necrotizing enterocolitis, dermatitis, malabsorption, sudden infant death syndrome, and toxigenic diarrhea in early childhood, as well as inflammatory bowel disease, nephritis, and autoimmunity in adulthood (Walker, 1985). Human milk is a natural means of passively assisting a sensitive neonate against the hazards of a deficient GI immune system. Ingestion of colostrum decreases antigen penetration (Udall et al., 1979). Intake of colostrum apparently enhances the maturation of mucosal epithelial cells and accelerates the development of an intact mucosal barrier (Heird and Hansen, 1977). Secretory IgA exists in milk and can prevent uptake of bacteria and luminal antigens (Walker, 1985). The capacity of colostrum to prevent cow’s milk allergy apparently is related to the IgA content (Savilahti et al., 1991). In addition to antibodies, human milk contains viable leukocytes as well as many other substances that impede bacterial colonization and prevent antigen infiltration. However, care must be taken by the nursing mother to minimize ingestion of allergenic foods that could be carried over into breast milk to sensitize an infant (Gerrard and Shenassa, 1983).

46

JAMES J. PESTKA

B. DETECTION AND AVOIDANCE OF FOOD ANTIGENS AND ALLERGENS Based on a survey of adults with allergies, Ericksson (1978) found that 24% of individuals asked believed that they experienced allergic symptoms after handling or ingesting food; 25% of parents interviewed believed that their children experienced adverse food reactions (Kayosaari, 1982; Bock, 1987). However, only one-third of these perceived food allergies could be verified by controlled clinical trials (Atkins et af., 1985). Because of the large number of people with food allergies (1-2% of the general population) and the even greater number with perceived food allergies, Metcalfe (1992) emphasized the critical need for proper classification of food-related diseases to facilitate development of rational approaches to and policies for dealing with suspected food allergies in the public and private sectors. Concurrent with this need is the need to label foods appropriately to alert the segment of the population that is susceptible to reaginic and nonreaginic hypersensitivities or various enteropathies when exposed to certain food proteins. Taylor (1992) highlighted certain processing and distribution errors that currently make avoiding certain food proteins through labeling problematic (Table XVI). He further suggested that, to avoid these oversights, food processors should establish quality assurance programs that are based on immunoassay procedures that are capable of detecting small amounts of food proteins in a complex matrix. Although clinicians use the sera of food-allergic patients to detect food (Porras et a / . , 1985; Yunginger et al., 1988; Keating et af., 1990; Gem et al., 1991), these sera are unlikely to become available for wide usage. A more practical approach would be to develop specific immunoassays for specific foods that would indicate allergenic potential, for example, the gluten assay described by Skerritt and Hill (1991).

TABLE XVI FACTORS AFFECTING ENTRY O F TRACE ALLERGEN A N D ANTIGEN LEVELS INTO A FOODa

Source of food ingredients unclear Inadequate cleaning of equipment Use of re-work Inadvertent/intentional addition of unlabeled ingredient Bulk distribution Based on Taylor (1992).

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C. DEVELOPMENT OF HYPOALLERGENIC FOODS Another goal for certain food products might be deliberate reduction of allergenic potential. Hypoallergenic foods are those that are completely incapable of inducing an allergic response in an individual (Taylor, 1992). Currently, direct challenge of an allergic person is the best means of assessing whether a food contains an active allergen. These approaches have been used for casein and whey hydrolysate formula (Walker-Smith et al., 1989; Merritt et al., 1990; Sampson et al., 1991a),rice (Yoshizawa and Arai, 1990), and edible oils from peanuts, soybeans, and sunflower seeds (Taylor et al., 1981; Bush et al., 1985; Halsey et al., 1986). Also, immunoassays may be used to evaluate the reduction of allergenicity in a food. For example, human serum IgE has been used to assess the allergenicity of peanut products (Nordlee, 1981),milk proteins (Pahud et al., 1985; Asselin et al., 19891, and rice proteins (Wantanabe et al., 1990). In the future, developing libraries of human monoclonal antibodies that react with key allergenic epitopes in foods may be possible to facilitate studies on the effect of processing on reduced allergenicity . Animal studies also may provide insight into the effect of processing on food allergens. Poulsen et al. (1990) compared intestinal anaphylactic reactions in sensitized mice challenged with untreated bovine milk and homogenized bovine milk. When given orally, homogenized milk resulted in IgE production in 10 of 14 mice, whereas untreated milk resulted in IgE production in only 1 of 12 mice. In contrast to untreated milk or saline, homogenized milk caused a large increase in the mass of the proximal gut segment of orally sensitized mice; only mice both sensitized and challenged orally with homogenized milk showed degranulation of mast cells in the intestinal wall. These observations suggest that homogenization of bovine milk may render the milk more aggressive in its ability to induce intestinal reactions. The study suggests that mice may be an attractive experimental animal model for mimicking the intestinal anaphylactic reactions of humans allergic to cow’s milk or other proteins. D. CONTROL OF MICROBIAL FLORA I . Reduction or Elimination of Microbial Pathogens For immunosuppressed populations, insuring that certain categories of food are pathogen-free, using appropriate processing techniques such as pasteurization or retorting, may be desirable. Appropriate packaging, labeling, and distribution must complement the development of such prod-

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ucts, and could be achieved best by implementing Hazard Analysis Critical Control Point procedures. 2. Ingestion of Probiotic Cultures

Based on theories on the beneficial effects of lactic acid bacteria on intestinal flora that were developed by Metchnikoff many years ago (Hughes and Hoover, 1991), interest in the effects of ingesting fermented milk products has been widespread. Probiotic agents have been defined as “a live microbial feed supplement which beneficially affects the host animal by improving its microbial balance” (Fuller, 1991). The underlying concept is that colonization of the gut by beneficial bacteria would impede attachment and entry of various pathogenic bacteria. Thus nonspecific immunity would be enhanced. Other benefits of probiotic cultures include improvement of lactose tolerance of milk products, anti-tumorigenic activity, reduction of serum cholesterol, and synthesis of B-complex vitamins (Gilliland, 1990). Lactobacillus spp. and Bifidobacterium spp. have been considered for use as probiotics in humans. Colonization of the gastrointestinal tract is thought to be host specific (Barrow et al., 1980; Tannock et al., 1982; Lin and Savage, 1984; Isolauri et al., 1991). Some researchers have reported that, on ingestion, only a small number of Lactobacillus strains survive at the terminal ileum (Bouhnik et al., 1992). However, Lidbeck et al. (1987) showed that fecal counts of Lactobacillus acidophilus increased after ingestion. Goldin et al. (1992) found that a newly isolated human strain of Lactobacillus (GG) was recoverable in the feces of human volunteers up to 7 days after ingestion and that the strain was resistant to the effects of ampicillin. Also, ingested lactobacilli have been suggested to alter fecal bacterial enzyme activities (Pedrosa et al., 1990). Bijidobacterium species are part of the major anaerobic microflora of the colon (Mitsuoka, 1984). Several fermented dairy products have been introduced commercially that may have probiotic effects (Colombel et al., 1987; Marteau et al., 1990; Hughes and Hoover, 1991). Using an intestinal perfusion technique in humans, Pochart et al. (1992) demonstrated the survival of bifidobacteria in the upper gastrointestinal tract. A streptomycin-resistant Bifdobacterium species was used to monitor survival and colonization of ingested bifidobacteria in humans (Bouhnik et al., 1992). The investigators concluded that exogenously administered Bifidobacterium spp. did not colonize the colon but that high concentrations of the exogenous bifidobacteria were compatible with metabolic probiotic activities.

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In evaluating the probiotic efficiency of an organism, knowing the effective therapeutic dose is critical (Saxelin et al., 1991).elements et al. (1983) detected batch to batch variation in probiotic efficiency of freeze-dried lactic acid bacteria products. To assess attachment capacity of some common dairy strains of bacteria, Elo et al. (1991) used a human colon carcinoma line. When compared with an adhesive strain of E. coli, Lactobacillus casei GG had potent attachment capacity whereas attachment by L . acidophilus, Lactobacillus bulgaricus, and various Bifidobacterium strains was weak or absent. Obviously, maintaining viability in any probiotic fermented dairy product that is marketed will be critical. Such maintenance will require intensive study on effects of processing, additives, and packaging in storage. Hughes and Hoover (1991) extensively reviewed research on the challenges and opportunities of commercial BiJidobacterium fermented milk products.

3 . Immunostimulatory Properties of Ingested Bacteria Intestinal microflora have a key role in the development of a normal immune response. For example, Bartizal et al. (1984) examined the effects of diet (chemically defined as opposed to natural ingredient), age, and microbial flora on the tumoricidal activity of NK cells from the spleens of mice. Germ-free mice raised on a chemically defined diet had significantly greater NK cell activity than their germ-free or “clean-conventional” (i.e., barrier-maintained) counterparts that were raised on a sterilized naturalingredient diet. NK activity of germ-free mice was increased dramatically after their alimentary tract was colonized with a complex intestinal flora. Conventional mice raised under clean (barrier) conditions had significantly lower NK cell activity than non-barrier-maintained mice. Changing germ-free mice from a chemically defined diet to a sterile natural-ingredient diet did not enhance NK cell activity. No significant differences in NK activity were evident among mice of different ages. These authors suggested that diet and microbial flora can modulate the NK cell activity of mice. Several investigators have hypothesized that organisms present in fermented dairy products may have immunostimulatory properties. Perdigon et al. (1986) reported that feeding of L. casei and L . acidophilus enhances peritoneal macrophage function and splenic lymphocyte activation in mice. Injection of mice with heat-killed L. casei LC99018 similarly was found to activate macrophage functions associated with microbicidal activity (Saito et al., 1987) and to augment host resistance to Listeria mono-

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cytogenes (Nomoto et a/., 19851, Pseudomonas aeruginosa (Miake, 19851, and transplanted tumors (Matsuzaki et a/., 1985). Antitumor activity also has been described for Lactobacillus strains by Friend and Shahani (1984) and for Streptococcus thermophilus by Kaklij et al. (1990). Lactic cultures stimulate cytokine production in some experimental models (De Simone et al., 1986; Yokukura et al., 1986; Nanno et a/., 1988).

E. AUTOIMMUNE THERAPY BY ORAL TOLERANCE INDUCTION Autoimmune diseases such as rheumatoid arthritis and multiple sclerosis appear to be initiated when systemic cell-mediated and humoral immune defenses target the body’s own tissue. Treatment of these diseases in humans typically involves use of immunosuppressive drugs that make the patient more susceptible to infection. Other approaches under study involve blocking the disease with monoclonal antibodies or synthetic peptides (Marx, 1991). As discussed earlier, oral tolerance involves suppression of systemic cellular and humoral immune responses by feeding a protein antigen. Several intriguing animal studies have been reported that show that simple ingestion of protein antigens that induce the aberrant anti-self responses actually can suppress autoimmunity via oral tolerance. For example, one animal model for multiple sclerosis involves injection of rats or guinea pigs with myelin basic protein, a component of the nerve fiber membranous sheath. This injection induces a systemic immune response directed specifically against the myelin sheath. Feeding of the myelin basic protein can suppress the onset of the experimental disease as well as ameliorate an ongoing response (Khoury et a/., 1990; Brod et al., 1991; Miller et a/., 1991; Whitacre et al., 1991). The mechanism for this protective response may involve the generation of T suppressor cells or the specific deletion of autoimmune T effector cells (Marx, 1991; Edgington, 1992). Deliberate induction of oral tolerance also has been tested in the inhibition of autoimmune effects in other models. Oral administration of insulin suppresses disease in a model for the autoimmune form of diabetes associated with insulitis (Zhang e f al., 1991). Nussenblatt et a/. (1990) demonstrated that oral administration of certain retinal antigens suppresses the eye inflammation associated with experimental autoimmune uveitis. Similarly, collagen-induced arthritis (a model for rheumatoid arthritis) can be suppressed by ingestion of collagen (Zhang et al., 1990).

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Suppression of autoimmunity by deliberate induction of oral tolerance might constitute a low technology and low cost alternative to the treatment of these diseases. Currently, human clinical trials are underway to test the efficacy of antigen feeding on multiple sclerosis, rheumatoid arthritis, and uveitis (Marx, 1991). An obvious research question that arises from these studies relates to the role antigens in normal foods have in protecting against or promoting certain autoimmune diseases.

F. NUTRITIONAL THERAPIES I.

Intestinal Diseases

Patients who have diseases of the small or large intestine, for example, ulcerative colitis, Crohn’s disease, and intestinal cancer, typically have impaired function of the digestive tract. The dysfunction impairs normal nutrient ingestion or absorption and predisposes the patient to malnutrition. Ultimately, this condition diminishes capacity of the intestine to respond appropriately to microbial and antigenic challenge in an effective and coordinated fashion. Diehl et al. (1983) reviewed approaches for intravenous hyperalimentation to diminish these effects. The general approach for preventing malnutrition requires the development of a profile of an individual patient’s nutritional state using anthropometric, biochemical, and immunologic tests to assess the body’s fat stores as well as somatic and visceral protein mass and immunocompetence. Ultimate success of parenteral nutrition support in intestinal diseases depends on proper placement of the catheter, maintenance of an aseptic environment, and surveillance of the patient’s metabolic status (Grant, 1980). Specific adjunctive nutritional therapies involving the enteral route also might be designed to bolster the immune system in certain instances of intestinal disease (Alexander and Peck, 1990) and cancer (Lowell et al., 1990).

2. AIDS-Related and Other Immunosuppressed Conditions A close relationship exists between adequate nutrition, gastrointestinal immune function, and long-term survival of AIDS patients (Simon et al., 1991). Crocker (1989) indicated that AIDS-related gastrointestinal disease is very common, and presents a challenge to all nutritional support clinicians because of long-term problems such as related weight loss, diarrhea, and malabsorption. The course of AIDS-related gastrointestinal disease is

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complicated by factors such as decreased food intake (resulting from fatigue and malaise), increased metabolic demand and nutritional requirements, and identifiable gastrointestinal pathology. Typically, chronic gastrointestinal dysfunction is caused by recurring opportunistic pathogens that are recalcitrant to chemotherapy. Chlebowski et al. (1989) investigated the clinical course of 71 patients with AIDS to determine relationships among nutritional status, gastrointestinal symptoms, and survival. Weight loss was observed in 98%, hypoalbuminemia was present in 83%, and gastrointestinal symptoms were present that included pharyngitis (54%), diarrhea (42%), nausea (23%), dysphagia (21%), and anorexia (18%). The degree of body weight loss and serum albumin level were associated strongly with survival. The authors concluded that (1) nutritional status may represent a major determinant of survival in AIDS and (2) the rate of albumin decrease may predict survival of individual patients with AIDS. Since even clinically stable AIDS patients have been diagnosed as chronically malnourished, patient care and long-term management of them ultimately must focus on fluid and electrolyte balance, nutritional support, and symptom control (Crocker, 1989). These persons are prone to rapid nutritional deterioration during disease exacerbations. Unfortunately, because of the eventual mortality associated with AIDS, physicians hesitate to prescribe aggressive nutritional support, particularly parenteral nutrition. The use of nutritional support as adjunctive therapy early in the course of disease also will be an issue with respect to the use of agents such as AZT, which are prescribed on a frequent basis for persons with AIDS. Although improving nutrition has not been shown to reverse any of the cellular manifestations of AIDS, nutritional support via counseling, food programs, or intervention with enteral or parenteral nutrition appears to improve strength and endurance, and enhance the overall quality of patient life. 3 . Immune Complex and Autoimmune Disorders

Skoldstam and Magnusson (1991) found that otherwise healthy and well-nourished patients with rheumatoid arthritis show significant clinical improvement when practicing prolonged fasting for 7 to 10 days. However, the improvement is reversible and is lost when eating is resumed. Although of little therapeutic value, the anti-inflammatory effect of short-term fasting might provide insight into some nutritional approaches to dealing with autoimmune diseases.

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RESEARCH NEEDS

Elucidation of the cellular and molecular basis of gut immune function will facilitate development of novel approaches for its homeostatic maintenance through diet as well as of novel food products that benefit general or select populations. Key basic research areas are identification of unique and nonunique leukocytes in the intestine, as well as clarification of how these cells function at the lurninal interface in the presence of food constituents and varied microflora. Such clarification requires an understanding of the mechanisms by which these sets of cells communicate with and regulate one another via cell surface molecules, cytokines, and neuroendocrine mediators. With increased understanding of GI immune function should come new methods of immunization against pathogenic microorganisms. A novel immunogenic approach also has been described for protection against intestinal carcinogens (Keren et al., 1986; Silbart and Keren, 1989),which involves conjugation of the carcinogen 2-acetylaminofluorine to cholera toxin and direct immunization of the intestine. Sensitive and accurate measures of immune function in the gastrointestinal tract are needed to elucidate more fully the effects of food constituents and contaminants. For example, experimental rodent models perhaps can be assessed best by monitoring leukocyte profiles by multiparameter flow cytometry. Specific leukocyte function can be measured by determining cytokine production in specific gut lymphoid tissue via immunoassay or the polymerase chain reaction technique. Results in experimental animal models then must be validated in humans using appropriate in v i m approaches with isolated human lymphocytes (Wood et al., 1992). One promising approach that may make possible the testing of hypotheses related to food and gut immunity involves the repopulation of immunodeficient mice with human lymphocytes (Gardner and Luciw, 1989). Additional information is needed on the molecular basis of the survival, uptake, and antigenicity of macromolecules in the intestinal tract. These questions will become even more pressing with the introduction of plant, animal, and microbial foods that have been modified by recombinant DNA techniques. One fundamental question is how multifactorial nonspecific immunity inhibits or favors survival and uptake of specific proteins. Another question relates to biotechnology-derived food products. For example, developing a transgenic cow that releases human Igs in its milk is theoretically possible (Lo et al., 1991). What effect would these human Igs have on adults and developing infants that ingest such milk? Another problem that has not been addressed adequately is the potential for sur-

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viva1 and uptake of DNA in the intestine. In preliminary work, we have demonstrated the rapid translocation of an orally presented plasmid into the blood of a mouse (L. Rasooly and J. Pestka, unpublished observations). Because of the large number of perceived food allergies, proper classification of food-related diseases and ingredient labeling are critical. Food processors need quality assurance programs that are based on sensitive immunoassay procedures capable of detecting small amounts of suspect food proteins in a complex matrix. Over the long term, the food industry must develop and identify foods that are devoid of or reduced in allergenic potential. Currently, direct challenge of sensitive persons and immunoassay with human IgE have been used to verify absence of allergenicity in a variety of food products. Also, immunoassays may be used in evaluating the reduction of allergenicity in a food. In the future, developing libraries of human monoclonal antibodies that react with key allergenic epitopes in foods may be possible, and may facilitate studies on the effect of processing on reduced allergenicity. Kessler et al. (1992) identified a regulatory framework for U.S. Food and Drug Administration evaluation of the safety of foods developed from biotechnology. High on this list is the potential of a donor food protein to be allergenic. One example would be the introduction of a peanut allergen into corn, thus making the new variety of corn allergenic to people allergic to peanuts. The authors suggested that employing recombinant DNA techniques to determine whether an allergenic determinant has been transferred from the donor to the new variety would be valuable. Since relatively few proteins are capable of eliciting an IgE response, elucidation of the structural requirements for allergenicity is both needed and feasible. As noted earlier, interaction of exogenous chemicals with lymphoid tissue may induce undesirable immunotoxic effects such as immunosuppression, uncontrolled proliferation, impaired host resistance, allergy, and autoimmunity. Chemicals that are potentially immunotoxic in the gut might be found among natural components, microbial products (including mycotoxins), additives, growth promoters, animal drugs, and various contaminants. Most information relative to immunotoxicity involves injection followed by assessment of systemic immunity. However, foodborne chemicals are most likely to have their greatest effect on GI immune function before they are absorbed and metabolized. Thus, studies on the effects of ingestion of chemicals at realistic levels on gut immunity are most useful. The potential for dysregulation of GI immunity by a foodborne chemical has been highlighted by the discovery that dietary

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exposure to vomitoxin increases serum IgA and induces IgA nephropathy in a rodent model (Dong et al., 1991). Progress has occurred in evaluating probiotic and immunologic effects of certain commensal and dairy strains of Lactobacillus and Bijidobacterium.A major problem in interpreting some experimental data is that the assays of immunologic function use leukocytes of systemic origin (e.g., peritoneal macrophages, blood lymphocytes, or spleen lymphocytes). Also, in some cases, cultures were injected into the animals. The specific effects of ingestion of lactic cultures on leukocytes found in the GI immune system must be elucidated. Additional research is needed on the role food antigens play in protecting against o r promoting certain autoimmune diseases (Marx, 1991). For example, can regular ingestion of a food that contains collagen reduce the severity of rheumatoid arthritis? Such a notion could lead to specific dietary recommendations for autoimmune individuals as well as to “designer’’ foods that enhance the ability of such individuals to cope with autoimmune diseases. Antigens might occur naturally in a food, might be added to the food, or might be recombinant proteins expressed in appropriate microbes, plants, or animals that are ingested normally. Using this final approach must be done cautiously since correct dosing for oral tolerance induction is critical to triggering the protective effect; overexposure actually may diminish the desired effect (Edgington, 1992). The specific effects of malnutrition and overnutrition on gastrointestinal immune function need to be investigated more fully. Hopefully, new dietary approaches can be used to maximize gut immunity in the face of a growing population that exhibits immunosuppression (including the elderly and AIDS patients) and chronic intestinal disease. In conclusion, the last two decades of immunologic research have expanded our understanding of gastrointestinal immunity greatly and have suggested possible routes for enhancing gut immune function. Although some study has been done of the interaction between food and GI immune function, the gap between basic immunology and the food and nutritional sciences is widening. This difference exacerbated by a general tendency in the popular literature and press to promote panaceas for stimulating immunity via special diets or foods. Equally disturbing is the potential for alarmism relative to adverse effects of food constituents, as exemplified by the promotion of pseudotests for immune function. With respect to gastrointestinal immunity, only through application of the basic research on topics described in this chapter can food and nutritional scientists offer

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rational and reliable approaches to enhancing the health of general and select populations via food and diet. ACKNOWLEDGMENTS This work was supported by Public Health Service Grant ES-03358, the National Kidney Foundation of Michigan, and the Michigan State University Agricultural Experiment Station. We acknowledge the kind assistance of Mary Rosner in manuscript preparation and Chris Oberg for illustrations.

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Cunningham-Rundles, C., Brandeis, W. E., and Good, R. H. (1979a). Bovine antigens and the formation of circulating immune complexes in selective immunoglobulin A deficiency. J. Clin. Invest. 64, 272-279. Cunningham-Rundles, C., Brandeis, W. E., Safai, B., O’Reilly, R., Day, N. K.. and Good. R. A. (1979b). Selective IgA deficiency and circulating immune complexes containing bovine proteins in a child with chronic graft vs host disease. Am. J. Med. 67,883-889. Czerkinsky, C., Prince, S. J., Michalek, S., Jackson, S., Russell, M. W., Moldoveau. Z., McGhee, J. R., and Mestecky, J. (1987). IgA antibody producing cells in peripheral blood after antigen ingestion: Evidence for a common mucosal immune system in humans. Proc. Natl. Acad. Sci. 84,2449-2453. Dean, J. H. and Murray, M. J. (1991). Toxic responses to the immune system. In “Casarett and Doull’s Toxicology” (M. 0. Amdur, J. Doull, and C. D. Klassen, eds.). pp. 282333. Pergamon Press, New York. Dean, J. H., Luster, M. I., Boorman, G. A., Luebke, R. W., and Lauer. L. D. (1980). The effect of adult exposure to diethylstilbestrol in the mouse: Alterations in tumor susceptibility and host resistance parameters. J. Reticuloendorhel. Soc. 28, 571-583. Delafuente, J. C. (1991). Nutrients and immune responses. Rheum. Dis. Clin. North A m . 17 (2), 203-212.

De Simone, C., Bianchi, B., Salvadori, R., Negri, R., Ferrazzi, M., Baldinelli, L., and Vesely, R. (1986). The adjuvant effect of yogurt on production of gamma interferon by Con A-stimulate$ human peripheral blood lymphocytes. Nutr. Reports Int. 33,419-433. Diehl, J. T., Steiger, E., and Hooley, R. (1983). The role of intravenous hyperalimentation in intestinal disease. Surg. Clin. North Am. 63, 11-26.

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Walker, R. I., and Owen, R. L. (1990). Intestinal barriers to bacteria and their toxins. Annu. Rev. Med. 41,393-400. Walker, W. A. (1983). Gastrointestinal transport of macromolecules in the pathogenesis of food allergy. Ann. Allergy 51,240-245. Walker, W. A. (1985). Absorption of protein and protein fragments in the developing intestine: Role in immunologic/allergic reactions. Pediatrics 75, 167-171. Walker, W. A. (1987). Pathophysiology of intestinal uptake and absorption ofantigens in food allergy. Ann. Allergy 59,7-16. Walker-Smith, J. A., Digeon, B., and Phillips, A. D. (1989). Evaluation of a casein and a whey hydrolysate for treatment of cow’s milk sensitive enteropathy. Eur. J . Pediatrics 149,68-7 I . Watanabe, M., Miyakawa, J., Ikezawa, Z., Suzuki, Y.. Hirao. T., Yoshizawa. T.. and Arai. S. (1990). Production of hypoallergenic rice by enzymatic decomposition of constituent proteins. J. Food Sci. 55,781-783. Wells, C . L., Maddaus, M. A., and Simmons, R. L. (1988). Proposed mechanisms for the translocation of intestinal bacteria. Reu. Infect. Dis. 10,958-979. Whitacre, C. C., Gienapp, I. E., Orosz, C. G., and Bitar, D. M. (1991). Oral tolerance in experimental autoimmune encephalomyelitis. 111. J . Immunol. 147 (7), 2155-2163. Wood, S. C., Karras, J. G., and Holsapple, M. P. (1992). Integration of the human lymphocyte into immunotoxicological investigations. Fund. Appl. Toxicol. 18,450-459. Yokokura, T., Nomoto, K., Shimizu, T., and Nomoto, K. (1986). Enhancement of hematopoietic response of mice by subcutaneous administration of Lactobacillus casei. Infecr. Imrnun. 52,156-160. Yoshizawa, T., and Arai, S. (1990). Production of hypoallergenic rice by enzymatic decomposition of constituent proteins. J. Food Sci. 55,781-783. Yunginger, J. W.. Sweeney, K. G., Sturner. W. Q., Giannandrea, L. A., Teigland. J. D.. Bray, M., Benson, P. A., York, J. A., Biedrzycki. L., Squillace, D. L.. and Helm, R. M. (1988). Fatal food-induced anaphylaxis. J. Am. Med. Assn. 260, 1450-1452. Zeitz, M., Schieferdecker, H. L., James, S. P., andRiecken, E. 0. (1990). Special functional features of T-lymphocyte subpopulations in the effector compartment of the intestinal mucosa and their relation to mucosal transformation. Digestion 46, 280-289. Zhang, Z. Y., Lee, C. S., Lider, O., and Weiner, H. L. (1990). Suppression of adjuvant arthritis in Lewis rats by oral administration of type I1 collagen. J . Immunol. 145 (8), 2489-2493.

Zhang, Z. J., Davidson, L., Eisenbarth, G., and Weiner, H. L. (1991). Suppression of diabetes in nonobese diabetic mice by oral administration of porcine insulin. Proc. Natl. Acad. Sci. U.S.A. 88 (22), 10252-10256.

ADVANCES IN FOOD AND NUTRITION RESEARCH. VOL. 31

EFFECT OF CONSUMPTION OF LACTIC CULTURES ON HUMAN HEALTH MARY E L L E N SANDERS Microbiology Consultant Littleton. Colorado 80122

I. Introduction 11. General Physiology A. Gastrointestinal Ecology B. Fecal Recovery C. Adherence of Probiotic Cultures in the Human Intestine D. Production of Antimicrobials 111. Health Targets A. Lactose Digestion B. Diarrhea C. Cholesterol Reduction D. Cancer Suppression E. Immune System Stimulation F. Constipation G. Vaginitis IV. Safety Issues V. Considerations for Strain Selection VI. Research Needs VII. Conclusions References

I. INTRODUCTION

The opinions of experts on the role of lactic acid bacteria in promoting health in humans are diverse. Some are very optimistic; some are guarded. Consider these examples: Yogurt . . . is indicated as the best diet for balancing and preserving the biological functions of the gut. All these biological effects of yogurt are of great importance for the 67 Copyright 8 1993 by Academic Press. Inc. All rights of reproduction in any form reserved.

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normalization of the autochthonous flora and the consequent increase of the body’s natural defense against infectious diseases and putrefactive and fermentative processes. (Bianchi-Salvadori, 1986)

.

[Lactic acid bacteria] impart nutritional and therapeutic benefits to the consumer. . The antimicrobial substances produced by these bacteria control the proliferation of undesired pathogens. . . . Their anticholesteremic properties assist in lowering serum cholesterol. It has been suggested that the tumor suppression trait of these microbes reduces the incidence of colon cancer. (Fernandes et al., 1987) There appears to be much potential for the use of bifidobacteria with other beneficial organisms as dietary adjuncts in cultured dairy products. Research has shown that in some cases bifidobacteriacan be used to control enteric infections, lower serum cholesterol levels, improve infant formulas, and make milk products more nutritious and more easily digestible by lactose-intolerant people. (Laroia and Martin, 1990) The therapeutic value of [Lacrobacillus acidophilus] has since been established for many diseases and disorders of the digestive tract. (Danielson et al., 1989) Consumption of cultured dairy products containing viable [lactic acid bacteria] alters favorably the gastrointestinal microecology, conferring protection to the host. (Fernandes and Shahani, 1989)

. . . It becomes . . . more and more important that we consume products containing the special lactobacilli, bifidobacteria, and yogurt cultures in foods on a daily basis. Those individuals who do so will help keep their immune systems activated to combat the influx of undesirable organisms and possible pathogens into their gastrointestinal system via the food chain. (Sellars, 1989) These generalized conclusions are remarkable because even the most basic of premises on which these conclusions are based-including the likelihood of intestinal implantation of dietary cultures, the role of diet on intestinal microflora, and the role of intestinal flora on human health-are still matters of intense scientific debate. Consider the guarded opinions of the researcher about substantiated health benefits, that are observed more commonly in the literature: The mechanisms of the beneficial effects of bifidobacteria are still insufficiently known, and further research and development are needed. (Kurmann and Rasic, 1991)

. . . An abundance of research results to support [health] claims of the current products have (sic) not been provided. (Kim, 1988) Many health promoting benefits . . . are purported to be gained by eating these fermented foods. Yet these claims are not fully substantiated by research and many conflicting opinions exist over what actual benefit is derived from eating these foods. (Truslow, 1986)

.

. . The belief that the consumption of lactobacillus-containing (sic) preparations promotes health is based largely on anecdotal information. The scientific literature is

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distressingly sparse concerning controlled, scientifically valid investigations of fermented milk products and their influence on health. (Tannock, 1990) Some evidence has accumulated to show the antimicrobial and anticholesteremic activities of these microorganisms [bifidobacteria]. The evidence is scant, however, and the therapeutic value of this important group of bacteria has not been conclusively established. (Modler et a / . , 1990) Colonization of the gut by bifidobacteria taken orally has not been clearly demonstrated. Recent evidence . . . has suggested that colonization can take place only when a human or animal subject has been fed his (sic) own strain of bifidobacteria; thus, inclusion of generic strains of bifidobacteria in dairy products may have no therapeutic value. (Modler et al., 1990) According to present-day reports on antitumor activity of fermented milks, it is not justified to claim such an effect. (Driessen and de Boer, 1989)

. . . Given the variation in life-styles and genotypes among the human population, measures aimed at stimulating the activities of certain members of the normal flora of the gastrointestinal tract by the use o f . . . dietary additives are unlikely to be practical or worthwhile. . . . The successful implantation of bacterial strains which are antagonistic to pathogens or potential pathogens in groups of adult humans is likely to be extremely difficult. (Tannock, 1984) Unfortunately, there is not good scientific evidence that the ingestion of milk fermented by lactobacilli is any more beneficial to health than simply ingesting plain, pasteurized milk. For every article in the scientific literature that claims beneficial results from the ingestion of fermented milk, another article will provide evidence to the contrary. Most of the reported studies have not been adequately controlled, statistical analysis of the results is rarely made, and the conclusions are largely speculative. (Tannock, 1984)

The controversial nature of this field demands care in summarizing even peer-reviewed research papers. Many published studies fall far short of providing direct evidence for a healthful effect of lactic cultures on human health. Many papers cited as proof focus on animal models, in uitro experiments, or insufficiently controlled human studies with few subjects, or pay little attention to substantive rather than statistical significance. Lactic acid bacteria have been used to promote health for hundreds of years. Perhaps their most obvious effect is the inhibition of pathogen growth in traditionally fermented food systems, accomplished primarily by the acidogenic nature of these cultures. These bacteria subsequently may exert .positive benefits after consumption as residents of or while travelling through the gastrointestinal tract. These traits generally are attributed to lactic acid bacteria of the intestinal tract, primarily Lactobacillus acidophilus and Bijidobacterium, but also may include other lactobacilli, Streptococcus salivarius thermophilus, and Enterococcus. Stated healthful effects of lactic cultures include improved absorbability

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of nutrients, alleviation of lactose intolerance symptoms, metabolism of some drugs, reduction of serum cholesterol, reduction of enzymes implicated in carcinogen production, improvement of intestinal motility, stimulation of the immune system, reduction of tumor incidence, creation of an antagonistic environment for intestinal pathogens by production of inhibitors, blocking adhesion sites from pathogens, inactivating enterotoxins, alleviating constipation, and relieving vaginitis. This impressive array of potential benefits from these organisms has led to the marketing of many products for the stated or implied health benefits derived from the viable cultures contained within them. These products include nonfermented milk, fermented milks, yogurts, dried cultures, soft drinks, and candy. Although many products are formulated to use these intestinal lactic acid bacteria as supplements, international interest also exists in the production of milk products fermented with intestinal lactics (Gurr, 1984; Kurmann, 1988). This type of product is unique because the probiotic culture must be functionally fermentative and impart good flavor to the product in addition to promoting health. In practical terms, developing high populations of desirable microbes in products in which their growth was encouraged would be easier. However, problems with acid sensitivity could reduce populations during storage (Misra and Kuila, I99 1a,b) . In addition to original research papers, several comprehensive reviews have been published that discuss this field in a general fashion (Sandine et al., 1972; Shahani and Chandan, 1979; Shahani and Ayebo, 1980; Rasic, 1983; Friend and Shahani, 1984; Gurr, 1984; Lemonnier, 1984; Fernandes et al., 1987; Bourlioux and Pochart, 1988; Kim, 1988; de Simone et al., 1989; Fuller, 1989,1991;Goldin, 1989,1990;Gorbach, 1989,1990;Kroger et al., 1989; Sellars, 1989,1991; Alm, 1991; Renner, 19911, or focus specifically on lactose intolerance (Savaiano and Levitt, 1987; Savaiano and Kotz, 19881, gastrointestinal microbiology (Gorbach, 1971 ; Brown, 1977; Savage, 1977,1983a,b;Tannock, 1983,1984,1990;Bianchi-Salvadori, 19861, antimicrobial activities (Fernandes and Shahani, 1989), anticarcinogenic properties (Fernandes and Shahani, 1990), vaginal health (Redondo-Lopez et al., 19901, bifidobacteria (Poupard et al., 1973; Rasic and Kurmann, 1983; Bezkorovainy and Miller-Catchpole, 1989; Mitsuoka, 1989; Reuter, 1989; Laroia and Martin, 1990; Mitsuoka, 1990; Modler et al., 1990; Sandine, 1990; Hughes and Hoover, 1991; Kurmann and Rasic, 1991; Misra and Kuila, 1991a), L. acidophilus (Sandine, 1979,1990; Fonden, 1989; Gilliland, 1989), and strain selection for dietary adjuncts (Gilliland, 1979; Klaenhammer, 1982). These reviews should be consulted for additional perspective on this field.

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II. GENERAL PHYSIOLOGY

A.

GASTROINTESTINAL ECOLOGY

Although the human body is composed of loi3 eukaryotic cells, an estimated prokaryotic cells colonize the body surfaces and gastrointestinal tract (Savage, 1977). Therefore, only 10%of the cells in the human body are eukaryotic. The influence of this sizable prokaryotic population is undoubtedly significant, but exactly how these microbes influence health is the subject of much debate. Studies with germ-free animals have shown that colonization is not necessary for survival. However, germ-free animals are much more likely to succumb to infection than their colonized counterparts (Collins and Carter, 1978), suggesting that the normal gastrointestinal flora (without dietary supplementation) play a significant role in interfering with the establishment of intestinal pathogens (Tannock, 1984). The distribution of gastrointestinal microbes occurs horizontally, from the center of the lumen to the depths of the crypts, and vertically, from the esophagus to the anus (Savage, 1977). The specific occurrence of microbes depends on a variety of factors including age, health state, use of antimicrobial drugs, and, perhaps to some extent, diet (Hentges, 1980). The influence of diet on the normal gastrointestinal flora is still uncertain, prompting Tannock (1983) to state that his treatise on the effect of diet and stress on gastrointestinal microbiota was “of a speculative nature.” Summaries of studies on the flora of the gastrointestinal tract can be found in several reviews (Gorbach, 1971,1986; Brown, 1977; Savage, 1977). These surveys are consistent in reporting the relatively low level of bifidobacteria (6-1 1%) and even fewer lactobacilli (0-1.3%) in the feces of healthy human adults. The exception to this condition is the dominant presence of bifidobacteria in breast-fed infants (Stark and Lee, 1982). When attempting to design intestinal lactic cultures that will exert a positive effect on human health, it is reasonable to consider the extent of influence that would be expected to be exerted by microbes that may be minor components of the gastrointestinal population. This question is even more pertinent in light of the widely held belief that a dietary culture, even one possessing in uitro adhering capabilities, is highly unlikely to displace any bacterial strain that colonizes a healthy human intestinal tract (Savage, 1977). This theoretical criticism of dietary supplementation with cultures perhaps is addressed best empirically. Human gastrointestinal microbiology is notably a difficult field to study because of limits on direct experimentation (Savage, 1983a) and the difficult physiological requirements of

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the intestinal microbes. As indicated by Tannock (1984), “All investigators approaching the study of the normal flora of the gastrointestinal tract must sooner or later be horrified by the complexity of an ecosystem that contains about 500 species of bacteria, most of which are technically difficult to work with under laboratory conditions.” Analysis of fecal flora is a common technique used to evaluate the effect of feeding lactic cultures, but can never be a good index of the true character of the gastrointestinal microbial ecosystem (Savage, 1977). Savage (1983a) reviewed techniques for measuring intestinal colonization and concluded that no technique could be relied on to give acceptable data on microbial colonization of humans. Savage (1977) challenged the value of population studies and suggested that the biochemical activities of the gastrointestinal microbiota may be more significant than their numbers. Perhaps even low populations of microbes can exert significant biochemical influences in vivo.

B. FECAL RECOVERY Although of questionable value, studies abound that examine the microbial content of human feces after feeding with probiotic cultures. Among these studies are those of Lidbeck et al. (1987), who attempted to determine the effect of L. acidophilus supplementation on the flora of the intestinal tract by conducting fecal analyses. The effect of feeding on the microbial content of saliva also was determined. The results showed that feeding 500 ml/day of milk fermented by L. acidophilus (Arla Acidofilus) had little effect on the microbial content of saliva, but had a detectable effect on microbial content of feces. The major effect of L . acidophilus feeding was a decrease in Escherichia coli levels in 6 of 10 subjects and an increase in lactobacilli in feces. After supplementation was stopped, the flora returned to preexperimental levels. Lidbeck et al. (1988) also studied the effect of feeding L . acidophilus NCDO 1748 fermented milk to 10 healthy volunteers after oral administration of the antibiotics enoxacin (active against gram-negative aerobic bacteria) and clindamycin (active against anaerobic bacteria). Microbial analysis was conducted on feces throughout antibiotic treatment and Lactobacillus supplementation. The authors concluded that “although there was a partial restoration of the intestinal microflora due to the reestablishment of lactobacilli and enterococci, L. acidophilus administration could not accelerate the normalization of most other strongly suppressed microorganisms in the intestine.” Additionally, Lidbeck et al. (1991) studied the effect of consumption of fermented acidophilus milk on

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dietary intake, fecal flora, and aqueous fecal bile acids in 14 colon cancer patients. Only minor microbiological changes occurred. Goldin et al. ( 1992) followed the excretion of Lactobacillus GG in feces 3 and 7 days after subjects consumed GG. GG was administered as a yogurt for 7 days (daily dose: 3.6 x 10" bacteria), as a concentrate for 28 days (daily dose 4 x 10" bacteria), or as a whey drink for 35 days (daily dose 1.6 x 10" bacteria). Strain GG increased 4-6 log cycles during GG feeding in almost all subjects. Levels of recovered GG fell after feeding stopped, but these lactobacilli were still present at 104/gfeces after 7 days. This type of experiment has been done with bifidobacteria as well. Bouhnik et al. (1992) fed a strain of Bijidobacterium, tagged with streptomycin and rifampicin resistance markers, via a fermented milk product to 8 healthy volunteers, 3 times daily for 8 days. This strain increased to levels of over 108/gfeces, but gradually disappeared after ingestion ceased. The authors concluded that Bijidobacterium fed to humans does not colonize the colon, but populations could reach levels high enough to exert metabolic probiotic effects. Kageyama et al. (1984) studied the effect of bifidobacteria on fecal flora of leukemia patients. Fecal flora were identified from 56 patients (32 males, 24 females, aged 20-60) with six different types of leukemia. Patients consumed 200 ml Morinaga Bifidus milk daily for 3 months. The milk contained about 107/mleach of Bijidobacterium and L. acidophilus. (Note that the title of the paper in question and the discussion throughout implied that only Bijidobacterium was administered, when in fact equal quantities of both microbes were consumed.) This study presented data that showed differences in fecal flora between patients consuming the Morinaga milk product and patients not consuming the milk. All subjects were undergoing antileukemic drug therapy. Tabulated data showed a higher level of E. coli, Bacteroides, and Veillonella during the administration of antileukemic drugs without Bijidobacterium, but unclear data presentation left a lack of understanding of what composed the reported numbers. Data on other minor intestinal bacteria were presented also. In this case, 10 control cases were used, but whether these controls were leukemic patients was unclear. Among the 56 cases undergoing drug therapy, some variation in minor fecal constituents was noted also. A legitimate decline in fecal levels of Candida was observed in patients administered the milk product. Marginal differences in urine indican and blood endotoxin occurred in patients treated with antileukemic drugs and bifidobacteria. In summary, this study did show an effect of administration of Morinaga Bifidus milk containing both L. acidophilus and Bifidobacterium on fecal content of leukemic patients receiving drug therapy. The extent of this effect could not be determined fully since no statistical analyses were done and since controls

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for some of the experiments were unclear. Further, no effort was made to follow the numbers of infections in these patients so true clinical effects could be determined. Clearly an effect on fecal populations was observed; how this change affected the clinical state of the patients was left unexplored. Numerous similar studies performed over the years have proven that certain lactic cultures can survive passage through the intestinal tract, whereas others cannot. Important factors include resistance to physiological levels of intestinal bile acids and feeding of the culture under conditions (usually in conjunction with food) that help neutralize stomach acid at or above pH 4. After consumption of a bacterium, recovery from the feces is certainly not evidence of implantation, even if recovery persists for a period after consumption has stopped, nor is recovery an indication that the probiotic culture is likely to impart a desirable influence. The administered microbes may reside exclusively in the large intestine, where many important benefits cannot be realized. Continued fecal recovery of these microbes after feeding has stopped simply may be because of residence time in the large intestine that exceeds microbial generation time, and may have little to do with colonization. C. ADHERENCE OF PROBIOTIC CULTURES IN THE HUMAN INTESTINE Adherence refers to the ability to stick to solid surfaces. Lactic cultures might be expected to have a greater impact on human health if they were capable of adhering to portions of the epithelial surfaces of the human intestine. Adherence differs from colonization because the latter implies the ability to adhere and also replicate. Mechanisms of adherence are diverse and complicated. However, retention of bacteria in the human intestine can result from specific adherence to epithelial cells, from nonspecific adherence to other intestinal bacteria, or from entanglement in mucus. Direct studies on adherence in humans are invasive and therefore essentially impossible to do. However, the contents of the human ileum, colon, and feces have been examined microbiologically. The presence of microbes in these intestinal or fecal contents is not proof that colonization has occurred, although absence suggests that colonization has not occurred (assuming suitable microbiological techniques). The body of scientific research on adherence deals with animal models, so their significance to humans could be questioned. As a comment on in uitro studies of adherence to human fetal intestinal cells, Tannock (1990) writes, “These observations presumably have little significance, since association of lactobacilli with intestinal epithelia in vivo has not been demonstrated.” Still, efforts continue toward identifying in uitro models that may mimic the in

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uiuo human environment. Experimentation with Caco-2 polarized human intestinal epithelial cells in culture (Chauviere et al., 1991) and other improved cell lines hopes to establish such a model. Confirmation of results from these in uitro systems with clinical data is necessary before these results would be considered valid for humans. The intestines of different species vary in chemical composition and nutrient availability (Tannock, 1990). Therefore, the observation of host specificity of adherence (Wesney and ,Tannock, 1979; Tannock et al., 1982; Lin and Savage, 1984) is not surprising. This host specificity can be achieved by specific adhesins and receptors on bacterial and host cells and/or by nutritional or physiological adaptation to different cell types or gut environments. Conway and Kjelleberg (1989) identified an extracellular protein from Lactobacillus fermentum that mediates host-specific L . fermentum adhesion and patented a process that uses this protein to promote adhesion (Conway and Kjelleberg, 1988). Sat0 et al. (1982), using mucosal epithelial cells from pig embryo ileum on glass slides, concluded that a Bifdobacterium strain required its polysaccharide fraction to adhere. The significance of this study is completely dependent on how well this in uitro model using pig intestinal cells represents a human system. Conway et al. (1987) studied the survival of lactic acid bacteria in the human stomach and their adhesion to intestinal cells as a means to identify cultures suitable as dietary adjuncts. Cultures were screened for survival in gastric juice aspirated from healthy humans (21-42 years old). Also, a Lactobacillus preparation was intubated directly into stomachs of volunteers, with and without skim milk. At 20-min intervals, gastric fluid was removed and bacterial survival was recorded. Lactobacillus acidophilus strains ADH and N2 were used. Strain-dependent survival in gastric juice occurred, but the stomach presented a challenge for culture survival. These results support the need to consume these microbes with food or in an encapsulated form. Goldin et al. (1992) reported that GG survived ex uiuo in human gastric juice at pH 3.0. Below this pH, survival was poor, suggesting that ingestion of cultures with food to raise the pH is important for gastric survival. These data are consistent with the results of Conway et al. (1987). Clements et al. (1983)verified survival of ingested lactobacilli from LactinexTM and Infloran Berna'" by sampling jejunal fluid in uiuo. Conway et al. (1987) studied adhesion using radioactive cells. Strain ADH was more resistant than strain N2 to low pH and gastric juice exposure in uitro and in uiuo. The cell wall of ADH was also resistant to lysis and bound significantly better to human ileal cells than did other strains. All strains (two L . acidophilus, one L . delbrueckii ssp. bulgaricus, and one S . saliuarius thermophilus) bound to pig ileal cells, but in the same strain-specific pattern seen with human cells. The addition of 1% skim milk increased the binding of ADH to human ileal cells by a factor of

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3. Since the survivability of cultures in phosphate-saline buffer mimicked results obtained with human gastricjuice, the authors recommended phosphate buffer for the selection of resistant strains. The impact of these results is minimized by the fact that only two L. acidophilus strains were evaluated. Further, the data from the adhesion assay has questionable physiological significance because of its in uitro nature, although an in uiuo source of the intestinal cells was used. The test determining survival in human gastric juice may be a useful screen for dietary adjuncts in the future. However, results should be generated with more than two strains to determine if phosphate-saline buffer is a reasonable substitute for gastric juice in general. Other authors have speculated on the role of carbohydrates in adherence of lactobacilli (Brooker and Fuller, 1975). Costerton et al. (1978) noted that extracellular glycocalyx is frequently expendable for bacteria that are removed from their natural habitat to be cultivated as pure cultures in the laboratory. Meaningful research requires preserving intestinal strains in their physiologically active state. The best method for such preservation is a subject for future research. As difficult as obtaining reliable information on adherence in human systems is, adherence is commonly believed to be required for a probiotic culture to exert most positive effects. Otherwise, residence time of the culture is believed to be too short. Clearly the ability to adhere is required for long-term implantation in the gut. However, even for adhering microbes, displacement of the established flora of the healthy adult intestine is difficult (Tannock, 1990), except with microorganisms that have an adaptive mechanism that directs displacement of other microbes or after a specific change in the physiological state of the person. Collectively, these facts suggest that adherence cannot be insured by simply feeding microbes that possess the potential to adhere. However, since the intestinal flora of an adult is not necessarily constant because of the influence of certain dietary, physiological, and symptomatic or asymptomatic disease states, chances may exist for alteration of the intestinal flora throughout the lifetime. Adherence may result if continued consumption of a suitable strain occurs during one of these opportunistic times. The newly adhering strain may be displaced, however, once the physiological condition returns to normal. Also, transient microbes may exert positive effects without adhering, which appears to be the case with yogurt bacteria that aid lactose digestion. Studies on fecal recovery of microbial dietary supplements show recovery of the fed microbe for days or even weeks after feeding has stopped. Although not a permanent condition, these studies suggest that residence times for these microbes may be sufficient to exert an effect. One could speculate that, if continuously consumed, transient populations of

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the dietary culture could possibly lower intestinal pH, produce antimicrobial compounds, neutralize toxins, utilize nutrients, and conduct other biochemical activities that could influence gastrointestinal ecology. D. PRODUCTION OF ANTIMICROBIALS The ability of lactic cultures to flourish at the expense of many other bacteria has been observed in food systems and is widely suspected to occur in the human intestine. In food systems, lactic acid bacteria create an environment that is inhospitable to many bacteria, including pathogens, by lowering the pH, decreasing the redox potential, and producing antimicrobial compounds including organic acids, hydrogen peroxide, and proteinaceous bacteriocins. In uiuo, the mechanisms and extent of competitive exclusion are much less clear. One aspect of lactic culture biochemistry that has attracted much research interest is the production of bacteriocins (Klaenhammer, 1988). The in uiuo significance of these compounds is not clear. Much of the literature published on bacteriocins is presented from the perspective of use of these substances or the producing microbes in food systems, not in humans (Babel, 1977; Gibbs, 1987). Characteristics of the bacteriocins required for these two different activities would be vastly different. However, claims continue to be made for the general antipathogenic effects of bacteriocin-producing lactic cultures in uiuo, although wellcharacterized bacteriocins purified from lactobacilli have been shown repeatedly to have a narrow spectrum of activity against closely related species and do not inhibit common gram-negative pathogens (Klaenhammer, 1988). Still, selection on the basis of bacteriocin-like activity is advised regularly as a screen for cultures that may be used for probiotic applications (Gilliland and Walker, 1990). Silva et a / . (1987) studied a low molecular weight protease (I 1000) and heat stable compound concentrated from Lactobacillus GG. This compound inhibited all 54 strains of 7 genera (E. coli, Streptococcus, Pseudomonas, Salmonella, Bacillus fragilis, Clostridium, and BiJdobacterium) tested. These properties are not consistent with those of a bacteriocin and seem more similar to those of an organic acid. When identical concentrations of pure lactic and acetic acids present in the GG extract were tested, no zones of inhibition were present. The authors concluded that the compound produced by GG must be distinct from lactic or acetic acid. Unfortunately, the organic acids were not tested in combination although they were present together in the GG extract. The authors did not eliminate the possibility of synergism with multiple organic acids or with other possible extract components. The involvement of lactic, acetic, or other organic acids in inhibition activity has not been clarified. The molecular weight,

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the spectrum of activity, and the physical stability of the compound all suggest the role of an organic acid. This antibacterial activity for a Lactobacillus strain is an observation of questionable significance. In the years since the publication, no follow-up on further characterization of the compound has been made. Meghrous et al. (1990)examined 13 strains of bifidobacteriafor bacteriocin production. One producer was found with a spectrum of activity that was narrow and limited to closely related gram-positive bacteria. Inhibition of clostridia in laboratory assays and in food systems by nisin, an antibiotic produced by Lactococcus lacris subsp. lactis, suggests the possibility that this compound may be effective against an intestinal pathogen such as Clostridium difficile.However, since L . lactis does not survive in the intestine, natural nisin-producing strains are not likely to be useful therapeutically. Further, nisin is degraded readily by proteolytic enzymes present during food digestion. Possibly, the genes that encode this bacteriocin could be transferred into bacteria that are likely to survive the stomach and intestinal environments, and possibly even implant in the intestine. However, no evidence exists that this bacteriocin is effective in an animal system. In addition to nisin, other bacteriocins from lactic cultures have demonstrated in uitro activity against pathogens, including Listeria (Bhunia et a f . , 1988; Harris et a!., 1989; Spelhaug and Harlander, 1989; Ahn and Stiles, 1990), Campylobacter (Lyon and Glatz, 1991), and Vibrio (Lyon and Glatz, 1991). In general, however, in uitro evidence suggests that bacteriocins of lactic acid bacteria are primarily effective against other lactic acid bacteria. The presence of some in uitro activity against some intestinal pathogens provides encouraging evidence that certain cultures may possess useful antipathogenic bacteriocin activity in uiuo. However, the effect of these bacteriocin-producing lactobacilli on other gastrointestinal microbes has never been studied in uiuo. The genetics of bacteriocin production in lactobacilli has progressed enough that comparative clinical studies of strains that are isogenic except for bacteriocin production genes could and should be conducted to determine the usefulness of this phenotype. Bacteriocin production by probiotic strains could have a negative effect by inhibiting or displacing native “desired” lactobacilli, not pathogens, in the gastrointestinal ecosystem. Ill.

HEALTH TARGETS

Research on the effects of consumption of lactic cultures on human health has focused on a few specific health targets, including lactose maldigestion, diarrhea, reduction of serum cholesterol, cancer, immune

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system stimulation, constipation, and vaginitis. This research is performed mostly from an empirical perspective, with little attention to culture selection, mechanism of effect, or degree of benefit that could be expected by the consuming public. Discussions of these specific health targets follow. A.

LACTOSE DIGESTION

Reviews on lactose intolerance have been prepared by Savaiano and Levitt (1987), Scrimshaw and Murray (1988), and Savaiano and Kotz (1988). The presence of lactose maldigestion in the majority of the human population (worldwide levels of about 70%) is well documented (Scrimshaw and Murray, 1988). Lactose-intolerant people may avoid milk and other dairy foods because of intolerance symptoms and therefore may consume suboptimal levels of calcium. This response is unnecessary and unfortunate (Gallagher et al., 1974;Gilliland and Kim, 1984),and provides excellent justification for the development of milk products that are digested more easily by lactase-deficient people. Note that some people may have milk protein allergies, which differ from lactase deficiency, that may prevent them from consuming milk products. Although lactose maldigestion may be prevalent worldwide, the symptomatic expression of lactose intolerance is less so. Most lactase-deficient people can consume one glass of milk per day asymptomatically (Savaiano and Kotz, 1988). Further, 85% of those individuals reporting discomfort with the consumption of milk and milk products characterize their symptoms as mild or state that they would not be deterred from drinking milk as a result (Scrimshaw and Murray, 1988). However, a greater percentage of people believe they are lactose intolerant than can be substantiated clinically. For example, when assessing individuals who claim to be intolerant of milk, 64% were shown to be lactose digesters (Rosado et al., 1987). Also, the incidence of symptoms in subjects fed lactose-free placebos also could reach 35% (Savaiano and Levitt, 1987). Therefore, lactose intolerance has a substantial psychological component. Dietary practices also can influence the expression of lactose intolerance. That a given quantity of lactose is tolerated more easily when consumed in milk or dairy products than when consumed in water is commonly accepted (Scrimshaw and Murray, 1988), although some dairy products are tolerated better than others. Yogurt, which may contain almost as much as or more lactose than milk, is tolerated better than milk. Some research has suggested that starter culture lactase may digest the lactose once the yogurt is consumed (Lin et al., 1991). Also, lactasedeficient people have been shown to be able to adapt to lactose by regular consumption of lactose-containing foods (Scrimshaw and Murray, 1988). The mechanism of this adaptation is not clear although, in animal studies,

80

MARY ELLEN SANDERS

three- to sixfold increases in the fecal microbial levels of j3-galactosidase occur. A change in the enzymatic activities of the intestinal bacteria or microbial populations toward greater ability to digest lactose may result in more tolerance to lactose. People clinically identified as lactose intolerant based on a standard lactose test may not show symptoms in day-to-day life since the quantity of lactose ingested at any one time in a normal diet is usually much less than that ingested in the lactose-tolerance test (Scrimshaw and Murray, 1988). These facts demonstrate that lactose deficiency exists as a multidimensional issue for humans. However, nonallergic people can consume at least some dairy foods, providing dietary options for good nutrition and much needed calcium. The choices and quantity of dairy foods that are available to lactoseintolerant individuals could be increased with proper formulation of culture-containing milk products. Although Lin et al. (1991) found that laboratory-preparedacidophilus milk containing lo8cfu/ml (counts almost two log cycles greater than those of commercially prepared acidophilus milk) showed only marginal effects on breath hydrogen values, similar milks prepared with yogurt cultures ( L . delbrueckii bulgaricus and S . salivarius thermophilus) elicited significantly lower breath hydrogen values in subjects. Of three L. acidophilus strains tested, one strain did show efficacy in alleviating symptoms, although yogurt cultures were better. These results clearly show that milk containing the proper cultures can be tolerated more readily by lactose-intolerant people. Improved lactose tolerance appeared to be due to in uiuo autodigestion of lactose by microbial lactase. The survival of intracellular microbial j3-galactosidase appears to be due to two factors (Savaiano and Levitt, 1987): the protective encasement of the enzyme by the microbial cell and the effect of the excellent buffering capacity of ingested dairy products on stomach pH. These conditions provide a mechanism for the microbial lactase to reach the intestine in an active form to digest the lactose. The results of Lin et al. (1991) also demonstrate the importance of strain selection, since certain microbes have greater efficacy in this role than others. In this regard, criteria for the selection of strains for their effectiveness in relieving lactose intolerance symptoms may be very different from and, in some cases, contradictory to those for selection for other proposed health benefits. For example, bile tolerance, a trait considered important for intestinal survival of probiotic cultures, may protect against bileinduced permeabilization of the microbe in the intestine, preventing intracellular microbial lactase from coming into contact with and digesting lactose. In fact, L. acidophilus NCFM, selected partly for its bile resistance for the Sweet Acidophilus'" product, performed worst in trials conducted by Lin et al. (1991).

81

LACTIC CULTURES AND HUMAN HEALTH

Martini et al. (l991a) published a paper describing the clinical effects of different species of lactic acid bacteria on lactose intolerance. In the first study, 7 lactase-deficient subjects were fed the following 5 meals in a random blind format: ( 1 ) yogurt made with undefined Yoplait starters; (2) yogurt made with S . salivarius thermophilus strain 3641 and L. delbrueckii bulgaricus strain 11842; ( 3 ) yogurt made with S . salivarius thermophilus strain 3641 and L. delbrueckii bulgaricus strain 880; (4) yogurt made with S . s. thermophilus strain TS2B and L. delbrueckii bulgaricus strain 11842; and (5) whole milk supplemented with 2% nonfat dry milk. Each meal contained 18 g lactose. Peak hydrogen excretion was about threefold higher with milk than with any of the yogurts. Fivefold more hydrogen was excreted over an 8-hr period after consumption of the milk than after consumption of any of the yogurts. No significant differences in any of the four yogurt patterns was observed, although a wide range of total (per g product) and specific (per mg protein) p-galactosidase levels was exhibited (Table I), implying that even the yogurt with the lowest 0-galactosidase activity contained sufficient enzyme for efficacy. A second study in this paper attempted to differentiate between the contributions of different strains to the alleviation of lactose-intolerance symptoms. In this experiment, 12 lactase-deficient subjects were fed the following meals: ( 1 ) low-fat milk; (2) yogurt made with L. delbrueckii bulgaricus and S . salivarius thermophilus; ( 3 ) milk fermented with only S . salivarius thermophilus; (4) milk fermented only with L . delbrueckii bulgaricus; (5) milk fermented with Bijidobacterium bijidum; and (6) milk fermented with L. acidophilus. Each meal contained 15 g lactose, but bacterial concentrations in the different products varied. Breath hydrogen was produced in decreasing levels from the following products: ( I ) milk; TABLE I

P-GALACTOSIDASE LEVELS OF FOUR YOGURT PRODUCTS FED To LACTOSEDEFICIENT SUBJECTS'

Yogurt starters

Total P-galactosidase activityh

Specific P-galactosidase activity"

Yoplait ST 3641 + LB I1842 ST 3641 + LB 880 ST TSZB + LB 11842

7.00 2.30 3.42 4.96

5.03 3.70 4.03 6.68

Reprinted with permission from Martini et a/. (1991a). Activity is pmol ONPG hydrolyzed/min/g product. ' Activity is pmol ONP released/min/mg protein.

82

MARY ELLEN SANDERS

(2) milk fermented with Bijidobacterium; (3) milk fermented with L. acidophilus; (4) milk fermented with S . salivarius thermophilus; ( 5 ) milk fermented with L. delbrueckii bulgaricus; and (6) milk fermented with both L. delbrueckii bulgaricus and S. salivarius thermophilus. The difference in breath hydrogen excretion among subjects consuming milk and those consuming milk fermented with both yogurt strains was ninefold. This study was the first published attempt to differentiate among the effects of different lactic cultures on lactose digestion. These results support the conclusions that, for given lactose loads, the presence of specific cultures enhances lactose digestion. Yogurt bacteria, alone or in combination, appeared to be the preferred bacteria for this application. The products containing L. acidophilus and Bijidobacterium were less effective, although populations of L. acidophilus were lower in the final product. This study suggests the importance of strain selection for a lactose-intolerance application. Martini et al. (1991b) determined the effect on lactose digestion of meal consumption and extra lactose consumption as well as yogurt consumption. In the first study, 12 lactase-deficient subjects were fed whole milk, commercial yogurt, a standard breakfast meal, or the same meal plus yogurt. Consumption of the meal with the yogurt did not affect peak breath hydrogen production, but did delay it I hr. In the second study, 10 lactasedeficient subjects were fed yogurt (10 g lactose), yogurt with added lactose (0-20 g), or milk (20 g lactose). The P-galactosidase activity contained in yogurt did not aid the digestion of lactose added in the form of additional milk or crystalline lactose. This study confirms that the lactose in yogurt is better tolerated than the lactose in milk. Yogurt will reduce lactose rnaldigestion, with or without concomitant meal consumption, but Pgalactosidase from yogurt cannot assist in the digestion of additional lactose consumed simultaneously. de Vrese et al. (1992) studied the effect of p-galactosidase-producing microbes contained in kefir fed to minipigs. Since, unlike yogurt, kefir does not contain free galactose (because of the presence of galactoseconsuming P-galactosidase-negative yeast), in vivo lactase activity was assayed by determining blood plasma levels of galactose (rather than breath hydrogen). This approach offers confirmation of the breath hydrogen method as a means to assay microbial lactase delivery. Two groups of five 64-pound pigs were given I liter kefir containing 100 mmol lactose per 1iter.and either fresh (70 U lactase activity) or heated (0.1 U lactase activity) kefir grains. Plasma was analyzed for galactose immediately before and up to 7 hr after feeding. The largest differences in galactose concentrations were measured 30- 180 min after feeding; pigs fed unheated kefir grains showed 30% higher plasma galactose levels 90 min after feeding. This study showed, in a minipig model, that microbial P-galactosidase

LACTIC CULTURES AND HUMAN HEALTH

83

significantly affects plasma galactose levels. Since the kefir contained no free galactose, the rise in plasma galactose was from increased intestinal lactose hydrolysis. Even greater differences in galactose levels might have been expected had baseline lactase levels been lower, as in lactose-intolerant individuals. This study did not differentiate between pgalactosidase activities of the mixed Lactobacilfuslyeast kefir culture, although the yeast p-galactosidase activity was a minor component. Although the study was conducted in an animal model, the results presented in this paper provide supporting evidence that consumption of dairy products containing p-galactosidase-containing microbes can decrease the physiological effects of lactose consumption by lactose maldigesters. Lactobacillus acidophilus cultures were effective in decreasing breath hydrogen levels of lactase-deficient subjects in a study by Kim and Gilliland (1983), in contrast to results of Lin et a f . (1991), McDonough et al. (1987), Payne et al. (1981), Welsh et a f . (1981), and Newcomer et al. (1983). The Kim and Gilliland (1983) study was criticized because of the short (3-hr) time of delay prior to sampling breath hydrogen. Sampling times of up to 8 hr are recommended (Savaiano and Levitt, 1987). Savaiano (personal communication) suggests that microbiological diversity of the culture called NCFM may account for some discrepancies between results from different laboratories. McDonough et al. (1987) did report an enhanced decrease of breath hydrogen when Sweet Acidophilus'" milk was sonicated prior to feeding, presumably predisposing the microbes to release lactase intestinally, Payne et al. (1981) reported a failure of commercially prepared Sweet Acidophilus'" milk to decrease breath hydrogen or to alleviate gastrointestinal symptoms. This study was criticized because the authors failed to analyze the microbiological characteristics of the milk prior to use. Since viable counts of lactobacilli in commercial unfermented acidophilus milk are frequently below 107/ml, perhaps the cell levels were lower than needed for efficient alleviation of symptoms. Savaiano and Kotz (1988) speculated that the failure of unfermented acidophilus milks to aid lactose digestion was the result of the use of frozen concentrate starter cultures. The concentration, freezing, and storage conditions may inactivate lactase. Combined with relatively low levels of culture in commercial milks, these processing steps may render the products ineffective against lactose maldigestion. Also, the bile resistance of commercial strains protects the culture from lysis once consumed, creating a barrier to lactase release. The contribution of bifidobacteria to the alleviation of lactose intolerance is currently speculative, since few publications are available on this topic. Roy et al. (1992) described the a-and p-galactosidase activities of the large colony type of Bijidobacterium infantis strain ATCC 27920. Using X-gal staining of polyacrylamide gels of cell-free extracts, three

84

MARY ELLEN SANDERS

bands of j3-galactosidase and one band of a-galactosidase activity were found. The study further characterizes the pH, temperature, and cation concentration optima of the enzymes, as well as their kinetic characteristics. The optimum pH (6.0-7.0) and temperature requirements (40") of the enzymes were consistent with human intestinal characteristics. The presence of these enzymes suggests that B. infantis may aid in the digestion of complex carbohydrates and may help alleviate lactose-intolerance symptoms in humans. The presence of some clinically useful j3-galactosidase activity in a Bijidobacterium strain is confirmed by Martini et al. ( 1991a). Clearly, from this and other work, certain cultures used in fermented dairy products can promote tolerance in lactose-intolerant people to lactose in milk. Mixed results have been obtained with consumption of Sweet Acidophilus'" milk, but the overwhelming evidence suggests that this commercial product is not very effective at decreasing breath hydrogen. The current commercial formulation of Sweet Acidophilus'" milk is not ideally suited to alleviate lactose-intolerance symptoms, but newer products produced with L. acidophilus and bifidobacteria blends of cultures have not been tested publicly. An unfermented culture-containing milk could be formulated to increase lactose digestion if the cultures used for this product contained suitably selected strains.

B. DIARRHEA Many studies have been conducted over the years to determine the effect of live lactic cultures on limiting the course of diarrheal diseases. Studies vary with culture used, dose used, target group (healthy vs diseased, pediatric vs adult), cause of diarrhea (viral or bacterial), and experimental format. Some experiments yielded positive results, some negative. Some conclusions appeared overextended; some were made carefully. All negative results could be discounted by criticizing culture selection or levels. Some positive results could be discounted because of poorly conducted experiments or marginal significance of results. However, a few well-done experiments suggest that, in carefully defined subpopulations, bacterial therapy can limit the course of diarrheal diseases. Additional research should be conducted on well-defined and properly selected cultures and their effect on the course of diarrheal diseases. The following discussion supports these conclusions. Colombel et al. (1987) conducted a double-blind placebo-controlled study in 10 healthy erythromycin-treated adults. Subjects were fed yogurt containing BiJdobacterium longum or a placebo yogurt in conjunction with erythromycin over two 3-day periods. Fecal weight, stool frequency,

85

LACTIC CULTURES AND HUMAN HEALTH

abdominal complaints, and presence of fecal clostridial spores were measured on days l and 3 of the test periods. Fecal weight (145 g/day vs 208 g/day), stool frequency (1.2/day vs 1.9/day), and abdominal complaints (1 person complaining vs 6) were reduced when patients consumed culture-containing yogurt but not the placebo (Table 11). Also, clostridial spores dropped significantly in subjects consuming bifid-yogurt. Spores were present in 7 of 15 subjects taking the placebo yogurt and in only 1 subject consuming bifid-yogurt. The authors concluded that bifid-yogurts could reduce antibiotic-induced alterations of intestinal flora, resulting in a decrease of gastrointestinal disorders. This study was conducted with a limited group (only 10 subjects) and should be considered only a pilot experiment, but the experimental design warrants acceptance of the results as valid. Siitonen et al. (19%) also studied diarrhea in healthy human volunteers treated with erythromycin. Of 16 subjects, 8 received yogurt made with strain GG and 8 received pasteurized yogurt. No significant difference was found in levels of total fecal lactobacilli or GG after 7 days of treatment, although 125 g yogurt containing GG was administered twice daily. Although no significant difference in Lacrobacillus counts was found, the duration of diarrhea was significantly shorter in the GG yogurt group (2 days) than in the placebo yogurt group (8 days). Authors report a lower incidence of stomach pain (23% in the GG group vs 39% in the placebo TABLE I1 EFFECT O F CONSUMPTION OF BIFIDOBACTERIUM LONGUM ON ERYTHROMYCIN-

INDUCED GASTROINTESTINAL EFFECTS I N

10 HEALTHY SUBJECTS'

Placebo

Stool weight (g/day) Stool number (/day) Abdominal discomfort Clostridial spores present

Yogurt

One day before erythromycin

Three days after erythromycin

One day before erythromycin

Three days after erythromycin

I lO(21)

208 (29)

132 ( 1 1 )

145 (16)

1 .O (0.15)

1.9 (0.35)

1.2 (0.13)

1.2 (0.13)

-

6

-

1

8

7

8

1

" Reprinted with permission from Colombel ef al. (1987).

86

MARY ELLEN SANDERS

group), diffuse abdominal pain, and nausea (data not shown), but statistical evaluation of these data was not provided. Clements et al. (1983) tested the ability of consumed commercial dried preparations of lactobacilli to prevent enterotoxigenic E . coli (ETEC)induced diarrhea in 23 healthy adults. Double-blind studies were conducted with two lots of Lactinex'" ( I .2 and 9.2 x lo8 viable lactobacilli per I oz package). No significant differences were found between the Lactinex'" and placebo groups with respect to ETEC attack rate, incubation period, duration of diarrhea, volume and number of diarrheal stools, and stool culture results. However, diarrhea resulting from treatment of the ETEC infection with neomycin was reduced in frequency and severity with one, but not a second, lot of Lactinex'". The authors concluded that LactinexTh'played no role in prevention or treatment of diarrhea caused by ETEC. The effect of Lactinex'T on neomycin-associated diarrhea was significant, but results were less than convincing because of the lot-to-lot variability of the culture. Since ETEC infection was induced in volunteers, results did not depend on a low frequency of symptom development in subjects. These results, however, do not support any prophylactic effect on ETEC-associated diarrhea. An earlier study by the same group (Clements et al., 1981) on LactinexTh'with 48 human volunteers showed a similar lack of efficacy. A study by Newcomer et al. (1983) to determine the effect of unfermented acidophilus milk on symptoms included 61 patients with irritable bowel syndrome with lactase sufficiency, 18 patients with lactase deficiency, and 10 healthy controls. The culture used in this study was NCFM, the acidophilus culture used to make Sweet Acidophilus'" milk. Milk contained at least 4 x lo6 cfu/ml and was consumed at a level of 720 ml/day for the irritable bowel group and between 0.25 and 4.5 8-oz glasses per day for the lactase-deficient group. The subjects were given milk randomly with or without added culture for a 10-wk period consisting of 2-wk periods in which milk was alternated with no milk. Cumulative indices were obtained for symptoms including abdominal paidcramps, diarrhea, bloating/gas, gurgling, and constipation based on subjective analysis by the subjects. No statistically significant difference was found between symptoms reported by the groups, for both lactase deficiency and irritable bowel syndrome subjects. This work can be criticized for the low levels of Lactobacillus used. However, the choice of bacterial levels can be defended easily since it was based on concentrations present in commercially available Sweet Acidophilus'" milk. Irritable bowel subjects consumed milk with each meal. This study clearly showed that no benefit for this type of diarrheal disease was obtained from Lactobacillus strain NCFM.

LACTIC CULTURES AND HUMAN HEALTH

87

The four papers discussed next present experiments that were conducted with pediatric cases. Pearce and Hamilton (1974) studied the effect of feeding a dried S . salivarius thermophilus (50-60%), L . acidophilus (35-45%), and L . delbrueckii bulgaricus (5%) preparation or a placebo to 94 children aged 3 years or younger who were hospitalized over a 10-monthperiod with acute-onset diarrhea. Each dose of culture contained at least lo8 viable bacteria in 400 mg (culture prepared by FermalacRougier Laboratories, Montreal). From 3 to 8 daily doses were administered, depending on the child's weight. The study was a double-blind design. No significant difference was found between placebo and treatment groups in the number and size of stool or the duration of diarrhea. The results did not differ when the data from short- and long-term cases were analyzed separately or together. This study is well controlled, with a large number of subjects, but shows no effect of this lactic culture treatment on alleviating diarrheal symptoms. Negative results could be attributed to strain selection, since no mention of the strain characteristics was made. Hotta et al. (1987) ran an uncontrolled study of 15 children under the age of 7 with intractable diarrhea (diarrhea that had an undetermined microbial cause, intractable clinical course, and high mortality) for at least 7 days prior to treatment. All children had various underlying diseases and all had been treated with various antibiotics (thought to cause the diarrhea). Clostridium difJicile was not found to be linked to these cases of diarrhea. Treatment included oral administration of (1) Bijidobacterium breve ( 109/g); ( 2 ) B . breve and Lactobacillus casei (each at 109/g); and/or (3) bifid-yogurt (MILMIL) containing B . breve ( 10'o/lOO ml), B . bijidum ( 10'o/lOOml), and L . acidophilus ( 109/100ml). Culture was administered at 3 g per day and yogurt at 60-600 ml per day. Of the patients, 10 received B . breve alone (BBG-01), 1 received the combination of B . breve, B . bijidum, and L . acidophilus in a yogurt product (MILMIL), 3 received L . casei and B . breve (BLG-B), and 1 received yogurt and the L . casei/B. breve combination culture. The B . breve strain was isolated from healthy breast-fed infants. No comment was made on the origin of the B . bijidum strain. Duration of treatment was not mentioned. In an average of all subjects, the duration of diarrhea prior to culture administration was 5-70 days (mean of 25.3 days); after treatment, duration was 3-14 days (mean of 7.0 days) (Table 111). Studies of fecal flora during treatment and recovery showed that fecal flora had stabilized in most, but not all, patients and contained large levels of bifidobacteria after diarrheal symptoms stopped. The aut h m attribute patient recovery to the bifidobacteria contained in the oral preparations and recommend viable bacterial preparations for diarrhea resulting from antibiotic therapy. This study was quite extensive in analy-

88

MARY ELLEN SANDERS

TABLE 111 EFFECT OF BlFlDO A N D ACIDOPHILUS ON DURATION OF DIARRHEA I N PATIENTS TREATED WITH VARIOUS ANTIBIOTICS"

Patient

Bacterio-therapyb

Duration of diarrhea before treatment (days)

Duration of diarrhea after treatment (days)

7 30 35

7 14 7 7 8 7 6 3 7 4 10 4 10 7 4

~

1

2 3 4 5 6 7 8 9 10 II 12 13 14 15

BLG-B BLG-B + MILMIL MILMIL BBG-01 BLG-B BBG-0 1 BBG-01 BBG-OI BBG-01 BBG-01 BLG-B BBG-01 BBG-01 BBG-01 BBG-OI

5 35 25 I1 10

25 9 70 7 30 40 40

Reprinted with permission from Hotta et al. (1987). For abbreviations. see text.

sis of patient physiological state, fecal flora, and diarrheal symptoms. Unfortunately, the lack of controls prevents a confident conclusion about the cause of the cure. However, the results are sufficient to warrant additional studies with adequate control groups and a greater number of subjects. Isolauri et al. (1991) studied 71 children between 4 and 45 months of age with acute viral (mostly rotavirus) diarrhea (less than 7 days of duration) and 8 control children with no gastrointestinal symptoms for the effect of Lactobacillus GG on shortening the course of diarrheal symptoms. Subjects were divided randomly into three groups to receive different dietary treatments. Group 1 (24 subjects) received strain GG-fermented milk, 125 g with cfu twice daily; Group 2 (23 subjects) received strain GG as a freeze dried powder with cfu twice daily; Group 3 (24 subjects) received fermented and pasteurized yogurt, 125 g twice daily (placebo). Each diet was given for 5 days. Oral rehydration of patients was conducted prior to nutritional therapy. All feeding products were obtained from Valio Dairies (Finland). The mean duration of diarrhea was reduced (95% confi-

LACTIC CULTURES AND HUMAN HEALTH

89

dence limit) in the groups receiving strain GG as a powder or a fermented product (1.4 days) compared with those receiving the placebo (2.4 days). Results were somewhat more pronounced when data from only patients with confirmed rotavirus infection were analyzed. This study showed that the duration of diarrhea could be reduced by feeding Lactobacillus GG with normal diet immediately after rehydration. This study was well controlled and conclusions seem warranted. Microbial treatments for pediatric diarrhea have not been limited to lactobacilli and bifidobacteria. Bellomo et al. ( 1980) treated pediatric diarrhea of various causes (104 patients from 1 month to 9 years old) with capsules of lyophilized Enterococcus faecium strain SF68 (3.8 x lo7 total cells; 53 treated) or L. acidophilus (5 x lo8),L. delbrueckii bulgaricus (5 X lo8), and L. lactis (4 X lo9) (51 treated). Patients were divided into two groups and were treated with one of the bacterial preparations. No untreated control patients were used. Treatment was for 3-10 days (treatment continued several days after recovery) with 1-2 capsules given per day, depending on the age of the child. Since most diarrheal disorders are self-limiting, all patients recovered within 4 days of treatment. However, in the group receiving the Enterococcus preparation, 62% recovered completely after 2 days of treatment, whereas only 35% recovered in the Lacrobacillus-treated group. These results suggest that Enterococcus strain SF68 may be useful in the treatment of miscellaneous diarrhea in children, and certainly was more effective than the Lactobacilluscontaining preparation used in this study. Unfortunately untreated illnesses and the varied treatment times make this study less than ideal. The data indicate at best a decrease of diarrhea duration of 1-2 days. Beck and Necheles (1961) ran an uncontrolled study of the effect of dried L. acidophilus (BacidTM)on 59 patients with various abdominal symptoms (constipation, antibiotic diarrhea, pancreatitis, diverticulitis, mucus colitis, ulcerative colitis, and colostomy-associated diarrhea). The authors concluded that, in most cases, a rapid improvement of symptoms occurred. Since no controls were run and since the target illnesses were a broad range of different diarrheal diseases, this conclusion is difficult to accept. This study is discussed because it is referenced frequently in support of the efficacy of Lactobacillus treatment for diarrheal diseases, and constitutes one source of unsubstantiated claims. Gorbach et al. (1987) treated 5 patients with relapsing C. difJicile colitis with 10'' (daily dose) viable Lactobacillus GG in skim milk for 7-10 days. Patients experienced 2-5 relapses over a 2- to I0-month period prior to treatment. After Lactobacillus treatment, no relapses occurred for 4 months to 4 years (Table IV). The authors stated that their experience supports the efficacy of treatment of C. difficile relapsing colitis

90

MARY ELLEN SANDERS

TABLE IV TREATMENT O F RELAPSING CLOSTRIDIUM DIFFICILE COLITIS WITH

LACTOBACILLUS

GG"

Patient (agekex)

Relapses (number)

Duration of illness (months)

Toxin titer before

A (35/M) B (93/M) C (241F) D (73/F)

2 3 3 4

2 4 3 6

111250 111250 111250 111250

E (5O/M)

5

10

Positive (no titer)

Toxin titer after 0 0 0 1/10 (delayed) 0

Follow-up with no relapses 16 mo 1 Yr 4 Yr 4 mo

4 Yr

Reprinted with permission from Gorbach et al. (1987).

with Lactobacillus GG. However, this study was not a controlled study and was done with only 5 patients. At best, this study suggests that further research may be warranted. Pozo-Olano et al. (1978) studied 50 travelers to Mexico for the effect of four daily tablets containing 3-6 X lo8 lactobacilli/tablet on diarrhea incidence and duration. The identification of the species, strain, or origin of the lactobacilli was not included in the publication. Subjects were divided randomly into two groups-one receiving the Lactobacillus preparation and the other receiving a placebo. The incidence of diarrhea in the experimental group was low (16 cases overall) and of short duration (most cases lasted only 2 days). No difference was detected between the two groups in incidence and, since no prolonged diarrhea occurred, no results could be generated on the effect of preparations on duration. Salminen et al. (1988) conducted a study on 21 gynecological cancer patients to determine whether oral administration of live L. acidophilus strain NCDO 1748 could alleviate intestinal side-effects of internal and external irradiation of the pelvic area. The test group received 150 ml of a fermented lactase-treated milk product containing 2 x lo9 /ml lactobacilli and 6.5% lactulose (added to support intestinal growth of the Lactobacillus strain). The control group received nothing. This experimental design did not control for effects of the fermented milk rather than the culture. Also, the lack of double-blind format brings into question the subjective reporting by both groups of their symptoms. Diet and antidiarrheal drugs were not controlled in the patients, but nutritional counseling was given to all subjects. The incidence of diarrhea was smaller in the yogurt group (10 reported incidences) than in the control group (34

LACTIC CULTURES A N D HUMAN HEALTH

91

reported incidences), but no difference was observed in vomiting, nausea, abdominal pain, loss of appetite, or weight loss. The yogurt group reported more flatulence than the control group, likely caused by the lactulose in the yogurt. Although the study lacked some desirable controls, the results were sufficiently significant to warrant further research on this population subgroup. Of 14 studies reviewed that tested the effectiveness of a bacterial preparation (Lactobacillus, bifidobacteria, or enterococci) on alleviating diarrheal symptoms, results of 5 were negative, of 6 were questionably positive (suspect due to experimental set-up or data analysis), and of 3 were positive. In this last group, the major effect was on decreasing diarrheal duration or incidence. Preliminary evidence on the effectiveness of limiting C. difficife,rotavirus, and erythromycin-induced diarrhea suggests that these conditions may be worthy subjects for further study. Cfostridium d$$cile colitis can be especially persistent and difficult to treat (George, 1980), so alternative therapies could be useful. Overall, the positive results on limiting diarrheal diseases with lactic cultures are encouraging. Additional research will be necessary to clarify and define the parameters for effective bacterial therapy. How probiotic microbes diffuse diarrheal illness is not known. One proposed mechanism is competitive colonization. Competitive colonization occurs when one intestinal microbe interferes with the colonization of another. However, documentation of this phenomenon by observing lactic cultures displacing pathogens or preventing pathogen adherence has been difficult experimentally. In uitro tissue culture studies and animal model studies have attempted to shed some light on the mechanism of lactic culture interference with intestinal pathogens. Chauviere et al. (1991) studied the competitive exclusion of ETEC by heat-killed lactobacilli in a tissue culture of human enterocyte-like Caco2 cells. Heat-killed L. acidophifus LB (a human fecal isolate) cells were combined with E. cofi cells and added to the tissue culture plates. After incubation for 3 hr, the monolayers were washed and adhering E. coli were quantitated. High concentrations of lactobacilli ( lo9 /ml) were needed to induce a 75% inhibition of ETEC adherence. Adherence limitation was proportional to Lactobacillus concentration. Yamakazi et a f . (1985) challenged germ-free mice monoassociated with B. longum with 10'' viable cells/mouse of E. cofi or endotoxin. Monoassociated mice showed greater survival than germ-free mice. When observed 18 hr after challenge with E. coli, only 4 of 11 germ-free mice survived, whereas all 10 mice monoassociated with Bijidobacterium survived. Although these results are unambiguous, their significance is less clear. Whether the effect is caused by a direct protective action of the

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Bifdobacterium or by immune system stimulation that accompanies intestinal colonization is not known. The significance of these results for fully colonized humans is not known either. Tomoda et al. (1988) studied a group of patients undergoing drug therapy for leukemia who showed a higher level of Candida in their feces than did normal controls. Two groups were generated from 49 patients with > lo5 Candidalg feces. In these groups, 28 individuals were fed Bifdobacterium for 3 months as dried bacteria or in milk; the other patients were not treated. Of the 21 untreated patients, l l developed diseases due to Candida; the same result occurred for 12 of 28 treated patients. In the Bifdobacterium-treated group, half the patients showed a decrease in Candida levels to < 104/g feces; these patients showed a significantly decreased frequency of Candida infection. The analysis of these data is questionable, however, because bacterial counts were expressed only as < 104/gor > 105/g.Whether the group of patients whose count fell below 104/gsimply had lower Candida counts initially was not clear. The authors chose an arbitrary cut-off point on which to base their interpretations. If the Bifdobacterium-treated patients whose Candida counts dropped to less than 104/g were compared with the nontreated group with similar counts, the Bifdobacterium-treated patients had a higher incidence of Candida infections (14% vs 6%). No data were presented on untreated patients with initial counts above lO’lg that spontaneously fell to lower levels. If, in fact, a strict correlation between population of Candida and Candida infection is known, then more comprehensive data are needed to analyze exactly how Bijidobacterium affects Candida counts. C. CHOLESTEROL REDUCTION The use of certain lactic acid bacteria to assimilate cholesterol in uitro (Gilliland ef al., 1985; Lin et al., 1989; Gilliland and Walker, 1990), or to lower blood cholesterol in uiuo (Danielson et al., 1989) has been studied. The proposed benefit is based on the opinion that decreasing blood cholesterol lowers the risk of heart disease in humans. The importance of monitoring dietary cholesterol for the general public, although widely believed, has been challenged by some individuals in the medical community, primarily for overextension of results obtained from a minor fraction of the population to the general public (Ahrens, 1985). Therefore, the issue of lactic cultures and their effect on cholesterol has two facets: (1) the ability of lactic cultures to reduce effectively and substantively the amount of cholesterol absorbed by consumers and (2) the effect this cholesterol reduction may have on the health of the consumer.

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Discussion of the second point is beyond the scope of this chapter. However, since cultures could be marketed based on the widely held belief that decreasing dietary cholesterol is healthful, the remaining discussion will focus on the scientific evidence available on the effect of lactic cultures on cholesterol. The following studies were conducted in human subjects. Jaspers et al. (1984) studied the effect of consuming 681 g of three nonfat yogurts on total cholesterol, high-density lipoprotein (HDL) cholesterol, lowdensity lipoprotein (LDL) cholesterol, serum lipoproteins, and serum triglycerides of 10 healthy male subjects. Yogurts were differentiated by cultures used for fermentation: two unidentified yogurt cultures from Chr. Hansen’s Lab and a blend of strains (from K. Shahani, University of Nebraska) L. delbrueckii bulgaricus 202 and S . salivarius thermophilus EBC. These investigators also measured the levels of uric, orotic, and hydroxymethylglutaric acid in the three yogurts to address claims by previous investigators (Richardson, 1978) that these acids may serve as hypocholesterolemic agents in milk. Although some significant differences in total cholesterol, serum lipoproteins, LDL cholesterol, and HDL cholesterol were observed at certain points during the study, no significant change persisted throughout the course of the study, suggesting that any hypocholesterolemic effect of yogurt consumption is transient. In addition, no significant effect on serum triglycerides occurred. The authors also determined that concentrations of uric, orotic, and hydroxymethylglutaric acids in the yogurts were insufficient to affect cholesterol levels. Although this reference is cited frequently in support of a hypocholesterolemic effect of yogurt, the transient nature of the effect suggests that the effect is marginal. Lin et al. (1989) tested the effect of LactinexTM,a tablet containing L. acidophilus strain ATCC 4962 and L. delbrueckii bulgaricus strain ATCC 33409, on serum lipoprotein concentrations in 334 human subjects. The daily dose in the treatment group was approximately 8 X lo6 viable lactobacilli. Analysis was done of total cholesterol, LDL, HDL, and very low density lipoprotein (VLDL), and triglycerides. Several factors in the experimental design suggest that the results of this experiment are meaningful: the double-blind design, the large number of subjects, the lack of variability in assay results, and the inclusion of all subjects in both treatment and control groups. Results failed to show any effect of culture consumption on reduction of serum lipoprotein levels. Hepner et al. (1979) conducted two 12-wk studies on 54 healthy subjects with cholesterol levels higher than 200 mg/100 ml (17 and 34 individuals in studies I and 11, respectively) to evaluate the effect of 2% milk, yogurt, and pasteurized yogurt on serum cholesterol, triglycerides, and

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diet. Commercially available Dannon yogurt at a rate of three 240-ml cups per day was used. Bacterial strains were not identified. Subjects were encouraged to maintain their regular diet throughout the course of the experiment. Dietary logs indicated that calorie, protein, and carbohydrate intakes increased and cholesterol, fat, and fiber intakes were not changed. In Study I, the 12-wk experiment was divided into three 4-wk feeding trials, consisting of sequential 4-wk periods of yogurt feeding, control feeding, and milk feeding in one group, and of milk feeding, control feeding, and yogurt feeding in a second group. No group was used during the study to control for variations in sampling or cholesterol determination. When yogurt feeding came first, a significant drop (99% confidence limit) in serum cholesterol occurred after the first week. The levels remained constant through the end of the 4-wk period. This result is consistent with a 5-7% drop in serum cholesterol in the first week seen by Rossouw et a / . (1981), although the first week was used to establish baseline levels of serum cholesterol and no milk product was fed until the second week. The data imply that the effect observed by Hepner et al. (1979) may not be caused by yogurt but by spontaneous modification of dietary intake induced by participation in the study and keeping dietary records (Rossouw et a/., 1981). During the subsequent control period, serum cholesterol rose; cholesterol leveled off during the milk feeding (a nonstatistically significant increase of serum cholesterol was observed during the milk feeding period). When milk feeding came first, no significant difference was detected in serum cholesterol levels at the end of the milk feeding period or the control period. However, serum cholesterol levels fell significantly (99% confidence) during yogurt feeding. Study I1 was designed to differentiate between effects of eating yogurt, pasteurized yogurt, 2% milk, and a normal unsupplemented diet. Subjects were fed the dairy supplements constantly. Pasteurization of the yogurt did not affect the hypocholesterolemic effect in Study 11, indicating that viable starter cultures did not mediate any effect. Although a significant difference was detected between cholesterol levels of subjects eating the control diet those of subjects eating the diet supplemented with the nonpasteurized yogurt, problems with the control group make this result suspect. The serum cholesterol levels of the control group were significantly higher after 4 and 6 weeks (95% confidence) than at the beginning of the study. Further, the control group was used only for half the duration of the experiment (6 weeks). N o significant effect on serum triglycerides was detected in either study. The authors conclude from these studies that yogurt and, to a

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lesser degree, milk exert a hypocholesterolemic effect. The investigators did not address problems with the control group for Study I1 in which a significant rise in cholesterol levels was observed. Since statistical comparison of the data was done with the control group, the unexplained elevated serum cholesterol levels increased the difference between control and experimental groups, with the resulting emergence of “statistically significant” data. The decrease in serum cholesterol levels observed with yogurt feeding in Study I reflected a drop from 202 mg/dl to 191 mg/dl, a result of questionable importance. Although statistically significant data were reported in this publication, the authors do not address the substantive significance of this finding. The reduced cholesterol level persisted for 6 weeks, in contrast to the results of another study that found the hypocholesterolemic effect to be temporary (Jaspers et al., 1984). Issues of proper control of experiments suggest the need for confirmation of results found in this study. Further, since cultures did not mediate the effects, this study is of marginal significance to marketing of culture-containing milks. This study is used frequently, however, as a reference to substantiate a hypocholesterolemic effect of yogurt. Rossouw et al. (1981) studied the effect of consuming 2 liters per day of skim milk, yogurt, or whole milk on serum total cholesterol, triglycerides, and HDL and LDL cholesterol in 32 adolescent boys. Statistical differences in serum total cholesterol correlated strongly with the differences in total dietary fat and cholesterol intake in the whole milkand yogurt-modified diets, and could not be attributed to a hypocholesterolemic “milk factor.” Some effect was observed with skim milk, however, so the authors do not dismiss the possibility of a hypocholesterolernic factor in skim milk. All measured parameters returned to baseline levels within the follow-up week of unsupplemented diet, with the exception of the serum total cholesterol of the skim milk group. The duration of this effect was not determined. Clearly, no hypocholesterolemic factor was identified in the culture-containing milk product. Further, this experiment was conducted with adolescent boys with n o evidence of hypercholesterolemia. Since dietary control of serum total cholesterol is a concern only in the minority of individuals with the genetic inability to regulate serum cholesterol effectively, the significance of this study can be questioned. Thompson et al. (1982) tested the effect of consuming 1 liter of low fat (2%), whole, skim, or Sweet AcidophilusTMmilk, yogurt, or buttermilk on total, LDL, and HDL cholesterol in 68 healthy volunteers with normal or low blood lipids. Dietary records on non-milk foods consumed by

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subjects were not kept. This study showed no effect of consuming these milk products on blood lipids, indicating neither a hypocholesterolemic nor a hypercholesterolemic effect of nonfat or whole milk products. Bazzare et al. (1983) found a positive effect of yogurt feeding and calcium supplementation in 16 female subjects (but not in 5 male subjects) on decreasing total cholesterol levels and increasing HDL: total cholesterol ratios. Renner (1991) stated that Lactobacillus GG was shown to decrease cholesterol in healthy human volunteers, but no retrievable reference was provided. Gilliland and Walker (1990) proposed a strategy to select an L . acidophilus culture that would produce a hypocholesterolemic effect in humans. These researchers advised that L . acidophilus strains be selected based on human origin, the in uitro ability to “assimilate” cholesterol, growth in the presence of bile, and bacteriocin production. Experiments designed to differentiate among cholesterol assimilation abilities of different L . acidophilus strains showed that the measurement of this ability was time dependent and therefore somewhat arbitrary, depending on the time chosen for assay. Experimentation also has been done in animal systems. In rabbits, Kiyosawa et al. (1984) found a total aorta cholesterol-lowering effect of both skim milk and yogurt, implying that the milk, not the cultures, exerted some effect. Gilliland et al. (1985) studied some strains of L . acidophilus isolated from pig feces. When grown in MRS broth with oxgall and pleuropneumonia-like organism (PPLO) serum fraction as the cholesterol source, the cholesterol was reduced in the broth and increased in the bacterial cells. The effect was dependent on the presence of oxgall. One strain each, designated cholesterol assimilating and cholesterol nonassimilating, was used in a feeding trial of 18 5-wepk-old piglets. All pigs were fed a cholesterol-containing diet (1.8 g/kg body weight) during the experimental period. In addition, the pigs were divided into three groups, fed 50 ml 10% nonfat milk solids or nonfat milk solids plus 5 x 10’O of one of the two L . acidophilus strains. Blood samples were taken and analyzed for total serum cholesterol. After 10 days, the control group and the group fed the nonassimilating strain showed elevated but not statistically different total cholesterol levels (74.44 and 73.48 mg/dl, respectively). The group fed the cholesterol-assimilating strain showed significantly lower total cholesterol levels (62.29 mg/dl) (Table V). Danielson et al. (1989) isolated three strains of L. acidophilus from porcine feces. All three were able to reduce water-soluble cholesterol (polyoxyethanyl cholesteryl sebacate) from MRS broth with 0.2 or 0.4% oxgall by 30-80% in 24 hr. Since strain LA16 was considered best at in uitro cholesterol assimilation and other screenines. this strain was used

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TABLE V I N F L U E N C E OF FEEDING LACTOBACILLUS ACIDOPHILUS CELLS ON SERUM CHOLESTEROL LEVELS I N PIGS ON A

HIGH-CHOLESTEROL

DIET".^

Group

Day 0

Day 5

Day 10

Control P47" RP32"

52.23 ( I .88)c' 55.58 (4.70)c' 52.84 (3.00)c1

69.10 (3.91)CD' 72.01 (3.02)c2 61.48 (3.30)D'

74.44 (4.64)C* 73.48 (4.68)c2 62.29 (4.91)D'

" Reprinted with permission from Gilliland e t a / . (1985). Each value represents the mean cholesterol (mg/dl) from six pigs; numbers in parentheses represent the standard deviation. Values in the same column followed by different superscript letters are significantly different (P<.05): values in the same row followed by different superscript numbers are significantly different (P<.05). There were no significant interactions among days and treatments (P>.05). ' L . acidophilus strain P47 was selected for its ability to grow in the presence of bile and its lack of ability to remove cholesterol from the growth medium. L. acidophilus strain RP32 was selected for its ability to grow well in the presence of bile and to assimilate cholesterol from the laboratory medium.

"

to feed 8 boars previously on a high cholesterol diet for 56 days. Serum lipids (cholesterol, triglycerides, HDL, and LDL) were tested. Only total cholesterol was different between the test and control groups (99% confidence level); the control and yogurt-fed groups showed 91.1 and 81.5 mg/dl levels of cholesterol, respectively. Large fluctuations in data occurred on a daily basis. The authors also reported a lowering of LDL, but this report was at the 92% confidence level, a level not commonly considered significant in scientific work. The authors concluded: Our data when applied to the correlation equations derived from the Framingham study indicate that the risk of coronary heart disease would be reduced by 21% if adult males consumed AY [acidophilus yogurt] in combination with high-cholesterol diets, assuming the AY were made from strains of LA [Lactobacillus acidophilus] that assimilate cholesterol. (Danielson et a / . , 1989).

This conclusion is an overextension of results, at best, in light of the use of an animal model and the questionable significance of results. The studies reviewed so far focused on the lactobacilli. With regard to bifidobacteria, Modler et al. (1990) conclude that no direct evidence implicates bifidobacteria in cholesterol reduction. The phenomenon of cholesterol assimilation by lactic cultures can be criticized based on the failure of the authors to address the issues of

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specificity and physiological significance of the proposed mechanism. Rasic et a / . (1992) attempted to differentiate in vitro the cholesterolassimilating properties of several bacteria, including strains of L. delbrueckii bulgaricus, S . salivarius thermophilus, B . bifidum, and L . acidophilus. Differences were observed in cholesterol uptake in broth as measured by the difference in the quantity of cholesterol in spent broth compared with a control. Although statistically significant, whether these differences were due to various levels of growth in the broth or to other factors in the assay was not clear. The assay for cholesterol assimilation used by Gilliland et a / . (1985) was based on the disappearance of cholesterol from an aqueous medium and its concomitant association with the bacterial culture. The association of the cholesterol molecule with the culture may have had more to do with the affinity of the hydrophobic cholesterol molecule for the cell membrane in an aqueous environment than with any specific or special activity of the culture. In this case, the physiological significance of the Gilliland et al. (1985) results is questionable, although the one cholesterol-assimilating strain selected in this study did perform better in pigs than did the cholesterol-nonassimilating strain. Further, cholesterol is absorbed primarily in the sparsely colonized ( 9 104/mlof intestinal fluid) upper small intestine; any microbial effect on cholesterol would require action at this point. Cholesterol that has reached the large intestine is likely to be excreted. Assimilation of cholesterol by lactobacilli in the large intestine would have no physiological impact. Therefore, although some significant results have been obtained with limited animal feeding trials, the meaning of the results and the scientific rationale for the experiments is debatable. The metabolic capability of some lactic strains to deconjugate bile acids may provide a legitimate mechanism for a role in cholesterol assimilation in humans, assuming adherence in the upper small intestine or regular consumption with a transient effect. Conversely, a lactic culture metabolite may influence cholesterol absorption. However, definitive mechanistic or clinical research is insufficient at this time. This research area is still in its infancy. Care must be taken in interpreting results from some published papers since, because of fundamental differences between cholesterol metabolism in animals and in humans, results from animal studies are not directly applicable to humans. Also, a statistically significant effect on cholesterol must be given meaning biologically, that is, the difference in cholesterol levels that must be achieved to yield a substantive effect on human health must be determined.. These issues have not been addressed adequately by either the medical community or the cultured dairy products industry.

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D. CANCER SUPPRESSION The reports of anticancer effects of fermented dairy products or lactic acid bacteria take four general forms: ( I ) in uitro studies on the inhibition of mutagen activity; (2) in uiuo decrease of fecal enzymes involved in conversion of procarcinogens to carcinogens, presumably with an effect on intestinal cancer rates; (3) in uiuo studies on tumor suppression or incidence in lab animals; and (4) epidemiology correlating cancer and certain dietary regimes. Direct experimental evidence for cancer suppression in humans as a result of consumption of lactic cultures in fermented or unfermented dairy products has not been obtained. Since no direct clinical studies have been conducted in humans, the following discussion will focus on the information available from indirect experimentation. This discussion is intended to provide insight into the oftenclaimed anticancer effects of dairy cultures. The repeated and continued use of nonhuman models to address this question is likely due to the difficulty, expense, and questionable likelihood of positive results of doing such studies in human populations in which cancer rates are low and effects may be unremarkable. The in vitro studies do provide a simple method to screen strains for implied optimal benefits; however, until such studies are correlated to efficacy in humans, their value is questionable. To be considered seriously for extrapolation to human systems, results from animal studies must correlate with epidemiological studies in humans. Drasar and Hill (1972) noted that intestinal bacteria can modify the intestinal chemical environment in humans. Specifically, the degradation of different bile salts into potential carcinogens by intestinal bacteria was studied (Hill et al., 1971); a correlation between these bacteria, the presence of these compounds, and the incidence of colon cancer was found. Although this study was preliminary, these authors felt that further study of the role of intestinal bacteria in colon cancer was warranted. Although the relevance of this observation to human disease was uncertain, the study provided a rational basis and justification for the examination of the effects of lactic cultures on potential cancerpromoting metabolic activities in the intestine. Modler et al. (1990) offered the opinion that large bowel cancer could be influenced directly by reducing intestinal pH, thereby preventing the growth of putrefactive bacteria. Goldin er al. (1980) studied the effect of L. acidophilus on the presence of four fecal enzymes in human omnivores. The four enzymes studied-P-glucuronidase, nitroreductase, azoreductase, and steroid

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7-a-dehydroxylase-were chosen based on their postulated involvement in activation of procarcinogens to carcinogens. [A preliminary study on these enzymes in rats was conducted earlier (Goldin and Gorbach, 1977).] Seven subjects consuming 4 ml 10” cfu/ml/day of L. acidophilus had significantly reduced nitroreductase (2.03 to 1.1 1 U) and P-glucuronidase (5.50 to 4.39 U) levels. The other enzyme levels were not affected by the Lactobacillus culture feeding. The remaining experiments in this study dealt with the effects of diet on the fecal enzymes and did not involve administration of L. acidophilus. The authors acknowledged that the correlation between enzyme levels and both carcinogen product formation and colon cancer incidence were not evaluated. Therefore, the effect of enzyme concentration on colon cancer risk was considered “a matter of speculation.” Whether a twofold decrease in nitroreductase or a 20% decrease in P-glucuronidase is sufficient to affect incidence of disease in humans is unknown. Note that the authors did not specify the strain used in this study, except to identify it as having been provided by Dr. Marvin Speck at North Carolina State University. This information suggests that NCFM was the likely strain. The strain dependency of fecal enzyme reducing ability is suggested in a later paper (Marteau et al., 1990). A commercial fermented milk containing L . acidophilus strain A1 (107/g),B. bifdum strain Bl (108/g), and mesophilic culture MO (108/g) was fed to 9 healthy subjects. Authors examined levels of fecal P-glucuronidase, nitroreductase, and azoreductase. Only nitroreductase levels decreased (95% confidence limit). Authors cited strain differences with the Goldin et al. (1980) study as an explanation of inconsistencies between the publications. In addition, levels of L. acidophilus fed to subjects were lower in the Marteau et al. (1990) study (109/day) than in the Goldin et al. (1980) study (4 x lO’’/day), and cultures were combined in the Marteau study. In evaluating the significance of reduced levels of fecal nitroreductase, the authors stated, “the exact role of this enzyme in colon cancer in humans is still unknown.” Goldin and Gorbach (1984a) studied the effect of feeding L. acidophilus strains NCFM and N-2 ( lo9 /day) on the activity of three bacterial enzymes-P-glucuronidase, nitroreductase, and azoreductase-in 21 healthy volunteers. The protocol for this experiment included establishment of a 4-wk baseline, then a 4-wk period of feeding plain milk, then another 4-wk baseline, then a 4-wk period of feeding milk supplemented with L. acidophilus, and finally a 4-wk baseline. Three fecal samples were taken during each 4-wk period. The results were not strain dependent, so data from both strains were combined. A significant decline in specific activity of all three enzymes occurred in all 21 subjects after 10

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days of Lactobacillus feeding. A reversal of the effect was observed within 10-30 days of stopping Lactobacillus feeding. This study provided evidence that feeding one of at least two strains of L . acidophilus caused a decrease in fecal enzymatic activity in healthy subjects. Milk alone did not cause this effect. Continuous consumption of these bacteria would be necessary to maintain the effect. The authors offered the reduction of these enzymes as a possible explanation for the reduced colon cancer incidence in rats given feedings of viable L . acidophilus (Goldin and Gorbach, 1980). However, they also recognized that the effect of Lactobacillus feeding on human colon cancer is entirely speculative since the ongin of the carcinogens causing this disease in humans is unknown. Goldin et al. (1992) provided evidence that consumption of Lactobacillus strain GG can mediate a reduction in fecal p-glucuronidase levels in humans. In this study, 8 control subjects were given either S . salivarius thermophilus or L . delbrueckii bulgaricus and 8 test subjects were given GG. The daily dose of microbe was 10". p-Glucuronidase levels fell to 80% of baseline values only in subjects consuming GG. The effect of intestinal flora on levels of p-glucuronidase, nitroreductase, and azoreductase was studied by Goldin and Gorbach (1984b). Human subjects received frozen concentrates of > 10" L . acidophilus strain NCFM/day. The experimental protocol for the human study (7 healthy subjects) included 1 month control (normal diet, no supplements), 1 month daily ingestion of culture, followed by 1 month control. The study was not blinded. Fresh fecal samples were tested for p-glucuronidase, nitroreductase, and azoreductase activities. Statistically significant differences (P <0.01) were found in p-glucuronidase and nitroreductase but not in azoreductase levels. A subsequent study with 21 subjects (data not shown) showed significant differences in all three enzymes during the culture feeding period. Several rat studies were conducted also that showed that, in a rat system, treatment with certain antibiotics decreased fecal enzyme activity, presumably by interference with intestinal bacterial enzyme activity. Ayebo et al. (1980) tested the effect of feeding 12 subjects unfermented acidophilus milk or low fat milk on p-glucuronidase and P-glucosidase activity levels in feces. The results depended on the sequence of consuming the two test products. The results of this paper do not contribute convincingly to knowledge in this area. McConnell and Tannock (1991) studied azoreductase activity in the ceca of mice. The mice used in the study contained a complex intestinal microflora (verified by determining 26 microflora-associated characteristics) but were either Lactobacillus- free (RLF) or colonized with specific Lactobacillus strains (RLFL). This experimental design allowed careful control of

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animal factors so any effect from the Lactobacillus strains could be determined readily. RLFL mice were colonized with two strains of L . delbrueckii and one of L . fermentum. Lactobacilli are numerically important members of the microflora of proximal regions of the gastrointestinal tract of mice. Azoreductase activity was reduced by 31% in mice harboring lactobacilli compared with azoreductase activity in Lactobacillus-free mice (99% confidence). The mechanism of azoreductase inhibition was not determined, but authors speculated that the bile salt hydrolase activity of lactobacilli increased the concentration of unconjugated bile acids in the intestine, resulting in inhibition of azoreductase activity. The authors cautioned that the biological significance of a 31% inhibition of azoreductase activity to the well-being of mice (or humans) is not known. These studies show that feeding certain cultures can result in a decrease of fecal enzymes that may be involved in carcinogen generation. On the whole, the studies are reasonably well controlled and the mechanism for the effect (biochemical activity of fecal microbes) is plausible. The criticism of this work rests with the lack of information on how or whether a reduction in these enzyme activities affects cancer rates in humans. Until this relationship is known, the impact of these results on human health cannot be ascertained. Further, the papers published to date do not consistently find reductions in the same enzymes, although findings with p-glucuronidase and nitroreductase are most consistently positive. Clearly, repeated experimentation is required to be assured of a repeatable effect with a given dose of a specific culture. In vitro studies on cancer suppression include model systems that use a decreased mutagenicity of known mutagens on bacterial cells (Hosono et al., 1986,1990; Zhang et al., 1990), a protective effect on tissue cell viability (McGroatry et a[,, 1988), determination of unbound mutagen after exposure to lactic culture cells in human gastric juice (Zhang and Ohta, 1991a), and binding of mutagens by cell wall fractions (Zhang et al., 1990; Zhang and Ohta, 1991b). In general, these studies show that cultures can decrease mutagenicity, mutagen levels, and cell viability. Some mutagenic pyrolyzates (including 3-amino-1 ,Cdimethyl-[ 'HIpyrido[4,3-b] indol and 3-amino-I-methyl-[ SH]pyrido[4,3-b]indol) bind to whole cells (live or dead) or cell wall fractions in water or in gastric juice (Hosno et al., 1986,1990; Zhang et al., 1990; Zhang and Ohta, 1991a,b). Results were generated with a diversity of strains, including L . acidophilus, B . bijdum, Lactococcus lactis cremoris, Enterococcps faecalis, L. delbrueckii bulgaricus, and S . salivarius thermophilus. Some strain dependency and mutagen specificity existed, but overall results were positive. Levels of mutagens decreased. A decrease of bacterial reversion rates was observed also, implying that mutagens were less

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potent. The danger in extrapolating these results to health claims for humans is that little is known about the fate of mutagens in the human gastrointestinal system. The reversibility of mutagen binding to cultures in vivo is unknown. Further, the biologically significant levels of mutagens and of lactic acid bacteria in the human system are unknown. In a different model system, McGroatry el al. (1988) tested the viability of human bladder tumor cells treated with five live or killed lactobacilli cell preparations. Tumor cell viability was reduced by four of five culture supernatant fluids. Heat treatment inactivated the cell lethality effect, suggesting the involvement of other than organic acids in the effect. However, protease treatment was not done. Concerns with this study include specificity of the effect for the cell line tested, the role of organic acids in cell killing (experiments were pH adjusted, but supernatant fluids were not dialyzed to remove undissociated acids or lethal compounds in MRS broth that were shown to be present), and, finally, the meaning of this study as it relates to in vivo effects on tumors. Strains of lactobacilli and E. coli were active in degrading nitrosamines (Rowland and Grasso, 19751, demonstrating the role of intestinal flora in degrading procarcinogens. Studies on the effect of lactic acid bacteria on cancer in animal models are also prevalent. Bijidobacterium bijidum was shown to grow selectively in necrotic tumors in mice, although not in normal tissue (Kimura et al., 1980). This specific association with malignant cells suggested that this strain could be used in the delivery of radioisotopes or anticancer drugs. Kato et al. (1981) studied the antitumor activity of L. casei strain 9018. Assay for antitumor activity included mouse survival when inoculated with tumor cells (sarcoma-180 cells, L1210 leukemia cells, and MCA K-1 cells) and growth inhibition of tumor cells in tissue culture (EL4 and P815 cell lines). Strain 9018 apparently was selected from 28 lactobacilli screened at an earlier date, but these results are not included in this publication nor is a reference provided. Strain 9018 administered to mice intravenously gave a dosage-dependent suppression of sarcoma- 180 tumors (tumor cells were implanted subcutaneously). Tumor weights in control and experimental animals were 2.64 and 0.64 g, respectively. Mice receiving an intraperitoneal administration of 9018 had a greater chance of surviving. The effect was greater when inoculation with 9018 came before tumor cell injection. Tumor inhibition also was observed with intravenous inoculation of 9018. Unfortunately, these data were not analyzed for statistical significance. The authors attributed these effects to an immunostimulation, although such an effect was not measured. This theory was supported by evidence that the antitumor effect was diminished by carrageenan, known to be toxic to macrophages.

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Sekine et al. (1985), using whole peptidoglycan isolated from B. infantis strain ATCC 15697, reported that a single subcutaneous injection significantly suppressed tumor growth. Also, five intralesional injections resulted in 70% tumor regression in mice. This peptidoglycan preparation was concluded to stimulate the host-mediated response, leading to tumor suppression or regression. Since in viuo treatment with chemically complex substances induces undesirable side-effects, limiting their clinical application, efforts were made to study purified cell wall components. The whole peptidoglycan used in this study demonstrated a tertiary structure the authors described as “bag shaped” that was composed chemically of polymerized polysaccharide and peptidoglycan active units. Interference of the tertiary structure with sonication decreased the effectiveness of the preparation. The authors speculated that the complete structure may be more effective because of increased persistence in the tissue, a screening effect protecting the peptidoglycan and polysaccharide from enzymatic degradation, or enhanced recognition by macrophages. Another paper addressing antitumor action of cell wall preparations (from L. delbrueckii bulgaricus) was published by Bogdanov et al. (1975). Microscopic degeneration of sarcoma S-180 tumor cells was noted in mice inoculated intravenously with one of three cell wall preparations. This paper reported detailed chemical characterization of the glycopeptides, but presented little information on the antitumor activity. Kohwi et al. (1978) showed the potential of two BiJidohacterium species, infantis and adolescentis, injected either subcutaneously or intraperitoneally into BALB/c mice to inhibit 3-methylcholanthrene-inducedtumors. Significant tumor regression and enhanced survival was observed in mice receiving intraregional injection of viable or killed B. infantis or B . adolescentis. The response was dependent on the number of inoculated tumor cells. This paper is similar to other reports of antitumor activity by lactic cultures. The primary limitation, related to a culture-added milk product, is that the studies deal with culture injection into mice, not bacterial ingestion by humans. Koo and Rao (1991) examined the effect of feeding bifidobacteria (isolated from the feces of CF, mice) and neosugar on 1 ,2-dimethylhydrazine dihydrochloride (DMH)-induced tumor formation in mice. Three experimental groups of mice were established: (1) 20 mice injected subcutaneously with DMH, fed a diet supplemented with neosugar, and treated twice per week with lo9 bifidobacteria/mouse 1 wk after the last injection of DMH; (2) 21 mice injected subcutaneously with DMH but no neosugar or bifidobacteria; and (3) five mice injected with ethylene diamine tetraacetic acid (EDTA) only on an unsupplemented diet. Animals were sacrificed at

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18, 28, and 38 wk after the last DMH treatment. Analysis was conducted for cecal acetic acid content, aberrant colonic crypts, and fecal levels of aerobes, anaerobes, and bifidobacteria. Results showed aberrant crypts in none of the control animals but in 100%of DMH-treated animals. Analysis of aberrant crypts per colon showed lower levels in mice fed the bifidogenic diet; statistically different ( P < 0.01) levels were reached at the 38-wk sacrifice time. Mean bifidobacteria counts increased (P < 0.05). Fecal anaerobes increased from 0.20 to 0.92/aerobe. No significant change in acetic acid concentration was observed but, since total cecal weight in the experimental mice was higher, the total amount of acetic acid was greater. The cecal pH was lower (7.33 vs. 7.75) in mice on the bifidogenic diet, but only at the 18-wk. not at the 38-wk, sampling period. Colonic aberrance was confined to the more distal end of the colon in mice fed the bifidogenic diet. Mice on the bifidogenic diet showed an increased cecal weight, presumably because of an increased amount of fluid as a result of the high osmotic activity of neosugar. The authors speculated on the mechanism of protective effect of the bifidobacteria and neosugar diet. This treatment resulted in lower intraluminal pH (note that lower pH was not observed in mice sacrificed after 38 wk) and increased amounts of the antagonistic acetic acid, presumably because of antibacterial activity against certain microbes in the intestine. Epidemiological studies have shown an association between a reduced colon cancer risk and low fecal pH in humans. pH may influence certain colonic enzymatic reactions, including dehydroxylation, conversion of primary bile acids, and dehydrogenation. However, that the moderate differences in pH detected in this study could modulate these activities effectively is not clear. This study did not separate the effect of the bifidobacteria from that of the neosugar-containing diet. However, the statistical significance of the results provide a hypothesis for the cancerinhibiting effects of bifidobacteria. Shackelford et al. (1983) studied the effect of feeding skim milk fermented with either L. delbrueckii bulgaricus or S . salivarius thermophilus ad libitum on tumor progression in rats. Rats fed water without receiving injections for tumor induction and rats fed unfermented skim milk with tumor induction were used as controls. Strains used in fermentation were not identified. Experimental diets were fed for 2 wk. For the next 20 wk, tumors were induced by weekly injection of DMH. The experimental diets were fed continually for 36 wk to all surviving rats. Differences were found in survival rates (100% for water control, 73.3% for skim milk control, 93.3% for L. delbrueckii bulgaricus fed, and 90% for S . salivarius thermophilus fed), but not in the incidence of colon tumors. Interestingly, rats fed

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the S . salivarius thermophilus milk had a significantly increased number of ear duct tumors. These results suggest that consumption of fermented milk has some effect (although the data were not analyzed statistically) on DMH-induced mortality in rats. Goldin and Gorbach (1980) studied the effect of L. acidophilus strain NCFM on the incidence of intestinal cancer in rats. A group of 77 rats was divided into five subgroups: ( 1 ) grain fed; (2) beef diet; (3) beef diet plus acidophilus supplement; (4) beef diet; and (5) beef diet plus acidophilus supplement. All animals were given the tumor-inducing agent DMH for 20 wk. Groups 1 , 2, and 3 were sacrificed after 36 wk and groups 4 and 5 after 20 wk. Rats were examined for tumors in the small intestine and colon. The only positive result obtained was a reduction in the number of animals with colon cancer after 20 wk of acidophilus feeding (40 vs 77%). N o difference was observed in small intestine carcinomas after 20 or 36 wk, or in colon cancer after 36 wk. These results do not provide strong support for an anticancer effect of this L. acidophilus strain in a mouse model. In these studies, the distinction between a specific antitumor activity and an adjuvant effect is not always made, especially when antitumor activity is attributed to enhanced functioning of the immune system. Many species of bacteria have been shown to decrease tumor activity; this occurrence is not peculiar to lactic cultures. The question of specificity of any observed activity is not resolved. Perhaps many microbes administered similarly would produce the same results. The method of testing activity is frequently intravenous, intraperitoneal, or intralesional injection of lactic cultures, not oral consumption. Therefore, the results cannot be applied to a dietary supplement. Additionally, specially bred species of mice frequently are used for these studies. The correlation of events in these animals to humans is not known. Review articles are replete with general claims of anticancer effects of lactic acid bacteria. After reviewing the literature, these conclusions seem unreliable, unfounded, and perhaps irresponsible, especially when extrapolating the results to a human system. One of the few epidemiological investigations of the effect of lactic acid bacteria on cancer in humans was done by van’t Veer et al. (1989), who examined the diets of 133 Dutch women with confirmed cases of breast cancer. They found that the women with breast cancer consumed significantly less fermented milk than women in the control group (548 women without breast cancer). The mean milk consumption k standard deviation was 116 rt 100 versus 157 k 144 g/day (P <0.01). The authors concluded that consumption of a high amount of fermented milk may have a protective effect against breast cancer, but acknowledged that this conclusion

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needed to be substantiated by further research. One difficulty in interpreting this study is that it provides only correlational, not causative, results. In addition to the correlative nature of this study, the diets of subjects were not controlled. Subjects recalled dietary consumption of 236 food items for the 12 months prior to diagnosis. This method can be criticized because of errors in recall, although this study is defended because fermented milk consumption patterns are believed frequently to be life-long patterns. Therefore, recall data for this study may be sufficient. Since breast cancer is a slowly developing cancer that originates many years prior to detection, life-long consumption patterns are essential to the conclusions of this study and must be proven experimentally. E. IMMUNE SYSTEM STIMULATION The immune system functions to fight infection from microbial pathogens or from uncontrollably growing tumor cells. In this chapter, the separation of the anticancer and immune system stimulating effects of lactic cultures is sometimes arbitrary, since the stimulation of the immune system is one mechanism of fighting cancer. This section examines reports that document a change in humoral or cell-mediated immune system function as a result of exposure to lactic cultures. The studies concerning enhancement of host immunogenicity by treatment with lactic cultures have been done almost exclusively in animal models, primarily mice. Some of the few human studies have been published in nonrefereed books and therefore do not constitute reliable sources of information (Renner, 1991). This subject was reviewed by Tannock (1991). In evaluating this research, the significant effects that the gastrointestinal system has on normal stimulation of the immune system must be considered (Hanson et al., 1983). The epithelial surface of the gut is constantly in contact with various antigenic macromolecules: food antigens, toxins, and microbes. Such mucosal contact must not lead to disease. The gastrointestinal tract generates some nonspecific defenses against infection, for example, pH control; digestive juice products such as lactoferrin, lysozyme, acids, and lactoperoxidase; the glycoprotein physical barrier of the mucous layer; and peristalsis. More specific defenses in the gut immune response also exist (see the chapter by Pestka in this volume). Half the lymphocytes of the body are located in the intestinal wall. These lymphocytes primarily produce IgA and are located in the lamina propria and in groups of lymphoid follicles making up the Peyer’s patches throughout the small intestine. The secretory IgA system is a major component of the gut immune defense system. The secretory IgA antibodies act by binding microbes and toxins, thereby preventing muco-

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sal contact. Therefore, under normal conditions, a colonized gastrointestinal system is very active in stimulating the immune system. How specific lactic cultures may supplement this condition is unknown. Yasui and Ohwaki (1991) tested the response of mouse Peyer’s patch cells in vitro to B. breve and B . breve cell walls. These investigators found that both antibody production and Peyer’s patch cell proliferation increased with B. breve treatment. Perdigon et al. (1986) elicited stimulated levels of macrophage and lymphocyte activity in mice fed unfermented milk containing L. acidophilus strain ATCC 4356 and L. casei strain CRL 431. The authors advocated the use of mixed cultures to elicit the strongest stimulation of host immune response. A similar conclusion was obtained in a study by the same research group after feeding L. casei and L . acidophilus to mice infected with a lethal dose of Salmonella typhimurium (Perdigon et al., 1990). Pretreatment of 10 mice by feeding a mixture of the two lactic bacteria resulted in 100% survival after 21 days. Treatment with either culture alone (10 mice in each group) slowed the rate of death but, after 21 days, survival matched that in the control group (20%). Liver and spleen colonization with the Salmonella pathogen was inhibited in the group fed fermented milk. However, mice fed individually fermented milks showed a more rapid colonization of the liver and spleen by Salmonella (2-3 days for the mice fed L. casei or L . acidophilus fermented milks and 7 days for the control group). When mice were fed the milk fermented with both lactic cultures (although the rate of colonization was faster by 1 day), the Salmonella were cleared completely by day 7, whereas in the control group approximately 10’ Salmonella remained per organ. Significantly higher levels of circulating antibodies were found in mice fed the mixed cultured milk than in individual or control mice. Moineau and Goulet (1991) tested the effect of feeding seven different fermented milks on macrophage activity in mice. Macrophages were collected from the lungs of mice fed milk fermented with B. longum, L. delbrueckii bulgaricus, L . casei, L . helveticus, L . lactis cremoris, L . lactis, or S . salivarius thermophilus. From each feeding group, 6 mice were sacrificed after 3, 5, and 8 days of feeding. Pulmonary alveolar macrophages were harvested and were exposed to zymosan (a yeast cell wall preparation). Phagocytic activity of macrophages from each mouse was measured by counting the percentage of macrophages that ingested three or more zymosan particles. A statistically significant increase (95% confidence level) in the levels of activated macrophages was observed in mice fed B . longum (after 3 and 5 days), L. casei (after 5 days only), and L. helveticus (after 8 days only). The greatest stimulation increase was from 38% (unfermented milk control at 5 days) to 54% (B. longum feeding after 5 days). The biological significance of this level of enhancement in macro-

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phage activity was not discussed. The authors attributed the immunostimulation to a hydrolyzed milk protein in the fermented milks, although no direct evidence was provided for this conclusion. Unfermented culture-containing milks were not run as controls, nor was any attempt made to isolate an immunostimulating peptide($ from the efficacious milks. Ueda (1986) documented specific immune response to bifidobacteria fed intragastrically to germ-free mice. IgA antibody was detected after feeding bifidobacteria. In addition, evidence of cell-mediated immunity to the bifidobacteria was obtained. Since this paper documented these findings in germ-free mice, the significance to human health is minimal. Halpern et af. (1991) reported an interesting rise in lymphocyte y interferon (IFNy) production in young human adults consuming 2 cups of yogurt daily. The purpose of this study was to determine whether high levels of yogurt consumption (450 g/day for 4 months) showed a detrimental health effect on 24 young adults (20-40 years old) as determined by extensive blood analysis. One control group ( N = 24) consumed heattreated yogurt; a second control group ( N = 20) consumed no yogurt. The experiment lasted 4 months. Analysis included blood count (platelets, white and red blood cell, hemoglobin, and differential) and blood chemistry (urea nitrogen, creatinine, sodium, potassium, chloride, C02 , anion gap, calcium, phosphorus, uric acid, ionized calcium, iron, total protein, albumin, globulin, cholesterol, and alkaline phosphatase. In addition, IFNy was determined in serum samples and in peripheral blood lymphocytes at some sampling times. No changes occurred in blood count. Blood from subjects consuming heated or unheated yogurt contained higher ionized calcium. IFNy was not detected in the serum of any subjects at any time. At the 120-day sampling period, subjects were tested for lymphocyte IFNy production also. Interferon production was markedly higher in lymphocytes obtained from subjects who consumed live active culture yogurt (20.9 IU/ml) than in those consuming heated yogurt (1.7 IU/ml) or in the control group (5.2 IU/ml). This observation suggests that yogurt consumption may be associated with an improved immune defense. Although the mechanism for this effect is unknown, binding of lactic acid bacteria to human peripheral lymphocytes may mediate the effect. Yamazaki et af. (1985) followed the development of the immune responses of germ-free mice during monoassociation with Bifidobacterium. Intragastric inoculation resulted in low levels of bifidobacteria ( 102-104cfu per organ) in the liver, kidneys, and mesenteric lymph nodes through week 2 after bifidobacteria feeding, but not after 4 weeks. Cell-mediated immunity, but not humoral, correlated well with the cessation of translocation. This paper was well written and the data well presented, but the paper does

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not contribute to an understanding of the role of bifidobacteria in human health. The intestinal mucosa provides an effective barrier against the penetration of microbes to internal organs. Although the nature of this barrier is not well understood, translocation is more common in germ-free animals. Therefore, the normal gastrointestinal flora play an important role in fighting infection. Current research has suggested that some strains of lactobacilli and bifidobacteria or their cellular components are able to stimulate macrophage production in animals. The significance of this observation is not clear. Most infectious diseases encountered by humans are contracted from the large surface areas of the mucosal membranes, which may explain the large concentration of immunocompetent cells associated with these tissues. This evidence prompted de Simone et af. (1989) to offer the hypothesis that exposure of the intestinal lining to the bacteria contained in fermented foods may induce a local immunomodulation. Unfortunately, experiments have focused on animal or tissue culture models, providing little data that can be applied directly to humans. In a review article, Tannock (1984) provided a similar speculation with respect to the normal intestinal flora as a whole. He suggested that flora antigens prime the immunologic tissues of the host so a degree of nonspecific resistance toward infection is produced, although direct evidence in humans is not cited. Any protective effect, however, can be overcome by the entry of large numbers of pathogens. The extent of immune system stimulation that results from consumption of lactic cultures by an individual with a fully colonized gastrointestinal system has not been determined. Further, Tannock (1991) stated, “Caution in the use of gram-positive bacteria or their products as nonspecific stimulators of the immunological system is advisable, since there is the potential for the development of autoimmune disease in some individuals.” F. CONSTIPATION Few studies have attempted to address the effect of lactic cultures on constipation. Graf (1983) studied 25 adult outpatients with moderately severe constipation. Patients were given either ordinary fermented milk or acidophilus fermented milk for 4 weeks each. Patients recorded abdomen and bowel behavior (cramps, flatulence, borborygmus, character of the stool). Patients also timed their bathroom stays, allowing calculation of mean time of passing stools. No statistical differences existed in any of the numerical results. This study shows no difference between acidophilus and “regular” fermented milk with respect to alleviating constipation. Unfortunately, no baseline information was provided, so no comment can

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be made on the effect of fermented milks compared with unfermented milk on constipation. Constipation affects a majority of hospitalized geriatric patients, many of whom resort to medication for relief. Alm et al. (1983) studied 42 hospitalized geriatric patients (36 women and 6 men, ages 50-95) and the effect of acidophilus milk on constipation. All patients had used several types of laxative regularly for a long time. Most had moderate to severe mobility limitations. These subjects were divided into two groups. The experimental period for Group 1 was 56 days and for Group 2, 41 days, with intermittent workout periods of 36 and 40 days, respectively. Fermented acidophilus milk was given ad libitum at breakfast during the experimental period. The experimental protocol was crossover, so patients served as their own control. The results showed that consumption of 200-300 ml acidophilus milk per day reduced the need for Laxatives in constipated, hospitalized elderly patients. The acidophilus milk preparation was more effective than a buttermilk preparation. Unfortunately, no unfermented milk controls were employed and statistical analysis of the data was not conducted. Although defecation rates were calculated, the results were difficult to interpret since different laxatives were administered as needed during the study. However, the results consistently showed that the need for laxatives was reduced. This study shows a marginal beneficial effect on constipation from consumption of fermented acidophilus milk. Seki et al. (1978) studied the effect of Bifidobacteriurn on constipation in aged people. In this study, 18 constipated seniors were given milk cultured with bifidus or lactic cultures to determine the effect on constipation. A 10-day prefeeding period was followed by 10-day lactic milk feeding period. Finally, the bifidobacteria-containing milk was fed for 10 days. Stool frequencies during these three periods were 4.8,5.7, and 7.1, respectively. Unfortunately, this experimental design does not distinguish between an effect of the bifidus milk and a postfeeding effect of the lactic milk. From these few references, assessing the effect of lactic cultures on constipation is difficult. However, a clinical study targeting this health effect would be relatively inexpensive to conduct and could be done readily in humans.

G. VAGINITIS Although vaginal inoculation is not the intent of a culture-containing fluid milk product, some evidence exists to support the importance of certain lactobacilli in maintaining vaginal health. The presence of lactoba-

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cilli in a healthy vagina is well established. Eschenbach et al. (1989) showed that hydrogen peroxide-producing lactobacilli were recovered from the vagina of 96% of 28 nonpregnant women without bacterial vaginosis, compared with only 6% of 67 women with bacterial vaginosis. The authors speculated that hydrogen peroxide production by lactobacilli was important in inhibiting vaginal pathogens. Hillier et al. (1992)conducted a cross-sectional study with 275 pregnant women to determine the role of hydrogen peroxide in the control of genital microflora. 60% of the women testing positive for lactobacilli contained hydrogen peroxide-producing lactobacilli. These women were less likely to have symptomatic candidiasis, bacterial vaginosis, or vaginal colonization by Gardnerella vaginalis, Bacteroides spp., Peptostreptococcus spp., Mycoplasma hominis, Ureaplasma urealyticum, or viridans streptococci. These studies mark the beginning of epidemiological data that support a role of specific lactobacilli in maintaining vaginal health. However, the authors are careful to note that “no studies to date have proven that instillation of exogenous lactobacilli into the vagina results in colonization or decreases the incidence of genital infection.” Klebanoff et al. (1991) show in vitro inhibition of G . vaginalis (a causative agent for bacterial vaginosis) and Bacteroides bevidus by a hydrogen peroxide-producing L . acidophilus strain, but not a hydrogen peroxide-negative strain. Unfortunately, since a hydrogen peroxide-negative mutant of the producer strain was not used, this study was not controlled for strain differences other than hydrogen peroxide production. Hughes and Hillier (1990) studied commercial preparations used to recolonize the vagina. Products tested were dairy products (yogurts and acidophilus milk), powders, tablets, and capsules. These authors evaluated an assortment of 16 of these commercial products for lactobacilli, hydrogen peroxide production, and microbial contaminants to determine their appropriateness for use in vaginal recolonization with lactobacilli. Populations in these products ranged from 10’ to lO’/g (dairy products were not enumerated). Discrepancies between claims of microbes contained in the product and those actually found were common for most of the nondairy products. Of the 16 products, 10 contained hydrogen peroxide-producing lactobacilli. Only 5 products contained L . acidophilus, although 13 products claimed it was the dominant microbe. Some contaminating microbes were isolated from 11 of 16 products. The authors concluded that this type of commercial product is unlikely to recolonize vaginal flora. This report clearly indicates the need for proper strain selection for specific applications (vaginal Lactobacillus isolates would be preferred) and improved quality control for some commercial products. Hilton et al. (1992) tested the effect of ingested yogurt on preventing candidal vaginitis. Candidal vaginitis is a common cause of gynecologic

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infection, especially in pregnant women and in women receiving antibiotic or corticosteroid therapy. This study assessed the effect of daily ingestion of L. acidophilus yogurt on vulvovaginal candidal infections in women with recurrent candidal vaginitis. Patients for the study were not on antibiotic therapy and did not regularly ingest more than 16 oz yogurt weekly before the study. Patients did not alter dietary or sexual practices during the study. Patients served as their own controls and consumed 8 oz yogurt daily for 6 months and no yogurt for 6 months. N o effort was made to make this study blind to the patients or interviewer, but all laboratory samples were blinded. Columbo plain yogurt containing lo8 L. acidophiluslml was used. Moderate hydrogen peroxide production was generated by the yogurt L. acidophilus. The study was completed by 13 women (33 began the 7 u)

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FIG. I. Candida infections and colonizations. (Top) Number of Candida infections per 6 months in individual patients. (Borrorn)Number of colonizations per 6 months in individual patients. Reprinted with permission from Hilton et. a / . (1992).

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study). The mean number of infections per 6 months was 2.5 in the control arm and 0.38 in the yogurt arm (P = 0.001). Figure 1 contrasts the number of infections and number of colonizations in patients consuming or not consuming yogurt. Because they experienced significant relief as a result of yogurt consumption, 8 additional women refused to enter the control arm of the study. These women had a chronic and prolonged history of vaginitis prior to the study. The authors acknowledged the need for a follow-up study using yogurt with killed culture as a control. This study is one of the few that provide statistically valid results with human subjects. The relationship between rectal and vaginal colonization is discussed, and suggests a mechanism for vaginal influence of consumed microbes. Klebanoff and Coombs (1991) evaluated in uitro the killing effect of a hydrogen peroxide-producing strain of L . acidophilus LB+ on human immunodeficiency virus (HIV). The peroxidase-hydrogen peroxidehalide system and hydrogen peroxide alone are viricidal to HIV. This finding suggests that people lacking hydrogen peroxide-producing lactobacilli may be at greater risk of infection by HIV.Obviously, this study must be supported by epidemiological research. IV. SAFETY ISSUES

Fermented milk products have been consumed by humans for thousands of years. Their safety as part of a varied diet is unquestioned. The addition of concentrated lactic acid bacteria that do not survive or implant in the human gastrointestinal system likewise presents no safety concerns. Our experience with consumption of lactic acid bacteria of intestinal origin, such as L. acidophilus or Bifidobacterium, is not as extensive, which has raised the issue of how addition of these bacteria will affect the gut ecosystem. Our knowledge of genetic transfer for many intestinal bacteria has progressed to the extent that transformation, conjugation, and transduction is well documented in some strains. In the laboratory, transfer of genetic material occurs among members of a species, between members of different species and genera, and even between gram-positive and gramnegative bacteria. Evidence of gene transfer in uiuo is not as widespread, but McConnell et al. (1991) clearly demonstrated the transfer of pAMP1 from Lactobacillus reuteri to E . faecalis in the digestive tract of infant mice. In this same study, transfer of pAMP1 from E. faecalis to L . fermentum in the intestinal tract of adult mice was reported. Transmissible genetic elements that encode antibiotic resistance and virulence factors are harbored by some intestinal bacteria, as are genes that mediate ad-

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herence. Clearly, the potential exists that these genes could be transferred from intestinal bacteria to the probiotic bacteria in humans, generating the potential to shift the genetic complement of lactic acid bacteria. The risk of this event increases with the microbial number and capability of colonization. However, the safety risks of this occurrence are difficult to assess. Consumption of a diversity of strains of lactobacilli and bifidobacteria of intestinal origin has taken place for decades. Metchnikoff ’s turn-of-the-century praise of the healthful attributes of a Bulgarian fermented acidophilus milk launched the concept of intestinal lactics as health-promoting inoculants in culture-containing milk products. Morinaga, Inc. has sold a bifid-containing milk product since 1974. Marschall Products has sold cultures for Sweet AcidophilusTMmilk since 1974. Hansen’s has sold Nutrish a/B (a bifidobacteria and L. acidophilus culture blend for fluid milk) since 1987. These products have been marketed and consumed with no apparent negative health impact. However, continuing to develop model systems that enable researchers to measure genetic events in supplemented gastrointestinal systems would appear to be prudent. V.

CONSIDERATIONS FOR STRAIN SELECTION

The commercial use of strains as human probiotics has begun. The challenge facing the health food and culture-containing milk industries is the development of a rational basis for the selection of strains for these applications. Unfortunately, in many commercial applications, the only bases for strain selection are genus, species, and the ability to grow under commercial fermentation conditions. Responsible selection of cultures requires that selection be based on functional criteria, however difficult they may be to define or measure. Several examples of suggested strain selection strategies or criteria are discussed in the literature (Gilliland, 1979; Klaenhammer, 1982; Tannock, 1984; Conway et al., 1987; Driessen and de Boer, 1989; Gorbach and Goldin, 1989; Gilliland and Walker, 1990; Misra and Kuila, 1991a). In addition, an international trend is developing toward the production of fermented milk products using intestinal microbes not only as dietary supplements, but to execute the fermentation of milk (Gurr, 1984). These cultures would require additional fermentative attributes, such as acceptable rate of acid production and proteolytic activity, the generation of acceptable texture in the final product, and resistance to inhibitors that may be found in the milk or during processing (phage, salt, high temperatures).

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In developing a strain selection strategy, the investigator must be mindful that relatively simple in uitro assays provide tempting strain differentiations, but may not have any physiological meaning when applied to human systems. Further, not all desired characteristics may be found in one strain. Strains that possess unique attributes may need to be blended to achieve the optimal culture. Much scientific scrutiny and marketing input will be necessary to develop a final recommendation of desirable microbial dietary adjuncts. Some attributes that have been suggested to be useful in differentiating and selecting strains for commercial use follow. 1. Ability to deconjugate bile acid. This attribute may relate to a culture's ability to reduce total cholesterol (Driessen and de Boer, 1989) or reduce azoreductase activity (McConnell and Tannock, 1991). 2. Human intestinal cell adherence in tissue culture (Klaenhammer, 1982; Gorbach and Goldin, 1989). 3. Production of high levels of lactic or other organic acids. The production of organic acids has been suggested to relate to the ability of probiotic cultures to inhibit putrefactive or potentially harmful bacteria in the intestine. The in uivo production of organic acids could lead to a decrease in intestinal pH, altering the intestinal environment. 4. Ability to grow at 37". This characteristic would be necessary in a strain selected for physiological activity and survival. 5 . Bile resistance. This trait is important for intestinal survival. 6. Desirable fermentative characteristics. This attribute is necessary if the microbe will be used in the production of a fermented product. 7. High level of lactase (not phospho-P-galactosidase). 8. Ability of culture to pass through the stomach with healthful activities intact. 9. Ability to release lactase in the intestine. 10. Demonstrated clinical efficacy against target conditions (lactose intolerance, diarrhea, etc .). 11. In uitro resistance to human gastric juice or phosphate saline at low pH (Conway et al., 1987). This characteristic would reflect the ability of the culture to survive ingestion. 12. Retention of all desirable traits through culture preparation, use, and storage of the product (Misra and Kuila, 1991b).

VI.

RESEARCH NEEDS

Conducting research in the area of health benefits of lactic cultures is a complex matter fraught with technical, economic, and philosophical dif-

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ficulties. Technical difficulties include development of model systems to answer appropriate questions. Such development is difficult because work is done either in humans (an expensive and sometimes unfeasible option) or with model systems that must be developed impeccably to have significance in humans. The economic difficulties parallel the technical concerns, since research of this type can be quite costly. The food industry is traditionally a small investor in research and development, with somewhat limited opportunities to recoup investment costs from the products of research. An additional complication is the unclear competitive advantage that this technology offers, considering the current regulatory climate, at least in the United States, that prevents labeling of food with health claims. A push to legally define a new class of foods as “nutraceuticals” has emerged (Pszczola, 1992), providing a legitimate marketing outlet for health claims on some foods. The fate of such a movement is hard to predict. The philosophical questions are no less complex or pertinent. The presence of much poorly conducted research leaves responsible investigators with a poor choice: let the poorly conducted work stand in the body of published works unchallenged or conduct work to refute the poor published conclusions, a task with little glory. As stated by Lederberg (1992) about another field, published work that is flawed by intellectual confusion and messy experimental reporting is worse than useless:

. . . It creates an aura of disreputability that repels younger investigators, and it establishes spurious theoretical paradigms. In addition, what incentive is there to clean up messy work? If it is disconfirmed, that is a social service that tends to glean little credit for the labor invested. If by some chance it should be confirmed, it will merely exalt the reputation of the first claimant, and for limited deserts. The current body of scientific research in the area of health benefits of lactic cultures has been suggested to have essentially “put the cart before the horse.” Tannock (personal communication) states that researchers have made an initial assumption that lactic acid bacteria are beneficial to humans, and have designed experiments to prove their point. Instead, he suggests that researchers ask much more fundamental and less complex questions that may lead to more legitimate conclusions: 1. Do lactic acid bacteria alter, to a statistically significant degree, the biochemistry of the intestinal ecosystem of their host? 2. What is the biological significance of any changes to the host? 3. How are the biologically significant changes, if any, produced? 4. Are any observed changes beneficial or detrimental to the host? 5. Are the effects relevant to the food industry?

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6. Can these lactic acid bacterial mechanisms be translated to commercial reality? This approach is different from the one that has guided research in this area to date. Despite the more logical sequence of the research program just outlined, an empirical approach to research in this field is likely to continue to be pursued. Certainly this approach has been used in much of the clinical work completed to date. Research needs for specific target areas have been suggested by a panel of experts convened on the subject (Sanders, 1993). These needs include the following objectives for specific health targets. I . Lactose intolerance Identify strains containing acid-resistant P-galactosidase. Conduct additional clinical studies expanding the diversity and number of the subjects studied. Address microbiological issues related to culture and P-galactosidase survival. Define culture conditions affecting levels of p-galactosidase produced by the bacteria. The dependence of the physiological state of the microbe on media, growth phase, and storage conditions must be clarified. Determine the relative importance of lactobacilli vs. S . saliuarius thermophilus for lactase delivery. 2. Diarrheal diseases Determine properly the effect of lactic cultures on diarrhea. The investment would be monumental, largely because of the great diversity of causative agents of diarrheal diseases and their pathogenic mechanisms. Clearly, lactic cultures could not be expected to limit this disease in general. However, specific targets may be able to be defined to determine efficacy, a task that would be performed best at a dedicated clinic by experts in recognizing diarrheal causes. Select strains properly prior to clinical evaluation. To screen candidate strains for potential in uiuo antipathogenic activity (e.g., ETEC) one could look for (1) adherence in the best available in uitro or animal assays and (2) in uitro production of bacteriocin-like inhibitory substances that may act in uiuo. Although these in uirro activities are not necessarily indicative of in uiuo efficacy, follow-up clinical studies would be necessary to determine usefulness of both the selected strains and the in uitro assays. Conduct deliberate infection studies with enterotoxigenic E. coli

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and determine the effect of lactic cultures on symptom limitation. Target prevention, not cure. Research that shows positive effect of cultures on decreasing the duration and/or severity of diarrhea may be indicative of positive effects on prevention, as well. The optimal prophylactic culture may be mixed, with different strains targeted toward different ailments blended into one preparation. Focus predominantly on diseases of the small bowel (because of relatively small populations of lactobacilli in large bowel) for example, rotavirus, E. coli, or traveler’s diarrhea. Further investigate pseudomembranous colitis caused by C. d$jicile infection, a large bowel disease. This disease is also difficult to study since most cases are treatable by a second antibiotic. Rarely cases are persistent, and these subjects tend to be scattered, but this problem is an important hospital problem, with some indication of successful treatment with Lactobacillus. Consider that cultures for certain applications should be resistant to therapeutic antibiotics (but sensitive to multiple others). Otherwise, the lactic culture administered during therapy would be killed. This consideration is not important in a prevention application, however, because no antibiotic therapy is being used. Perform epidemiology studies that may give an indication of the likelihood of success of this research. 3. Small bowel overgrowth Study the effect of lactic culture consumption on conditions resulting from small bowel overgrowth. Clinical models are being defined currently. Explore bowel motility and stasis further in these patients. 4. Cancer or antitumor effects Include effect on primary tumor growth as well as on metastasis in studies concerning Lactobacillus modulation of turnorigenesis. Use patients with small bowel overgrowth, for example, individuals with renal failure or achlorhydria or the elderly, as models for the effect of lactic cultures on mutagen uptake and cancerpromoting enzyme levels. Perform animal studies to gain a sense of the mechanism of action of lactic cultures on the levels of these enzymes. Repeat fecal enzyme studies to gain consensus on results.

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Confirm in uiuo mutagen binding study. Coordinate with the ongoing massive longitudinal studies that are being conducted already on thousands of people to obtain epidemiological data for potential correlation of decreased levels of fecal enzymes and cancer incidence and/or cultured dairy food consumption and cancer incidence. These studies may be done less expensively if they can be piggybacked onto existing studies. 5. Adherence Correlate in uitro adherence capability in uiuo. Define adherence factors. Perform competitive exclusion studies with adhering and nonadhering strains in in uitro model systems. These studies may not be as useful since studies to date suggest that the organism added first to these systems is the one that excludes the other. Therefore, the pathogen could just as likely exclude the lactic culture, which justifies continuous feeding to initially occupy sites and potentially decrease pathogenicity. Challenge humans with enterotoxigenic E. coli after administration of adhering L. acidophilus. Quantify symptoms and adherence. Determine if adherence or transient passage is responsible for positive effects in lactase delivery, small bowel overgrowth, levels of cancer-promoting enzymes, or effects on intestinal pat hogen population. Use recombinant DNA techniques to characterize strains. Safety should be studied. Investigate colonization of the human large intestine by biopsy with focus on diseased humans (e.g., use studies of patients with pseudomembranous colitis). 6. Immune system stimulation. Use focused animal studies prior to human studies until working hypothesis is obtained. Immune system stimulation is too broad a topic. Target specific subpopulations, such as the elderly or immunosuppressed, when advanced to human studies. 7. Cholesterol reduction Feed strains selected for in uitro cholesterol binding capabilities to ileostomy patients. Check for cholesterol levels from ileum contents to determine if cholesterol absorption is changed. Rescuing the bacteria with cholesterol bound may be possible, providing mechanistic evidence for cholesterol absorption by the lactobacilli.

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VII. CONCLUSIONS

Considerable interest has been generated in the role of lactic cultures in minimizing the proliferation of undesirable physiological states brought on by lactose maldigestion. diarrhea, high serum cholesterol, cancer, and low immune system activity. Some of the literature suggests that these microbes also may act to help alleviate symptoms of high blood pressure (Furushiro et al., 1990), liver disease (Macbeth et al., 1965; Muting er al., 1968). small bowel overgrowth (Simenhoff, personal communication), and constipation. However, the evidence for these effects is minimal. Unfortunately, a faction in the scientific and marketing world chooses to attribute health-promoting capabilities to intestinal lactic cultures in general, with little regard for strain selection issues or critical analysis of published research, especially as applied to human systems. A careful examination of the scientific literature leads to more guarded conclusions on health-promoting characteristics of lactic cultures. Responsible promotion of health-promoting bacterial inoculants requires more evidence than that some strains of lactic cultures under some conditions have been shown to have some effect. The exact strain or strain characteristics that will cause an effect must be defined. The conditions necessary for causing the effect, the level of effect, and the importance of the effect to the overall health of the human must be known. For most applications, this information is not available. Additional research must be done to explore some basic questions on mechanisms of effects. Empirical research should use the best strains available on targets that have been indicated by all preceding research to be the most likely to be successful and on targets that are consistent with the marketing objectives for the product. REFERENCES Ahn. C. and Stiles, M. E. (1990). Plasmid-associated bacteriocin production by a strain of Carnobacrerium piscicola from meat. Appl. Environ. Microbiol. 56, 2503-25 10. Ahrens. E. H.,Jr. (1985). The diet-heart question in 1985: Has it really been settled? Lancer ii, 1085-1087. Alm, L. (1991). The therapeutic effects of various cultures-An overview. In “Therapeutic Properties of Fermented Milks” (R. K. Robinson. ed.). pp. 45-64. Elsevier Applied Science, New York. A h . L., Humble, D., Ryd-Kjellen, E.. and Setterberg, G. (1983). The effect of acidophilus milk in the treatment of constipation in hospitalized geriatric patients. I n “Nutrition and the Intestinal Flora” (Bo Hallgren. ed.), pp. 131-138. Symposia of the Swedish Nutrition Foundation XV. Almqvist & Wiksell International. Stockholm.

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Poupard, J. A., Husain, I., and Norris, R. F. (1973). Biology of the bifidobacteria. Bacreriol. Rev. 37, 136-165. Pozo-Olano, J. D., Warram, J. G., Jr., Gomez, R. G., and Cavazos, M. G. (1978). Effect of a lactobacilli preparation on traveler’s diarrhea: A randomized, double blind clinical trial. Gastroenterol. 74,829-830. Pszczola, D. E. (1992). The nutraceutical initiative: A proposal for economic and regulatory reform. Food Technol. 46,77-79. Rasic, J. L. (1983). The role of dairy foods containing bifido- and acidophilus bacteria in nutrition and health? N. Europ. Dairy J . 4,2-10. Rasic, J. L., and Kurmann, J. A. (1983). “Bifidobacteria and Their Role. Microbiological, Nutritional-Physiological, Medical, and Technological Aspects and Bibliography.” Birkhauser Verlag, Basel. Rasic, J. L., Vujicic, I. R., Skrinjar, M., and Vulic, M. (1992). Assimilation of cholesterol by some cultures of lactic acid bacteria and bifidobacteria. Biotechnol. Bull. 14,39-44. Redondo-Lopez, V., Cook, R. L., and Sobel, J. D.(1990). Emerging role of lactobacilli in the control and maintenance of the vaginal bacterial microflora. Rev. Infect. Diseases l2, 856-872. Renner, E. (1991). Cultured dairy products in human nutrition. Int. Dairy Fed. Bull. 255, 2-24. Reuter, G. (1989). BiJidobacterium characteristics and classification. In “Les Laits Fermentes. Actualite de la Recherche,” pp. 49-58. John Libbey Eurotext, Montrouge, France. Richardson, T. (1978). The hypocholesteremic effect of milk-A review. J. Food Protect. 41, 226-235. Rosado, J. D., Lindsay, H. A., and Solomons, N. W. (1987). Milk consumption, symptom response, and lactose digestion in milk intolerance. Am. J . Clin. Nutr. 45, 1457-1460. Rossouw, J. E., Burger, E.-M., van der Vyver, P., and Ferreira, .I. J. (1981). The effect of skim milk, yogurt, and full cream milk on human serum lipids. Am. J. Clin.Nutr. 34, 351-356. Rowland, I. R., and Grasso, P. (1975). Degradation of N-nitrosamines by intestinal bacteria. Appl. Microbiol. 29,7-12. Roy, D., Blanchette, L., Savoie, L., and Ward, P. (1992). a-and p-galactosidase properties of Bifidobacterium infantis. Milchwissenschafr 47, 18-21. Salminen, E., Elomaa, I., Minkkinen, J., Vapaatalo, H., and Salminen, S. (1988). Preservation of intestinal integrity during radiotherapy using live Lactobacillus acidophilus cultures. Clin.Radiol. 39,435-437. Sanders, M. E. (1993). Summary of conclusions from consensus panel of experts on health attributes of lactic cultures: significance to fluid milk products containing cultures. J. Dairy Sci. In Press. Sandine, W. E. (1979). Roles of Lactobacillus in the intestinal tract. J. Food Protect. 42, 259-262. Sandine, W. E. (1990). Roles of bifidobacteria and lactobacilli in human health. Contemp. Nutr. 15(1). Sandine, W. E., Muralidhara, K. S., Elliker, P. R., and England, D. C. (1972). Lactic acid bacteria in food and health: A review with special reference to enteropathogenic Escherichia coli as well as certain enteric diseases and their treatment with antibiotics and lactobacilli. J . Milk Food Technol. 35,691-702. Sato, J., Mochizuki, K., and Homma, N. (1982). Affinity of the BiJidobacterium to intestinal mucosal epithelial cells. BiJidobacteria Microflora 1 , 5 1-54. Savage, D. C. (1977). Microbial ecology of the gastrointestinal tract. Ann. Rev. Microbiol. 3, 107- 133.

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Savage, D. C. (1983a). Associations of indigenous microorganisms with gastrointestinal epithelial surfaces. I n “Human Intestinal Microflora in Health and Disease” (D. J. Hentges, ed.), pp. 55-78. Academic Press, Orlando, Florida. Savage, D. C. (1983b). Morphological diversity among members of the gastrointestinal microflora. International Rev. Cytol. 82,305-334. Savaiano, D. A., and Levitt, M. D. (1987). Milk intolerance and microbe-containing &iry foods. J. Dairy Sci. 70,397-406. Savaiano, D. A., and Kotz, C. (1988). Recent advances in the management of lactose intolerance. Contemp. Nutr. 13(10). Scrimshaw, N. S., and Murray, E. B. (1988). The acceptability of milk and milk products in populations with a high prevalence of lactose intolerance. Am. J . Clin. Nutr. (Suppl.)48, 1083-1 159. Seki, M., Igarashi, M., Fukuda, Y., Simamura, S., Kawashima, T., and Ogasa, K. (1978). The effect of Bifidobacterium cultured milk on the “regularity” among on (sic) aged group. Nutr. Foodst. 31,379-387. (in Japanese) Sekine, K., Toida, T., Saito, M., Kuboyama, M., Kawashima,T., and Hashimoto, Y. (1985). A new morphologically characterized cell wall preparation (whole peptidoglycan) from Bifidobacterium infanfis with a higher efficacy on the regression of an established tumor in mice. Cancer Res. 45, 1300-1307. Sellars, R. L. (1989). Health properties of yogurt. I n “Yogurt: Nutritional and Health Properties” (R.C. Chandan, ed.), pp. 115-144. National Yogurt Association, McLean, Virginia. Sellars, R. L. (1991). Acidophilus products. In “Therapeutic Properties of Fermented Milks” (R. K. Robinson, ed.), pp. 81-116. Elsevier Applied Science, New York. Shackelford, L. A., Rao, D. R., Chawan, C. B., and Pulusani, S. R. (1983). Effect of feeding fermented milk on the incidence of chemically induced colon tumors in rats. Nutr. Cancer 5 , 159-164. Shahani, K. M., and Ayebo, A. D. (1980). Role of dietary lactobacilli in gastrointestinal microecology. Am. J. Clin. Nutr. 33,2448-2457. Shahani, K. M., and Chandan, R. C. (1979). Nutritional and healthful aspects ofcultured and culture-containing dairy foods. J . Dairy Sci. 62, 1685-1694. Siitonen, S., Vapaatalo, H., Salminen, S., Gordin, A., Saxelin, M., Wikberg, R., and Kirkkola, A.-L. (1990). Effect of Lactobacillus GG yogurt in prevention of antibiotic associated diarrhoea. Ann. Med. (Helsinki) 22,57-59. Silva, M., Jacobus, N. V., Deneke, C., and Gorbach, S. L. (1987). Antimicrobial substance from a human Lactobacillus strain. Antimicrob. Agents Chemother. 31, 123 I1233. Spelhaug, S. R., and Harlander, S. K. (1989). Inhibition of foodborne bacterial pathogens by bacteriocins from Lactococcus lactis and Pediococcus pentosaceous. J . Food Protect. 52,856-862. Stark, P. L., and Lee, A. (1982). The microbial ecology of the large bowel of breast-fed and formula-fed infants during the first year of life. J . Med. Microbiol. 15, 189-203. Tannock, G. W. (1983). Effect of dietary and environmental stress on the gastrointestinal microbiota. I n “Human Intestinal Microflora in Health and Disease” (D. J. Hentges, ed.), pp. 517-539. Academic Press, New York. Tannock, G. W . (1984). Control of gastrointestinal pathogens by normal flora. I n “Current Perspectives in Microbial Ecology” (M. J. Klug and C. A. Reddy, eds.), pp. 374-382. American Society of Microbiologists, Washington, D.C. Tannock, G. W. (1990). The microecology of lactobacilli inhabiting the gastrointestinal tract. Adu. Microb. Ecol. 11, 147-171.

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Tannock, G. W. (1991). The normal microflora of the gastrointestinal tract and non-specific resistance to disease. Proc. Nurr. SOC.New Zealand 16, 123-132. Tannock. G. W., Szylit. 0.. Duval, Y., and Raibaud, P. (1982). Colonization of tissue surfaces in the gastrointestinal tract of gnotobiotic animals by Lactobacillus strains. Can. J . Microbiol. 28, 1196-1 198. Thompson, L. U . , Jenkins, D. J. A.. Amer, M. A. V.. Reichert, R., Jenkins, A., and Kamulsky, J. (1982). The effect of fermented and unfermented milks on serum cholesterol. Am. J. Clin. Nufr. 36, 1106-1 1 1 I . Tomoda, T., Nakano, Y., and Kageyama. T. (1988). Intestinal Candida overgrowth and Candida infection in patients with leukemia: Effect of BiJdobarrerium administration. BiJidobacreria Microflora 7,71-74. Truslow, W. W. (1986). Lactobacillus and fermented foods. J. Holisric Med. 8, 36-46. Ueda. K. ( 1986).Immunity provided by colonized enteric bacteria. BiJdobacreria Microflora 5,67-72. Van? Veer. P.. Dekker, J. M., Lamers, J. W. J., Kok, F. J., Schouten, E. G., Brants, H. A. M., Sturmans, F.. and Hermus, R. J. J. (1989). Consumption of fermented milk products and breast cancer: A case-control study in the Netherlands. Cancer Res. 49, 4020-4023. Welsh, J. D.. Payne. D. L., Manion, C.. Morrison, R. D., and Nichols, M. A. (1981). Interval sampling of breath hydrogen as an index of lactose malabsorption in lactase-deficient subjects. Digestive Dis. Sci. 26, 681-685. Wesney, E., and Tannock, G. W. (1979). Association of rat, pig, and fowl biotypes of lactobacilli with the stomach of gnotobiotic mice. Microb. Ecol. 5 , 35-42. Yamazaki. S ., Machii, K.. Tsuyuki, S.,Momose, H.. Kawashima, T., and Ueda, K. (1985). Immunological responses to monoassociated BiJdobacrerium longurn and their relation to prevention of bacterial invasion. immunology 56,43-50. Yasui. H.. and Ohwaki, M. (1991). Enhancement of immune response in Peyer's patch cells cultured with BiJidobacrerium breve. J . Dairy Sci. 74, 1187-1 195. Zhang, X. B., and Ohta, Y. (1991a). In uirro binding of mutagenic pyrolyzates to lactic acid bacterial cells in human gastric juice. J. Dairy Sci. 74,752-757. Zhang. X. B.. and Ohta. Y. (1991b). Binding of mutagens by fractions ofthe cell wall skeleton of lactic acid bacteria on mutagens. J. Dairy Sci. 74, 1477-1481. Zhang, X. B.. Ohta. Y., and Hosono, A. (1990). Antimutagenicity and binding of lactic acid bacteria from a Chinese cheese to mutagenic pyrolyzates. J. Dairy Sci. 73,2702-2710.

ADVANCES IN FOOD A N D NUTRITION RESEARCH. VOL. 37

DEFINING THE ROLE OF MILKFAT IN BALANCED DIETS LOUISE A. BERNER Nutrition Consrtltunt San Luis Obispo, Culiforniu 93401

I. Introduction 11. Background Information-Dietary Fat Consumption and Composition A. Trends in Fat and Milkfat Consumption B. Milkfat Composition and Triglyceride Structure C. Fat and Cholesterol Content of Commonly Consumed Dairy Foods 111. Dietary Fat, Dairy Products, and Coronary Heart Disease A. Effects of Dietary Fat Type and Source on Serum Lipoproteins and Apolipoproteins B. Effects of Dietary Fat Type on LDL Modification C. Effects of Dietary Fat Type on Thrombosis Tendency D. Other Potential Determinants of Response to Dietary Fat E. Influence of Milk and Other Dairy Products on Serum Lipids and Risk of Coronary Heart Disease F. Summary IV. Dietary Fat, Dairy Products, and Cancer Risk A. Population Studies B. Animal Studies C. Summary V. Milkfat as Part of the Total Diet A. Assessment of Overall Impact on Health B. Fitting Dairy Foods into Diets That Meet Current Fat Recommendations C. Summary VI. Conclusions and Research Needs References

I.

INTRODUCTION

The literature on the influence of dietary fats on the risk of chronic diseases is large and rapidly expanding. Over the last several decades, by far the most attention has been focused on the effects of different dietary 131 Copyright 0 1993 by Academic Press. Inc. All rights of reproduction in any form reserved.

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LOUISE A. BERNER

fats on serum lipoprotein levels (often with a single fat source providing all or nearly all fat intake). The general conclusion has been that serum lipids, especially total and low-density lipoprotein (LDL) cholesterol, are elevated by diets rich in total fat and saturated fatty acids (SFAs). In more recent years, researchers have begun to address more complicated questions: How do individual fatty acids influence risk of coronary heart disease (CHD)? How do dietary fats and fatty acids impact aspects of lipid metabolism and atherogenesis other than blood lipoprotein levels (e.g., apolipoprotein metabolism, LDL modification, thrombosis)? How do dietary fats impact carcinogenesis, including metastasis of tumors? How do different dietary components (including fatty acids) interact to influence lipid metabolism and disease risk? Do genetic markers of disease risk and diet sensitivity exist that will permit recommendations to be targeted to individuals rather than-or in addition to-to the population as a whole? The understanding of dietary fat-disease relationships is incomplete, so dietary guidelines can be expected to evolve as researchers find answers to these questions. Nevertheless, government and health professional groups have been increasingly vocal and consistent over the last 15 years in recommending that United States citizens curtail intakes of fat and cholesterol to decrease risk of CHD, certain cancers, and perhaps other chronic diseases. In 1977, a United States Senate committee issued “Dietary Goals for the United States,” which included a recommendation for Americans to reduce total fat, SFA, and cholesterol intakes (US.Senate, 1977). Several years later, the Surgeon General gave similar advice regarding fat and cholesterol consumption (Surgeon General, 1988). The Food and Nutrition Board’s Committee on Diet and Health [National Research Council (NRC), 19891, the Dietary Guidelines committee (U.S. Department of Agriculture and U.S. Department of Health and Human Services, 1990), and the National Cholesterol Education Program Expert Panel on Population Strategies (Carleton et al., 1991) reiterate the same basic advice and include the now-common dietary fat and cholesterol guidelines: no more than 30% of energy (en%) from fat, no more than 10 en% from SFAs, and no more than 300 mg cholesterol daily. The dietary fat guidelines must be translated into food choices to be practical for consumers. Because many dairy foods are perceived as high in fat, and because milkfat is rich in SFAs, standard fat (i.e., not lowfat or fat-modified) dairy foods are targeted by many nutritionists for elimination from diets in an attempt to meet the guidelines just stated. For example, the Committee on Diet and Health recommends substituting whole milk dairy products with low- or nonfat dairy foods. However, practical questions remain. Can standard fat dairy foods be part of

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healthful diets? Do dairy foods have the impact on health predicted by their fat content and composition? Is obtaining fat from a variety of dietary sources important? The purpose of this chapter is examining the evidence relating milkfat and dairy food consumption to risk of chronic diseases, especially CHD and cancer. An effort is made to provide a perspective on the health implications of milkfat in balanced varied diets. The following areas are addressed: composition and consumption of dietary fats; effects of dietary fats on CHD risk (with emphasis on milkfat); effects of dietary fats (especially milkfat) on cancer risk; and overall impact of milkfat in a mixed-fat diet.

II. BACKGROUND INFORMATION-DIETARY FAT CONSUMPTION AND COMPOSITION

Trends in the consumption of dietary fats must be discussed before the nutritional implications of milkfat and other fats can be evaluated. In addition, to compare and understand the effects of various food fats on lipid metabolism and health, reviewing their fatty acid composition and triglyceride structure is necessary.

A. TRENDS IN FAT AND MILKFAT CONSUMPTION

Two general lines of evidence support food and nutrient consumption trends. Disappearance data estimate foods and nutrients available in the food supply, whereas intake data are derived directly from surveys of individual food consumption (usually 24-hr recalls or several-day dietary records). Both types of information are discussed in this section. I . Disappearance Data The Human Nutrition Information Service and Economic Research Service of the U.S.Department of Agriculture (USDA) compile food and nutrient disappearance data annually. Although the data often are referred to as food consumption data, they are actually estimates derived by subtracting exports, industrial uses, year-end inventories, and farm seed/feed uses from data on food production, beginning inventories, and imports (Putnam, 1990). The disappearance figures generally overestimate actual consumption because they do not account for spoilage and waste. Still, the numbers are often useful for showing trends in food and nutrient intake over time and for comparing nutrient contributions of various food groups.

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LOUISE A. BERNER

The nutrient contributions of dairy foods (excluding butter) and fats and oils (including butter) to the United States food supply in 1968 and 1988 (the year for which the most recent published data are available) are shown in Table I. Dairy products contributed about 12% of the fat in the United States food supply, whereas fats and oils contributed 47% and meat, poultry, and fish contributed 32% (Fig. 1). As mentioned, butter is included in the fats and oils group, but represents only a small and declining proportion of that group. The availability of butter in the food supply was 5.7 lbs (2.6 kg) per person in 1966-1968 and 4.6 Ibs (2.1 kg) TABLE I NUTRIENTS AVAILABLE FOR CONSUMPTION IN UNITED STATES FOOD SUPPLY,

1968 AND 198P Percentage

Dairy foods (excluding butter)

Fats/oils (including butter)

Nutrient

1968

1988

1968

Food energy Protein Total fat Saturated fat Monounsaturated fat Polyunsaturatedfat Cholesterol Carbohydrate Vitamin A Carotenes Vitamin E Vitamin C Thiamin Riboflavin Niacin (preformed) Vitamin B6 Folate Vitamin BI2 Calcium Phosphorus Magnesium Iron Zinc Copper

10 20 12 19 9 3 14 6 17 3 4 4 9 35 2 10 9 17 76 34 20 2 17

10 20 12 20 8 2 15

Data from Raper (1991).

4

5 16 2 3

3 8 33 2 10 8 18 75 34 19 2 19 4

17

1988 20

-

-

40 31 42 58 6 -

41 32 48 69 5

-

14

11

4 60

2 67

-

-

-

ROLE OF MILKFAT IN BALANCED DIETS

135

Fats and oils 47% ( I d . b u m . tallow, lard)

gs 2%

umes and nuts 4%

airy (exd. butter) 12%o FIG. 1. Fat in U.S. food supply. Data from Raper (1991).

per person in 1986-1988. At the same time, salad oils, cooking oils, and vegetable shortenings were available in the food supply at 29.1 Ibs (13.2 kg) per person in 1966-1968 and 46.6 Ibs (21.2 kg) per person in 1986-1988 (Putnam, 1990). Waste of fats and oils, particularly frying fats in restaurants, is considerable. In fact, about 10% of the 1988 disappearance of fats and oils is estimated to be such waste, so the availability of frying fats probably is overestimated more than that of other fat sources. Nevertheless, in 1988 butter constituted only 4.6 Ibs (2.1 kg) per person of a total fat and oil availability of 62.7 Ibs (28.5 kg) (Putnam, 1990), a figure that translates to about 8% of total fats and oils when the total fats and oils category is adjusted for estimated waste of frying fats and oils. Since fats and oils as a group contributed 47% of total fat in the United States food supply, we can assume that butter contributed about 3.8% of total fat (i.e., 8% of 47%). Thus, all dairy foods combined (including butter) contributed approximately 15.8% of the fat available for consumption in the United States. This contribution is small in comparison with the contributions of fat and oils and meat, fish, and poultry. Dairy products (excluding butter) also contribute about 20% of the SFAs in the food supply; meat, fish, and poultry (excluding animal fats such as tallow and lard) supply 40% and fats and oils supply 32%. In 1988, cholesterol in the food supply was provided by dairy products (15%), eggs (33%), and meat, fish, and poultry (47%) (Raper, 1991). Although dairy products (excluding butter) supply 10% of the energy and 12% of the fat in our foods, they also provide generous amounts of numerous essential nutrients. As shown in Table I, dairy foods consistently have supplied these levels of essential nutrients: 75-76% calcium, 16-17% vitamin A, 33-35% riboflavin, 20% protein, 10% vitamin Bg, 17-18% vitamin B I 2 ,34% phosphorus, 19-20% magnesium, and 17-19% zinc (Raper, 1991). Moreover, dairy products are good sources of several nutrients such as vitamin D, niacin equivalents, and potassium that are

136

LOUISE A. BERNER

not accounted for by the USDA data. On the other hand, fats and oils as a group provide few nutrients other than energy, fat, and vitamin E (Table I). Thus, the consumer who avoids dairy foods to decrease fat or cholesterol intake pays a high cost in nutrient trade-offs, which does not occur with fats and oils. The consumption of vegetable shortenings and oils has grown steadily. Animal fat contributed 17% of the total visible fat in the food supply in 1988, a reduction from 28% in 1968 (Putnam, 1990). Nevertheless, although the proportion of fat from animal products has decreased dramatically, all animal sources still contributed more fat than all vegetable sources (53% vs 47%) in 1988 (Raper, 1991). USDA disappearance data also indicate trends in the per capita “use” of different dairy products. Use of whole milk declined steadily over the last two decades, from 27.5 gallons (104.5 liters) per person in 1971 to 12.9 gallons (49.0 liters) in 1986-1988. Low-fat milk, skim milk, and yogurt consumption increased during that same period from 3.1 to 11.6 gallons (1 1.8 to 44.1 liters), 1.3 to 1.7 gallons (4.9 to 6.5 liters), and 0.5 to 4.5 lbs (0.23 to 2.0 kg) per person, respectively. At the same time that consumers were showing preference for lower-fat fluid milks, cheese use rose from 10.1 to 23.5 lbs (4.6 to 10.7 kg) per person and cream use rose from 5.8 to 7.1 lbs (2.6 to 3.2 kg) per person (Putnam, 1990). This information helps explain why the contribution of dairy products to total dietary fat has remained constant over the past two decades although whole milk use declined dramatically. 2. Intake Data a . Fat Consumption. Stephen and Wald (1990) compiled and analyzed data from 171 studies in which dietary fat intake was determined; all studies were conducted in the 1920s or later in the United States. The analysis showed that fat contribution to energy intake was 40.5% in 1950-1959,39.9% in 1960-1969,37.8% in 1970-1979, and 37.5% in 19801985. [Note that disappearance data show considerably higher and less variable percentages of energy from fat, e.g., 43% in 1968 and 42% in 1988 (Raper, 1991).] At the same time that fat contribution to energy was decreasing, the mix of fatty acids changed quite dramatically. During the same four time periods, the percentage of energy from SFAs fell (16.6%, 15.8%, 13.8%, and 11.8%), as did the percentage of energy from monounsaturated fatty acids (MUFAs) (20.8%, 16.4%, 14.0%, and 12.4%). Energy from polyunsaturated fatty acids (PUFAs) increased (4.3%, 3.7%, 5.1%, 5.4%). The number of subjects studied in the most recent years examined, 1980-1985, was much smaller than in the three

ROLE OF MILKFAT IN BALANCED DIETS

137

preceding decades. Therefore, the figures for 1980-1985 may not reflect accurately the fat intake of the United States population. Nevertheless, the numbers do provide convincing evidence that fat intake as a proportion of energy has decreased over the past few decades, and that the proportion of SFAs and MUFAs has decreased while that of PUFAs has increased. The authors concluded that “if the trends shown in this study . . . continue in the same manner ,into the late 1980s and 1990s, a fat intake of 30-35% energy is likely for 1990” (Stephen and Wald, 1990). As mentioned, however, their conclusion is based in part on figures from 1980-1985, a time period in which relatively few subjects were studied. A survey of nationally representative samples of 2000 households has been conducted annually since 1957 by MRCA Information Services. In 1986-1988, data from over 10,000 individuals indicated a mean of 36 en% from fat (Lyle et al., 1992). Several government surveys (some included in the analysis by Stephen and Wald) also shed light on actual fat and fatty acid intake in the United States population. The 1987-1988 Nationwide Food Consumption Survey indicated that respondents over age 15 consumed an average of 37-38 en% as fat, 13-14 en% as SFAs, 14 en% as MUFAs, and 6.3 en% as PUFAs (Ganji et al., 1992). Data from the 1985 Nationwide Food Consumption Survey -Continuing Survey of Food Intakes I1 (CSFII) showed that women ages 19-50 consumed an average of 36.8 en% from fat, and 13.3 en%, 13.6 en%, and 7.4 en% from SFAs, MUFAs, and PUFAs, respectively. From the same survey, children consumed an average of almost 35 en% as fat, and 13.7 en% as SFAs, 12.7 en% as MUFAs, and 5.9 en% as PUFAs (NRC, 1989). Block and Subar (1992) reported nutrient intake data from the 1987 National Health Interview Survey, which included a food-frequency questionnaire given to over 20,000 adults in the United States. The authors pointed out that the data are particularly useful for looking at true distributions of nutrient intakes in the population since the questionnaire focused on usual intakes. [On the other hand, 24-hr recalls tend to overestimate the true distributions of usual nutrient intakes because “on any given day some individuals will eat very little food, for example, whereas others will eat an unusually large amount” (Block and Subar, 1992).] Mean intakes and ranges of intake of fat, SFAs, oleic acid, and linoleic acid for adult men and women interviewed in the 1987 survey are listed in Table 11. Overall, data indicate a trend toward decreasing percentage of energy from fat over the past few decades. Another important piece of information is that energy intakes have declined also (NRC, 1989; Stephen and Wald, 1990). Thus, the absolute intake of fat (g/day) must be declining at an even faster rate than total energy intake. Note that this observation is

TABLE I1 MEAN AND RANGES OF FAT AND F A l T Y ACID INTAKES FOR MEN A N D WOMEN, FROM THE

1987 NATIONAL HEALTH INTERVIEW

SURVEY^"^ Women

Men Percentile Nutrient and age group Fat (g) 18-34 35-49 50-64 65-79 80+ Fat (en%) 18-34 35-49 50-64 65-79 80+

Percentile

Mean

10th

25th

50th

75th

90th

Mean

10th

25th

50th

75th

90th

I16 104 90 76 74

61 54 46 40 41

81 71 61 52 51

108 97 71 71

147 129 I14 93 88

183 163 I45 120 114

68 65 58 51 50

37 35 32 28 29

48 46 40 36 36

63 61 54 48 46

82 79 71 62 62

104 I03 92 79 76

37 38 37 37 37

28 29 28 26 29

33 33 32 32 33

37 38 37 37 38

41 43 42 42 41

44 46 46 46 45

36 38 37 35 34

28 29 27 26 25

33 33 32 30 30

37 38 37 35 34

41 42 41 40 39

44 46

84

46

44 43

Saturated fat (g) 18-34 35-49 50-64 65-79 80+ Oleic acid (g) 18-34 35-49 50-64 65-79 80+ Linoleic acid (9) 18-34 35-49 50-64 65-79 80+

43. 38 33 27 26

22 19 16 13 14

29 25 22 17 18

40 35 30 25 24

53 48 42 34 32

68 60 54 45 40

24 23 20 17 17

12

43 38 34 29 28

22 20 17 14 IS

30 26 23 19 20

40 36 31 26 27

54 48 43 35 34

69 62 55 46 43

24 24 21 19 19

20

9 7 6 5 5

12 10 9 7 7

18 14 12 I1

25 20 17 14 13

33 26 23 19 18

13 11 10 8 8

15

13 12 11

Data from Block and Subar (1992). All values rounded to the nearest whole number.

10

8 9

17 16 13 12 12

22 21 18 16 16

30 28 25 21 22

38 37 33 28 27

13 13

17 17

11 10

I5

23 22 19 17 17

30 29 26 23 23

38 38 34 30 30

11 10

16 14 12 10

21 18 16 14 13

11 10

II

13 13

6 5 4 4 4

8 7 6 6 5

9 8 7

10

140

LOUISE A. BERNER

at odds with the disappearance data, which show that more fat was available per person in 1988 than in 1968 (Raper, 1991). The MRCA database (which includes all household members) shows an average daily energy intake of 1768 kcal (7.4 MJ) for 1986-1988 (Lyle et al., 1992). Stephen and Wald (1990) determined an average energy intake of only 1571 kcal (6.6 MJ) for women ages 18-65 and 2476 kcal (10.4 MJ) for men ages 18-65 in their analysis of surveys conducted from 1970-1979. In 19501959, the numbers for women and men in the same age group were considerably higher-1857 kcal (7.8 MJ) and 2857 kcal (11.9 MJ), respectively (Stephen and Wald, 1990). The consequences of an ever-declining energy intake deserve consideration. Current recommendations are to limit fat intake to 30 en% or less and SFAs and PUFAs to less than 10 en% each (NRC, 1989). The trends in fat and fatty acid intake discussed here suggest that consumers have attempted to follow such guidelines. However, if energy intakes decrease further, trying to make the foods Americans like a part of recommended diets will be increasingly difficult. More important is that meeting recommended levels of essential nutrients (such as calcium and zinc) by diet alone may be increasingly difficult. A reasonable alternative approach to meeting current dietary fat recommendations would be keeping fat intakes at current levels and increasing total energy intake with low-fat and nonfat foods (fruits, vegetables, grains, nonfat dairy foods). Of course, this approach requires a simultaneous increase in energy expenditure. Unfortunately, the primary message to consumers currently is that they are choosing too many fatty and high-calorie foods, rather than that their activity levels are too low. A shift in emphasis may be prudent, especially for women and adolescent girls whose energy intakes already are low. b . Dairy Product Contributions to Fat and Other Nutrients. Several investigators have examined the contributions of various foods or food groups to nutrient intakes. Using National Health and Nutrition Examination Survey I1 (NHANES 11) data (1976-1980) for adults ages 19-74, Block et al. (1985a) determined that whole and low-fat milk beverages, cheeses, butter, and frozen desserts contributed approximately 16% of total fat intake, 26% of SFA intake, and 13% of cholesterol intake. Meats were the largest contributors to total fat and SFA intakes, and ground beef was the largest single contributor (Block et al., 1985a). Contributions of selected items are shown in Table 111. One shortcoming of the data is that food items used as ingredients in food mixtures (e.g., cheese or meat in pizza; oil in fried chicken) were not accounted for as individual food items. Thus, knowing the total contribution of individual foods to nutrient intakes is difficult.

ROLE OF MILKFAT IN BALANCED DIETS

141

TABLE 111 CONTRIBUTIONS OF SELECTED DAIRY PRODUCTS, MEATS, A N D EGGS TO TOTAL FAT, SATURATED FATTY ACIDS, A N D CHOLESTEROL IN DIETS OF ADULTS SURVEYED IN NHANES I I (1976-1980)"

(%)

Saturated fatty acids (%)

Cholesterol

Product

Total fat Whole milk, whole milk beverages Cheeses, excluding cottage Butter Ice cream, frozen desserts 2% Fat milk Hamburgers, cheeseburgers, meat loaf Hot dogs, ham, lunch meats Beef steaks, roasts Pork, including chops, roast Bacon and sausage Chicken or turkey, including fried chicken Eggs

5.98

9.11

5.41

4.54 2.39 2.05 I .37 7.02

7.28 3.67 3.11 2.48 9.31

3.05 1.70 1.71 0.79 7.28

6.39 5.45 3.97 2.64 2.09

7.04 7.28 3.99 2.65 1.80

4.29 8.69 3.61 1.10 4.30

4.63

4.52

35.88

(%)

Data from Block er a/. (1985a).

Krebs-Smith el al. (1990, 1992) analyzed data from the CSFII 1985 survey to find out how different food groups and individual foods contributed to women's intakes of energy, macronutrients, cholesterol, and fiber. Contributions of four major food groups to energy, fat, and cholesterol are shown in Table IV. The four groups were chosen because,

TABLE IV CONTRIBUTION OF FOOD GROUPS TO INTAKES OF ENERGY, FAT, SATURATED FAT, AND CHOLESTEROL BY WOMEN AGED 19-50, ~~~~~

~

198Pb

~

Food group

Energy (%)

Total fat (%)

Saturated fat (%)

Cholesterol (%I

Milk products Meat, fish, poultry Eggs Fatsloils

14 18 2 12

19 26 4 30

33 21 3 21

15 36 42 4

~~~~~~~~

~

Adapted with permission from Krebs-Smith ef a / . (1990). The figures for each food group include items used as ingredients in food mixtures.

142

LOUISE A. BERNER

collectively, they provide most of the fat and cholesterol in our diets. Note that the values for each food group include items that commonly are found in food mixtures. For example, the cheese from pizza was included in the milk products group. Clearly, milk products made only a small contribution to cholesterol intakes of the women. The contribution of this food group to total fat intake was exceeded by both the meat and the fats and oils groups (keep in mind, of course, that the fats and oils group supplies few nutrients other than fat). The single food item making the largest contribution (9.2%) to fat intake was salad dressings; margarine was next at 7.9% (Krebs-Smith et al., 1992). However, milk products actually contributed a higher proportion of SFAs than any other food grouping (Krebs-Smith et al., 1990). Cheese was the single food item contributing the highest proportion of SFAs to the women's diets (13.4%), although ground beef and other beef cuts combined contributed the same percentage (Krebs-Smith et al., 1992). Among other milk products, whole milk beverages contributed 7.8%; butter, 5.1%; frozen desserts, 3.9%; cream, sour cream, and cream cheese, 3.4%; and low-fat milk (2% fat milk), 3.4% of total SFA intake. In a group of 200 adolescents consuming 34 en% as fat and 12.5 en% as SFAs, dairy foods also supplied the major portion (about 35%) of SFA intake (Witschi et al., 1990). In one study of fifth-twelfth graders in Texas, participants were divided according to dietary fat intake. In a group with >38 en% as fat and almost 16 en% as SFAs, beef was the single food category providing the highest proportion of SFAs. However, in a group eating less than 30 en% as fat and less than 10 en% as SFAs, pizza contributed the highest proportion of SFAs (McPherson et al., 1990). Much or most of the SFAs in the pizza were likely to be from cheese. Dairy foods may provide a larger proportion of total and saturated fat in low-fat diets than in higher fat diets. The estimates of dairy product contribution to SFAs by Krebs-Smith et al. (1990, 1992) and Witschi et al. (1990) exceed those made by Block et al. (1985a). Possible explanations are that Krebs-Smith et al. and Witschi et al. studied particular population subgroups rather than a cross section of the entire population, or that Block et al. failed to include dairy products present as ingredients in food mixtures in their milk group contributions. Block et al. (1985b) assessed food sources of several vitamins and minerals for adults participating in NHANES 11. These investigators found that milks, cheeses, and frozen desserts contributed approximately 30% of riboflavin, 17% of potassium, and 55% of calcium intakes (Block et al., 1985b). Krebs-Smith et at'. did not report how food groups contributed to intake of vitamins and minerals by women participating in the 1985 CSFII study, but did note that whole milk beverages contrib-

ROLE OF MILKFAT IN BALANCED DIETS

143

uted 7.8% of SFA intake and 16% of calcium intakes (Krebs-Smith et al., 1992). Pennington and Young (1991) examined mineral intakes as part of the Total Diet Study by the Food and Drug Administration (FDA) and found that dairy products provided the following for adults: 42-46% of calcium, 18-23% of phosphorus, 13-15% of potassium; 10-13% of magnesium; and 10-12% of zinc intakes. For adolescents and children, dairy foods contributed much greater percentages of these nutrients. As part of the Bogalusa Heart Study, Nicklas ef al. (1992) examined 24-hr dietary recall data collected in 1973-1982 for 871 I0-year-olds. The percentage of energy from fat in the children’s diets ranged from about 18% to more than 60%. The sample was divided into four categories according to en% from fat (<30%, 30-35%, 35-40%, and >40%). As expected, total fat, SFA, and energy intakes were greater in the high-fat than in the low-fat groups. Although dairy foods made a larger contribution to the percentage of total fat consumed by the lowest fat intake group, children with the highest fat intakes consumed more dairy products (on average, 96 calories more from dairy products per day). Meat consumption was dramatically different among the groups: meat contributed more fat (8.1 vs 40.5 g/day) and more energy (129 vs 530 kcallday) to diets as fat increased from <30 en% to >40 en%. Although the children with diets containing <30 en% as fat ate fewer dairy foods and fewer meat products than the higher fat intake groups, they also consumed more candy and beverages with sugar. These food choices help explain why the percentage of children consuming less than two-thirds of their recommended dietary allowances (RDAs) for vitamin B6, vitamin BIZ,vitamin E, thiamin, riboflavin, niacin, calcium, phosphorus, magnesium, and iron was higher in the lowest fat intake group than in the highest fat intake group (Nicklas et al., 1992). In these children, then, serious trade-offs occurred for self-selected diets with less than 30 en% from fat. On the other hand, McPherson et al. (1990) assessed diets of 138 children and adolescents and found that intake of vitamins and minerals per 1000 kcal was not lower in subjects eating lower fat diets. However, these researchers neglected to report the absolute intakes of micronutrients that could have differed since energy intake was higher in those consuming a greater percentage of energy from fat. Reviewing more current data, for example, from the NHANES 111, to assess fat intakes and food contributions to total fat and fatty acids will be interesting. As people try to follow more “prudent” diets, what are the nutritional trade-offs? Will the shift in the proportions of fat and fatty acids obtained from various foods continue? These questions will be put into perspective when the health implications of fat intake (type, source, and amount, with emphasis on milkfat) are discussed in Sections I11 and IV of this chapter.

144

LOUISE A. BERNER

B. MILKFAT COMPOSITION AND TRIGLYCERIDE STRUCTURE Milkfat commonly is referred to as a “saturated” fat, but, as discussed here, is considerably more complex than any one-word descriptor implies. Most of the lipid in milk is present in milkfat globules that are surrounded by membranes. The milkfat globule membrane, because of its high content of phospholipids, prevents the globules from coalescing and, thus, maintains emulsion stability (Jensen et al., 1991). The lipid distribution in the membrane, globule core, and skim milk portion can be altered during processing of milk to make different products (e.g., during fat removal, homogenization, heating, churning), so the proportion of membrane material varies among different dairy foods (Walstra and Jenness, 1984). Because the lipid compositions of the fat globule core and of the membrane differ (the latter being rich in phospholipids and cholesterol) differences exist in lipid composition among different dairy products. However, the differences are minor because the fat globule core contributes a much greater proportion of the lipid in dairy products than does the membrane. Thus, in this chapter, the composition of milkfat will be considered equivalent among different dairy foods, unless otherwise noted. Distinction of the terms milkfat and butter or butterfat is important, however. Throughout this chapter, “milkfat” is a generic term referring to the fat in all dairy foods (including butter), whereas reference to butter or butterfat indicates that butter per se is the fat source used in the cited study. Sources of milkfat other than butter (e.g., whole milk, yogurt, cheese) also will be specified when appropriate. 1 . Fatty Acid Composition

a . Predominant Fatty Acids. Milkfat composition is highly complex: 400 different fatty acids have been identified, either conclusively or tentatively, to date (Jensen et al., 1991; Jensen, 1992). To evaluate the nutritional impact of dietary fats, concentrating on the dozen or so most prominent fatty acids in milkfat and other food fats is sufficient. The major fatty acids in selected dietary fat sources are shown in Table V (developed from USDA data). Milkfat is unique in several respects. First, butyric acid constitutes over 3% by weight of the major fatty acids in milk, whereas no other common food fat contains this short-chain fatty acid. Second, milkfat also contains appreciable amounts of other short and medium chain SFAs: butyric, caproic, caprylic, and capric acids (C4-Cl0) make up about 9.2% by weight of the major milkfat fatty

TABLE V APPROXIMATE FATTY ACID COMPOSITION OF SELECTED FATS AND OILS‘

Weight % of major fatty acids ~

Fat source Milkfat Beef tallow Lard Chicken fat Menhaden oil Coconut oil Palm kernel oil Palm oil Cocoa butter Soybean oil Cottonseed oil Corn oil Safflower oil Olive oil Canola oil Peanut oil

4:O

6:O

8:O

~

1O:O

~

12:O

14:O

16:O

18:O

16:l

18:l

18:2

18:3

20:4

3.0 0.9 0.2 0. I

10.6 3.9 1.4 0.9 8.7 17.8 17.4 1.o 0.1 0.1 0.8

27.7 26.0 24.9 22.6 16.6 8.7 8.6 45.5 26.6 10.8 23.7 11.4 6.5 11.5 4.2 10.0

12.8 19.8 14.1 6.3 4.1 3.0 3.0 4.5 34.7 4.0 2.4 1.9 2.3 2.3 1.9 2.3

2.3 4.4 2.8 6.0 11.5 -

26.6 37.7 43.1 39.1 15.9 6.2 12.1 38.3 34.1 23.8 17.8 25.3 12.2 75.8 58.7 47.1

2.3 3.2 10.7 20.4 2.4 1.9 1.7 9.5 2.9 53.4 53.9 60.7 77.4 8.3 21.2 33.6

1.6 0.6 1.0 1.0 1.6

trh -

47.4 49.7 0.1

-

-

-

-

-

0.1

-

0.1

-

0.3 0.2 0.2 0.8 0.4 0.8 0.2 0.1

-

0.2 0.1 7.1 0.2 0.7 0.4 0.6 9.7 -

~~

205

22:6

-

0.1 1.3

-

0.1

-

-

a All values calculated from USDA Handbooks 8-1.8-4, or the 1989 and 1990 supplements to those handbooks. Values may not add to 100% because traces of other fatty acids are present. tr, Trace amounts.

146

LOUISE A. BERNER

acids. Third, myristic acid constitutes about 10% of milk fatty acids; of the other common food fats, only beef tallow (with about 4%) and coconut and palm kernel oils (with 17-18%) supply appreciable amounts of this fatty acid. Fourth, the content of PUFAs in milk is low. A final observation is the striking range of fatty acids in milkfat. No single fatty acid stands out as the predominant fatty acid, although palmitic and oleic acids are present in the greatest amounts. Of the major fatty acids in milkfat, 9.2% are SFAs with 10 carbons or less, 54% are SFAs with 12-18 carbons, about 29% are MUFAs, and less than 4% are PUFAs. A single value is given for the amount of each fatty acid in Table V, but literature values for the fatty acid composition of milkfat (and other dietary fats) obviously vary somewhat because of variations in methodology as well as actual differences attributable to feed sources, breed, and so forth. Iverson and Sheppard (1986) compiled data from several researchers who determined the major fatty acids of butter as methyl esters. The mean weight % of butyric and oleic acids, for example, ranged from 2.97 to 4.10% and 23.03 to 25.44%, respectively. Similar ranges were determined for all fatty acids considered. The cause of the variation, although undetermined, surely is multifactorial (breed, feed, season, analytical technique, etc.). For the volatile short-chain fatty acids, significant loss occurs when they are determined as methyl esters rather than butyl esters (Iverson and Sheppard, 1986). Thus, literature values for the weight % of short-chain fatty acids may be underestimated because analyzing fatty acids as methyl esters has been common. Jensen (1992) has identified the lack of a modem database on fatty acids in various milk products, especially with respect to short-chain SFAs, longchain PUFAs, and trans fatty acid isomers. Application of modem analytical methods to determine the fatty acid composition of milkfat and other food fats should be encouraged. b. Trans Isomers. An important consideration about fatty acid composition is whether unsaturated fatty acids are present as cis or trans isomers. In most unprocessed foods, the proportion of trans fatty acids is very low to nonexistent. However, during partial hydrogenation of oils to make margarines, shortenings, and frying oils, some of the cis fatty acids are converted to trans isomers (Hunter and Applewhite, 1991). In addition, trans fatty acids occur naturally in small amounts in fats from ruminant animals (e.g., beef tallow, milkfat) because of hydrogenation in the rumen (Hunter and Applewhite, 1991). The percentage of trans fatty acids in selected fat sources is listed in Table VI. Obviously, animal fats have low levels of trans fatty acids compared with partially hydroge-

ROLE OF MILKFAT IN BALANCED DIETS

I47

TABLE VI TRANS FATTY ACIDS I N COMMON FATS/OILS~

Product Vegetable oil/shortening, partially hydrogenated, commercial Vegetable shortening, household Margarines Butter Lard Beef tallow

Trans fatty acids (% of total fat) 13-42 14-18 7-3 1 2-3 0

3-6

Compiled from Enig ef al. (1983, IM),Hunter and Applewhite (1991), Jensen et al. (1991). and Slover ef al. (1985).

nated vegetable oil products. Most of the trans fatty acids in the food supply are trans monoenes, although trans dienes are present at significant levels in partially hydrogenated vegetable oils, especially when the levels of trans monoenes are lower (Enig et al., 1990). Commercial frying and baking oils contain significant amounts of trans fatty acids; therefore baked goods, fried snack foods, and fried fast-food items contain significant amounts of trans fatty acids. For example, Hunter and Applewhite (1991) cite an average of 9.8% of fat in chips and extruded snacks as trans fatty acids, whereas Enig et al. (1990) have found ranges of 0.8-54% for corn and corn/cheese snacks and 3-37% for doughnuts, french fries, fried chicken, and fried fish. Currently, nutrient databases and nutrition labels on foods do not distinguish trans from cis unsaturated fatty acids, although, as discussed later in this chapter, doing so may be desirable. Clearly, for many food items, including trans fatty acid isomers in nutrient databases will be a difficult task because of the wide ranges of trans isomers reported among brands. c . n-3 Fatty Acids. A final consideration about the fatty acid composition of milkfat and other foods is where the double bonds are located in PUFAs. In most food fats and oils, n-6 PUFAs predominate (primarily linoleic acid), but fish oils typically contain 20-25% of their fat as n-3 fatty acids, particularly eicosapentaenoic (20 :51-3) and docosahexaenoic (22 :6n-3) acids. Two commonly used vegetable oils, soybean and canola, contain a significant proportion of n-3 fatty acids; about 7 and lo%, respectively, of their fat exists as a-linolenic (18 :3n-3) acid (Hunter, 1990). Margarines, vegetable shortenings, butter, and milk products contain a small proportion of a-linolenic acid, usually a small percentage or

148

LOUISE A. BERNER

less (Hunter, 1990). Thus, milkfat does contain some a-linolenic acid, but more significant dietary sources of n-3 fatty acids are fish, fish oils, and unhydrogenated soybean and canola oils.

2. Triglyceride Structure The fatty acids in milkfat are not distributed randomly on the triglyceride (TG) molecule. In particular, as shown in Table VII, the short-chain fatty acids show a distinct preference for the sn-3 position of the TGs (Breckenridge and Kuksis, 1968; Parodi, 1982; Jensen et al., 1991). In fact, all butyric acid is located at the sn-3 position. In addition, myristic acid is found predominantly in the sn-2 position, palmitic and stearic acids mostly in the sn-1 and 2 positions, and oleic acid in the sn-1 and 3 positions (Parodi, 1982; Jensen et al., 1991). Barbano and Sherbon (1975) fractionated milkfat into high melting and medium-low melting TGs. Stereospecific analysis showed that in the high-melting TGs, which made up less than 25% of milkfat, palmitic acid was found preferentially at the sn-2 position whereas stearic and elaidic (trans 18: 1) predominated at the sn-3 position. Hence, the positional distribution of fatty acids in milkfat TGs can vary among different milkfat fractions. Although certain fatty acids clearly are found preferentially at specific positions on milkfat TG molecules, the actual number of individual triaTABLE VII POSITIONAL DISTRIBUTION O F FATTY ACIDS IN MILKFAT TRIACY LGLY C E R O L S ~

Mol % Fatty acid

Trig1yceride

sn-l

sn-2

sn-3

-

35.4 12.9 3.6 6.2 0.6 6.4 5.4 1.2 23.1 2.3

~

4:O 6:O 8:O

1o:o 12:o 14:O 16:O 18:O 18:1

18:2 a

11.8 4.6 I .9 3.7 3.9 11.2 23.9 7.0 24.0 2.5

1.4 I .9 4.9 9.7 34.0 10.3 30.0 1.7

Reprinted with permission from Jensen et al. (1991).

0.9 0.7 3.0 6.2 17.5 32.3 9.5 18.9 3.5

149

ROLE OF MILKFAT IN BALANCED DIETS

cylglycerols in milkfat is enormous. Because of the complexity, the major stereospecific TGs of milkfat have not been identified completely (Small, 1991). Theoretically, since milkfat has some 400 different fatty acids, there could be 40O3or 64 million different triacylglycerols (Jensen et al., 1991). Some TG species are more predominant than others. The major TG species in several fats and oils are listed in Table VIII. Note that, for butter, the “major” individual triacylglycerols listed still represent only a few mol % of the total so, from a practical point of view, knowing the individual TG species in milkfat may not be of great value. For example, the most predominant triacylglycerol in milkfat, calculated using the 1-random-2-random-3-randomhypothesis (which shows good but not exact agreement with analytical results), is 16 :0-16 :0-4 :0, estimated to be present only at about 4 mol % (Jensen et al., 1991). In other words, 96% of milkfat consists of a myriad of other TG species, each present in even smaller amounts. Thus, for nutritionists, using information about the positional distribution of major milkfat fatty acids, as discussed earlier and shown in Table VII, may be more feasible and important. Understanding the nutritional significance of having short-chain fatty acids at the sn-3 position, myristic acid in the sn-2 position, and so on, rather than undertaking the seemingly endless task of learning the nutritional significance of individual milkfat triacylglycerols, may be worthwhile. TABLE VIII THREE MAJOR TRIACYLGLYCEROL SPECIES OF SELECTED FATS A N D OILSO

Fat/Oil Butterfat Lard Beef tallow Cocoa butter Coconut oil Palm kernel oil Palm oil Corn oil Olive oil Soybean oil Low-erucic acid rapeseed oil (canola)

Three major triacylglycerols 16:0-16:0-4:0 18:0-16:0-18: 1 16:0-18:1-18: 1 16:0-18:1-18:0 12:o-12:o-12:o 12:o-12:o-12:o l6:0-18: 1-16:O 18:2-18:2-18:2 18:1-18:1-18: 1 18:2-18:2-18:2 18:1-18:1-18: 1

16:0-16:0-10:0 18:1-16:0-18:2 16:0-18: 1-16:O

16~0-18:1-16:O 18: I- 16:O- 18: 1

18:0-18: 1-18:O

l6:O-18: 1-16:O 1o:o-12:o-14:o 18: 1-12:0-18: 1 16:0-18:1-18:2 18:2-18:2-16:0 18:1-18:2-18:1 18:2-18:2-16:0 18:1-18:1-18:3

1o:o-12:o-12:o 14:0-18:1-12:0 16:O- 18: 1- 18: 1 18:2-18:1-18:2 18:1-18:1-16:0 18:2-18:2-18:1 18:2-18:1-18:1

16:0-18:1-18:0

a Adapted with permission from Small (1991). Annual Review in Nutrition 11. Copyright 01991 by Annual Reviews, Inc.

150

LOUISE A. BERNER

3. Other Lipid Components

At least 96% of milk lipid is triglyceride. The remaining lipids, mostly phospholipids and cholesterol, are associated primarily with the milkfat globule membrane in fresh unprocessed milk. (During processing, some of the membrane material may move to the skim milk phase.) Phospholipids make up about 1% by weight of total milk lipids, whereas cholesterol is present at less than 0.5% by weight of milk lipids. Phosphatidylethanolamine, sphingomyelin, and phosphatidylcholine each make up 26-3 1% by weight of milk phospholipids, phosphatidylinositol and phosphatidylserine each constitute about 5%, and other phospholipids exist in trace amounts (Jensen et al., 1991). C. FAT AND CHOLESTEROL CONTENT OF COMMONLY CONSUMED DAIRY FOODS The energy, fat, and cholesterol contents of usual servings of some common dairy products are summarized in Table IX. The table contains several points of interest. First, dairy foods are a diverse group, with wide-ranging fat contents. In low-fat and skim milks, cottage cheeses, yogurts, ice milks, and fat-reduced products such as low-fat cheeses and frozen desserts (not shown in the table), fat contributes one-third or less (only a small percentage in nonfat products) of the energy content. In whole milk, creams, full-fat cheeses, and ice cream, fat contribution ranges from nearly half to almost all the energy. However, most dairy foods are high-moisture foods, so the absolute amount of fat, even in servings of full-fat milk and cheeses, is less than 10 g (which translates to about 10% of a daily recommended fat intake limit of 78 g, based on 30 en% from fat and a 2350-calorie diet). Dairy foods can contribute significant amounts of fat to the diet, depending on the product chosen, the frequency of consumption, and the total energy intake of the individual. Dairy foods are not cholesterol rich, as shown in Table IX. A generous serving (1 tablespoon) of butter, an ounce of cheddar cheese, or an 8-oz glass of whole milk contains 30-33 mg or about 10% of the recommended daily intake limit. On the other hand, one large egg contains over 200 mg and a 3-oz serving of meat, fish, or poultry has 50-90 mg cholesterol. This background knowledge of the actual consumption of different food fats and their chemical compositions should help put into perspective the following discussion of dietary lipids as they impact CHD and cancer risk.

TABLE IX ENERGY, FAT, AND CHOLESTEROL IN TYPICAL SERVINGS OF DAIRY FOODS'

Product Milk (Fat %) Whole (3.3%) Lowfat (2%) Lowfat (1%) Nonfatkkim Cream Half and half Heavy, whipping Sour, cultured Cheese American Cheddar Cottage, regular Cottage, lowfat Mozzarella, part-skim

Swiss

Serving size

(g)

Percentage of recommended allowance'

Cholesterol (mg)

Percentage of recommended allowanced

8.2 4.1 2.4 0.6

10.5 6.0 3.1 (1

33 18 10 5

11.0 6.0 3.3 I .7

6 21 5

2.0 7.0 I .7

27 30 34

9.0 10.0 11.3 3.3 5.3 8.7

Percentage of kcal as fatb

Fat

Weight (g)

kcal 150 125 104

Ic

244 245 245 245

90

48 33 20 6

1T IT IT

15 15 12

20 52 26

75 94 85

1.7 5.6 2.5

2.2 7.2

1 02 1 02

28 28 225 226 28 28

106

74 73 38 12 56

8.9 9.4 10.1 2.3 4.5 7.8

11.4 12.0 12.9 2.9 5.8

Ic I C I C

Ic Ic 1 02

I oz

I I4 232 164

72 I07

64

3.2

10.0

10

16 26

continues

TABLE IX Continued ______

Product

Percentage of recommended allowance'

Cholesterol (mg)

2.8 0.4

3.6 <1

11 4

3.7 1.3

47 60

14.3 23.7

18.3 30.4

59 88

19.7 29.3

27 18 100

5.6 4.6 11.4

7.2 5.9 14.6

18 13 31

6.0 4.3 10.3

Serving size

Percentage of kcal as fatb

Weight (g)

kcal

lc Ic

227 227

194 127

13 3

lc lc

133 148

269 349

lc

131 175 14

184 223 102

Fat (g)

Percentage of recommended allowanceJ

Yogurt,wladded milk solids Lowfat Nonfat Ice cream Regular (10% fat) Rich (16% fat) Ice milk Hard (4.3% fat) Soft serve (2.6% fat) Butter

lc 1T

Data from or calculated from USDA (1988). The percentage of kcal from fat is included here because it is easily calculated from new nutrition labeling information, although it is recognized that recommended energy intake from fat (i.e., <30 en%) applies to diets and not to individual foods. Calculated as a percentage of 78 g fat, which represents the amount of fat in a diet with 2350 kcal and 30 en% as fat. Calculated as a percentage of 300 mg cholesterol, the recommended daily limit.

ROLE OF MILKFAT IN BALANCED DIETS

I53

Ill. DIETARY FAT, DAIRY PRODUCTS, AND CORONARY HEART DISEASE

This section reviews evidence linking dietary fatty acids and fat sources to serum lipid levels and other factors influencing CHD risk [serum apolipoprotein levels, low-density lipoprotein (LDL) modification, tendency for thrombosis]. Special emphasis is placed on the effects of milkfat from butter and other dairy foods. Coronary heart disease, a major public health problem that results from atherosclerosis, is a multifactorial disease. Genetic predisposition, gender, advancing age, high serum total cholesterol, high serum LDL cholesterol, low serum high-density lipoprotein (HDL) cholesterol, cigarette smoking, high blood pressure, obesity, and inactivity are among the many factors that influence the development of atherosclerosis and CHD (Carleton er al., 1991). Risk factors for CHD are summarized in Table X. Gender, advancing age, and genetic composition are three factors we cannot change whereas high blood cholesterol, smoking, high blood pressure, excess weight, diabetes, and inactivity are modifiable risk factors (Carleton er af., 1991). Dietary habits can influence several of the CHD risk factors listed in Table X. Of particular relevance to this chapter is the widely accepted view that diets rich in SFAs have a negative influence on several CHD risk factors compared with diets rich in PUFAs or MUFAs. SFA-rich diets have been shown to raise serum total and LDL cholesterol levels in humans and animal models, although evidence now shows that all SFAs are not equivalent in their effects (Kris-Etherton e f af., 1988; Grundy and

TABLE X RISK FACTORS FOR CORONARY HEART DISEASE

Elevated total blood cholesterol Elevated LDL blood cholesterol Low HDL blood cholesterol Male sex Advancing age Genetic predisposition; family history of early CHD Cigarette smoking High blood pressure Obesity Inactivity Diabetes mellitus

154

LOUISE A. BERNER

Denke, 1990; McNamara, 1992). Less is known about how different dietary fats and fatty acids influence several aspects of lipoprotein and apolipoprotein metabolism, modification of LDL molecules (which in turn influences the atherogenicity of LDLs), and thrombosis. In other words, the dietary fat-CHD story has not yet been completed. Several questions, important from both a practical and a scientific viewpoint, are raised by a critical review of the literature. First, do dietary or human conditions exist under which SFAs (in general) or milkfat (in particular) do not have a negative influence on heart disease risk factors? Second, is evidence available that a balance of dietary fatty acids, including saturates, has a positive influence on any risk factors for CHD? Finally, what impact do dairy foods, including standard-fat products, have on serum lipids and other CHD risk factors when consumed at levels typical in the United States? Is the impact of dairy foods different from that predicted by their fat content alone? The current state of knowledge about the answers to these questions is addressed and summarized in the following discussion.

A. EFFECTS OF DIETARY FAT TYPE AND SOURCE ON SERUM LIPOPROTEINS AND APOLIPOPROTEINS Lipids such as triglycerides and cholesterol are transported in the body as part of lipoproteins, complexes of lipids and proteins (apolipoproteins). The major classes of plasma lipoproteins and their characteristics are summarized in Table XI. The reader is referred to several excellent reviews for detailed discussions of the synthesis, degradation, and overall functions of the blood lipoproteins (Kris-Etherton et al., 1988; Grundy and Denke, 1990; Grundy, 1991; McNamara, 1992). LDL particles are the major cholesterol transport lipoproteins and promote the formation of atherosclerotic lesions, although the mechanisms have not been defined completely (Carleton et al., 1991). HDLs, on the other hand, function to transport cholesterol from peripheral tissues to the liver via pathways known as “reverse cholesterol transport;” this function of HDLs is thought to explain inverse correlations between serum HDL levels and atherosclerosis (Franceschini et al., 1991). Thus, understanding the influence of dietary fats and fatty acids on the levels and metabolism of serum LDLs, HDLs, and their major apolipoproteins is important. The Expert Panel on Population Strategies for Blood Cholesterol Reduction of the National Cholesterol Education Program (NCEP) reviewed the strong evidence relating high serum total and LDL cholesterol levels to CHD risk (Carleton et al., 1991). In fact, desirable blood

ROLE OF MILKFAT IN BALANCED DIETS

I55

cholesterol levels not only help prevent the formation of atherosclerotic plaque, but can stimulate regression of atherosclerosis when blood cholesterol levels are lowered (Yamamoto, 1991). Total and LDL cholesterol levels, respectively, have been classified by panel consensus as desirable if they are <200 and <130 mg/dl ( 4 . 1 5 and c3.36 mmol/L), borderline high if they range from 200 to 239 and 130 to 159 mg/dl (5.17-6.18 and 3.36-4.11 mmol/L), and high if they are >240 and >160 mg/dl (>6.21 and B4.13 mmol/L) (Expert Panel, 1988; Carleton et al., 1991). High blood TG levels also are associated with increased CHD risk, although the data are not as strong as for total or LDL cholesterol (Carleton ef al., 1991). A review of data from the Framingham Study indicates that elevation of blood TGs is a particularly significant CHD risk factor in women, as well as in men with low HDL cholesterol levels (Castelli, 1986). For the most part, high fasting TG levels are caused by elevated very low density lipoprotein (VLDL) levels (Castelli, 1986). As mentioned earlier, HDL cholesterol levels are related inversely to CHD risk because of the role of HDL in reverse cholesterol transport (Carleton et al., 1991; Franceschini et al., 1991). An HDL cholesterol level less than 35 mg/dl has been established as a risk factor for CHD (Expert Panel, 1988; Carleton et al., 1991); evidence suggests that the HDL;! subfraction is the most important fraction to consider (KrisEtherton et al., 1988). Several other lipoproteins or their components also have been associated with CHD risk. Postprandial cholesterol-rich lipoprotein particles (such as chylomicron remnants) have been suggested to be atherogenic (Carleton et al., 1991). Lipoprotein(a), which is made up of a protein called apo(a) attached to an LDL particle, has been associated with increased risk of CHD in some studies (Carleton et al., 1991) but not all (Jauhiainen et al., 1991). The major apolipoproteins associated with LDL and HDL, respectively, are apo B and apo A-I; these apoproteins are useful in assessing CHD risk (Kris-Etherton et al., 1988; Jauhiainen et al., 1991). Based on their interpretation of many published studies, the NCEP and other health professionals recommend a “Step 1” diet for all Americans over the age of 2 to lower blood cholesterol levels in the population. This diet’has 30 en% or less from total fat, less than 10 en% from SFAs and PUFAs, dietary energy levels to maintain desirable weight, and less than 300 mg cholesterol daily. This recommendation implies a need for some changes in current American diets, because average intake of energy from fat is 36-37%, average intake of SFAs is 13-14 en%, and cholesterol intakes average 304 mg for women and 435 mg for men (Carleton et d., 1991). [The importance of dietary cholesterol in determining

TABLE XI CHARACTERISTICS OF THE PLASMA LIPOPROTEINS"

Class of lipoprotein Characteristic

Chylomicron

VLDL

IDL

Density (g/rnl) Electrophoretic mobility Origin

(0.95 Origin

0.95-1.006 Pre-p

I .006-1.019 pre-p to p

Intestine

Liver and intestine

Physiological role

Transport of dietary TG"

Transport of endogqnous TG

In circulation secondary to catabolism of other lipoproteins Liver LDL precursor

Relative atherogenicity

0

+

+++

60 12 10

40 30 20 10

9-100, CI, CII,

9-100, E

Composition (%) triglyceride cholesterol phospholipid protein Major apolipoproteins

90 5 3 2 A-I, A-IV. 9-48. CI, CII. CIII

18

CIII, E

" Reprinted with permission from Kris-Etherton et al. (1988). TG, Triglycerides.

LDL

HDL

I .O 19- I .M3

1.063-1.210

P

a

Liver and intestine Liver Major cholesterol transport lipoprotein

++++ 10

50

15 25 9-100

Reverse cholesterol transport Negatively correlated with atherogenicity 5 20 25 50 AI, A-11

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serum cholesterol levels will not be discussed in any depth in this chapter because fat composition is generally assumed to be the more important dietary factor and because dairy foods do not contribute much cholesterol to typical diets in the United States. Dietary cholesterol may receive renewed attention, however, based on a meta-analysis by Hopkins (19921, who found that responsiveness to dietary cholesterol intake was heavily dependent on baseline cholesterol intake. If baseline intake was near zero, a small increase in dietary cholesterol could have a large effect on serum cholesterol; conversely, a person averaging 400 mg or so daily would not improve cholesterol levels without drastically curtailing cholesterol intake nor would an adverse effect be seen from higher cholesterol intakes. Dietary cholesterol may play a more or less important role than previously thought, depending on individual responsiveness and baseline intake.] Grundy (1991) estimated that lowering intake of SFAs to 7 en% (in other words, cutting intake of SFAs in half) would lower serum cholesterol levels by 20 mg/dl or about 10%.This estimate is based on equations developed several decades ago by Keys et al. (1957, 1965) and Hegsted et al. (1965), whose studies will be discussed later. The NCEP Expert Panel on Population Strategies expects a 10% or greater reduction in average blood cholesterol levels in the population, with a subsequent decrease in CHD of 20% or more, if everyone adopts the recommended diet (Carleton et af., 1991). The idea that a 2% decrease in CHD rates will be seen for every 1% decrease in blood cholesterol derives from several epidemiological and clinical trials, especially the Lipid Research Clinics Coronary Primary Prevention Trial (LRCT), almost all of which were conducted in hypercholesterolemic men (Expert Panel, 1988; NRC, 1989). The mean blood cholesterol level on entry into the LRCT was 292 mg/dl (7.55 mmol/L). The “two-for-one” prediction of decreased CHD risk with reduced plasma cholesterol is cited widely, yet some questions exist about how the data apply to the total population. For example, data from the 1976-1980 NHANES I1 indicate that the mean blood cholesterol for the United States adult population was 210215 mg/dl(5.43-5.56 mmol/L) (Expert Panel, 1988). In addition, some of the trials tested effectiveness of cholesterol-lowering drugs rather than diet. Nevertheless, the NCEP and others believe recommended diet changes will have a major impact on serum cholesterol levels and CHD risk in the entire United States population. Relatively little argument exists that lowering elevated blood cholesterol levels into the desirable range should decrease CHD, although one comprehensive meta-analysis concludes that “lowering serum cholesterol concentrations does not reduce mortality and is unlikely to prevent

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coronary heart disease” (Ravnskov, 1992). Even if reducing serum cholesterol levels is accepted to be beneficial, a number of researchers believe that current estimates of the potential effectiveness of dietary changes are unrealistic. For example, Ramsay et al. (1991) analyzed numerous published trials in which fat-reduced diets were used in an attempt to lower total cholesterol levels. These investigators concluded that cholesterol levels fell only 0-4% in response to a Step 1 diet, and that such a response is too small to be effective in subjects with high cholesterol levels. The authors suggested that many experts have unrealistic expectations from diet interventions because of “overreliance on short-term experiments, controlled studies of rigorous diets in ‘captive’ populations, and uncontrolled observations” (Ramsay et al., 1991). Grover et al. (1992) and Browner et al. (1991) used computer models to estimate the long-term effectiveness of serum cholesterol reduction by diet. These researchers used computer modeling because the length of clinical trials is limited by practical necessity, although their models were based on outcomes of clinical trials and population studies. Browner et al. (1991) determined that, if dietary fat intake was restricted to 30 en%, CHD mortality rates could be reduced by 5-20%; most of the benefit would be experienced by men over 60 years old and women over 70. Those statistics sound dramatic but translate to an increase in life expectancy of only 3-4 months. In addition, the researchers noted that they assumed a “best-case” scenario (e.g., the analysis assumed that the whole population would achieve the desired fat intake for the rest of their lives, and that the change in fat intake would be 100% efficacious). Grover et al. (1992) computed changes in life expectancy as a result of dietary fat reduction and medication in adults aged 35-65 with blood cholesterol levels of 200-300 mg/dl, with and without the additional risk factors of smoking and hypertension. For nonsmokers and nonhypertensives, the increase in life expectancy because of diet changes alone was 11 days to 4 months. The greatest benefit was seen for smokers and hypertensives who were treated with both diet and cholesterol-lowering drugs. Thus, a population-based approach may not be ideal if, in fact, only a certain subpopulation would receive the bulk of the benefit. Still, the population-based diet recommendations of the NCEP appear to be widely accepted possibly because they are identical to guidelines from numerous other groups (e.g., NRC, 1989). Also, people express a feeling that the recommended diets “might help but can’t hurt.” A shortcoming of many epidemiological studies and computer modeling studies (such as those just described) and of some clinical trials is that these studies only account for the effect (or predicted effect) of dietary fat changes on total serum cholesterol levels, not on HDLs or other lipoproteins or apolipoproteins known to be associated with in-

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creased or decreased CHD risk. Keeping this criticism in mind, the effects of specific dietary fats and fatty acids on serum lipids and apolipoproteins are reviewed next. 1 . Saturated and Unsaturated Fatty Acids

a . General Observations. In the 1950s and 1960s, numerous landmark studies on how dietary fat source and saturation affect serum cholesterol levels were carried out (e.g., Ahrens et al., 1954, 1957; Keys et al., 1957, 1965; Malmros and Wigand, 1957; Hegsted et a f . , 1965). In general, serum total cholesterol was the endpoint measured; serum TGs were measured in some experiments. These studies are important to review because many of the results, ideas, and equations generated from these experiments are accepted today. Ahrens et a f . (1954) studied 6 subjects under strict metabolic ward conditions to see whether replacing animal fat with vegetable fat influenced patient blood cholesterol levels. The subjects ate low-fat solid foods such as fruits, vegetables, spaghetti, jams, and hard candies, supplemented with fat- and protein-rich formulas. The fat in the formulas was mostly from butter and eggs (animal fat diet) or from margarine, salad oils, and peanut butter (plant fat diet). The patients consumed 45-52 en% as fat during the 3- to 10-wk test periods. Serum cholesterol was lowered 17-26% after switching to the plant fat diet. At least two factors make these experiments of questionable relevance today: the high fat intake of the subjects and, more importantly, the confounding effect of very high dietary cholesterol levels on the animal fat diet compared with the plant fat diet. When the animal fat diets were fed, the subjects had cholesterol intakes of 1000-1800 mg/day (although the researchers probably overestimated the actual cholesterol intake somewhat, since newer data have lowered food cholesterol values). Therefore, the results do not necessarily indicate the effect of fatty acids from plant rather than animal sources. Also, the feeding of formula diets instead of real foods is a concern. A diet without solid foods is not physiological; serum lipid values tend to drop regardless of the fat source in liquid diets. For example, in a recent study, formula diets rich in SFAs, MUFAs, or PUFAs were fed to young normolipidemic volunteers. Significant and rather large reductions from baseline levels occurred in total cholesterol, LDL cholesterol, and apo B, even on the SFA-rich formula (Becker et al., 1983). Use of liquid formulas is an experimental variable that must be considered when evaluating studies. Ahrens and co-workers continued to probe the effects of dietary fat source and saturation on blood cholesterol in a series of experiments. They fed liquid formula diets to have optimal control over fat composi-

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tion of the diets; sources of fat included animal fats, cocoa butter, corn oil (used in many studies), and numerous other vegetable oils. These researchers concluded that the effect of a fat was largely a function of its iodine value (i.e., its degree of unsaturation); more-unsaturated fat sources depressed blood cholesterol relative to more-saturated fats (Ahrens et al., 1957). On the basis of their results, the researchers posed a series of questions that were yet unanswered. Is the lipid-lowering effect of unsaturated fatty acids dependent on the number of double bonds or is it peculiar to certain fatty acids? How do monoenes, dienes, and trienes compare? Do all saturated fatty acids have the same effect? What is the effect of fatty acid isomers produced during hydrogenation? More than 30 years later, lipid researchers are still sorting out the answers. Malmros and Wigand (1957) performed numerous experiments with healthy adult subjects who received meals under controlled conditions. A very low fat basic diet was fed, and various fats were added during preparation of foods to bring the fat level to about 40 en%. Energy intakes were rather high; each subject received about 150 g/day of the various fats and 3375 calories (14.12 MJ) daily. The investigators concluded that corn, safflower, olive, rapeseed, and whole oil lowered serum cholesterol relative to miikfat and coconut oil. Keys et al. (1957) and Hegsted et al. (1965) conducted a series of studies in institutionalized schizophrenic men with initial blood cholesterol levels averaging about 225 mg/dl (5.81 mmol/L). Basic low-fat diets were fed, and various test fats (numerous vegetable oils, butter, sardine oil, lard) were incorporated into recipes to bring fat levels to 38-42 en%. Test diets generally were fed for 4-wk periods. Neither group kept dietary cholesterol levels constant among the dietary treatments, although Hegsted et al. (1965) did look at the effect of dietary cholesterol per se by comparing vegetable oil-based diets in which the dietary cholesterol level was varied purposely by adding egg yolk. As a result of their comprehensive studies of male mental patients, the following equation was developed by Keys et al. (1957) to predict the effect of fat quality and dietary cholesterol on serum cholesterol levels: AChol (mg/dl) = 2.74AS - 1.31AP Hegsted et al. (1965) developed other equations: AChol (mg/dl) = 2.328s

+ 0.32AM - 1.46AP + 0.065AC + 0.83

or AChol (mg/dl) = 8.45ASI4+ 2.12ASI6- 1.87AP + 0.056AC - 6.24

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For both groups, S was SFAs as a percentage of energy, P was PUFAs as a percentage of energy, M was MUFAs as a percentage of energy, C was dietary cholesterol (mg), SM was myristic acid as a percentage of energy, and SI6was palmitic acid as a percentage of energy. Hegsted et al. (1965) first developed a best-fit equation by grouping all fatty acids into one of three major types-saturates, monounsaturates, or polyunsaturates. Their equation had significantly better fit when dietary cholesterol was included as a variable. These investigators recalculated equations after dividing SFAs into five classes: those with fewer than 12 carbons, lauric acid, myristic acid, palmitic acid, and those with 18 or more carbons. The second Hegsted equation listed earlier, in which myristic and palmitic acids were the only SFAs considered and MUFAs were excluded, had an even better fit than the first equation. Thus, Hegsted and colleagues (1965) concluded that myristic acid and, to a lesser extent, palmitic acid and dietary cholesterol had the most significant blood cholesterol-raisingeffects whereas PUFAs had the opposite effect. About two-thirds of the total variance in serum cholesterol level could be explained by changes in dietary myristic acid alone. Stearic acid and saturates with 10 carbons or fewer had little influence on blood cholesterol levels (Hegsted et al., 1965). The original Keys equation did not assign any weight to dietary cholesterol but indicated that a decrease in SFAs was twice as effective as an increase in PUFAs in lowering blood cholesterol levels. After subsequent experiments testing serum cholesterol response to dietary cholesterol, Keys et al. (1965) modified their equation as follows: AChol. (mg/l) = 2.7AS - 1.3AP + 1.5AZ where Z is the square root of dietary cholesterol measured in mg/lOOO kcal . Keys et al. (1957, 1965) and Hegsted et al. (1965) were careful to point out limitations of their equations. Keys and co-workers (1957) noted that applicability was limited to the conditions of their experiments (men, test diets fed 2-4 wk, initial serum cholesterol levels less than 300 mg/dl, etc.). These researchers also mentioned that the equation did not work equally well for all SFAs (Keys et al., 1965). Hegsted et al. (1965) emphasized the descriptive nature of the equations and the fact that the regression coefficients for the fatty acids could change significantly if other variables (such as dietary carbohydrates) were added. Nevertheless, the equations still receive wide use, for example, by forming the basis of Grundy’s assertion that a decrease in SFA intake from 14 to 7 en% will cause an average decrease in blood cholesterol of 20 mg/dl in the United States population (Grundy, 1991). Also, these equations likely

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LOUISE A. BERNER

are the basis for the NCEP assertion that recommended dietary fat changes will lead to a 10% decrease in average blood cholesterol level in the United States (Carleton et al., 1991). Grande et al. (1970) confirmed that serum total cholesterol levels were lower when men were fed diets rich in stearic acid rather than palmitic acid, although serum TGs changed in the opposite direction. On the other hand, McGandy and colleagues (1970) reported that stearic and lauric acids both were hypercholesterolemic compared with unsaturated fatty acids, a result that contrasted with earlier findings by this group that stearic acid in particular was neutral in effect (Hegsted er al., 1965). The investigators explained that earlier conclusions about stearic acid were based primarily on data from cocoa butter, but that semisynthetic fats made by transesterifying safflower or olive oils with specific fatty acids (such as stearic acid) gave different results. The researchers reasoned that triglyceride structure may be important to consider, since the positional structure of the semisynthetic fats differed from that of natural fats such as cocoa butter (McGandy et al., 1970). Using the semisynthetic fats, this group also found that myristic acid was not much more hypercholesterolemic than palmitic or stearic acid, again at odds with their earlier calculations. The conflict was attributed to a heavy reliance on data using one fat, coconut oil (rich in myristic acid), as a saturated fat source in the earlier studies. However, the suggestion that stearic acid is not hypercholesterolemic compared with some other SFAs has been supported in studies in which cocoa butter (Kris-Etherton et al., 1990, 1991) or a synthetic fat made by randomly reesterifying completely hydrogenated soybean oil with high-oleic safflower oil (Bonanome and Grundy, 1988) was the major source of stearic acid. Many human studies have confirmed or extended findings about how SFAs and n-6 PUFAs influence serum lipids. Clearly, substituting diets high in PUFAs for typical American diets or feeding diets with increasing PUFAISFA ratios (often as high as 2.0 or even higher; levels that are difficult if not impossible to attain with self-selected foods) to adults causes total serum cholesterol levels, LDL cholesterol levels, and often VLDLs and TGs to drop significantly (Shepherd et al., 1978, 1980; Becker et al., 1983; Baudet et al., 1984; Jackson et al., 1984; Mattson and Grundy, 1985; Grundy et al., 1986; McNamara et al., 1987; Mensink and Katan, 1989; Fumeron et al., 1991; Garcia et al., 1991), and seems to occur whether the subjects are initially hypercholesterolemic (Baudet et al., 1984; Mattson and Grundy, 1985) or not (Shepherd et al., 1978, 1980; Becker et al., 1983; Baudet et al., 1984; Garcia et al., 1991). At the same time, however, diets very high in PUFAs (or low in SFAs) tend to lower HDL cholesterol and/or apo A-I levels (Shepherd et al., 1978, 1980;

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Schonfeld et al., 1982; Baudet et al., 1984; Jackson et al., 1984; Mattson and Grundy, 1985; Mendis and Kumarasunderam, 1990; Fumeron et al., 1991; Garcia et al., 1991). Brinton et al. (1990) suggested that the mechanism for dietary effects on HDL cholesterol levels differs from the mechanism by which HDL cholesterol levels differ among individuals. These investigators propose, therefore, that the lowering of HDL cholesterol levels by adopting “prudent” diets may not be of importance. At present, however, no evidence suggests that the mechanism of HDL cholesterol reduction (rather than the HDL cholesterol level itself) impacts the risk of CHD. The paradox of simultaneous decreases in LDL and HDL cholesterol on “antiatherogenic” diets often is trivialized by the explanation that LDL levels fall more than HDL levels do with highPUFA diets. In some cases, LDL and total cholesterol levels have fallen significantly while HDL and/or apo A-I levels have not (McNamara et al., 1987; Mensink and Katan, 1989; Wardlaw and Snook, 1990). However, in other studies the HDL/LDL ratios remained unchanged as the dietary PUFA/SFA ratio changed (Jackson et al., 1984) or HDL levels fell to undesirable levels following high-PUFA diets (Shepherd et al., 1978). A major criticism of most studies in which PUFA/SFA ratios are varied is that the high PUFA/SFA diets fed are impractical; indeed, no populations naturally consume diets so high in PUFAs. . As early as 1965, investigators suggested that the PUFA/SFA ratio “should be discarded even though it has the merit of simplicity” (Hegsted et al., 1965). Certainly, varying the PUFA/SFA ratio can have an impact on serum lipoprotein levels, as noted in the preceding paragraph, but the utility of the PUFA/SFA ratio breaks down for two main reasons: (1) it ignores the role of MUFAs, as discussed in a later section of this chapter and (2) not all SFAs-or PUFAs, for that matter-have equivalent effects on serum lipids (Grundy and Denke, 1990). As discussed earlier, stearic acid apparently is not hypercholesterolemic (Hegsted et al., 1965; Bonanome and Grundy, 1988), a fact that may help explain why, in 19 normolipidemic males, a beef fat-rich diet (relatively high in 18:O) resulted in lower levels of total cholesterol, LDL cholesterol, and HDL cholesterol than a coconut oil-rich diet (Reiser et al., 1985). However, both beef fat and coconut oil are considered “saturated” fats. A study of 12 middle-aged men compared the effects of two other SFAs, lauric and palmitic acids, on serum lipoproteins. Dougherty and Iacono (1992) used palm kernel oil as the major source of lauric acid, which constituted 5 en% of the diets or about 10 times the level of lauric acid in diets in the United States. Palmitic acid from a variety of foods replaced lauric acid in the comparison diet; both diets had 10 en% from saturated fatty acids and 9 en% each from monounsaturated and polyun-

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LOUISE A. BERNER

saturated fatty acids. Palmitic and lauric acids were equivalent in their effects on serum lipoproteins, although the investigators pointed out that intakes of palmitic acid are much greater than intakes of lauric acid in typical diets in the United States (Dougherty and Iacono, 1992). In contrast, Denke and Grundy (1992) found that lauric acid was modestly (but significantly)less cholesterolemic than palmitic acid when liquid formula diets were fed to volunteers. A synthetic high-laurate oil was the sole source of fat in the lauric acid-rich formula (supplying over 17 en% from lauric acid), whereas palm oil was the sole fat source in the palmitic acid-rich formula (supplying over 17 en% from palmitic acid). The practical significance of this study is questionable because of the unrealistically high levels of lauric acid in the high-laurate diets, the use of liquid formulas, and the use of a synthetic randomized fat (with an unnatural triglyceride structure) as a source of lauric acid. Sundram et al. (1991) reported that total and LDL cholesterol levels were higher and TGs lower when normocholesterolemic young men were given diets containing 8 en% from lauric plus myristic acids rather than palmitic acid. If, as reported by others, lauric acid is either equivalent to palmitic acid in its effects on serum lipoproteins (Dougherty and Iacono, 1992) or less cholesterolemic than palmitic acid (Denke and Grundy, 1992), then myristic acid might be concluded to be the most cholesterolemic saturated fatty acid. Animal experiments generally confirm findings from human studies, and some add insight into mechanisms for hyper- or hypocholesterolemic effects of dietary fats. Feeding SFA sources (butter, lard, coconut oil) rather than more unsaturated fats (corn oil, safflower oil, olive oil) led to increases in total and/or LDL cholesterol levels in several species of monkey, swine, and rabbit (Rude1 et al., 1981; Kim et al., 1984; Johnson et al., 1985; Masi et al., 1986; Nicolosi et al., 1990; Pronczuk et al., 1991). The major mechanism cited for how diets rich in SFAs raise LDL. cholesterol levels is that LDL receptor activity is suppressed by SFA feeding (Spady and Dietschy, 1985; Nicolosi et al., 1990). Woolett et al. (1992) showed in hamsters that, compared with laboratory chow diets, hydrogenated coconut oil-rich diets suppressed LDL receptor activity and increased LDL cholesterol production rate whereas safflower oilrich diets increased receptor-dependent LDL transport. Nicolosi et al. (1990) reported a decrease in non-receptor-mediated LDL apo B fractional catabolic rate in monkeys fed coconut oil in place of corn oil. Coconut oil cannot be assumed to represent all “saturated fats” when defining the mechanism for dietary fat effects on serum lipids, however. For example, Khosla and Hayes (1991) fed diets rich in palmitic acid (from 90% palm oil and 10% soybean oil) or lauric and myristic acids

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(from 90% coconut oil and 10% soybean oil) to rhesus monkeys. LDL apo B was higher in the coconut oil-fed monkeys than in the palm oil-fed monkeys because of an increase in the direct production of LDL apo B. In animal models, SFA-rich diets cause increases in HDL levels compared with high-PUFA diets (Rudel et al., 1981; Johnson er al., 1986; Nicolosi et af., 1990). Thus, both positive and negative effects occur when replacing PUFAs with SFAs in animal studies. The mechanism by which SFAs increase HDL levels (or PUFAs decrease them) is not clear. However, Rudel e f al. (1981) showed that safflower oil-fed monkeys produced larger chylomicrons with less surface material than did monkeys fed butter or lard; therefore, these investigators hypothesized a shortage of HDL precursor material in PUFA-fed animals. In later studies, the research group reported that monkeys fed PUFA-rich diets secreted fewer HDL precursor particles from liver to plasma than did SFA-fed animals (Johnson et af., 1986). In addition to data differentiating effects of SFAs from those of PUFAs, evidence from animal studies (as with humans) indicates that not all SFAs are equivalent. Grande (1962) fed dogs with diets containing equivalent amounts of SFAs, MUFAs, and PUFAs but different types of SFA. Diets rich in lauric and myristic acids (from coconut oil) were hypercholesterolemic compared with diets rich in palmitic and stearic acids (beef fat plus totally hydrogenated corn oil); the lowest cholesterol levels were seen when diets rich in caprylic and capric acids (synthetic medium-chain triglycerides) were fed. Almost three decades later, Hayes et al. (1991) confirmed that lauric and myristic acids were more hypercholesterolemic than palmitic acid in three monkey species. HDL cholesterol did not vary with dietary fat source except in cebus monkeys, in which a decline in HDL cholesterol paralleled a decline in LDL cholesterol. When data for all three monkey species were pooled, plasma apo A-I was not affected by dietary fat whereas apo B generally varied with LDL cholesterol (Hayes et af., 1991). In a separate study of normocholesterolemic rhesus and cebus monkeys, Khosla and Hayes (1992) found no effect on serum lipoprotein levels when purified cholesterolfree diets differing only in palmitic and oleic acids were fed. Again, the data suggest that palmitic acid is not hypercholesterolemic under these conditions. Hayes and Khosla (1992) offered an intriguing analysis of animal and human studies, proposing that myristic acid is consistently hypercholesterolemic, linoleic acid is consistently hypocholesterolemic when present at levels high enough to “counteract” effects of myristic acid, and oleic acid is consistently neutral. On the other hand, palmitic acid is neutral in normocholesterolemic subjects who consume less than 300 mg cho-

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LOUISE A. BERNER

IesteroUday but hypercholesterolemic in people with high baseline serum cholesterol levels (>200 mg/dl) and more generous intakes of dietary cholesterol. These researchers point out that linoleic acid above 5-6 en% (close to current consumption levels) would cause little additional improvement in cholesterol levels given typical myristic acid intakes in the United States (Hayes and Khosla, 1992). Does this analysis suggest that current diets already might be approaching “optimal” levels in terms of effects on serum lipids, at least for normocholesterolemic individuals? Although SFAs often are considered hypertriglyceridemic as well as cholesterolemic, Lai et al. (1991) and Ney et al. (1991) reported that different SFA sources have different effects on TG metabolism and HDL composition in rats. For example, both the plasma TG pool and the rate of TG secretion from liver to plasma were greater in animals fed beef fat than in animals fed other saturated and unsaturated fat sources (Lai et al., 1991). Clearly, not all SFAs or SFA sources have equivalent effects on blood lipoprotein levels. Moreover, as discussed earlier, the effect of specific fatty acids may depend on the other fatty acids present (Hayes et al., 1991). The net change in serum lipoprotein levels when polyunsaturated vegetable oils rather than animal fats or tropical oils provide most of the dietary fat is a beneficial one, although unanswered concerns about iowering HDL levels by PUFAs still exist. On the other hand, less is known about what happens after very modest changes in dietary fat sources or when an increase in one type of fatty acid (for example, myristic or palmitic) is accompanied by a generous intake of another fatty acid (for example, linoleic acid or an 0 - 3 fatty acid) that potentially can modulate its cholesterolemic effect. Currently, the consensus seems to be that lauric, myristic, and palmitic acids are the hypercholesterolemic SFAs (NRC, 1989; Grundy, 1991). Still, the relative importance of each of these fatty acids has not yet been established firmly, nor is it known whether the effects of the offending fatty acid are dependent on its positional distribution on triglycerides, on certain other fatty acids present in the diet, or on the genetic make-up of the consumer. 6 . Butter or Other Dietaty Fats. Butter is referred to as a “saturated” fat because approximately two-thirds of its major fatty acids are saturated, although only about 41% of these are fatty acids currently considered hypercholesterolemic(i.e., C12, C14, and C16). Nearly 60% of butterfat fatty acids are thought to have little or no effect on blood cholesterol levels. Over the years, many researchers have used butterfat as a fat source in

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human experiments. Numerous studies in which the effect of butterfat on serum lipids and lipoproteins was compared with that of other common food fats are summarized in Table XII. Subjects in most of the experiments had initial cholesterol levels of about 220 mg/dl (5.69 mmol/L), although in a few cases the mean prestudy levels were higher or lower. Note that butter (or the fat source with which it was compared) supplied 20-40% of total energy and 50-100% of the fat in the diet. Without question, butter is hypercholesterolemic compared with most other food fats when fed at such high levels. Total and LDL cholesterol levels were raised significantly in nearly all studies listed in Table XII; HDL levels also tended to increase significantly when diets rich in butter were eaten. Triglyceride levels increased on butter-rich diets compared with diets rich in fish oils.(Childs et al., 1990), sunflower and peanut oils (Baudet et al., 1984; Baudet and Jacotot, 1988; Fumeron et al., 1991), and corn oil (Wardlaw and Snook, 1990). In the studies in Table XII, the potential beneficial effects of butterfat-rich diets on HDL levels are outweighed by negative effects .on total and LDL cholesterol levels, although some question still remains about implications of diets low in SFAs and high in PUFAs because of the adverse effects on HDL and apo A-I levels (Fumeron et al., 1991). The experiments summarized in Table XI1 all have the drawback that 20-40 en% was from butter. In reality, however, butter and all other sources of milkfat combined contribute an average of only 16-19% to total fat intake in the United States (Block et al., 1985a; Krebs-Smith et al., 1990) or 6-7 en% if we assume an average of 37 en% from fat. Conclusions about the hypercholesterolemic effects of butter or milkfat compared with other food fats are drawn almost exclusively from studies in which single fat sources provided nearly all fat intake. Such conclusions ignore the fact that the American diet is highly varied and that very high PUFA intakes (such as those achieved by feeding corn or safflower oils as the primary sources of dietary fat in clinical trials) are not found among free-living populations. Hegsted et al. (1965), in their landmark studies, did assess the effects of blends of fat sources as well as of single fat diets. For example, these investigators tested 50-50 blends of butterfat with corn, safflower, and olive oils. The 50-50 butterfat-corn oil and butterfat-safflower oil blends actually lowered serum cholesterol levels relative to a control diet that was representative of a “usual American diet.” The butterfat-olive oil blend was hypercholesterolemic compared with the control diet, but much less so than butterfat alone. Similar results were seen with several other butterfat-vegetable oil blends. In all cases, as expected, blending butter with less saturated fats blunted the hypercholesterolemic effect

TABLE XI1 COMPARISON OF BUTTERFAT WITH OTHER FOOD FATS,FED AS PRIMARY SOURCES OF DIETARY FAT: EFFECTS ON BLOOD LIPIDS AND LIPOPROTEINS

Effect of butter on" Study

Subjects"

Butterfat (en%)

Comparison fat (same en%)

TotalC

LDLC

HDLC

TG

Comments

Malmros and Wigand (1957)

12 m/f 4 wk

40

Corn oil

Keys et al. (1957)

12-21 m 2-4 wk

28

inc

Total fat was 40 en%, 3/4 fat was test fat

Hegsted et al. (1%5)

9-10 m 4 wk

38

Cottonseed, corn, olive, sunflower, sardine oil, lard Coconut oil Safflower or olive oil Cocoa butter

NS inc inc

Dietary cholesterol not equal among test diets

Baudet et

69 f

20

Sunflower oil Palm oil Peanut oil

inc inc inc

01.

(1984)

5 mo

Cholesterol lower with milkfat than with self-selected diet

inc inc inc

inc inc inc

inc dec inc

Total fat was 30 en%, 2/3 fat was test fat

McNamara et al.

25m 6 wk

35

Vegetable oils and margarines

inc

inc

NS

NS

20 f

16

Sunflower oil

inc

inc

inc

Canola oil

inc

inc

NS

Peanut oil

NS

NS

dec' inc" dec' inc" dec'

(1987)

Baudet and Jacotot

6 wk

(1988)

Exact diets not specified, but fat "primarily" from dairy or vegetable oils Total fat 30 en%

inc

Childs et al. (1990)

8m 3 wk

36

Pollock oil Tuna oil Salmon oil

inc inc inc

NS inc inc

inc' incd NS

inc inc NS

Pollock oil is rich in EPA; tuna and salmon rich in DHA

Wardlaw and Snook

20 m

34

5 wk

Corn oil High-oleic sunflower

inc inc

inc inc

NS NS

inc NS

Total fat was 40 en%, 85% fat was test fat

20

Sunflower margarine

inc

inc

inc'

inc

Total fat was 40 en%

(1990)

Furneron et al. (1991)

36 m 3 wk

Number and sex of subjects; length of study. C, Cholesterol; TG, triglyceride; inc, increase ( p < 0.05); dec, decrease ( p < 0.05); NS, no significant change; -, no value reported. HDL2 only. HDL3 only. HDL2 and HDL3.

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LOUISE A. BERNER

associated with high butter intakes, yet butterfat intakes were still very high in these experiments (19 en% or more in the blended-fat diets). Little attention has been given to the impact of butterfat in realistic mixed-fat diets. Studies of monkeys or swine in which butter was the principal or sole dietary fat in place of vegetable oils show results similar to those of clinical trials because total, LDL, and HDL cholesterol levels are generally higher (Rude1 et al., 1981; Kim et al., 1984; Johnson et al., 1985). In rats, however, butterfat feeding is less likely to be hypercholesterolemic than vegetable oils (Larking and Sutherland, 1977; Lai et al., 1991; Ney et al., 1991). When compared with certain other SFA sources, butter has, in some instances, been shown to be less cholesterolemic and/or triglyceridemic. For example, in rats, plasma TGs and VLDLs tended to be higher in animals fed beef tallow and coconut oil than in those fed butter (Lai et al., 1991; Ney et a / . , 1991), and plasma total cholesterol levels were higher in rats fed tallow or palm oil than in those fed butter (Ney et al., 1991). A particularly well-designed experiment was performed in three species of monkey by Pronczuk et al. (1991). The researchers raised cebus, rhesus, and squirrel monkeys throughout their lives (8-12 years) on diets with either corn oil or coconut oil (no dietary cholesterol) as the fat source. As expected, the animals fed coconut oil had elevated total plasma cholesterol, LDL cholesterol, plasma TGs, and apo B levels. In short-term (8-wk) experiments, the monkeys were switched to diets with one of the following fat sources: their original fat source plus cholesterol; lard; butter; tallow; or fish oil plus the original fat source in a 2-to-1 ratio. Cholesterol was added to the plant fat diets to make it approximately equivalent among diets. Relative to the corn oilbased diets, butter feeding caused the greatest increases in plasma LDL cholesterol, TGs, and apo B, but compared with feeding the coconut oil-based diets, butter feeding induced significant decreases in total plasma cholesterol, LDL cholesterol, HDL cholesterol, and apo A-I. Of the other fat sources, fish oil was the most hypolipidemic (Pronczuk et al., 1991). Thus, the effect of butter as a single fat source can be positive or negative, depending on the point of reference. Because of the dramatic effects of fish oil (i.e., lowering of total cholesterol, HDL cholesterol, and apo A-I in all monkeys and lowering of LDL cholesterol and TGs in monkeys originally fed coconut oil), seeing whether addition of modest amounts of fish oil to the butter diets would influence the outcome would have been interesting. These experiments with single fat sources logically could lead to exploration of effects of various fat mixtures.

ROLE OF MILKFAT IN BALANCED DIETS

171

2. Role of Monounsaturated Fatty Acids The preceding discussion focused primarily on comparisons between SFA- and PUFA-rich dietary fat sources. Over the years, much attention has been paid to effects of varying dietary PUFA/SFA ratios, yet in the typical American diet MUFAs (predominantly oleic acid) make as large a contribution to energy intake as do SFAs and a much larger contribution than PUFAs (NRC, 1989; Ganji et al., 1992). In some parts of the world, for example, the Mediterranean, MUFAs make up a large portion of energy intake from fat. In addition to attempting to discern the cholesterolemic effects of MUFAs, distinguishing between cismonounsaturates (the predominant monounsaturated fatty acids, found naturally in plant and animal fats) and trans-monounsaturates (the major trans fatty acids found in most partially hydrogenated vegetable oil products) is important. a. Cis Monounsaturates. The Keys and Hegsted equations treated MUFAs as neutral in terms of their effect on total serum cholesterol. In more recent years, numerous clinical trials have been conducted to ascertain how MUFA-rich diets compare with PUFA-rich or low-fat diets. Some researchers have concluded that MUFA-rich diets are more beneficial than PUFA-rich diets in their effects on blood lipid profiles (Mattson and Grundy, 1985; Mata et al., 1992). Mattson and Grundy (1985) compared diets very rich in SFAs, PUFAs, ‘or MUFAs obtained from palm oil, safflower oil, and high-oleic safflower oil, respectively. The PUFA and MUFA formulas lowered total and LDL cholesterol levels relative to the palm oil formula, but only the PUFA diet lowered HDL cholesterol significantly. Therefore, these investigators reasoned that MUFAs may be preferable to PUFAs because they were as effective in lowering the atherogenic cholesterol fractions but did not alter HDL cholesterol (Mattson and Grundy, 1985). Mata et al. (1992) also found that MUFA-rich diets had beneficial effects on blood lipid profiles of normolipidemic women. The subjects received diets (36 en% from fat) rich in SFAs for 4 wk, followed by sequential 6-wk periods of diets rich in MUFAs and PUFAs. The MUFA- and PUFA-rich diets lowered total and LDL cholesterol similarly, but HDL cholesterol and apo A-I levels were higher after the MUFA-rich diet. Other research groups found that MUFA-enriched diets were as effective or nearly as effective as PUFA-enriched diets in lowering total and LDL cholesterol levels (Mensink and Katan, 1989; Berry et al., 1991; Wahrburg et al., 1992). In these studies, HDL levels were unchanged by

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LOUISE A. BERNER

any of the diet interventions, although Wahrburg et al. (1992) found that apo A-I levels were higher when a MUFA-rich diet was fed. The differences in observed effects of MUFA- and PUFA-rich diets on HDL levels may relate to the very high levels of PUFAs fed in the study by Mattson and Grundy (1985) or to the lower baseline cholesterol levels of the subjects in the later experiments (Mensink and Katan, 1989; Berry et al., 1991; Wahrbwg et al., 1992). Two clinical trials suggest that MUFA-rich diets may not be advisable. Chang and Huang (1990) reported that 8 normolipidemic young men had lower TG and LDL cholesterol levels after eating diets rich in soybean and coconut oils than after eating diets rich in soybean and olive oils. The PUFA/SFA ratios of the two diets were identical at 1.0, but the diets had different PUFA + MUFA/SFA ratios. Dreon and colleagues (1990) fed diets with 30 en% as fat but rich in PUFAs (primarily from safflower and corn oil) or MUFAs (primarily from olive and peanut oils) to 34 adults. The only significant differences in lipoproteins were that HDL2 level was 50% higher on the PUFA diet and HDL3 was 7% higher on the MUFA diet, a result that is not favorable for MUFAs. Also, apo B was significantly lower on the PUFA than on the MUFA diet (Dreon et al., 1990). The subjects studied by Chang and Huang (1990) and Dreon et al. (1990) had low cholesterol levels compared with those in the studies by Mattson and Grundy (1985), which may help explain why MUFAs do not consistently appear to improve plasma lipid profiles. In fact, this explanation may apply to the myriad of dietary fat-serum lipid studies conducted in normolipidemic young subjects. An important issue is whether individuals with elevated initial blood cholesterol levels respond the same as normal individuals do to dietary treatment. In spite of varying results from human studies, the bulk of evidence suggests that MUFA-rich diets do not have adverse effects on blood lipids. Groups such as the Committee on Diet and Health (NRC, 1989) and the NCEP Population Panel (Carleton et al., 1991) regard MUFAs as neutral or beneficial. Limits are placed on recommended SFA and PUFA intakes but MUFAs can “make up the difference” to account for up to a total of 30 en% as fat. In fact, some researchers have suggested olive oilor MUFA-rich, high-fat (38 en%) diets as realistic palatable alternatives to low-fat diets (Grundy, 1989; Sacks and Willett, 1991). Several studies support this suggestion. Grundy and co-workers (1988) compared wholefood diets high in both SFAs and cholesterol with diets low in cholesterol and ( 1 ) high in MUFAs from high-oleic safflower oil and peanut butter or (2) low in total fat and SFAs. The high-MUFA diet was as effective as the low-fat diet in lowering total and LDL cholesterol levels, but did not cause the significant reduction in HDL cholesterol caused by the low-fat

ROLE OF MILKFAT IN BALANCED DIETS

173

diet. The change in fatty acids themselves may have caused the reduction in serum cholesterol, since dietary cholesterol also was lowered from 900 to 200 mg/day in switching from the SFA-rich to the other diets. Nevertheless, the results do show that eating a low-fat (20 en%), low-cholesterol diet was not necessary to obtain optimal benefit to serum lipoproteins; a MUFA-rich diet at 40 en% was at least as desirable (Grundy et al., 1988). In a study of 36 healthy men, a MUFA-rich diet (38 en% from fat, 18 en% from MUFAs) was as effective as a Step 1 diet in lowering total and LDL cholesterol levels relative to an “average American diet” rich in SFAs (Ginsberg et al., 1990). Baggio et al. (1988) found that an olive oil-rich, high-fat diet (38 en%) lowered total plasma cholesterol, TG, and apo B levels relative to a low-fat diet (28 en%) without affecting HDL levels. However, the order of the two diets was not randomized in the study. Berry et al. (1992) gave further support to the idea that MUFA-rich diets are as good as or better than low-fat diets with a 12-wk, randomized, crossover design trial of 17 young men. Total and LDL cholesterol levels were significantly lower when the students ate a diet rich in MUFAs (32.5 en% from fat and 50.5% from carbohydrate) compared with a diet rich in carbohydrates (18.3 en% from fat and 64.9% from carbohydrates). Colquhoun et al. (1992) found significant decreases in LDL cholesterol and apo B when 15 women were switched from their typical diets (33 en% from fat, 13.6 en% from SFAs, 13.9 en% from MUFAs) to a higher fat diet enriched in MUFAs from avocados (36 en% from fat, 10.8 en% from SFAs, 20 en% from MUFAs) but not when they received low-fat diets (20.8 en% from fat, 7.2 en% from SFAs, 7.9 en% from MUFAs). Further, HDL cholesterol levels were lowered when the women switched to the low-fat but not the avocado-enriched diets. Results of trials comparing MUFA-rich and low-fat diets are summarized in Table XIII. The evidence strongly supports the idea that blood lipoprotein and apolipoprotein profiles are either unaltered or improved when MUFA-rich diets with total fat exceeding 30 en% replace low-fat diets. Olive oil, canola oil, peanut oil, and high-oleic sunflower or safflower oils (not readily available to consumers) are vegetable oils particularly rich in MUFAs. By altering feeding practices for dairy cows, oleic acid in milkfat can be increased 5 0 4 0 % and may approach 50% of milk fatty acids (Grummer, 1991). (See Section V of this chapter for further discussion.) About 48% of the MUFAs available in the United States food supply in 1988 came from fats and oils (including butter), yet nearly as large a contribution (45%) was made by dairy products, meat, poultry, fish, and eggs. As consumers decrease consumption of animal fats and SFAs, MUFA intake is also likely to decrease. In fact, Stephen and

TABLE XI11 EFFECTS OF MUFA-RICH COMPARED WITH LOWFAT DIETS ON SERUM LIPOPROTEINS A N D APOLIPOPROTEINS IN HUMANS

Effect of MUFA Compared with Lowfat Diet on” Study Grundy et al. ( I 988)

Subjects 10 men

X choli = 5.87 mmol/L crossover

Baggio et al. (1988) Ginsberg et al. (1990)

I 1 men

W choli

5.90 mmol/L crossover (not random) 24 men, I2/diet group TI choli = 4.58 mmol/L =

Diets” 40-7-27-6 (higholeic safflower + peanut butter) vs 20-7-7-6 38-10-25-3 (olive oil) vs 28- 12- 13-4 38-10-18-10 (mixed foods) vs

Cholesterol

LDL C

HDL C

apo B

apo A 4

NS

NS

inc

-

-

dec

dec

NS

dec

NS

NS

NS

NS

NS

NS

NS

dec

NS

-

-

NS

NS

inc

dec

NS

30-10-10-10

Berry et al. (1992)

17 men W choli = 4.00 mmol/L 2 crossovers

Colquhoun et al. ( 1992)

I5 women 51choli = 6.10 mmol/L crossover

32-7-17-7 (olive oil. avocado, almonds) and 18-5-7-6; each vs baseline values 37-11-20-6 (avocado) vs 21-7-8-6

Numbers for diets denote en% from total fat, SFAs, MUFAs, and PUFAs, respectively. Major sources of MUFAs for MUFA-rich diets noted parenthetically. C, Cholesterol; NS, no significant difference between high-MUFA and lowfat diets; dec, decrease on high-MUFA diet ( p < 0.05); inc, increase on high-MUFA diet ( p < 0.05); -, no measurement made.

”

ROLE OF MILKFAT IN BALANCED DIETS

175

Wald (1990) showed that intake of MUFAs in the United States has declined from about 20.8 en% in 1950-1959 to 12.4 en% in 1980-1985; over the same time period, a shift has occurred toward a higher proportion of fat from vegetable than from animal sources in the United States food supply (Raper, 1991).

b. Trans Fatty Acids. As discussed in the first part of this chapter, partial hydrogenation of vegetable oils to make shortenings, frying fats, and margarines results in the formation of varying amounts of unsaturated fatty acids with trans double bonds. Although trans dienes may be found at significant levels, trans monoenes constitute most of the trans fatty acids in hydrogenated vegetable oil products in the United States (Enig et a f . , 1990), so the effects of trans fatty acids on blood lipids are discussed here. Mattson et a f . (1975) reported that trans fatty acids did not have an adverse effect on blood cholesterol or triglycerides in normocholesterolemic men fed liquid formula diets. These researchers designed two liquid diets with approximately equivalent levels of different fatty acids, except one diet contained a test fat with all cis fatty acids and the other a test fat with 34% by weight of trans 18: 1, 9% by weight of cis,rrans 18:2, and 1% by weight of trans,trans 18:2 fatty acids. The test fats supplied 35% of energy intake. All 37 subjects drank the “cis” diet for 4 wk. Then 17 subjects continued for another 4 wk while 16 men switched to the “trans” formula for that period. No significant differences were seen in plasma cholesterol or TG levels between the men consuming “cis” and those consuming the “trans” formulas (Mattson et a f . , 1975). Drawbacks of the study included the use of liquid diets, the lack of a crossover design, and blood measurements limited to total cholesterol and TG levels. In two earlier studies comparing the cholesterolemic effects of butter and margarines, the investigators also concluded that the hydrogenation process, and therefore trans fatty acids, did not have an adverse effect on blood lipid levels (Beveridge and Connell, 1962; McOsker et a f . , 1962). However, neither of these studies was designed to test the effects of trans fatty acids per se. In both cases, liquid formulas were prepared in which the fat was supplied by butter or partially hydrogenated vegetable oil products. McOsker et al. (1962) found that butterfat caused a significant elevation in total cholesterol levels compared with cottonseed oil and several partially hydrogenated vegetable oil products (containing about 29% total trans fatty acids, by weight) when the fat sources made up 41% of the energy of formula diets. The researchers neglected to mention or account for differences in dietary cholesterol, however, and

I76

LOUISE A. BERNER

clearly the butterfat diets would have contained cholesterol whereas the other diets would have been devoid of it. The study by Beveridge and Connell (1962), in which they compared butter with corn oil and eight margarines varying in trans fatty acid content, had the same limitation. Also, the feeding periods were only 8 days, which is too short a time for blood lipid levels to stabilize. In a review of animal experiments, Kritchevsky (1982) concluded that trans fatty acids were slightly hypercholesterolemic compared with their cis counterparts, but were not atherogenic, at least in rabbits and vervet monkeys. Two well-designed human studies tested the effects of trans isomers of oleic acid on serum lipids. Mensink and Katan (1990) recruited normolipidemic adults and fed them, in random order, three diets identical in all respects except that 10 en% was provided as either oleic acid, trans isomers of oleic acid, or SFAs (mostly lauric, myristic, and palmitic acids). Three different margarine or shortening-typeproducts were made to supply the different fat types. The oleic acid was provided by oleicrich sunflower, olive, and canola oils, whereas the trans isomers were prepared by isomerizing oleic acid-rich sunflower oil. The trans fatty acid diets contained a variety of positional trans 18 : 1 isomers, the most prominent being trans-9-octadecenoic acid (elaidic acid), trans- 10octadecenoic acid, and trans-I 1-octadecenoic acid (vaccenic acid). Lightly hydrogenated palm and palm kernel oils provided the SFAs. Each margarine was incorporated into a bread recipe. A strength of the study was the use of real-food diets as well as the crossover design. A limitation may be that the three margarine products themselves did not reflect fats consumed normally because part of the production process involved interesterification of some of the fat sources. HDL cholesterol levels were significantly lower by 7 mg/dl (0.17 mmol/L) on the trans diet than on the other two diets (Table XIV). LDL cholesterol and triglycerides were elevated on both the SFA-rich and the trans diet relative to the oleic acid-rich diet. In addition, apo A-I was significantly lower on the trans diet than on the SFA- or oleic acid-rich diet, and apo B was raised by both the trans and the SFA diet relative to the higholeate diet (Table XIV). The researchers concluded that “the effect of trans fatty acids on the serum lipoprotein profile is at least as unfavorable as that of the cholesterol-raising saturated fatty acids, because they not only raise LDL cholesterol levels but also lower HDL cholesterol levels” (Mensink and Katan, 1990). In a subsequent study, the same laboratory confirmed the apparently adverse effects of trans fatty acids on blood lipoproteins (Zock and Katan, 1992). Three diets were fed to 56 normolipidemic adults, in ran-

I77

ROLE OF MILKFAT IN BALANCED DIETS

TABLE XIV EFFECT OF TRANS ISOMERS OF OLEIC ACID ON PLASMA LIPOPROTEINS AND APOLIPOPROTEINS~'

Diet Measurement

High oleic

High trans

High SFA

Total cholesterol (mmol/L) LDL cholesterol (mrnol/L) HDL cholesterol (mmol/L) APOA-I (g/L) Apo B (g/L)

4.46' 2.611 1.42I I .331 0.94'

4.722 3.042 1.25' 1 .242

5.003 3.143 1.42l 1.34' I.a3

1.072

Data from Mensink and Katan (1990). Within a row, values with different superscripts are significantly different from each other 2 most cases, p
dom order. Each diet contained approximately 40 en% from fat. One diet had 12 en% as linoleic acid from a commercial margarine, one had 12 en% as stearic acid from a mix of fully hydrogenated sunflower oil and other oils, and the third diet supplied 7.7 en% as trans fatty acids, primarily elaidic acid, produced by a controlled hydrogenation of high-oleic acid sunflower oil. Compared with the linoleic acid-rich diet, both other diets led to higher levels of total and LDL cholesterol, whereas HDL cholesterol levels were lower (Zock and Katan, 1992). Apo B levels were highest and apo A-I levels were lowest when the trans fatty acid diet was fed. Again, interesterification of the fat sources used in the stearic acidrich diet may be cause to question the relevance of the findings. Nevertheless, trans fatty acids, at least elaidic acid, appear to have an adverse effect on serum lipoproteins and apolipoproteins when fed at relatively high levels. In contrast, lower levels of trans fatty acids did not appear to be deleterious in another experiment. Diets with test fats from oil blends (which included partially hydrogenated oils) were compared with typical Australian diets. Although trans fatty acid intake was increased modestly by 4 en% on the test diets, LDL cholesterol decreased and HDL cholesterol levels did not change (Nestel et al., 1992). According to the researchers, this result was attributable to the high linoleic acid :palmitic acid ratio of the test diets; the trans fatty acids themselves were without effect. Current estimates of trans fatty acid intakes in the United States vary widely (Craig-Schmidt, 1992). Hunter and Applewhite (1991) calculated a trans fatty acid availability of 8.1 g/person/day in the United States whereas Enig et al. (1990) estimated an actual dietary intake range of

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LOUISE A. BERNER

11.1-27.6 g/person/day. Mensink and Katan (19%) fed trans fatty acids as 10% of energy; based on stated energy intake ranges, the subjects consumed a calculated 14.6-53.1 g trans fatty acids daily. Subjects in Zock and Katan’s study (1992) consumed an average of 24.5 g trans fatty acids per day, with a range of 11.2-35.8 g daily. These amounts of trans fatty acids are generally larger than, but not totally out of line with, high-end estimates of trans fatty acid intakes in the United States. At any rate, the studies have renewed interest in the health effects of trans fatty acids and may suggest some necessary modifications in diet recommendations in the future. The NCEP Population Panel recommends using “oils, margarines, and shortenings with vegetable oils containing primarily unsaturated fatty acids” instead of more saturated fat sources such as tropical oils or butter (Carleton et al., 1991). However, trans monoenoic fatty acids currently can be included in the category of “unsaturated” fatty acids; many margarines, shortenings, and commercial frying fats are high in trans isomers. Thus, this advice may need revision. Kummerow (1991) suggested that “the substitution of margarine for butter . . . would not provide physiological benefit” because, although butter has a high percentage of SFAs, many margarines and shortenings are high in isomeric fatty acids (both positional and geometric isomers). Whether or not this is true, more research is needed on the effects of typical levels of trans fatty acids on blood lipid profiles and CHD risk. Moreover, comparing effects of different trans fatty acids (e.g., vaccenic acid, which is the main trans fatty acid in milkfat, and the range of trans fatty acid isomers produced during partial hydrogenation of vegetable oils) will be important. 3. Role of n-3 Polyunsaturated Fatty Acids

Reviewing in depth the effects of n-3 fatty acids, particularly from fish oils, on serum lipids and other factors contributing to CHD risk is beyond the scope of this chapter. However, some general information will be provided because n-3 fatty acids may exert beneficial effects in typical mixed fat diets. The effects of fish oils on plasma lipoproteins are not consistent (Kinsella et al., 1990). In groups of hypercholesterolemic and normolipidemic patients, serum total cholesterol, LDL cholesterol, apo B, TGs, and HDL cholesterol fell similarly when either salmon oil or safflower oil replaced butter in the diet of study subjects (Friday et al., 1991). In hypertriglyceridemic patients, consumption of graded levels of fish oil (MaxEPA) caused significant lowering of total cholesterol and TGs, but raised LDL cholesterol (Harris et al., 1990). Kestin et al. (1990) compared the effects of diets rich in n-3 fatty acids from either plant

ROLE OF MILKFAT IN BALANCED DIETS

179

(a-linolenic acid) or marine [eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA)] sources with a linoleic acid-rich diet. In mildly hypercholesterolemic men, n-3 fatty acids from fish lowered TGs and VLDL cholesterol, but raised LDL cholesterol. On the other hand, the a-linolenic acid was without effect on blood lipids (Kestin et al., 1990). Therefore, fish oils appear to lower TG levels fairly consistently but the effect on serum cholesterol levels is more variable (Kinsella et al., 1990). Childs et al. (1990) provided evidence that the type of fish oil is important in determining effects on plasma lipids. When subjects were fed EPA-rich pollock oil, an unfavorable effect on cholesterol was seen because LDL cholesterol did not change and HDL cholesterol dropped significantly. On the other hand, DHA-rich tuna or salmon oils decreased LDL cholesterol more than HDL cholesterol. Again, all fish oils depressed serum VLDL and TGs (Childs et al., 1990). Unlike the case with plasma lipids, platelet aggregation and thrombotic tendency (discussed more in Section III,C) are consistently decreased when fish oils are consumed (Kinsella et al., 1990). The major n-3 fatty acids in fish oils, EPA and DHA, replace arachidonic acid in platelet phospholipids and therefore reduce production of thromboxane A2, a proaggregatory eicosanoid. At the same time, fish oils stimulate production of antiaggregatory prostaglandin 13. On the other hand, linoleic acid (an n-6 PUFA) can be converted to arachidonic acid and therefore can increase thromboxane A2 synthesis. Thus, n-3 PUFAs may counteract potentially negative effects of n-6 PUFAs on thrombosis. In addition, by modulating cell-cell interactions, n-3 PUFAs may help counteract negative effects of increased plasma LDL levels caused by certain SFAs (Kinsella et al., 1990). Inclusion of n-3 PUFAs from fish and plant sources may have beneficial effects in a balanced diet.

B. EFFECTS OF DIETARY FAT TYPE ON LDL MODIFICATION A relatively new and exciting area of research studies how dietary fat quality influences LDL modification. As reviewed by Steinberg et al. (1989), uptake of LDL by monocytes and macrophages to form foam cells and subsequently fatty streak lesions requires that LDL first be modified. The modification of LDL can involve peroxidation of PUFAs in LDL lipids, an event that requires low levels of copper or iron and can be inhibited in uitro by chelators such as ethylene diamine tetraacetic acid (EDTA) or antioxidants such as butylated hydroxytoluene (BHT) and vitamin E. Apparently oxidation of LDL occurs in the artery wall rather than in the circulation (Steinberg et al., 1989). In the Watanabe heritable hyperlipidemic (WHHL) rabbit, 15-lipoxygenase colocalizes

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LOUISE A. BERNER

with oxidized LDL in macrophage-rich areas of atherosclerotic lesions, suggesting a role for lipoxygenase in LDL oxidation (Yla-Herttuala et af., 1990). Treatments that prevent or reduce LDL oxidation (e.g., by inhibiting 15-lipoxygenase in macrophages) should make LDL less atherogenic. In animal models, probucol has proven to slow atherogenesis by preventing LDL modification without altering plasma LDL level (Steinberg et af., 1989). What is the potential for dietary fat to influence oxidative modification of LDL? In a pioneering study, Parthasarathy et al. (1990) fed rabbits diets in which high-oleic acid sunflower oil or conventional sunflower oil (rich in linoleic acid) was the dietary fat. The main differences in fatty acid composition between the two diets were in the levels of oleic and linoleic acids. LDLs isolated from the rabbits fed the high-oleic sunflower oil were enriched in oleic acid and were very resistant to copperinduced oxidation in uitro, compared with LDL from rabbits fed linoleate-rich sunflower oil. Also, after incubation with copper, the LDLs were not subject to degradation by mouse macrophages whereas the LDLs from rabbits fed conventional sunflower oil were. Thus, a dietary strategy to decrease LDL oxidation may be to feed oleic acid-rich diets (Parathasarathy et al., 1990). In a study of young healthy males, Berry et af. (1991) compared effects of diets rich in MUFAs (from olive oil, avocado, and almonds) with those of diets rich in PUFAs (from safflower oil, soy oil, and walnuts). The PUFA-rich diet was marginally better than the MUFA-rich diet at lowering total and LDL cholesterol levels, although levels decreased from baseline on both diets. However, the researchers also assessed the presence of thiobarbituric acid (TBA)-reactivesubstances in native LDL and after incubation of LDL with copper or smooth muscle cells from bovine aorta. In these in uitro experiments, TBA-reactive substances increased significantly in both native and “conditioned” LDL from subjects fed PUFA-rich relative to those of MFA-rich diets, suggesting a lower susceptibility of the LDL to oxidative stress (Berry et af., 1991). The same researchers found that LDLs from young men fed a low-fat, high-carbohydrate diet were metabolized by macrophages more extensively than LDLs obtained after the men were fed diets rich in MUFAs, implying that the MUFA-rich diet protected the LDL from macrophage uptake (Berry et af., 1992). Bonanome et af. (1992) studied 1 1 healthy males fed oleic acid- and linoleic acid-enriched diets for 3 wk each in a crossover design. Plasma LDLs were obtained and their susceptibility to lipid oxidation was measured in uitro. The resistance to lipid peroxidation of LDL was higher after the MUFA diet phase, and was related to the oleic : linoleic acid ratio of the LDL. Reaven et al. (1991) conducted a similar study and found that LDLs from subjects fed an oleate-rich

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diet were less degraded by macrophages than LDLs from subjects fed linoleate-rich diets. These studies are consistent in finding that oleic acid-rich diets have beneficial effects on LDL oxidation and uptake by macrophages. Compared with baseline values, LDL isolated from adults fed fish oilenriched diets for 4 wk had more TBA-reactive substances and were metabolized more by macrophages (Haratz et al., 1991). This study suggests indiscriminate use of fish oil supplements may have an adverse effect on atherosclerosis. On the other hand, another group of researchers suggested fish oil may have a beneficial effect. Hamsters were fed fish oil-rich diets; then copper-oxidized human LDLs were injected into the animals. After the fish-oil rich diet, the oxidized LDLs adhered less to the endothelium of arterioles and venules, possibly indicating that fish oil can protect against the earliest stages of atherogenesis (Lehr et al., 1991). Thus, the net effect of dietary fish oils on LDL modification and atherogenesis is unclear. Conceivably, diets enriched in SFAs might give rise to LDLs resistant to oxidation, as do oleic acid-enriched diets. In rabbits fed highcholesterol diets, metabolism of LDL and remnants by macrophages was greater and TBA-reactive substances were higher after safflower oil-rich diets than after butter-rich diets. In other words, having butter as the fat source instead of safflower oil protected against LDL modification and uptake by macrophages (Haratz et al., 1988). However, Steinberg et al. (1989) suggested that two factors are working together to enhance atherogenesis: (1) high levels of serum LDL and (2) oxidative modification of LDL, which would be more likely as total LDL levels increase. Therefore, if SFAs are fed at high levels that enhance LDL concentration, oxidative modification may not be improved significantly since more LDL would be available to undergo such modification. More likely, some optimal balance of SFAs, MUFAs, and PUFAs is required to achieve optimal benefits to both LDL levels and LDL oxidation. C. EFFECTS OF DIETARY FAT TYPE ON THROMBOSIS TENDENCY The most widely investigated relationship between dietary fats and CHD risk surrounds the influence of fats on serum lipoprotein levels (and, in turn, how lipoproteins such as LDLs contribute to atherosclerosis). An additional area of research concerns how fat type affects thrombosis tendency. Thrombosis, the formation of a clot from constituents of the blood, can lead to myocardial infarction when it occurs in coronary arteries. In fact, although atherosclerosis causes a decrease in blood flow to the heart, the actual thrombotic event is often lethal in patients with

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advanced atherosclerosis (Kinsella et al., 1990). The thrombotic process itself is highly complex, involving injury to the vascular endothelial surface, aggregation of blood platelets, and other factors initiating plasma coagulation (Mann, 1992). Ex vivo measures of platelet aggregation, platelet and endothelial lipids, synthesis of proaggregatory thromboxane A2 (TXA2), and production of antiaggregatory prostacyclin often are used as indicators of the thrombotic tendency in response to dietary fats. However, one of the conclusions of a workshop on dietary fatty acids and thrombosis was that “many currently used methods and surrogate endpoints for measuring thrombotic tendency are of questionable significance. . . . Valid indicators of thrombotic tendency must be identified and reliable methods for assessing these endpoints must be developed” (Hoak and Spector, 1992). Although this limitation should be kept in mind, a brief discussion of dietary fat and thrombosis follows. Fish oils exert an antithrombotic effect by reducing thromboxane synthesis and influencing metabolism of other eicosanoids in platelets (Kinsella et al., 1990). On the other hand, the NRC Committee on Diet and Health states, “There is no conclusive evidence that hemostatic variables such as platelet function and blood coagulation as measured in vitro are influenced by dietary intake of SFAs, MUFAs, or n-6 PUFAs” (NRC, 1989). Participants at a workshop concluded that “although the antithrombotic effects of n-3 fatty acids have been demonstrated and plausible mechanisms of action proposed, there is no clearly established prothrombotic effect of saturated dietary fatty acids” (Hoak and Spector, 1992). However, some publications make fairly strong statements that SFAs are proaggregatory and thrombotic (Renaud et al., 1986; Naughton et al., 1988). A review of a few human and animal experiments that focused on comparisons of SFA-rich fats with other fat sources suggests that the situation is complicated. In a long-term study of French farmers, Renaud et al. (1986) examined platelet function at baseline and 1 , 2, and 3 yr after half of the 98 men decreased saturated fat intake from about 16 en% to 10 en%. The men were told to eliminate butter and cream from their diets, and replace them with vegetable oils and soft margarines. The farmers also were counseled to remove visible fat from meat and to eat more fruits and vegetables. The control group was told to continue their regular dietary practices. At 1-yr intervals, the researchers performed several measures of platelet function, such as clotting time of platelet-rich plasma and platelet aggregation. Results showed that the farmers instructed to modify their diets did so; the PUFA/SFA ratio increased from 0.32 to 0.97 at the end of 1 yr and to 0.7 at the end of years 2 and 3. Plasma clotting time was prolonged significantly after 1 yr in farmers on the modified diets but was not different in the control group of farmers (Table XV). Aggrega-

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TABLE XV EFFECT OF DIETARY FAT MODIFICATION ON PLASMA CLOTTING TIME I N FRENCH FARMERS“

Usual diet group”

Modified-diet group

Measurement

Baseline

Year I

Baseline

Year I

Year 2

Diet PUFA/SFA ratio Plasma clotting time (sec)

0.33 243

0.36 240

0.32 268

0.97 348

0.69 356

~~

Data from Renaud et al. (1986). Diets were modified after the first year so comparable data for year 3 are not available in that group. a

”

tion of platelets in response to thrombin was depressed after 1 yr and in response to collagen after 3 yr of diet modification. On the other hand, aggregation of platelets in response to ADP increased significantly 1 yr after subjects switched to high-PUFA diets. The farmers were advised to decrease their intake of PUFAs during the next 2 years of the study because the researchers thought the increased platelet aggregation in response to ADP might have been a result of the high PUFA intake. Indeed, at the end of years 2 and 3, when the farmers consumed diets with a PUFA/SFA ratio of 0.7 instead of 0.97 at the end of year 1, no effect of dietary modification was seen on ADP-stimulated platelet aggregation. Renaud and co-workers (1986) concluded that SFAs had an adverse effect on platelet function and thrombotic tendency. Renaud and de Lorgeril(l989) warned that intakes of both SFAs and PUFAs should be low because high PUFA diets also adversely affect platelet reactivity. Kwon et al. (1991) examined platelet aggregation and fatty acid composition in healthy men fed diets with varying fat sources. First, all subjects received, for 3 wk, a baseline diet with SFAs at a level approximating the level in typical United States diets (13 en%). Then, half the subjects were fed a diet rich in safflower oil and half a diet rich in canola oil for the remaining 8 wk of the experiment. Although platelet aggregation decreased after 3 wk on either the safflower oil or the canola oil diet, the degree of platelet aggregation returned to baseline levels by the end of the 8-wk experiment. No persistent effect of fat type on platelet aggregability was measured, nor was there an effect of diet on TXB2 production in platelet-rich plasma (TXB2 is a product of TXA2 and was measured because it is not as labile). The investigators did note differences in platelet phospholipid fatty acids as a result of diet, and also noted associations between platelet aggregation and fatty acid content of platelet phospholipids. One interesting finding was that the amounts of palmitic

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and stearic acids in platelet phospholipids were associated with a decrease in collagen-induced platelet aggregation. Kwon et al. (1991) suggested this result as possible evidence that not all SFAs are prothrombotic. In three species of monkey, Pronczuk et al. (1991) compared the effects of different dietary fats on collagen-induced platelet aggregation. Fish oil clearly reduced platelet aggregation compared with all other fats, whereas long-term (8-12 yr) coconut oil feeding increased aggregation relative to corn oil and short-term (8 wk) butter feeding increased aggregation relative to lard and corn oil. Renaud and Gautheron (1975) fed rabbits diets with 10% butter or 5% butter plus 4.5% cocoa butter, coconut oil, olive oil, or corn oil. Cholesterol was added also at a level of 0.1% of the diets. The clotting time of platelet-rich plasma from the rabbits was highest to lowest in the following order: corn oil + butter, olive oil + butter, coconut oil + butter, butter alone, and cocoa butter + butter. The researchers suggested that stearic acid is the most thrombogenic dietary fatty acid, in part because the ratio of stearic:linoleic acid in the diet was associated with more rapid clotting (Renaud and Gautheron, 1975). On the other hand, Masi et al. (1986) reported that platelet aggregability did not differ among rabbits fed diets with butter, olive oil, or corn oil as the major fat source. Further, corn oil feeding reduced the release of prostacyclin from arteries relative to olive oil and butter. Thus, no consistent positive effect of corn oil (or negative effect of butter) on thrombotic tendency has been demonstrated. Foxall and Shwaery (1990) fed swine diets high in cholesterol with either butterfat or MaxEPA fish oil as the fat source. These researchers measured the ability of platelets from the swine to adhere to endothelial cells, a process that, in uiuo, presumably would promote thrombosis. No effect of dietary fat on platelet adhesion to endothelial cells was measured. However, the researchers did show that monocyte adhesion to porcine aortic endothelial cells was higher in swine fed fish oil than in those fed butterfat. This observation is not related to the thrombogenic effects of the fat sources, but monocyte adhesion to endothelial cells is an initiating event in fatty lesion formation (Foxall and Shwaery, 1990). Numerous groups have used rats as animal models in similar studies. When rats were fed butter-rich diets in place of low-fat diets or diets rich in corn oil or lard, the concentration of arachidonic acid (AA) in platelet phospholipids decreased whereas the levels of EPA increased (Morita et al., 1984; Naughton et al., 1988; O’Dea et al., 1988). The rat readily converts linoleic acid to AA, which explains why diets low in linoleate (e.g., when butter is the main dietary fat) lead to low levels of AA in platelet lipids. TXAz is produced from AA in platelets, so a decrease in AA levels would be expected to decrease levels of this proaggregatory

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substance and reduce platelet aggregation. However, although platelet phospholipid fatty acid levels can be altered by dietary fat in rats, platelet aggregation is not consistently affected. Morita et al. (1984) showed that rats fed butter and EPA had significant inhibition of collageninduced platelet aggregation compared with rats fed corn oil and EPA, yet fat source (butter or lard) and level (10, 30, and 50 en%) had no influence on platelet aggregation in another study (O’Dea et al., 1988). Moreover, O’Dea et al. (1988) found that arterial prostacyclin production decreased in rats as dietary butter level increased. Prostacyclin, an antiaggregatory compound, is formed from AA in endothelial cells; the decrease in prostacyclin production paralleled a reduction in AA in tissue and plasma phospholipids (O’Dea el al., 1988). Thus, feeding of butter and other fats has “the potential for complex ramifications in prostanoid formation” and tendency for thrombosis, but what those ramifications are is not clear at present. Even the feeding of fish oils, widely thought to decrease platelet aggregation and thrombotic tendency (Kinsella, 1990), can have varying effects depending on the specific fatty acids in the basal diet. For example, Garg et al. (1990) reported that fish oils were effective in reducing AA levels (presumably the means by which n-3 fatty acids are antithrombotic) as long as the basal diet was rich in SFAs (hydrogenated beef tallow) but not when the diet was linoleate rich (safflower oil). More experiments will be required before conclusions can be drawn about the thrombogenic nature of different fatty acids, particularly SFAs. D. OTHER POTENTIAL DETERMINANTS OF RESPONSE TO DIETARY FAT Several other factors in addition to chain length, saturation, and geometric isomers of unsaturated fatty acids may influence response to dietary fat. In this section, the importance of three potential factors will be considered: (I) triglyceride structure; (2) fat quantity compared with composition; and (3) individual genetic make-up. I.

Triglyceride Structure of Dietary Fats

Although hundreds or even thousands of TG species exist in many fat sources, all species have not been identified and quantified for most dietary fats (Small, 1991). As discussed for milkfat in the first part of this chapter, the distribution of fatty acids on the glyceride backbone has been shown not to be random (Mattson and Lutton, 1958). Relatively little is known about how TG structure affects lipoprotein composition and metabolism, although two reviews shed some light on the subject

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(Kritchevsky, 1988; Small, 1991). One reason dietary TG structure may influence lipoprotein metabolism is the way dietary fat is digested and absorbed. The action of digestive enzymes forms free fatty acids plus 2-monoacylglycerols, most of which are absorbed into the intestinal mucosa, resynthesized into TGs, packaged into chylomicrons, and secreted into lymph (Mattson and Volpenhein, 1964; Small, 1991). Hydrolysis and absorption of fed TGs is apparently more efficient when short-chain fatty acids are in the 1 and 3 positions, as shown by Jandacek et al. (1987) in a comparison of 2-linoleolyl-1,3-dioctanoyl glycerol and 2-linoleolyl-1,3dioleoyl glycerol. If the free fatty acids have 10 carbons or fewer, they are absorbed via the portal vein and are metabolized rapidly in the liver (Small, 1991); thus, they do not become part of serum lipoproteins. [Recall that the vast majority of short-chain fatty acids in milkfat is present in the 3 position of milkfat TGs. The mode of digestion of milkfat is unique because fatty acids with 4-10 carbons are cleaved from the 3 position of milkfat TGs by the action of gastric lipase, and are absorbed largely in the stomach (Jensen, 1992).] The TGs resynthesized in the intestinal mucosa mostly retain the original fatty acid in the 2 position (Akesson et al., 1978; Small, 1991). In relation to the fat digestion and absorption scheme just described, TG structure may have an important influence on serum lipids (and thus on CHD risk) in at least two conceivable ways. First, the fatty acids at the 1 and 3 positions which are cleaved from the glyceride backbone in the lumen of the small intestine may, in some instances, become unavailable for absorption (Small, 1991). For example, if the fatty acids are long-chain SFAs, especially stearic acid, their melting points are high and they may precipitate in the intestine. If enough calcium is present, calcium soaps with even higher melting points could form and precipitate, thus inhibiting absorption of the fatty acid (Small, 1991). Filer et al. (1969) fed infants formulas containing natural lard (palmitic acid is primarily in the 2 position of lard TGs) or randomized lard (with palmitic acid randomly distributed along the glycerol backbone). The infants fed the randomized lard excreted much more fat, especially palmitic acid, than did infants fed natural lard (Filer et al., 1969). This study lends support for the hypothesis that palmitic acid cleaved from the 1 and 3 positions of lard is less absorbable than palmitic acid present as a 2monoacylglycerol. Similarly, Mattson et al. (1979) showed that when synthetic TGs containing stearic and oleic acids were fed to rats, stearic acid was well absorbed when present in the 2 position but not the 1 and 3 positions, especially in the presence of magnesium and calcium. However, oleic acid was equally well absorbed, regardless of position on the TG. Cocoa butter, with 35% of its fatty acids as stearic acid, is

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digested poorly compared with corn oil and palm kernel oil (Apgar et al., 1987; Chen et al., 1989). Only a small percentage of the stearic acid is present in the 2 position of cocoa butter TGs (Kritchevsky, 1988), which might explain the poor digestibility. Poor absorption of long-chain SFAs may cause a fat to be less hypercholesterolemic. For example, cocoa butter is not hypercholesterolemic compared with many other fats (Kritchevsky, 1988). Also, plasma cholesterol levels were lower in rats fed tristearin than in those fed safflower oil or triolein (Feldman et al., 1979), perhaps, in. part, because of poor absorption of stearic acid or depressed cholesterol absorption (Feldman et al., 1979; Chen et al., 1989). A second possible way that TG structure may be important to lipid metabolism is by causing changes in chylomicron removal that ultimately could lead to changes in serum lipoprotein levels (Small, 1991). The fact that the fatty acid in the 2 position of fed TGs is largely preserved in that position in chylomicrons may be relevant here. Redgrave et al. (1988) fed a series of I ,2-dioleyl-3-acyl-sn-glycerols; the fatty acid in the 3 position was oleic, myristic, palmitic, stearic, arachidic (20 : 0), behenic (22 :01, or lignoceric (24:O) acid. Except for the SFAs with 20 or more carbons, which may have been absorbed poorly, the fatty acid composition of chylomicrons reflected the fatty acids fed. Chylomicrons from the fed rats were obtained and injected into other rats, and removal of the chylomicrons from the blood was measured. Uptake of chylomicron remnants from rats fed triolein was faster than from rats fed 00s and especially OOP, possibly because the different TG structures caused changes in remnant conformation and binding to hepatic receptors (Redgrave et al., 1988; Small, 1991). (00s refers to a TG with oleic acid at positions sn-1 and sn-2 and stearic acid at sn-3; OOP has palmitic acid at sn-3.) In a second experiment, the effects of feeding 00s and OSO were compared (Redgrave et al., 1988). As might be expected if some stearic acid from the 3 position was unavailable for absorption, less stearic acid was found in the 00s than in the OSO chylomicrons. Chylomicron remnant clearance and TG hydrolysis were much slower when chylomicrons from rats fed OSO were compared with those from rats fed 00s (Redgrave et al.,. 1991). If slow removal of chylomicron remnants has an adverse effect such as enhancing uptake of remnants into arterial walls (Small, 1991), then OSO might be expected to be more atherogenic than 00% and 00s and OOP more atherogenic than 000. Why stearic acid at the 2 position rather than the 3 position of the fed fat influenced clearance rate of chylomicrons is not known, nor do we know whether the same effect would be seen if other long-chain SFAs had been studied (e.g., OOP or OOM vs OPO or OMO). Such data may be useful because two of

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the “hypercholesterolemic” fatty acids in milkfat, lauric and myristic acids, are found predominantly at the 2 position of milkfat TGs; palmitic acid is distributed about equally between the 1 and 2 positions (Jensen et al., 1991). Also, palmitic acid is present primarily in the 2 position in lard (Filer et al., 1969; Yamamoto et al., 1971). Additional studies should be encouraged. Several researchers have attempted to assess the importance of TG structure to lipid metabolism by comparing effects of natural and randomized fats (Kritchevsky and Tepper, 1977; Myher et d., 1977; Mukherjee and Sengupta, 1981; Verdonk and Christophe, 1981; De Schrijver et al., 1991). Myher et al. (1977) found that randomized peanut oil was less atherogenic than natural peanut oil when fed to rabbits in a highcholesterol diet. The natural peanut oil had a greater proportion of linoleic acid in the 2 position and a smaller proportion of very-long-chain SFAs (arachidic, behenic, lignoceric) in the 3 position. The researchers hypothesized that the TG conformation made the native oil “metabolically more saturated” (Myher et al., 1977). On the other hand, Kritchevsky and Tepper (1977) found no differences in atherogenicity or serum cholesterol levels between native and randomized butter or native and randomized lard when these fats were fed to rabbits in highcholesterol diets. De Schrijver et al. (1991) compared lipid metabolism in rats fed native and randomized fish oil or native and randomized peanut oil and found no differences attributable to TG structure. Verdonk and Christophe (1981) fed native and randomized butter (60 g per day for 20 days) to 6 healthy volunteers; the randomized butter was hypocholesterolemic compared with natural butter. However, this conclusion is weakened because the investigators did not provide statistical analyses and all 6 subjects received the butters in the same order (Verdonk and Christophe, 1981). Mukherjee and Sengupta (1981) compared cholesterolemic effects of butter, butter-soy oil mixtures, and an interesterified buttersoy oil product. In both rats and humans, serum total cholesterol levels were lowest when the interesterified product was fed. Analyses of the new interesterified fat showed that some of the myristic acid from the sn-2 position of butter was shifted to the sn-1 and sn-3 positions of the new fat. Also, the new fat contained fewer trisaturated TGs than did butter. The researchers suggested that these changes were responsible for the hypocholesterolemic effect of the interesterified fat relative to butter alone or the butter-soy oil blend (Mukherjee and Sengupta, 1981). Unfortunately, details of the diet studies were not provided. The importance of TG structure to lipoprotein metabolism is largely unexplored. To date, results have been mixed but are suggestive enough to justify continued work on this complex subject.

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2 . Fat Quantity and Composition Health professionals have recommended reduction of total fat intake (i.e., less than 30% of kcal from fat) as well as changes in the quality of fat consumed (NRC, 1989; Carleton et al., 1991). However, questions remain about the relative effectiveness of changes in fat quantity compared with quality and about the extent to which total fat intake should be curtailed. One possible argument against low-fat diets comes from international epidemiological data showing that HDL levels decrease as the proportion of energy from total fat decreases (Knuiman et al., 1987). Grundy (1989) and Sacks and Willett (1991) suggest that diets with fat levels similar to those currently consumed in the United States (i.e., 35-40 en% from fat) might be appropriate, as long as MUFAs make up a large portion of the fat. The discussion of MUFA-rich diets vs. lowfat diets in Section II1,A suggests that low-fat diets may not be necessary or even desirable if the fatty acid composition of the diet is favorable (i.e., rich in oleic acid and limited in SFAs). Several researchers have compared effects of diets differing in fat quantity and composition on blood lipid and lipoprotein levels; McNamara (1992) provides a good review. Barr et al. (1992) compared effects of three different diets in young normolipidemic males. For 3 wk, 48 men received “average American diets” with 37 en% from fat and 16% from SFAs. Then, for the next 7 wk, the men were divided into three groups. One group continued on the average American diet, a second consumed a Step 1 diet with 30 en% from fat and 9% from SFAs, and the third group ate a modified Step 1 diet with 30 en% from fat and 14% from SFAs. Results showed that serum cholesterol and LDL cholesterol levels fell significantly from baseline only on the Step 1 diet low in SFAs (Barr et al., 1992). HDL cholesterol also was lower on the Step 1 low-SFA diet. These investigators concluded that lowering total fat without lowering SFA intake was not effective in improving blood lipid profiles, although mean baseline cholesterol levels were already less than 185 mg/dl (4.78 mmol/L) in these men. In contrast, Denke and Breslow (1988) found that low-fat diets with relatively high SFA intakes were as effective as low-fat low-SFA diets in improving blood lipids. In their study, 14 young volunteers were fed, in random order, one of three diets: (1) a high-fat very-high-SFA diet (42 en% from fat, 24 en% from SFAs); (2) an American Heart Association (AHA) Phase 2 diet (25 en% from fat, 6 en% from SFAs); and (3) an AHA Phase 2 diet with intermittent ingestion of high SFA meals (overall diet was 30 en% from fat, 13 en% from SFAs). Both reduced-fat diets lowered total cholesterol, LDL cholesterol, and apo B levels but only the traditional Phase 2 diet lowered

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HDL cholesterol (Denke and Breslow, 1988). Grundy et al. (1986) compared AHA Phase 1 with AHA Phase 3 diets (30 or 20 en% from fat, with equal distributions among SFAs, MUFAs, and PUFAs) in normolipidemic men. No benefit was seen from the Phase 3 diet; in fact, HDL cholesterol was lowered more on the Phase 3 diet than on the less severe Phase 1 diet. Both diets lowered total and LDL cholesterol from baseline levels (Grundy et al., 1986). Young men consuming a diet very low in fat and high in carbohydrate (18.3 and 64.9 en%, respectively) actually had higher total and LDL cholesterol levels than when they were given a diet rich in MUFAs (32.5 en% from total fat and 50.5% from carbohydrate). Both diets had similar levels of SFAs and PUFAs (Berry et al., 1992). In 14 mildly hypercholesterolemic women, lowering fat intake from 37 en% to 21 en% without changing the PUFAISFA ratio (0.45) effectively improved their serum lipid profiles (Schmeisser et al., 1992). Although the ratio of fatty acids was not changed, the lower total fat intake resulted in a lower SFA intake (from 16 to 8 en%). Therefore, attributing the results simply to a lowering of fat quantity is difficult. Collectively, these studies do not conclusively reveal either fat quantity or fat composition as the more important factor. Instead, they suggest that both fat quantity and composition are important, and that drastic lowering of total or saturated fat is not beneficial. Four of the five studies were conducted in normolipidemic young subjects, so the results may have limited applicability to the general population, especially to those people who would benefit most from serum cholesterol reduction. Boyd et al. (1WO) recruited women to participate in a 12-month randomized trial. Women in the control groups continued their usual dietary habits (37 en% from fat) whereas the other women reduced fat to about 21 en%. Changes in serum cholesterol levels over the course of the study depended on baseline cholesterol levels. After 12 months on the low-fat diets, cholesterol levels dropped 10% in the women with baseline cholesterol levels in the upper third but actually rose in the women with the lowest initial cholesterol levels. Thus, the Keys and Hegsted equations were not equally applicable to all women (Boyd et al., 1990). In other words, not everyone can expect the same benefit from a low-fat diet. An interesting finding in this study was the trend for cholesterol levels to move back toward baseline levels over the 12-month period in all groups, although the participants reported adhering to the low-fat diets over the entire year. Several questions can be raised. Is the effect of a low-fat diet transient? If the study had been carried out for another year, would all cholesterol values have returned to baseline levels? Is there a genetically programmed cholesterol “set-point”? Is long-term compliance with a low-fat diet the actual problem?

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Some idea of the relative importance of fat quantity and composition may be derived from comparisons of typical diets with vegetarian diets. Thorogood et af. (1990) compared serum lipid levels with dietary intakes in groups of vegans, lacto-ovo vegetarians, fish eaters, and meat eaters. Serum total and LDL cholesterol levels were significantly lower in the vegans than in the meat and fish eaters; they tended to be lower in the lacto-ovo vegetarians but the results were not statistically significant. Total fat intakes, as a percentage of energy, did not differ among the diet groups (although they tended to be lower in both vegetarian groups), but SFA intakes were significantly lower and PUFA intakes significantly higher in the vegan than in the other groups. Therefore, the researchers concluded that fat type and not amount was the important factor leading to different serum lipid levels. However, cholesterol intakes were lower and dietary fiber intakes higher in the vegan than in the other diets, so a number of dietary factors (in addition to potential differences in lifestyle) could account for the different serum lipids. A lacto-ovo vegetarian diet (30 en% from fat, 10% from SFAs) and a low-fat meat-containing diet (30 en% from fat, 10% from SFAs) were both effective in lowering serum cholesterol levels compared with a high-fat high-SFA diet, indicating that meat per se did not have an adverse effect on serum cholesterol levels. However, both prudent diets raised TG levels significantly relative to the high-fat high-SFA diet in this study of 26 men (Kestin et af., 1989). Masarei et al. (1984) studied effects of lacto-ovo vegetarian and omnivore diets in 19 men and 17 women. Both diets had about 40 en% from fat, but SFA intake was somewhat lower (14.6 vs 17 en%) and PUFA intake was much higher ( 1 1 vs 5 en%) in the Vegetarian diet. The diets had no effect on serum lipids in the women, but in the men total, HDL, and LDL cholesterol levels were lower when the vegetarian diet was consumed. Factor analysis showed that the changes in total and HDL cholesterol were primarily caused by an increase in fiber and PUFA intake on the vegetarian diet (Masarei et al., 1984). This study suggests that fat composition is more important than fat quantity in determining dietary effects on serum lipids. Again, however, another diinterpretation of the results. etary consituent-fiber-confounds No definitive answer is available for the question of relative importance of fat quantity and composition as they influence serum lipoproteins. Of relevance to this chapter are the possible cholesterolemic effects of milkfat in the context of low-fat diets. When Barr et af. (1991) studied a low-fat (30 en%) milkfat-enriched diet that was considerably higher in SFAs (14 en%) than recommended levels (
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Seventh-Day Adventists tend to have lower risk of CHD than nonvegetarian Adventists (Phillips et al., 1978), probably because the vegetarians obtain a larger proportion of their SFA intake from dairy foods than do the nonvegetarians, which makes speculating that dairy foods in the context of vegetarian diets do not have adverse effects on CHD risk tempting. However, Phillips er al. (1978) found that consumption of dairy products is actually higher among nonvegetarian Adventists. At present, no conclusive information is available on the impact of milkfat in the context of low-fat or otherwise modified diets. 3 . Genetic Factors

An ultimate goal in trying to understand how diet and genetics interact to influence CHD risk is the ability to tailor diets to individual needs. Identifying individuals who will benefit most from dietary modifications is particularly important. With the public health approach, everyone is encouraged to adopt diets low in fat, cholesterol, and SFAs, yet diet choices may have little meaningful impact on blood lipids in a significant portion of the population, so developing individualized diet recommendations has merit. Currently, targeting drug treatment to individuals based on profiles of serum lipids, lipoproteins, and apolipoproteins is possible; a limiting factor is that serum total cholesterol, and to a lesser extent HDL and LDL cholesterol, are the only fractions routinely measured (Cotton, 1988). However, targeting dietary advice to individuals is not yet a routine practice, except in cases of known disease. Many apparently healthy people probably would benefit from fat-modified diets because of underlying genetic defects in lipid metabolism, whereas others may not. Research clearly shows that hyper- and hyporesponders to dietary cholesterol and fat exist (McNamara et al., 1987; Grundy and Vega, 1988; Katan et al., 1988). However, how much of the variation in response to diet is because of genetic factors rather than natural biological fluctuations in serum lipid levels is not known. Berg (1991) proposes that “level genes” (genes influencing absolute levels of serum lipids or apolipoproteins) and “variability genes” (genes influencing to what extent diet or other environmental factors can cause variations in serum lipids or apolipoproteins) work together to determine individual risk of CHD. Identification of “variability genes” may allow prediction of a person’s responsiveness to diet. McBean (1990) reviewed the role of genetics in CHD, with special emphasis on the genetic control of serum lipoproteins. Genes and genetic mutations influencing levels and types of LDLs, HDLs, and so on have

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been described (McBean, 1990). Eventually, developing a panel of genetic markers for establishing CHD risk and sensitivity to dietary modifications may be possible. In the more distant future, even gene therapy to correct a deficiency in lipoprotein metabolism may be possible. Gene therapy was used to provide the LDL receptor gene to LDL receptordeficient rabbits (Anonymous, 1992). The approach has many drawbacks, however, and much more research is needed if this option is ever to be viable for human subjects. Rubin et al. (1991) compared responses to dietary fat in control mice and in transgenic mice expressing high amounts of human apo A-I. NonHDL cholesterol levels were similar in control and transgenic mice, whereas HDL cholesterol and apo A-I levels were at least twice as high in the transgenic mice as in the control mice, regardless of diet. Control mice fed atherogenic diets (one with butter and one with cocoa butter as the fat source) had much larger fatty streak lesions per area of aortic section than did the transgenic mice. In other words, expression of the human apo A-I gene essentially protected the mice from the early stages of atherosclerosis development that were induced in control mice by diets high in SFAs and cholesterol. This result raises the possibility that people genetically geared to produce high levels of apo A-I might not need to concern themselves with fat and cholesterol intake. Of course, much more work is needed before such a simple conclusion can be drawn in this complex area. Sorci-Thomas et al. (1989) found that, in monkeys, diets high in PUFAs reduced liver but not intestinal apo A-I mRNA concentrations. The investigators concluded that PUFAs altered the expression of the apo A-I gene in a tissue-specific manner. A complicated picture emerges: expression of the gene for apo A-I protects against diet-induced atherogenesis; at the same time, diet can alter the expression of the apo A-I gene. Agellon e f al. (1991) developed transgenic mice with the human cholesteryl ester transfer protein (CETP) gene (mice normally lack CETP activity). When the gene was inserted into mice, HDL levels decreased when the mice were fed either chow or high-fat diets. This study suggests that high levels of CETP are a cause of reduced HDL levels; thus, control of CETP activity could help preserve desirable levels of HDL. In humans with CETP deficiency, HDL levels are elevated (Brown et al., 1989; Inazu et al., 1990). Better understanding of the control of expression of this gene someday might lead to dietary advice designed to optimize HDL levels. More research is needed before relatively simple measures such as serum apolipoprotein levels can be used to predict a person’s genetically predetermined responsiveness to changes in dietary fat intake. Ulti-

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mately , tailoring dietary advice to individuals may replace the current practice of involving all people to benefit those individuals who do need to make dietary changes. E. INFLUENCE OF MILK AND OTHER DAIRY PRODUCTS ON SERUM LIPIDS AND RISK OF CORONARY HEART DISEASE Because butter is hypercholesterolemicwhen fed as a major portion of dietary fat intake, consumers have been told to choose low-fat or nonfat dairy foods in place of whole milk products. However, some reports claim that milk is hypocholesterolemic;in other cases, standard-fat dairy products do not raise blood cholesterol levels as would be predicted from their fat content and composition. In other words, the variety of dairy foods from which consumers derive most of their milkfat may not elicit the expected hypercholesterolemic response. Evidence from clinical trials, animal experiments, and population studies is discussed in this section. I . Intervention Studies a. Human Trials. The idea that milk or certain milk products may be hypocholesterolemic arose from the observation by Mann and coworkers that Maasai tribesmen in Kenya and Tanzania had low serum cholesterol levels and no CHD, yet subsisted on about 4 literdday of fermented whole milk with weekly consumption of large amounts of meat (Mann and Spoerry, 1974). In the first intervention study, 24 Maasai men were recruited and assigned to one of two dietary groups (Mann and Spoerry, 1974). One group was to consume 4-5 litedday of a fermented whole milk prepared by inoculating milk with a wild culture of Lactobacillus, whereas the other group was to consume the same milk plus a surfactant, Tween 20, which the researchers believed would increase absorption of dietary cholesterol and therefore raise blood cholesterol levels. The experiment was to last 4 wk, but the men drank very large quantities of the milk (more than 8 literslday) and gained weight so the experiment was ended at 3 wk. In spite of the weight gain, serum cholesterol levels dropped from their already low baseline levels in both treatment groups. In fact, cholesterol levels dropped more in the men who gained the most weight. Mann and Spoerry (1974) concluded that the surfactant did not influence blood cholesterol levels, and that there must be a hypocholesterolemic factor in milk. Lack of control of milk and meat intake and exercise during the study by Mann and Spoerry was a potential problem confounding interpreta-

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tion of results. However, a more important criticism was that the results may be peculiar to Maasai tribesmen, since they obviously have different diet and lifestyle habits than other populations. Thus, Mann (1977a) conducted several small feeding trials in U.S. adults. In four separate trials, subjects consumed either 2 liters/day of whole milk yogurt, 4 literslday of whole milk yogurt, 2 liters/day of skim milk yogurt, or 2 liters/day of whole fresh milk for 12 days. There was no control over intake of other foods except that participants were asked to keep their body weights, activity, and cholesterol intakes at their usual levels. Total serum cholesterol levels were measured before, during, and after the interventions. Consumption of whole and skim milk yogurt caused significant decreases in cholesterol levels, while whole fresh milk did not have a significant impact (Mann, 1977a). Actually, whole fresh milk appeared to lower serum cholesterol levels but the number of subjects (4) was too small for statistical significance. No dietary assessment was conducted, so it is impossible to know how nutrient intakes (especially fat and cholesterol) varied from the subjects’ typical intakes when the yogurts or milk were consumed. Nevertheless, the studies did show that consumption of large amounts of whole milk yogurt or whole milk had either a neutral or positive effect on blood cholesterol. The work by Mann sparked a number of communications, in the form of letters to editors, about what might be responsible for the hypocholesterolemic effect of milk. Calcium (Howard, 1977), a “nonprotein, dialysable, heat and acid stable, polar molecule” (Mann, 1977b), lactose (Helms, 1977), and a factor in the milkfat globule membrane (Howard and Marks, 1979) were suggested variously as hypocholesterolemic agents. Calcium and lactose subsequently were ruled out (Howard and Marks, 1977; Marks and Howard, 1977), although the experiments that did so were summarized in letters to editors and never appeared as peerreviewed comprehensive publications. Supporting a lack of a role for lactose, Stahelin and Ritzel (1979) summarized a study of 9 healthy subjects in a research note. When participants were fed 105 g dried cheese whey per day (of which 80 g was lactose) for 2 wk, no significant effect on total cholesterol, HDL cholesterol, or TG levels was detected. The observations by Mann also sparked a number of human trials, nearly all camed out in normolipidemic young adults (summarized in Table XVI). The first two studies listed in the table appeared as letters to the editor of Lancet, whereas the rest were peer-reviewed publications. All the studies except two involved the addition of rather large quantities of fluid milks or yogurt to the self-selected diets of the subjects. Meredith et al. (1989) and Keim et al. (1981) fed specially prepared menus to the participants under controlled conditions. As made clear in the table,

TABLE XVI EFFECTOF MILKA N D OTHERDAIRYPRODUCTS ON SERUM LIPIDSI N HUMANSUBJECTS Effect on" Study Howard and Marks (1977)

Subjects and design 16 mlf 8lgroup 2 wk

Diet comparisons

+ 4 pints whole milk/d vs baseline + 4 pints skim milkld vs

Total C

LDL-C

HDL-C

TG

Comments

dec

NS

No control group

dec

NS

NS

-

NS

-

baseline Antila et al. ( 1980)

18 m 9lgroup 5 wk

+ I .5 L buttermilkld vs baseline + 1.5 L cultured skim milk/d vs

No control group

baseline Hepner et al. (1979) Hepner et al. (1979)

Keim et al. (1981)

17 m/f crossover 4 wkltrt

+ 24 oz 2% fat yogurtld

dec

NS

+ 24 oz 2% fat milkld

decb

inc

36 m/f 5- 1 1/group 12 wk

+ 24 oz unpasteurized yogurtld

dec

NS

dec

NS

dec

NS

inc

NS

NS inc

-

9 mlf 2 wk

vs baseline + 24 oz pasteurized yogurtld vs baseline + 24 oz 2% fat milk/d vs baseline control (typical diet) vs baseline

+ 2 qt skim milkld vs baseline vs high or low Ca control

Control group only followed for 6 wk

Order of treatments not random

Hussi et al. (1981)

Rossouw et a / . (1981)

77 m 20lgroup 3 wk

+ 2.7 L skim milk/d vs control

NS

-

inc

NS

diet + 2.0 L 1% fat buttermilkld vs control diet

NS

-

NS

NS

32 teen m 10- 1 1/group 3 wk

+ 2 L skim milk/d vs baseline + 2 L I .8% fat yogudd vs

dec NS

dec NS

dec NS

dec NS

+ 2 L whole milk/d vs baseline

NS

NS

NS

dec

+ 1 L 2% milk/d vs baseline

NS

NS

NS

NS

Same results when subgroups fed other milk products

NS

NS

NS

NS

Fat and cholesterol intake same on both diets

+ 16 oz 2% fat yogurtld vs

NS

NS

NS

NS

baseline + 16 oz 2% fat milk/d vs baseline

NS

NS

NS

NS

Treatments not in random order: intake of fat, cholesterol same among all diets

Thompson et al. (1982)

68 mlf 3 wk

Massey (1984)

32 m 3 wk

McNamara et al. (1989)

18 m 4 wk/trt

Dietary fat, etc., equal between test and control

No control group

baseline

and follow-up

+ 1.5 L 2% fat milk/d vs no milk period

continues

TABLE XVI Continued Effect on" Study

Subjects and design

Diet comparisons

Total C

LDL-C

HDL-C

TG

Meredith et al. (1989)

10 f crossover 3 wkldiet

80 g cheddar cheeseld vs 280 g tofuld

inc

inc

NS

NS

Naito (1990)

10 f

+ 400 ml whole milkld vs

NS

NS

inc

NS

dec

-

-

dec"

Buonopane et al. (1992)

a

12 wk

baseline and follow-up

64 mlf 8 wk

+ I quart skim milk/d vs baseline

Comments Differences disappeared when fat and cholesterol intakes equalized Transient increase in total cholesterol after 4 , 8 wk No changes in cholesterol and TG in 18 control subjects

C, Cholesterol: TG, triglyceride; inc, increase ( p < 0.05); dec, decrease (p < 0.05): NS, no significant change; -, no value reported.

* Decrease in total C and increase in TG were seen only in half the subjects.

Decreases were seen only in a subgroup of the subjects (n = 39) with initial total C > 190 mg/dl.

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daily consumption of 3 cups or more of whole milk, 2% low-fat milk, skim milk, low-fat yogurt, or buttermilk (obtained as a by-product of butter made from cultured milk) either decreased or had no effect on serum lipids. In many cases, serum lipid levels after supplementation of diets with the milk products were compared with baseline lipid levels; no control groups were followed to see whether natural variations in lipid levels occurred during the period of study (Howard and Marks, 1977; Antila et al., 1980; Rossouw et al., 1981; McNamara et al., 1989). This design suffers because of the natural tendency for blood lipid levels to decrease when participants enroll in diet studies (Kris-Etherton et al., 1988), so the decreases attributed to milk products simply may have been due to this phenomenon. However, similar results were found in studies employing crossover designs, control diet groups, or follow-up blood lipid measurements (Hepner et al., 1979; Hussi et al., 1981;Thompson et al., 1982; Massey, 1984; Naito, 1990; Buonopane et al., 1992). A potential problem with interpretation of these studies is that, when subjects add generous portions of milk products to self-selected diets, obviously they must delete other foods. For most of the studies in Table XVI, the foods (and how much fat or what types of fat) that were deleted are not stated. Hussi et al. (1981), Massy (1984), and McNamara et al. (1989) did indicate that fat and cholesterol intakes were equivalent during test and control or baseline periods. In the studies in which whole milk was fed, the influence on intakes of fat, SFAs, or cholesterol of supplementing diets with whole milk is not clear (Howard and Marks, 1977; Rossouw et al., 1981; Thompson et al., 1982; Naito, 1990). Still, the conclusion that supplementing self-selected diets with whole milk as well as lower-fat milk products does not adversely affect blood cholesterol is a safe one. Meredith et al. (1989) found that women fed lacto-ovo vegetarian diets containing 80 g cheddar cheese/day had higher blood cholesterol levels than women fed the same diets with 280 g tofu instead of cheese. Fat, SFAs, and cholesterol intakes were higher in the cheese-containingdiets. When intakes of fat and cholesterol were made equivalent in the two diets, no differences in serum lipid levels were detected (Meredith et al., 1989). Thus, addition of fat from cheese to a vegetarian diet had a negative effect on blood lipids in these normolipidemic women. If cheese was substituted for other foods of animal origin instead of for tofu, as may be the case in more traditional diets, the result may not have been unfavorable for cheese. Another human trial addressed how diets low in fat but rich in milkfat compared with traditional American diets (Barr et al., 1991). A double-blind, randomized, crossover study was designed in which 25 normolipidemic, healthy men received three different diets:

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an average American diet with 37 en% from fat, 500 mg cholesterol, and 16 en% from SFAs; an AHA Step 1 diet with 30 en% from fat, 300 mg cholesterol, and 9 en% from SFAs; and a diet with 30 en% from fat, 300 mg cholesterol, and 14 en% from SFAs (mostly dairy foods). The dairy-rich diet lowered plasma total and LDL cholesterol levels relative to the average American diet, but raised them relative to the Step 1 diet (Barr et al., 1991). Unfortunately, the simultaneous changes in dietary cholesterol, total fat quantity, and fatty acid composition make the results somewhat difficult to interpret. The same research group also compared three Step 1 diets in which the majority of SFAs was provided either by dairy products, meats, or coconut oil. Total and LDL cholesterol levels were significantly lower when 24 normolipidemic young men ate dairy-enriched rather than coconut oil-enriched diets (Ginsberg et al., 1992). Total and LDL cholesterol levels were intermediate (but not significantly different from the other groups) when the meat-enriched diet was consumed. This randomized crossover design study suggests that milkfat and other sources of SFAs may not have equivalent effects on blood lipids. In a few studies designed specifically to measure the effects of dairy calcium on blood pressure or bone health, blood lipid measurements were made also. Baran et al. (1990) conducted a 3-yr study of bone loss in premenopausal women. Dietary calcium was increased in 20 women by an average of 610 mg/day by eating more dairy foods; control subjects were 17 age- and weight-matched women. Serum lipid measurements were made at baseline and 6, 12, 18, 30, and 36 months into the study. Although the dairy-supplemented group increased fat intake significantly, no significant changes occurred in total cholesterol, LDL cholesterol, or HDL cholesterol levels over the 3-yr study when compared to baseline levels or control subject levels. In a 12-wk study of the effects of CaC03 and dairy foods on blood pressure, 300 subjects randomly received either placebo, CaC03 (1000 mg Ca/day), or dairy foods supplying 1500 mg Ca/day. Although energy, protein, carbohydrate, and SFA intakes increased significantly in the dairy group, no significant differences in plasma lipids were measured among groups (Karanja et al., 1992). In another calcium-hypertension study, 200 volunteers consumed 1 qt/day skim milk and 2% fat milk, for 3 months each. Compared with baseline levels, no adverse effect on total and HDL cholesterol levels was seen for either milk (Bierenbaum et al., 1987). Apparently, dairy foods may not have the adverse effect on blood lipids that would be predicted from their fat content and fatty acid composition. b. Animal Studies. Experiments conducted in several animal species generally support findings from human studies. When rats were used

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20 1

as animal models, serum cholesterol levels were lowered by feeding skim milk (Nair and Mann, 1977; Kritchevsky et al., 1979; Marlett et al., 1981; Navder et al., 1990), whole milk (Kritchevsky et al., 1979; Chawla and Kansal, 1984), or whole milk fermented with Streptococcus thermophilus or Lactobacillus acidophilus (Rao et al., 1981; Chawla and Kansal, 1984) as supplements to chow or semipurified diets. Schneeman and coworkers did not observe a consistent hypocholesterolemic effect of whole milk in rats, but did observe a rather dramatic decline in serum TG levels in response to whole milk (Schneeman et al., 1989; Marquez-Ruiz et al., 1992). In one set of experiments, Schneeman et al. (1989) fed rats one of four diets: a skim milk-containing diet (5% total fat), a skim milk control diet with casein as the protein but no other milk fraction (5% total fat), a whole milk-containing diet (20% total fat), and a whole milk control diet with casein as the protein and lard and corn oil as the fat sources (20% total fat). Fasting serum TG levels were much lower on the whole milk than on the control diet (100 vs 178 mg/dl, p C 0.05). The rats fed whole milk also had lower liver TG and cholesterol levels than the control rats, so the lower serum TG levels were not the result of lipid accumulation in the liver (Schneeman et al., 1989). In a second set of experiments, these researchers compared the effects of the whole milkcontaining diet (20% fat from the milk) with two control diets. Both control diets had casein as the protein source and 20% fat from butter oil, but differed in carbohydrate source (lactose vs sucrose). Rats fed whole milk had significantly lower serum cholesterol levels than rats fed the casein-lactose control diets, and dramatically lower serum TG levels than both control diets (Schneeman et al., 1989). Thus, the hypotriglyceridemic effect of whole milk could not be attributed to fat type or lactose. Possible hypotriglyceridemic factors in whole milk that remain unexplored are whey proteins and fat globule membrane components such as phospholipids (which are present at only trace levels in butter oil). A role for milk proteins was ruled out in later studies (Marquez-Ruiz et al., 1992). Thus, the investigators hypothesized that some fat globule membrane component in whole milk may be responsible for its hypolipidemic effect. Another study lends support to this possibility, although the milk products were made from a combination of cow milk and buffalo milk. Abou-Zeid (1992) prepared an Egyptian cheese called Domiati with 50-50 cow milk-buffalo milk and then with buttermilk, replacing the milk mixture at levels of 20, 30, 40, 50, and 60%. The buttermilk was obtained as a by-product of manufacture of butter from buffalo milk. True buttermilk (as opposed to cultured buttermilk available in stores and made from low-fat or skim milks) is the liquid obtained after churning cream to make butter, and is rich in fat globule membrane material. Groups of rats were fed the various cheeses and a control (chow)

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diet. In rats fed the regular cheese and cheeses made with 20, 30, and 40% buttermilk, serum cholesterol levels increased relative to initial levels and relative to levels in rats fed the control diet, but serum cholesterol levels either did not change from initial levels or dropped when rats were fed cheeses made from 50 and 60% buttermilk (Abou-Zeid, 1992). The group of rats fed the 60% buttermilk cheese had serum cholesterol levels lower than those of the rats on the chow diet. Thus, enrichment of diets with fat globule membrane material may have had a beneficial effect on blood cholesterol. Unfortunately, no information was provided about feed intake and weight gain, so the reasons for the differences in serum cholesterol levels cannot be known with certainty. In rabbits, addition of milk (type not specified), yogurt, and calcium carbonate to a cholesterol-containing control diet lowered serum cholesterol levels. The degree of cholesterol lowering was greater in the yogurt group than in the milk group (Thakur and Jha, 1981). The researchers suggested that calcium could be a hypocholesterolemic factor in milk (Thakur and Jha, 1981). In other studies, rabbits fed skim milk also had lower blood lipid levels (total cholesterol, LDL cholesterol, VLDL cholesterol, and TGs) than rabbits fed milk-free control diets (Kiyosawa et al., 1984; Aggarwal and Kansal, 1991a). Moreover, aortic cholesterol concentrations were lower and atheromatous areas were smaller in skim milk-fed rabbits (Kiyosawa et al., 1984; Aggarwal and Kansal, 1991b). The apparent hypolipidemic effect of milk has been suggested to be due to inhibition of cholesterol or fatty acid synthesis caused by some component in milk. Kritchevsky er al. (1979) reported significant decreases in hepatic fatty acid synthetase (FAS) activity in rats fed whole or skim milks compared with a control (no-milk) group. On the other hand, Burton et al. (1992) did not observe inhibition of hepatic FAS activity in rats fed whole milk-containing diets rather than control diets (butter oil or butter plus casein plus lactose). A decrease in the activity of hepatic HMG CoA reductase (the rate-limiting enzyme in cholesterol biosynthesis) was noted in one study of rats fed whole or skim milk (Kritchevsky et al., 1979) but not in another study of rats fed skim milk or skim milk powder (Marlett et al., 1981). Although the effects of milk feeding on activity of enzymes involved in fatty acid or cholesterol synthesis are not consistent in animal studies, in uitro studies consistently have shown inhibition of cholesterol synthesis by some factor in milk. In liver slices, milk inhibited incorporation of [ I4C]acetate and [3H]mevalonate into sterols. The inhibitory factor in milk survived boiling and was found in the supernatant after precipitation of milk proteins (Boguslawski and Wrobel, 1974). In addition, incorporation of [I4C]acetate into sterols was lower in liver slices from rats fed

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203

milk than in those from rats fed a control diet (Boguslawski and Wrobel, 1974). Bernstein et al. (1976, 1977) also reported inhibition of cholesterol biosynthesis in rat liver homogenates by milk and cultured buttermilk, whereas Ahmed et al. (1979) found that cholesterol biosynthesis was inhibited by skim milk in rat liver slices. Richardson (1978) reviewed in-depth such in uitro studies, as well as in uiuo tests of the hypocholesterolemic effects of milk. He concluded that orotic acid, present in cow’s milk at 73-122 mg/liter, might be responsible for the effect. Several studies indicated that orotic acid inhibited cholesterol synthesis in uitro (Bernstein et al., 1976, 1977; Ahmed et al., 1979), but when orotic acid was fed to rats in another study, serum cholesterol, LDL cholesterol, and liver lipids all increased (Navder et al., 1990). Further, orotic acid induces fatty livers in rats (Richardson, 1978), but feeding of milk to rats does not have the same effect (Kritchevsky et al., 1979; Chawla and Kansal, 1984; Schneeman et al., 1989). Therefore, orotic acid does not seem likely to be responsible for the hypolipidemic effect of milk. Nair and Mann (1977) suggested that hydroxymethylglutarate (HMG) may be the factor because it inhibits HMG CoA reductase, but evidence is lacking that HMG is present at meaningful levels in milk. Moreover, Bernstein et al. (1977) showed that milk did not inhibit HMG CoA reductase in rat liver. Still no consensus exists on the hypocholesterolemic or hypotriglyceridemic agent(s) in milk. 2 . Population Studies

Consumption of dairy products has been associated variously with an increase, decrease, or absence of change in CHD risk in several epidemiological studies. Positive correlations between intake of one or more dairy foods and CHD have been reported by several researchers. Over 14,000 subjects were studied as part of the Tromso Heart Study. Consumption of butter or hard margarine was associated positively with total serum cholesterol (Jacobsen and Thelle, 1987). Use of hard margarine rather than butter was not distinguished, however. Slattery and Randall (1988) compared trends in CHD mortality in the United States from 1909 to 1980 with food consumption trends during that same period. Food consumption trends were based on disappearance data and household food consumption data. They concluded that pertinent changes in food consumption or disappearance, which preceded a decline in CHD mortality by 10-20 years, were decreases in dairy product (especially whole milk and butter) and egg consumption and increases in margarine and poultry consumption. However, although whole milk and butter consumption did decline over

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the years of study, consumption of skim milk, low-fat milks, yogurt, and cheese increased to keep total dairy intake quite steady. Although the investigators concluded that “dietary substitutions toward less-saturated fatty acids support the hypothesized relationship between dietary fat and CHD,” no direct evidence that changes in milkfat intake led to changes in CHD mortality was presented, especially since cheese consumption rose dramatically during the period examined (Slattery and Randall, 1988). Trevisan et al. (1990a, b) analyzed food frequency and serum cholesterol data obtained from over 4900 men and women participating in the Italian Nine Communities Study. In one study, these researchers noted that increased intake of butter and margarine was associated with higher blood cholesterol levels, whereas intakes of olive oil and vegetable oils were associated inversely with serum cholesterol levels (Trevisan et al., 1990a). The study had two major weaknesses, however. First, the food frequency questionnaire asked whether the subject used the fat sources in “a large amount, a small amount, or not at all, both in cooking and not in cooking” (Trevisan et al., 1990a). These qualitative data are especially weak because interpretation of ‘‘large’’ or “small” may vary considerably from person to person. Second, confounding risk factors may have existed between the high-butter and high-oil groups that were not accounted for. The same research group also found that intake of “atherogenic” foods was correlated positively with blood cholesterol levels in the same Italian adults (Trevisan et al., 1990b). The list of atherogenic foods (high in cholesterol and SFAs) consumed most by the population included hard and soft cheeses, although most of the 14 foods on the list were meats (salami, pork, sausages, meat sauces, bacon, etc.). Milk and butter were not on the list. Thus, no direct or compelling evidence was presented in this study that milkfat intake (from cheeses, in this case) was associated with serum cholesterol. Instead, increased frequency of consumption of a variety of high-fat foods was associated with increased serum cholesterol. Again, the researchers obtained no information on portion sizes, just on frequency of intake of selected “atherogenic” foods (Trevisan et al., 1990b). Blankenhorn et al. (1990) examined 82 men enrolled in the placebo group of the Cholesterol Lowering Atherosclerosis Study in California. All men had previous coronary bypass surgery, and were counseled to achieve dietary goals of 26 en% from fat, 5 en% from SFAs, and less than 250 mg cholesterol per day. On entry into the study 24-hr dietary recalls were obtained. Similar recalls were obtained 1 and 2 yr later. Coronary angiography data also were obtained, on entry into the study and 2 yr later. Of the 82 subjects, 18 developed significant new coronary artery lesions at the end of 2 yr. New lesions did not develop in subjects who increased their protein intakes

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205

and decreased fat by substituting low-fat meats and dairy foods for traditional higher-fat products (Blankenhorn et al., 1990). How important changes in dairy food selections were to the overall changes in protein and total fat is not clear. Also, the researchers found that increased consumption of total fat, PUFAs, lauric acid, oleic acid, and linoleic acid significantly increased the risk of new lesions. These results do not concur with those of a host of other studies that suggest that PUFAs and MUFAs are not atherogenic, nor does the result with lauric acid make sense in light of the very low intakes of lauric acid in typical United States diets. Perhaps the small number of study subjects and use of 24-hr dietary recalls confounded interpretation of the data. Other researchers have not suggested positive association between milkfat intake and CHD risk. Snowdon (1988) reviewed data from over 27,000 Seventh-Day Adventists living in California. The participants had completed food frequency questionnaires in 1960; mortality from a variety of diseases including CHD was followed until 1980. Milk and cheese consumption were not associated with an increase in death from CHD (or other diseases), whereas egg and meat consumption were (Snowdon, 1988). A weakness of the study is that diet habits were assessed only in 1960, whereas mortality was assessed through 1980. The investigator also provided data showing that milk consumption is much higher among Seventh-Day Adventists than among non-Adventists, a fact that is interesting in light of the lower mortality rates for all causes of death among Seventh-Day Adventists, even in comparison with nonsmoking controls. Of course, the Adventist life-style also excludes alcohol and pork; consumption of other meats, eggs, and caffeine is discouraged (Snowdon, 1988). In a group of Japanese adults aged 40-69, studied from 1964 to 1983, incidence of CHD did not change although serum cholesterol levels rose significantly and intake of animal fat more than doubled (however, the change was not statistically significant) (Shimamoto et al., 1989). How much milkfat contributed to the increase in animal fat intake is not clear. These data should be interpreted cautiously, since serum cholesterol levels still averaged below 200 mg/dl (5.17 mmol/L) in 1983 and CHD incidence did rise significantly in women over 70. An epidemiological trial in England generated interest when a progress report showed that middle-aged men who drink milk were less prone to heart attacks than non-milk drinkers, and that men who eat butter suffered fewer heart attacks than consumers of margarine (Anonymous, 1991). Because this result goes against the grain of traditional “dietheart disease” thinking, the study is currently under scrutiny (Elwood, 1991) and has been criticized (Shaper et af., 1991). Shaper et al. (1991)

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suggest that characteristics peculiar to milk-drinkers or butter-eaters may confound interpretation of the results. In a study of 7735 middleaged British men (the British regional heart study), participants were asked if and how they used milk and what kind of spread they used. Data also were obtained on incidence of major ischaemic heart disease events over a 10-yr period. Similar to the results in the controversial progress report, heart disease incidence was greater in those who drank no milk or used it only in tea or coffee. Further, heart disease incidence was greater in men who ate only margarine than in those who ate only butter (Shaper et al., 1991). However, the apparent protective effects of milk and butter did not hold up when other risk factors (smoking, obesity, blood pressure, physical activity, and/or previous incidence of ischaemic heart disease) were accounted for. Therefore, Shaper et al. (1991) concluded that milk intake or spread choice had no significant protective effect on heart attack incidence in British men. An equally significant alternative interpretation is that milk drinking and butter use did not adversely affect heart disease incidence. The “French Paradox” has received much attention in the lay press over the past few years. Apparently, the French eat more milkfat (cheese, cream, butter) than Americans yet have a much lower incidence of CHD, although reliable data comparing French and American nutrient intakes have not been cited (Dolnick, 1990). (However, individuals have pointed out that life expectancy is not longer in France than in the United States.) The most popular theory is that the wine-drinking habit in France accounts for the difference in CHD incidence between France and the United States. Of course, other plausible explanations include life-style, other dietary factors, and genetics. Also, milkfat from cheese, a favorite in France, has been suggested not to be hypercholesterolemic as is milkfat from other dairy foods such as whole milk (Renaud and de Lorgeril, 1989). This suggestion is strange, however, because whole milk itself has not been shown to be hypercholesterolemicin numerous animal and human studies (discussed earlier). At any rate, the casual observation that a population can enjoy a diet rich in milkfat yet have a much lower incidence of CHD than the United States deserves attention from the scientific community. F. SUMMARY This review of the literature suggests that the optimal balance(s) of dietary fatty acids for maximal protection against CHD has not yet been defined. As depicted in Figure 2, each of the major types of fatty acids has both positive and negative aspects. Numerous groups have recom-

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Positive Effects Saturates

Monounsaturates

n-6 PUFAs

n-3 PUFAs

raise HDL-C inhibit LDL oxidation?

lower total cholesterol vs. saturates low= LDL-C vs. saturates no effect on HDL-C inhibit LDL oxidation

lower total cholesterol vs. saturates lower LDL-C vs. saturates

lower TG may lower total Cholesterol antithrombotic

I

I

I

I

OPTIMAL BALANCE(S) OF DIETARY FATTY ACIDS

I thrombogenic? raise LDL-c raise total

I

I

I

tram lower HDL-C rrum raise LDL-C

lower HDL-C enhance LDL oxidation enhance TXA 2 synthesis

may raise

cholesterol

Saturates

Monounsaturates

n-6 PUFAs

LDL-C may lower

HDL-C enhance LDL oxidation n-3 PUFAs

Negative effects

FIG.2. Scheme suggesting need for proper balance of fatty acids (as yet undefined) to decrease coronary heart disease risk.

mended a diet with less than 10 en% of SFAs and PUFAs, with MUFAs at a level so total fat intake does not exceed 30 en%. Although this prescription represents current consensus on how fatty acids influence serum total and LDL cholesterol levels, it has several weaknesses. First, the effects of fatty acids on HDL cholesterol and apolipoprotein levels are not considered. Second, the effects of fatty acids on parameters other than serum lipids and lipoproteins, for example, thrombotic tendency and LDL oxidation, have not been considered. Finally, grouping fatty acids into only three categories ignores the fact that not all fatty acids within a group (SFAs, MUFAs, or PUFAs) act similarly. Therefore, a challenge to lipid nutritionists is to define better the optimal balance(s) of fatty acids in an antiatherogenic diet. A parallel challenge will be to define better the optimal balance of fat sources (i.e., foods) in the diet. Butter, when fed as a major or sole portion of dietary fat, is hypercholesterolemic compared with most other food fats. On the other hand, numerous animal and human studies suggest that whole milk does not raise cholesterol levels and even may exert hypolipidemic effects. Con-

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LOUISE A. BERNER

sumers currently are advised to avoid full-fat dairy foods, and some avoid dairy products altogether in an effort to choose heart-healthy diets. The nutritional drawbacks of reduced dairy food consumption were discussed in Section 11; the benefits in terms of reduced CHD risk are unproven. From a practical standpoint, better defining the role of milkfat in typical balanced diets, rather than extrapolating results from subjects fed high-butter experimental diets to all Americans, will be important. Only partial responses to the questions posed at the beginning of Section 111 are available currently. First, some evidence suggests that “hypercholesterolemic” SFAs may not adversely affect blood lipids in certain instances, for example, in low-fat diets or in the presence of n-3 fatty acids. However, results are not strong or consistent. Milkfat, when consumed in milk or yogurt, apparently does not adversely influence CHD risk factors. Second, SFAs, including those from butterfat, have potentially positive effects on HDL cholesterol levels, sometimes on apo A-I levels, and perhaps on LDL modification. However, the best balance of fatty acids to keep HDL and apo A-I levels high while not raising other serum lipids or lipoproteins has not been determined. In fact, a universal consensus that focusing on HDL levels is important has not even been reached. Finally, the impact of dairy foods on blood lipids and CHD risk seems to diverge from the impact predicted by fat content and composition alone. The characterization of whole milk as an atherogenic food has not been substantiated. A hypolipidemic factor in milk has not been identified, however. How milk components and other diet factors (e.g., dietary fiber, n-3 fatty acids, calcium) can modulate the cholesterolemic effects of milkfat or other SFA sources has not been explored satisfactorily. IV. DIETARY FAT, DAIRY PRODUCTS, AND CANCER RISK

The effects of dietary fat in general, and milkfat or dairy products in particular, on cancer risk will be discussed in this section. Unlike Section 111, which emphasized clinical trials, this section focuses necessarily on epidemiological studies and selected animal research. The greatest limitations to understanding dietary fat-cancer relationships are that intervention trials are largely unfeasible at present and that biomarkers for risk of various cancers (akin to serum cholesterol as a biomarker for CHD risk) have not yet been developed or fully validated. Nevertheless, population studies and animal experiments will be described here, especially those specifically addressing the impact of milkfat or dairy product intake, and shortcomings of the experimental approaches will be mentioned. The major stages in the cancer process, as well as current ideas

ROLE OF MILKFAT IN BALANCED DIETS Initiation of primary tumor

4-- - - - -

209

Role for dietary fat?

I I I

4

-- ---

Promotion of primary tumor 4-

High n-6 PUFAs (animal models)

I I I

+

High totalfat (animal models, epidemiology)

-----

4Tumor Metastasis (Implantation,Survival, Proliferation)

High linoleic acid (animal models) Other?

FIG. 3. Stages in the cancer process and hypothesized roles for dietary fat.

about the impact of dietary fat on each of the stages, are shown in Figure 3. Keep in mind that the scheme is general, and only a limited number of cancer types are influenced clearly by dietary fat, according to results from animal or epidemiological studies. A. POPULATION STUDIES Several types of epidemiological studies have been carried out to test diet-cancer associations (Table XVII). International and intranational comparisons involve correlations between the estimated intakes of a nuTABLE XVII MAJOR TYPES OF EPIDEMIOLOGICAL STUDIES FOR ASSESSING DIETARY FATCANCER RELATIONS HIPS^

Classification

Description

Inter- and Intranational comparisons

Correlation between estimated national or regional nutrient “intakes” (usually disappearance) and cancer incidence or mortality rates from cancers in populations Cancer incidence or mortality rates from cancers in populations migrating from one environment to another, each environment (e.g., country) having its own characteristic cancer incidencehortality rate Nutrient intakes of cancer patients compared with control subjects Dietary assessment of a large number of people; follow-up comparisons of nutrient intakes in those who do and those who do not develop cancer

Migrant studies

Case-control studies Prospective (cohort) studies

a

Information from Willett (1990) and Goodwin and Boyd (1987).

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LOUISE A. BERNER

trient (such as fat) in several populations and the incidence of disease in the same populations; individuals are not studied (Goodwin and Boyd, 1987). Such correlation studies have several drawbacks: (1) the crude estimates of food and nutrient intakes; (2) differences in disease detection or reporting among countries; and (3) inability to identify and control for confounding factors such as genetic make-up or environment. Studies of cancer incidence with respect to dietary intakes in migrant populations rule out genetic differences as confounding factors (Willett, 1990), but environmental factors other than diet are not easy to control. Case-control studies, in which dietary intake of a group of cancer patients is compared with that of control subjects (matched on the basis of such factors as age, place of residence, and sex) are of stronger design but still suffer from potentially serious methodological limitations (Willett, 1990). Potential major problems are the.choice of controls who are not appropriately “matched” to cases; the fact that dietary information is obtained after the subjects already have cancer, although cancer induction is a long process; and low participation rates, especially of controls (Stemmermann et al., 1984; Willett, 1990). Finally, prospective (cohort) studies involve dietary assessment of a large group of people, all of whom are initially free of cancer and are followed until the time a significant number of cancer cases develops in the group. Comparison of nutrient and food intakes then is made between those who develop cancer and those who do not. Prospective studies generally have the fewest methodological limitations, although they require relatively large numbers of subjects followed over periods of several years or more, and thus are expensive to conduct (Willett, 1990). In this section, results of each of these types of epidemiological studies will be discussed. I . Fat Quantity Breast and colon cancers have received the most attention as cancers possibly influenced by fat intake, although scattered studies have been done on a variety of different cancers. In spite of the fact that casecontrol studies show weak, if any, influence of fat intake on cancer risk (Carroll, 1991; Kritchevsky and Klurfeld, 19911, major groups have concluded that dietary fat intake is a risk factor, at least for some types of cancer (Surgeon General, 1988; NRC, 1989; FDA, 1991). During preparation of a new comprehensive nutrition labeling and health claims proposal, the FDA concluded that the evidence relating dietary fat to risk of breast, colon, and prostate cancers is strong enough to allow a health claim to that effect (FDA, 1991). The recommendations of government groups apparently are based largely on animal studies and international

ROLE OF MILKFAT IN BALANCED DIETS

21 1

correlation studies, since other epidemiological data, at least in the case of breast cancer, do not provide strong support for a fat-cancer relationship (Goodwin and Boyd, 1987; Kritchevsky and Klurfeld, 1991; Byers, 1992). a. Breast Cancer. Goodwin and Boyd (1987) reviewed the evidence that dietary fat is related to breast cancer risk in humans. These investigators applied several major criteria that should be fulfilled to establish a link: consistency of the association, strength of the association, a relationship in time, a dose-response relationship, specificity of the association, and biological plausibility. The researchers concluded that “insufficient evidence existed to conclude a causal association existed between dietary fat and breast cancer risk.” Willett (1990) and Byers (1992) also reached the same conclusion. One of the critical studies to date was a prospective study of almost 90,000 American nurses followed over a 4-yr period (Willett et al., 1987). No relationship was seen between fat intake and breast cancer risk; in fact, a modest protective effect of fat was seen when comparing women in the highest and lowest quintiles of intake (Willett et al., 1987). In two other prospective studies, total fat intake was weakly but nonsignificantly (p > 0.05) associated with breast cancer risk in Finland and Canada (Knekt et al., 1990; Howe et al., 1991). However, both papers highlighted a relationship between fat intake and breast cancer risk in spite of the weak and statistically nonsignificant association. In both of these studies, risk of breast cancer was related inversely to carbohydrate intake (Knekt et al., 1990; Howe et al., 1991) and in one study it was related inversely to energy intake as well (Knekt et al., 1990). In a case-control study of women in western New York State, fat intake was not associated with risk of breast cancer (Graham et al., 1991), whereas the relationship was significant in a case-control study of Dutch women (Van’t Veer et al., 1990). Both research groups adjusted for potential confounders such as age, reproductive history, and energy intake. Boyd et al. (1989) compared fat intake in women with and without mammographic dysplasia, a condition associated with increased cancer risk. No difference in intake of any nutrient existed between women with and without dysplasia, except that alcohol intake was greater in the former (Boyd et al., 1989). However, only 30 cases and 16 controls were studied, so the small number of subjects may have limited the value of the study. At any rate, based on prospective and case-control studies mentioned here and in other reviews (Goodwin and Boyd, 1987; Carroll, 1991; Kritchevsky and Klurfeld, 1991), no good evidence supports that fat intake is associated directly with breast cancer risk.

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However, the notion persists that higher fat intakes increase breast cancer risk. Plausible biological mechanisms for the effect exist (see Carroll and Parenteau, 1991), and animal studies (discussed subsequently) and international comparisons support the idea. For example, Hursting et al. (1990) conducted an analysis of cancer incidence and dietary “intake” (Food and Agricultural Organization food balance sheets) in 20 countries. These researchers found that total fat intake was associated strongly with breast cancer risk, yet detection and reporting of breast cancer is hardly likely to be similar among the various countries studied (e.g., the United States, Romania, Hong Kong, and Yugoslavia). Moreover, the fat intake estimates used in international comparisons are very crude. Hursting et al. (1990) list the average per capita total fat “intake” in the United States as 164 g/day, a figure that is at least 1.5 times the actual value for United States citizens (and at least 2 times the actual value for United States women viewed separately). The “intake” data could be argued to be similarly overestimated for all countries. However, waste of foodstuffs (and therefore waste of dietary fat) likely would be greater in countries such as the United States, whereas home production of foods, not always accounted for in disappearance data, would be greater in less developed countries. In other words, a disproportionate overestimation of fat intake in some countries may have occurred, which obviously would skew the results of international correlation studies. One of the explanations for why international correlation studies find an association between fat intake and breast cancer whereas epidemiological studies of generally stronger design do not is that the range of fat intakes in the latter studies is too small. (This explanation has been offered with other cancers, too.) Disappearance data used in international comparisons show ranges from 15 en% as fat in countries such as Japan to close to 40 en% as fat in countries such as the United States (Goodwin and Boyd, 1987). On the other hand, in the largest prospective study to date, fat provided 32-44 en% (Willett et al., 1987). However, this smaller range of fat intakes is exactly the range relevant to the situation in the United States, since Americans are being advised to lower fat intakes from about 37 en% to 30 en%. Byers (1992) suggested that if lowering fat intake is of benefit to lowering breast cancer risk, the benefit will not be observed with the dietary changes currently being recommended in the United States. Further, no answer is yet available to whether more dramatic reductions in dietary fat would be beneficial. b. Colon Cancer. As noted in several reviews, the results of studies on dietary fat-colon cancer relationships are inconsistent (Carroll, 1991; Kritchevsky and Klurfeld, 1991). As with breast cancer, international

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213

comparisons generally show strong relationships whereas results of case-control studies are more varied. Byers (1992) concluded that a consistent increase in relative risk of colon cancer is seen when comparing the highest to the lowest quartiles of fat intake in numerous studies; West et al. (1989) and Willett et al. (1990) also concur with the prevailing view that high intakes of fat increase risk for colon cancer. Only a few cohort studies have been done. The large prospective study by Willett et al. (1990) of almost 90,000 women showed a significantly increased risk of colon cancer when comparing women in the highest four quintiles of fat intake (>58 g fat/day) with those in the lowest quintile of fat intake ( 4 8 g fat/day). No dose-response effect was seen, however, since the relative risk of colon cancer was 2.48 for the second quintile of fat intake, 1.88 for the third, 2.61 for the fourth, and 2.00 for the fifth (Willett et al., 1990). In a smaller prospective study of 7074 middle-aged men of Japanese ancestry living in Hawaii, a significant negative relationship between colon cancer and intake of total fat (expressed as en%) was actually seen (Stemmermann et al., 1984). Willett et al. (1990) criticized the latter study for the use of 24-hr dietary recalls rather than more comprehensive instruments for measuring dietary intake. However, Garland et al. (1985) had the benefit of 28-day dietary histories obtained from men in 1957-1959 who were then followed for 19 years. In this prospective study of 1954 men, intake of fat (en%) was not related significantly to colorectal cancer risk. Possibly gender differences exist in the response to dietary fat, since Willett et al. (1990) studied women and Garland et al. (1985) and Stemmermann et al. (1984) studied men. More research is needed to clearly establish the relationship between total fat intake and colon cancer risk.

2. Fat Composition and Source Most emphasis in this discussion is placed on the influence of milk products and milkfat on cancer risk, but a brief comparison of effects of different fat sources (SFAs vs PUFAs, animal vs vegetable fats) follows. a . General Observations. Among epidemiological studies that have found relationships between fat intake and cancer risk, no clear picture is available of the source of fat involved. Carroll (1991) and Byers (1992) concluded that, in epidemiological studies, total fat intake correlates better with cancer risk than does intake of any particular type of fat. In an international correlation study, Hursting et al. (1990) found that both SFA and PUFA intakes were associated with increased breast cancer risk, whereas MUFA intake was not. On the other hand, in a pro-

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spective study of Canadian women by Howe et al. (1991), MUFAs were the only component of fat significantly (although weakly) related to increased breast cancer risk. Results of case-control studies are conflicting and do not help clarify the picture (Gerber et a/., 1989; Van’t Veer et a/., 1990). Verreault et al. (1988) and Brisson et a/. (1989) compared breast tissue morphology and dietary intakes in Canadian women. Among postmenopausal women diagnosed with breast cancer, increased SFA intake was associated with an increased frequency of spread of cancer to lymph nodes, although the trend was not significant. PUFA intake, on the other hand, was associated with a statistically significant lower frequency of positive nodes (Verreault et al., 1988). These results do not strongly implicate SFAs as undesirable or PUFAs as protective against the spread of breast cancer, especially because the associations were not seen in premenopausal women and because the associations were nonsignificant or weak (p = 0.14 for the effect of SFAs; p = 0.03 for PUFAs). Among women serving as controls in a Canadian breast cancer study, increasing SFA (but not PUFA) intake was associated with high-risk features from mammography (Brisson et al., 1989). However, no direct assessment of a diet-cancer link was done and whether the women with “high-risk” mammographic features really will go on to develop breast cancer is unknown. Eid and Berry (1988) tried to circumvent one problem of epidemiologic studies, that of obtaining accurate dietary information from subjects, by instead analyzing adipose fatty acids as indicators of fatty acid intake. These investigators obtained samples from women with breast cancer and from women with benign tumors, and found no systematic differences in fatty acids or PUFA/SFA ratios in the adipose tissue. The researchers concluded that quality of fat consumed was not associated with risk of breast cancer (Eid and Berry, 1988). In their international comparison study, Hursting et al. (1990) found that intake of SFAs but not PUFAs was related to increased colon cancer risk. In agreement, Willett et al. (1990) showed that animal fat (from meat) but not vegetable fat was associated positively with colon cancer risk in their prospective study of nearly 90,000 women. Unlike their results for total fat, a consistent dose-response trend of increasing risk with increasing quintiles of animal fat intake was determined. These researchers suggested that their results also might indicate that some other factor in red meat is responsible for the increased colon cancer risk, since red meat consumption was shown to increase risk independent of fat intake. In contrast, Stemmermann et al. (1984) found that SFA intake was associated negatively with risk of colon cancer in their prospective study of men in Hawaii, with relative risks of 1.00, 0.51, 0.70, 0.61, and 0.44 from the lowest to the highest quintiles of SFA intake. West et al.

ROLE OF MILKFAT IN BALANCED DIETS

215

(1989) found that high intakes of PUFAs and MUFAs but not SFAs were associated with a significantly increased risk of colon cancer in both men and women from Utah. Epidemiological data do not consistently implicate any particular component of fat as increasing colon cancer risk (Byers, 1992), although the largest prospective study to date found a relative risk of 1.89 for women in the highest compared with the lowest quintile of animal fat intake (Willett et al., 1990).

b. Milk and Other Dairy Products. Many epidemiological studies have been carried out in recent years to determine whether intake of milk, other dairy products, or butter is associated with increased risk of cancer. Results of case-control studies, listed in chronological order, are summarized in Table XVIII. Dairy product intake, including butter, is not consistently associated with an increase or a decrease in risk of various cancers. For example, breast cancer was the cancer studied most frequently. Six studies revealed an increased risk of breast cancer with increasing frequency of dairy product intake (Talamini et al., 1984; Le et al., 1986; Toniolo et al., 1989; Mettlin et al., 1990; D’Avanzo et al., 1991; Kesteloot et al., 1991); six showed a protective effect (Isovich et al., 1989; Pryor et al., 1989; Van’t Veer et al., 1989, 1991; Knekt et al., 1990; Simard et al., 1990); and five showed no effect (Lubin et al., 1981; Mills et a / . , 1988; Pryor et al., 1989; Van? Veer et al., 1989, 1990). Phillips (1975) reported that milk drinking was associated negatively with colon cancer risk in a case-control study of Seventh-Day Adventists, although use of other dairy products (presumably higher in fat) was associated positively with risk. Hislop et a/. (1986) determined that whole milk drinking in adulthood increased risk, whereas whole milk drinking in childhood was protective against breast cancer. In most of the studies in Table XVIII, the dairy products considered were full-fat products such as whole milk, cheese, butter, or cream. The overall picture from case-control studies, then, does not suggest an increase in cancer risk with increased frequency of milkfat intake. Results of prospective studies also are conflicting. For example, Giovannuci et al. (1992) analyzed semiquantitative food frequency information from 7284 American men who had undergone recent colonoscopy and/or sigmoidoscopy and of whom 170 had documented colorectal adenomas (not cancer). Results indicated that intake of fat from dairy foods (as well as intake of total fat, red meat, animal fat, and SFAs) increased risk of colorectal adenoma. However, in a much larger prospective study of 88,751 middle-aged women with 150 cases of colon cancer, fat from all dairy sources was not related to colon cancer risk although intake of total animal fat was. Intakes of whole milk, cheese, and ice cream also were not related signifi-

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TABLE XVIII CASE-CONTROL STUDIES OF THE ASSOCIATION BETWEEN DAIRY PRODUCT INTAKE AND CANCER RISK

Dairy product Study Lubin er al.

Cancer type Breast

(+) Association

(-) Association

No association

Skim milk

Cheese, milk, cream, butter Cheese

-a

(1981)

Cramer et al.

ovarian

( 1984)

Talamini et al.

Breast

( 1984)

Hislop et al.

Breast

( 1986)

Leer al. (1986)

Breast

La Vecchia er al. Colon Rectal (1988) Mills et 01. (1988) Breast Bladder Slattery et al. (1988) Tuyns et al. ( 1988)

Franceschi er al. ( 1989)

Isovich et al.

Whole milk, butter Milk, dairy products Whole milk (as adult) Cheese, fat level in milk Butter

-

-

Breast

-

Lung

Whole milk

Mettlin er al.

Prostate

Whole milk

(1989)

Breast

-

( 1989)

Breast

( 1989)

Van? Veer et al.

Breast

(1990)

Farrow and Davis (1990)

Colon Rectal Pancreatic

-

Butter Cheese

Whole milk products 2% Fat and skim milks

-

Milk, cheese, Yogun

Butter, ice cream, cream

-

Butter, milks, high-fat cheese

-

( 1989)

Benito et al.

Milk, cheese Milk, cheese, butter Milk, cheese

Milk

Colon and Milk rectal Non-Hodgkin’s Milk, butter lymphoma

Mettlin (1989)

Toniolo er al.

-

-

( 1989)

Pryor et al.

Whole milk (as child) Yogurt

Dairy products

-

Gouda cheese, fermented milks

-

-

Milk Dairy products

-

Milk, cheese

217

ROLE O F MILKFAT IN BALANCED DIETS

TABLE XVIII Continued Dairy product Study

Cancer type

(+)

Association

(-)

Association

Mettlin and Piver Ovarian Whole milk (1990) Mettlin et a/. Numerous sites Whole milk vs. no milk (1990) Negri et a/. Colorectal (1990)

Reduced fat milks 2% Milk vs. no milk

Simard et a / . ( 1990)

Breast

Milk, butter, cream, milk desserts

Van? Veer et a / .

Breast Breast

Total milk consumption

-

Milk, other dairy products

-

(1990) D'Avanzo et al.

No association

-

Fat from milk products, cheese

-

Butter

(1991)

La Vecchia et al. Prostate (1991b) La Vecchia et a / . Oral and pharyngeal (1991a) Van? Veer ef al. Breast (1991) a

Milk

Cheese, butter -

Milk, cheese Fermented milks

-, None of the dairy products studied fit in the category.

cantly to colon cancer risk (Willett et al., 1990). In light of this finding, making note of the growing body of evidence associating high calcium and/or vitamin D intakes with decreased risk of colon cancer in humans may be important (Garland et al., 1985; Lipkin and Newmark, 1985; Garland and Garland, 1986; Sorenson et al., 1988). Protective factors such as vitamin D in fortified milks and calcium in a variety of dairy products could help explain why animal fat from meats but not dairy foods has been associated with increased colon cancer risk. Ursin et al. (1990) conducted a prospective study of over 15,000 Norwegian adults. These investigators examined associations between milk intake (type not specified, but the researchers noted that most milk in Norway is whole) and incidence of all cancers for which they had data. No relationship was seen between milk consumption and total cancer incidence. When specific cancers were analyzed, milk consumption was

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LOUISE A. BERNER

associated positively with 4 of 24 cancers, a result that easily could be attributed to spurious associations. The strongest positive association with milk consumption was for cancers of the lymphatic organs, but only 37 such cancers were found among the 15,914 people studied. Several international and regional comparisons of dairy product disappearance (a surrogate for “intake”) and cancer risk also have been carried out. Cramer (1989) noted a positive correlation between per capita milk “intake” and ovarian cancer when data from 27 countries were compared. He hypothesized that galactose was the “toxic” factor in milk, since lactase persistence (and therefore digestion of lactose to yield glucose and galactose) also was correlated with ovarian cancer. However, detection and reporting of ovarian cancer are highly unlikely to be similar among all countries studied, a serious potential confounder of all international comparisons of diet and disease. Kesteloot et al. (1991) found positive correlations between per capita food supplies of dairy fat plus lard and total, breast, prostate, rectal, colon, and lung cancers. Again, differences in cancer detection and reporting among the 36 countries included in the analysis were not addressed. Moreover, possible confounders such as smoking, alcohol intake, fruit and vegetable intake, vitamin C intake, and beta-carotene intake were not controlled for. Ghadirian et al. (1991) found a positive association of milk “consumption” (FA0 food supply data) with pancreatic cancer mortality when they examined data from 29 countries. However, these investigators found no such correlations with any of the other cancers studied, including cancers of the esophagus, stomach, colon, rectum, bladder, breast, ovary, uterus, and prostate. Also, they did not control for effects of cigarette smoking and alcohol intake, two factors that the authors indicated are either known or purported risk factors for pancreatic cancer (Ghadirian e? al., 1991). Thus, although the authors highlight positive correlations between milk or milkfat and incidence of certain cancers in the three international comparisons noted here, the data are weak and unconvincing. Within-country studies of diet-disease relationships may be based on more reliable measures of dietary intake and more consistent disease detection and reporting. In a comparison of diet and cancer among counties in Sweden, food intake data were derived from a large food expenditure study in which subjects were asked to record all food expenditures over a 2-wk period (Rosen et al., 1988). The researchers also used data on milk sales as indicators of intake. Milk consumption (largely whole milk) based on sales data was protective against colorectal cancer, whereas the household expenditure data showed a very weakly protective (but not significant) effect of milk and cheese against stomach,

ROLE OF MILKFAT IN BALANCED DIETS

219

colorectal, and breast cancers. Fat intake was not associated with increased risk of these cancers in this study (Rosen et al., 1988).Gaskill et al. (1979)looked at breast cancer mortality and diet in different regions of the United States. Milk intake was determined from the 1965-1966 Household Food Consumption Survey. Breast cancer mortality was associated positively with weekly consumption of milk when the four regions of the United States (south, west, north-central, and northeast) were compared. Using per capita food demand estimates from each state, Gaskill et al. (1979) also found a positive association between breast cancer and milk “intake.” Similar examination of data for other cancer sites revealed positive associations of milk demand with mortality from ovarian cancer, rectal cancer, colon cancer, and testicular cancer, and a negative association between milk demand and cancers of the cervix, prostate, and respiratory organs. The authors pointed out that milk consumption may be a surrogate for some other factor that increases risk of selected cancers, yet they believe the data for breast cancer “may be of fundamental biological significance.” Potential problems are seen in each of these epidemiological studies, however, as mentioned at the beginning of this section. For example, the assessment of food and nutrient intakes was, in almost all cases, by food frequency questionnaires; information on portion sizes often was not ascertained, so absolute intake of foods and nutrients could not be determined with any accuracy. In two of the studies, participants were asked to recall adolescent food habits (Hislop et al., 1986;Pryor er al., 1989), which further complicates accuracy of dietary information. In international comparisons, food “intake” data were really disappearance, production, or sales figures. Inaccurate dietary intake data, among other factors, confound interpretation of results. Making a case for milkfat consumption as a risk factor for cancer based on the studies reviewed here is hard if not impossible. B. ANIMAL STUDIES Mammary tumors have been studied more than other cancers in rodents; in recent years, colon cancer also has received much attention. Thus, the focus in this discussion will be on these two types of cancer. Several general approaches have been taken to studying the effects of dietary fat on carcinogenesis in animal models. Most commonly, rodents have been treated with chemical carcinogens [e.g., 7,12-dimethylbenzanthracene (DMBA) for mammary tumors or 1,2dimethylhydrazine (DMH) for colon tumors] and fed diets with varied fat content or fat sources. Spontaneous tumor development was monitored

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LOUISE A. BERNER

in some studies. To study how fat affects tumor metastasis, the cancer process responsible for most cancer deaths in humans (Erickson and Hubbard, 1990),tumor cells have been transplanted into animals and the effect of diet on the spread of the primary cancer was assessed.

1 . Fat Quantity Animal studies reveal that dietary fat rather consistently enhances carcinogenesis in rats and mice by increasing the incidence of tumors, number of tumors, or tumor burden, or by shortening the latency period (see reviews by Birt, 1990; Carroll, 1991; Kritchevsky and Klurfeld, 1991; Zhao et al., 1991). Fat affects carcinogenesis at the promotion stage. Several studies suggest fat also may influence tumor initiation and tumor metastasis (Erickson and Hubbard, 1990). Rose et a f . (1991) found that a 23% corn oil diet, compared with a 5% corn oil diet, enhanced metastasis to the lung of human breast cancer cells transplanted into mice; other studies have shown similar results (Erickson and Hubbard, 1990). The level of fat has been shown to have to exceed some threshold level to affect carcinogenesis (Kritchevsky and Klurfeld, 1991). Because rodent diets are typically low in fat, how varying the percentage of fat in a rodent diet compares with typical variations in fat level of human diets is not clear. Comparing effects of rodent diets with about 5 and 20% fat has been common, although finding or hoping to achieve four-fold differences in total fat intake in humans would be unusual. Investigators are in general agreement that increasing dietary fat per se enhances carcinogenesis in animal models, but evidence also suggests that an increase in energy intake may account for part of the observed effects (Kritchevsky and Klurfeld, 1991). Zhao et al. (1991) carried out a meta-analysis of 14 studies in which the effects of fat on colon cancer in rats were assessed. Animals were fed ad libitum in all studies included in the analysis. These researchers found that incidence of chemically in-. duced colon carcinomas was enhanced significantly by fat intake and en% from fat but not by total energy intake. On the other hand, Clinton et al. (1992) reported that ad libitum energy intake, but not fat, enhanced carcinogenesis in rats treated with azoxymethane, a colon carcinogen and metabolite of DMH. Note that, in the meta-analysis by Zhao et al. (1991), the association between fat and cancer was seen only for Fischer 344 rats and not for the Sprague-Dawley strain. Clinton et al. (1992) used Sprague-Dawley rats, which may explain why they saw no effect of dietary fat level on colon carcinogenesis. Still, the strong influence of ad libitum energy intake on colon tumor incidence was striking in their study (Clinton et al., 1992). Welsch et al. (1990) studied the effects of fat

ROLE OF MILKFAT IN BALANCED DIETS

22 1

on chemically induced mammary cancer in Sprague-Dawley rats. These investigators fed one of four diets: 5% corn oil diet, ad libitum; 20% corn oil diet, ad libitum; and 5% or 20% corn oil diets, but restricted by 12% compared with the ad libitum diets. Energy intakes were identical in the two ad libitum diets (5% and 20% corn oil) and in the two restricted diets. Mammary carcinogenesis was enhanced only when the rats were fed the high-fat ad libitum diet. No enhancement was seen when the high-fat diet was fed in restricted fashion, although the rats receiving the 20% corn oil restricted diet obviously ate more fat than the rats fed the 5% corn oil restricted diet and also gained more weight. Because the number and weight of mammary tumors increased when rats were fed the 20% fat ad libitum rather than the 5% fat ad libitum diet, although energy intakes were identical, this study points to an independent effect of fat on carcinogenesis. However, by modestly restricting energy intake, the effect was abolished. Also, a separate analysis of data from rats in the 20% fat ad libitum group showed that rats consuming more energy developed more mammary carcinomas (Welsch et al., 1990). Thus, the “energy vs fat” dilemma has not been resolved. Perhaps the safest conclusion at present is that both factors are important, since energy restriction clearly inhibits carcinogenesis and high fat intake has been shown to enhance carcinogenesis independent of energy intake in ad libitum fed animals. Researchers should consider the importance of rat strain and ad libitum compared with restricted feed intake when designing future studies.

2. Fat Composition and Source Animal studies have been designed to compare effects of PUFAs and SFAs, n-6 and n-3 PUFAs, and cis and trans unsaturated fatty acids. n-6 PUFAs, notably linoleic acid, are more effective than SFAs in promoting chemically induced cancers, especially mammary tumors (Braden and Carroll, 1986; Carroll, 1991; Kritchevsky and Klurfeld, 1991). For example, Braden and Carroll (1986) showed that addition of 17% coconut oil to a 3% sunflower oil diet did not increase mammary tumor incidence or number in rats, whereas addition of an extra 17% sunflower oil did. Hubbard and Erickson (1991) determined that metastasis of mammary tumors in mice was dependent only on the level of dietary linoleic acid and was not enhanced by high levels of coconut oil or oleic acid. The idea that linoleic acid-rich oils are better promoters of cancer than fats rich in SFAs is not confirmed in all studies, however, perhaps because of differing essential fatty acid (EFA) requirements among different tumors (Birt, 1990). In contrast to results with n-6 PUFAs, n-3 fatty acids fed at fairly

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high levels consistently inhibit carcinogenesis in animal models, at least in part because of effects on eicosanoid metabolism (Braden and Carroll, 1986; Karmali, 1989; Birt, 1990). Selenskas et al. (1984) and Sugano et al. (1989) compared effects of trans and cis fatty acids on rat mammary and colon carcinogenesis, respectively. Both groups compared fats with nearly identical fatty acid composition, except the “trans” product had 38-39% (by weight) trans isomers of 18 : 1 and the other had all cis 18 : 1. No differences were seen in the effects of the trans and cis fats on colon or mammary carcinogenesis (Selenskas et al., 1984; Sugano et al., 1989). Selenskas and coworkers also compared diets with 5% and 20% corn oil to the 5% and 20% trans and cis fat diets. Tumor incidence was greater in the rats fed corn oil than in rats fed either the trans or the cis fats at the corresponding fat level, perhaps because the EFA requirement of the tumors was not met by the cis or trans fat diets. Little attention has been given to the effect of milkfat on carcinogenesis in animal models. Yanagi et al. (1992) reported that chemically induced mammary carcinogenesis was not enhanced in rats fed diets enriched in whole milk, skim milk, or cream. However, the diets were defined and described poorly, so assessing the study adequately is difficult. Newmark et al. (1984) hypothesized that dietary fat promotes colon cancer because high-fat diets increase levels of free fatty acids and bile acids in the colon. Fatty acids and bile acids have been shown to irritate colonic epithelium. The researchers further suggested that, in the presence of high levels of dietary calcium, calcium will form soaps with free fatty acids and bile acids, thereby detoxifying the irritants (Newmark er al., 1984). Although the hypothesis remains speculative, evidence suggests that elevated levels of dietary calcium prevent adverse effects of deoxycholic acid on colonic epithelium in mice (Wargovich et al., 1983) and decrease colon cancer risk in animal models and high-risk human subjects (Lipkin and Newmark, 1985; Appleton et al., 1987; Sorenson et al., 1988). One might surmise that milkfat in full-fat dairy products such as whole milk and cheese, which are also calcium rich, would not be likely to promote colon carcinogenesis in animal models. Evidence is lacking, however, that milkfat is a cancer promoter even in the absence of calcium. Behling et al. (1990) found no differences in incidence or volume of colon tumors in rats treated with DMH and fed one of four diets using butter as the fat source. The diets were lowfat; high-fat; high-calcium, low-fat; and high-calcium, high-fat. Neither a promoting effect of butter nor a protective effect of calcium was observed.

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3. Trace Lipids of Potential Importance

In addition to purported effects of total fat and fat composition on carcinogenesis in animal models, two minor lipid components have been shown to be potent anticancer compounds. These trace components are mentioned here because both are known to be present in milk or dairy products. Pariza and co-workers have identified and isolated isomers of linoleic acid containing a conjugated double bond system (designated conjugated linoleic acid or CLA) from several foods. The levels are particularly high in certain cheeses and processed cheeses (Ha et a/., 1989; Pariza and Ha, 1990; Pariza, 1991). CLA has been shown to inhibit cancer in animal models, apparently by acting as an antioxidant or by altering the tumor promotion process (Pariza, 1991). Pariza and Ha (1990) estimate that per capita consumption of CLA in the United States may be 1 g/day, but that endogenous synthesis may be a more important source of this anticarcinogenic compound. The significance of CLA in foods such as cheese remains to be determined. Other lipid components with anticancer activity are sphingolipids, present in the milkfat globule membrane. Sphingomyelin is present in similar concentration in bovine and human milks (Zeisel e f a / . , 1986). Sphingosine, the backbone of sphingolipids, has been shown to be a potent inhibitor of protein kinase C which is involved in such processes as cell growth and differentiation (Merrill and Stevens, 1989). Although evidence suggests that sphingosine has potential anticancer activity in a mouse skin cancer model, much work remains before the importance of sphingolipids in foods such as milk can be assessed (Merrill and Stevens, 1989; Merrill, 1992). C. SUMMARY Although international correlation studies show positive associations between total fat intake and risk of several cancers, results of casecontrol and prospective studies are inconsistent. In the case of milkfat and milk products, international comparisons again suggest an association between dairy product disappearance figures and cancer risk. However, the results from studies of stronger design do not reveal a consistent adverse or protective effect of milkfat. Animal studies generally support the idea that high levels of dietary fat, especially oils rich in n-6 PUFAs, enhance carcinogenesis. Use of animal models, usually mice or rats, is thought to be important to discern

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mechanisms for dietary fat effects (Kritchevsky and Klurfeld, 1991; Byers, 1992). However, such studies are of questionable relevance in establishing dietary fat-cancer links in humans and so are limited in usefulness, since the epidemiological data currently show so many inconsistencies. Intervention trials and additional long-term prospective studies are needed before dietary fat-cancer relationships can be understood more fully. Milkfat may be of special interest for further study. Since milkfat is rich in SFAs (and low in linoleic acid), and because dairy foods contain some potentially protective components such as calcium and CLA, welldesigned experiments are not likely to show a cancer-enhancingeffect of milkfat present in a variety of dairy foods. V.

MILKFAT AS PART OF THE TOTAL DIET

The purpose of this section is providing a perspective on the overall health implications of milkfat and milk products in typical varied diets and assessing how dairy foods, both traditional and modified, can fit into diets currently being recommended by a host of health professional and government groups.

A.

ASSESSMENT OF OVERALL IMPACT ON HEALTH

This chapter has provided analyses of the relationships between milkfat and CHD risk and between milkfat and cancer risk. Also critical are the implications of milkfat and dairy foods for other health issues. I . Diet and Disease

a . CHD and Cancer. An assessment of the overall impact of milkfat in American diets must take into account typical food and nutrient intakes rather than exaggerated experimental diets. Clinical trials indicate that diets high in butter or other fats rich in SFAs, typically fed as 20-40 en%, cause significant increases in serum cholesterol levels compared with diets high in MUFA- or PUFA-rich fats. This situation is unrealistic because people do not obtain 50-100% of their fat calories from single fat sources. In the case of milkfat, more effort should be made to delineate its impact in a varied diet and from a variety of dairy products. Evidence suggests that the experience with diets very high in butter or milkfat cannot be extrapolated to real life. For example, supplementing diets of animals or humans with whole milk (Howard and

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Marks, 1977; Kritchevsky et al., 1979; Rossouw et al., 1981; Chawla and Kansal, 1984; Schneeman et al., 1989; Naito, 1990) or adoption of dairyrich diets by humans for periods of up to 3 yr (Baran et al., 1990; Karanja et al., 1992) did not have the predicted hypercholesterolemiceffect. Animal studies indicate that carcinogenesis is enhanced when mice or rats are fed high levels of total fat or linoleic acid-rich oils. This situation also is unrealistic because laboratory rodents typically eat diets low in fat and fat levels are raised several-fold in experiments testing tumorpromoting effects of fat. Learning whether self-selected diets enriched in PUFAs (i.e., at levels reasonably achieved using real foods) are able to promote any cancers in humans, as they do in rodents, will be important since the trend in the United States has been for PUFA intake to increase at the expense of SFAs and MUFAs (Stephen and Wald, 1990). 6 . Gallstones. Much attention has been paid to dietary fat-cancer and especially to dietary fat-CHD relationships. Even if realistic changes in fat consumption could lead to changes in incidence of these chronic disease, improvement in one disease condition should not be at the expense of another. Sturdevant ef al. (1973) studied autopsies of older men who died during participation in an atherosclerosis prevention study and found that gallstone formation was more prevalent in men placed on a cholesterol-lowering diet (high in PUFAs) than in men in a control group (eating a “typical” American diet rich in animal fats). Scobey et al. (1985) confirmed the finding in an animal model: 5 of 8 monkeys fed a diet rich in PUFAs for up to 5 yr had gallstones, whereas 0 of 8 monkeys fed SFA-rich diets during the same time period had gallstones. In other studies of monkeys, however, Scobey et al. (1992) were unable to confirm a consistent adverse effect of PUFA-rich diets on either gallstone formation or indices of gallstone risk. Mott et al. (1992) found that decreasing dietary cholesterol and increasing dietary PUFAs had opposite effects on bile cholesterol saturation index in baboons. Specifically, bile cholesterol saturation, a presumed predictor of gallstone formation, was increased when dietary cholesterol was high and dietary PUFAs were high. The researchers concluded from their study that the net effect of cholesterol-loweringdiets (i.e., low in cholesterol and high in PUFAS) on gallstone formation is probably small (Mott et al., 1992). The effects of fat type on gallstone risk remain unclear but deserve further attention. c. Osteoporosis. In the United States, osteoporosis risk is now well established to be decreased by high lifetime calcium intakes (Berner et al., 1990; National Osteoporosis Foundation, 1991; McBean, 1992), yet

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some consumers avoid dairy foods, the major sources of dietary calcium, because of the perception that these foods are high in fat and calories and because health authorities recommend avoiding milkfat because of its SFA content. Hinders (1991) reported that elderly women on physicianprescribed cholesterol-lowering diets consumed 26% less calcium than elderly women on self-selected diets. On the other hand, results of two studies designed to assess whether counseling women to adopt very-lowfat (15-20 en%) diets could be successful do not suggest that low-fat diets necessarily lead to lower calcium intakes. Buzzard et al. (1990) and Gorbach et al. (1990) enrolled highly motivated women in programs to test the feasibility of an intervention trial on the effect of dietary fat on breast cancer risk (the actual intervention trial was canceled). Gorbach et al. (1990) collected 4-day food records at the beginning of the feasibility study and after 12 months. In the control group ( n = 114), mean calcium intake stayed about the same (710 and 704 mg/day), as did fat as a percentage of energy intake; in the intervention group ( n = 173), mean calcium intake rose slightly (from 717 to 750 mg/day) although energy intake decreased by more than 400 kcal/day and fat intake decreased from 39 to 22 en%. Buzzard et al. (1990) reported that calcium intakes dropped from an average of 687 to 628 mg/day after 17 women participated for 3 months in a similar low-fat intervention trial, although the drop was not statistically significant. [Note that women in both trials failed to meet recommended calcium intakes, although Buzzard et al. (1990) made a special attempt to counsel the women on high-calcium food choices.] Kristal et al. (1992) reported further on outcomes of one of the feasibility studies for the proposed intervention trial of dietary fat and breast cancer risk. These researchers studied food habits of 894 women who spent an average of 16 months adopting very-low-fat diets. The investigators determined that the women found it relatively easy to substitute most lower-fat foods for standard-fat foods (e.g., skim milk for whole milk); among the hardest habits to maintain were avoiding ice cream, avoiding sauces and table fats, and using low-fat instead of traditional cheeses. The studies just discussed suggest that maintaining calcium intakes when adopting diets including only reduced-fat dairy foods is possible, but that allowing the consumer to choose from a wide variety of dairy products is more feasible.

2 . Weight Control Reduction of CHD risk seems to be the primary goal of those who recommend reductions in dietary fat. The prevalence of obesity in the

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United States may provide another rationale for low-fat diets, including those with less milkfat, because nobody disputes that fat is more than twice as energy-dense as carbohydrate or protein. By choosing foods lower in fat, consumers can eat the same quantity of food at a lower energy cost or more food for the same energy cost. However, dispute surrounds the environmental factor(s) most responsible for obesity. For example, the presumption that overweight people consume more energy than normal weight individuals has not been confirmed consistently (NRC, 1989; Colditz et al., 1990; Klesges et al., 1992). Some studies point to physical inactivity as an important factor, whereas others suggest that fat intake, not total energy, is associated with higher body weight or greater propensity for obesity (Dreon et al., 1988; Romieu et al., 1988; Schutz et al., 1989; Klesges et al., 1992). Klesges et al. (1992) reported on a 3-yr longitudinal study of 142 men and 152 women. In the women only, energy intake predicted weight gain, and higher work and leisure activity were associated with smaller increases in weight. The researchers suggested that men and women may be affected differently by factors such as activity and energy intake. A consistent finding was that, in both men and women, increases in dietary fat intake predicted increases in body weight gain. Also, evidence suggests that “fat calories’’ are used more efficiently than calories from protein or carbohydrate (Donato, 1987; Leveille and Cloutier, 1987; Leibel, 1992). However, Leibel (1992) suggests the greater efficiency of energy utilization from fat may not be of clinical importance, because . . . whatever differences may characterize the short-term in uiuo handling of lipids versus carbohydrates or proteins, such differences do not appear to be reflected in the longer-term metabolic efficiency with which fat is used for structure, storage, or fuel.

Kendall et al. (1991) conducted a crossover study to determine whether women placed on low-fat diets would compensate for a reduction in fat level by eating more food. For 1 1-wk periods, 13 women were given either a low-fat diet (20-25 en%) or a control diet (fat as 3540 en%). The women were free to eat as much of the experimental diets as they wanted. When eating the low-fat diet, subjects did not fully compensate for a reduction in fat by increasing energy intake; in fact, they took in on average 290 kcal(l.22 MJ) less per day on the low-fat than on the control diet. Participants lost weight on both diets, but more so on the low-fat diet (on average, 1.28 kg or 2.82 lbs more). This study shows that weight loss can be achieved without dieting (Kendall et al., 1991). A troublesome finding was that weight loss on the low-fat compared with

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the control diet was not as much as predicted by theory, perhaps because of errors in measurement of food intake or differences in the efficiency of energy utilization. The understanding of causes of obesity, including genetic factors, is far from complete. A high-fat diet may prove to be one important and controllable factor. On the other hand, the potential role of physical inactivity should not be overlooked because women adopting very-low-fat diets have been shown to have correspondingly low (and not necessarily desirable) calorie intakes (not to mention that subjects on very-low-fat diets also have undesirable changes in serum HDL cholesterol levels, as discussed in Section 111). In two low-fat intervention feasibility studies described earlier, women who lowered fat intake from 38-39 en% to 22-23 en% also lowered energy intake by more than 400 kcal/day (1.67 MJ) so average energy intakes were only about 1,300 kcal or 5.4 MJ/day (Buzzard et al., 1990; Gorbach et al., 1990). This change seems to be regarded by the researchers as an accomplishment, but for several reasons such low energy intakes are not desirable. First, obtaining essential nutrients is difficult as energy intakes diminish. Buzzard et al. (1990) found that zinc and magnesium intakes declined when low-fat diets were adopted. Although Gorbach et al. (1990) did not report a decline in vitamin or mineral intakes, they did not evaluate several important nutrients such as zinc, folacin, vitamin B6, and fiber. These research groups studied highly motivated women; the typical American is unlikely to be so conscientious in food choices. Moreover, how long even highly motivated individuals could accept low-fat low-energy diets that also are consistently nutrient dense is not known. A second problem with low energy intakes is that people become more efficient at energy utilization; thus, weight gain is easier on readoption of higher energy intakes, as seen in the yo-yo dieting scenario (Steen et al., 1988). For these reasons, emphasizing significant increases in energy expenditure in addition to modest adjustments in fat and possibly energy intakes as part of a long-term strategy for weight control may make more sense. Collectively, evidence presented in this chapter suggests that the ageold messages of balance, variety, and moderation are supported by modern science. Figure 2 depicts the likely importance of balance and variety of fatty acids to decrease CHD risk. A parallel figure might be envisioned in the future for fatty acid balance and cancer risk, and perhaps for other chronic diseases as well. The optimal contribution to the diet from typical fat sources, including milkfat, is not yet known, but a strong rationale exists for maintaining variety and balance. Moderation becomes an operative word, because there are both reasons why it is healthful to adopt

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diets lower in fat as well as reasons that diets dramatically lower in fat (e.g., less than 25 en%) are not desirable. B. FITTING DAIRY FOODS INTO DIETS THAT MEET CURRENT FAT RECOMMENDATIONS Several government groups have recommended substantial changes in fat intake by Americans. In 1988, the Surgeon General recommended that most people “reduce consumption of fat (especially saturated fat) and cholesterol” by choosing foods low in fat, such as low-fat dairy products, and using food preparation methods that add little fat (Surgeon General, 1988). In 1989, the National Research Council’s Committee on Diet and Health recommended that United States citizens “reduce total fat intake to 30% or less of calories. Reduce saturated fatty acid intake to less than 10% of calories, and the intake of cholesterol to less than 300 mg daily” (NRC, 1989). The fat goals are to be achieved, in part, by substituting low-fat and nonfat dairy products for whole milk products. The latest “Dietary Guidelines for Americans” echoes this advice, suggesting also that Americans should “choose skim or low-fat milk and fat-free or low-fat yogurt and cheese most of the time” (USDA and USDHHS, 1990). Although some of these recommendations were challenged in this chapter, exploring how dairy foods fit into currently recommended diets is instructive. I.

Considerations with Dairy Foods Currently Available

Smith-Schneider e f al. (1992) used computer modeling to determine the dietary strategies that were most effective in helping a person meet recommended goals (as mentioned) for total fat, SFAs, and cholesterol. These investigators designed 7-day menus of commonly eaten foods (using standard-fat meat and dairy choices) for men and for women. The menus included popular high-fat foods such as french fries and cookies and did not exclude butter. Baseline menu energy intakes were 2560 kcallday (10.7 MJ) for men and 1604 kcal/day (6.7 MJ) for women. Then the investigators substituted one or more of the following for the full-fat versions: skim milk, low-fat milk, lean meats, medium-fat meats, or fatmodified dressings, sauces, and spreads. For men, any strategy incorporating lean meat exchanges (with or without any other changes in diet) was successful in achieving the dietary fat goals. Note that cheese was included in the “meats” category, so low-fat cheeses were included as lean meat exchanges (Smith-Schneider et al., 1992). (The researchers

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included mozzarella cheese, presumably part-skim, as a lean meat.) For women, only combinations of strategies, such as skim milk plus lean meats or lean meats plus fat-modified products, allowed them to meet recommended fat and cholesterol intakes. One important finding from the study was that omitting all standard-fat dairy foods was not necessary to meet recommended fat and cholesterol intakes, especially for men. A second finding was that energy intake from MUFAs declined along with declines in SFAs. This result is explained by the fact that about half of our MUFAs come from animal foods (Raper, 1991). A question can be raised about the implications of a decline in MUFAs concomitant with either an increase in or stable levels of PUFAs. Third, substitution of lower-fat milks for whole milk was a simple change for women that can help meet dietary fat guidelines. Unfortunately, computer modeling cannot predict whether diets that meet fat guidelines would be palatable and acceptable for most people. Women have difficulty substituting low-fat cheeses for full-fat versions (Kristal et al., 1992). Basch et al. (1992) studied diets of Latino children aged 4-7 and found that children obtained a large portion of their SFA intake from dairy foods, and that whole milk was the major source. These investigators determined that substitution of 1% fat milk for whole milk would have single-handedly achieved the goal of less than 10 en% from SFAs in all children except those in the highest two quintiles of SFA intake (Basch et al., 1992). If the children do not find lower-fat milks acceptable, it is questionable whether the risks of serious nutrient trade-offs by omitting milk are worth the supposed benefits. Fat and SFAs in selected full-fat dairy foods and the amounts “allowed” in diets of varying energy levels are listed in Table XIX. Also included are values for several fat-reduced dairy foods and a few popular full-fat foods from other food groups. If energy requirements are approximately 2500 kcal/day (10.46 MJ) or greater, fitting several servings of full-fat dairy foods into diets with less than 30 en% from fat and less than 10% from SFAs is relatively easy. In fact, at 3000 kcal/day (12.55 MJ), a person could choose each of the full-fat foods listed in the table and still reach only 85% of their SFA limit (the remaining 15% could be contributed by a variety of lower-fat foods). At 2500 kcal/day (10.46 MJ), three servings of standard-fat dairy foods (for example, two l-oz servings of cheddar cheese plus 1 cup whole milk) provide approximately 40% of the SFA limit and less of the total fat limit. For consumers with lower energy requirements, the situation is more challenging. Including several servings of full-fat dairy foods in a diet meeting the fat guidelines is still possible, but this would have to be at the expense of other full-fat food choices (e.g., meats, fried foods, salad dressings, and bakery items). A

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TABLE XIX COMPARISON OF FAT IN FOODS WITH FAT ALLOWANCESU

Energy intake level (kcal) 1500lday Fat Allowance @/day) Food Whole milk, I c Cheddar cheese, 1 oz Butter, 1 T Ice cream, 10% fat, 0.5 c Nonfat milk. I c Lowfat milk, I%, 1 c Lowfat yogurt, 8 oz Part-skim mozzarella, I oz Hamburger, 3 oz, + bun, fast food, plain Chicken breast, half, w/skin, roasted French fries, small order, vegetable oil Brownie, I d Italian dressing, 1 T Avocado, half

50

16 19 23 14
I5 24 20 14 30

2000/day

SFh Fat 17

SF

2500lday Fat

SF

67 22 83 28 Percentage of allowance" 20 12 I5 10 12 23 14 18 I1 14 28 17 21 14 17 18 11 14 9 11 1
30001day Fat

SF

100

33

8 9 I1 7
10 12 14 9 <1 3 4 6 8 5 9 9 2 7

Allowances based on 30% of energy from fat and 10%from saturated fat. SF, Saturated fatty acids. For foods, calculated using.USDA data for C 12 + C14 + C16, since these are regarded most widely as the cholesterol-raising saturated fatty acids. Rounded to nearest percentage point. For brownie, SF is all saturated fatty acids (no data for C12 + C14 + C16). a

logical strategy for individuals with lower energy intakes, then, would be to combine full-fat and fat-reduced products to meet the dual goals of (1) recommended intakes of fat and other essential nutrients and (2) a palatable enjoyable diet. The subpopulations who must take the most care in dietary selections to meet dietary guidelines are young children, women, adolescent girls, and elderly people, simply because they tend to consume less energy than adolescent boys and men. [Ironically, these are not the same subpopulations (i.e., young and middle-aged men) who have been the subjects of the vast majority of clinical trials on which dietary fat recommendations are based.] Again, we see that food choices are easier as energy intakes increase, adding one more argument for increased activity levels and against the trend for ever-decreasing energy intakes.

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Use of fat substitutes as ingredients in traditional foods is suggested as a means to achieve recommended fat intake levels. Many products are now available that use carbohydrate- or protein-based ingredients in place of fat. Lyle et al. (1992) calculated the potential benefits to consumers if fat-free products replaced all foods in several food categories. These researchers obtained data on current food intakes for the years 1986- 1988 from MRCA Information Services, a company that conducts quarterly food consumption surveys in nationally representative samples of United States households. Substituting nonfat products in seven product categories (cottage cheeses, cream cheeses, sour creams, dressings, frozen desserts, processed cheeses, and commercial sweet baked goods) at median levels of intake resulted in a decrease in dietary fat from 36 to 30 en%, a 3 glday decrease in SFA intake, and a 13 mglday decrease in cholesterol intake (Lyle et al., 1992). These changes, especially in SFA and cholesterol, are rather modest when one considers that researchers assumed nonfat products would substitute for all foods in the seven product categories listed. Such a scenario is highly improbable. Of greater potential benefit would be making modest adjustments in fat intake from meats, poultry, fish, fats, and oils-collectively the source of 79% of the fat in the United States food supply (Raper, 1991). Mela (1992) suggests that the nutritional benefits of fat substitutes are, for the most part, unproven. In addition, he points out that, although no important risks appear to be associated with fat substitutes, “large-scale introductions of fat substitutes into food products may have unanticipated effects on food selection and acceptance and micronutrient intakes” (Mela, 1992). Although Rolls and Shide (1992) agree that many of the long-term benefits of fat-reduced foods and fat substitutes are unproven, their review of the literature provides support for the potential benefits of such products in combatting obesity and several chronic diseases. Consumer demand for fat-modified foods will continue to insure a place for fat substitutes in a variety of products. Bruhn et al. (1992) found that a high proportion of consumers surveyed were interested in fat-reduced spreads, ice cream, and cheese that incorporated fat substitutes.

2 . Potential for Further Modification of Milkfat or Dairy Products The ultimate driving force for modification of dairy foods is market demand. Consumers are health conscious; considerations about nutritional quality of foods play an important role in shopping decisions (Anonymous, 1988). Perception of dietary fats as risk factors for heart disease, cancer, and high blood pressure has risen dramatically over the

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last decade (Blumenthal, 1989), yet consumer purchases clearly are not simple reflections of concern about excess dietary fat. The sensory properties of fats are important; more people rank taste as “very important” in determining food selection than any other factor (Anonymous, 1988; Drewnowski, 1990). Light et af. (1992) conducted consumer taste panels to see whether nutrition label information influenced how much a panelist liked ice cream and processed cheese slices. Presentation of nutrition label information did not change the sensory appeal of ice cream, but panelists did like normal fat processed cheese a little less and low-fat processed cheese a little more when nutrition information was available. Overall, the researchers found that fat level was the most important determinant of how much a product was liked (i.e., the panelists liked the normal-fat ice cream and cheese better than low-fat versions) (Light e f af., 1992). The contribution of milkfat and other fats to texture, flavor, and aroma of foods and palatability of the diet cannot be overlooked (Drewnowski, 1990). Another key factor influencing consumer demand for dairy products is price (Haidacher et af., 1988). Still, efforts have been and are being made to modify dairy products to meet demand for products low in fat, SFAs, and cholesterol (Ney, 1991). This section provides a brief overview of strategies for modifying milkfat or dairy foods to optimize nutritional properties or to expand the variety of products already available to consumers. In 1988, the Wisconsin Milk Marketing Board convened a panel of experts to discuss technologies for manipulation and use of milkfat. The panel agreed that an “ideal” fat from a nutritional viewpoint would have fewer SFAs (presumably CIz, C14, and C1& more PUFAs, and a higher proportion of MUFAs than milkfat (O’Donnell, 1989). The idea of manipulating single fat sources, such as milkfat, to have “ideal” nutritional profiles ignores the overall picture of a diet with multiple fat sources. However, trying to move single fat sources toward “ideal” fatty acid composition might help consumers meet recommended dietary fat guidelines. Numerous approaches are under study for modifying milkfat. a . Animal Husbandry Approaches. Changes in milkfat composition or fat content of milk are possible by altering feeding practices and through genetic selection or manipulation. The effects of feed on milkfat composition and concentration have been reviewed (Sutton, 1989; Grummer, 1991). Feeding cows diets rich in CI8 fatty acids (e.g., by supplementing feed with vegetable oils) will cause a decrease in the proportion of C4-C16 SFAs and a corresponding increase in 18:O and 18: 1 (Banks, 1991; Grummer, 1991). In fact, MUFA content of milkfat can be increased by as much as 80% (Grum-

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mer, 1991). This change in milkfat composition by feeding Cls-rich oils (regardless of the degree of unsaturation of the C1g fatty acids being fed) happens for several reasons. First, the CISfatty acids are hydrogenated in the rumen and can be converted to 18 : 1 fatty acids (cis and trans) by a desaturase present in the intestinal wall and in the mammary gland. Second, as more fatty acids reach the mammary gland via the bloodstream, de nouo synthesis of shorter chain SFAs decreases (Banks, 1991). This change in milkfat composition might seem desirable because the proportion of “hypercholesterolemic” saturates is decreased and Clg MUFAs are increased. However, a closer look reveals several potentially undesirable outcomes. Short-chain SFAs, which contribute to the functional and sensory properties of milkfat, and have no effect on blood cholesterol levels, are decreased. In addition, a significant part of the increase in MUFAs is accounted for by trans isomers of oleic acid, although the proportion of trans fatty acids can be limited by feeding fats rich in stearic or oleic acids rather than vegetable oils rich in linoleic or linolenic acids (Grummer, 1991). Ruminal disturbances may limit the widespread use of lipid supplementation of feed for cows (Banks, 1991; Grummer, 1991). Moreover, lipid supplements tend to decrease milk protein concentration (Sutton, 1989) which is not desirable because of the great demand for milk protein. Nevertheless, great potential exists for changes in feeding practices to have a positive impact on milkfat fatty acid profiles. Producing milkfat with increased proportions of 18 :2 and 18 :3 is also possible by “protected lipid” feeding. Vegetable oils rich in PUFAs can be encapsulated so the fatty acids are protected from hydrogenation in the rumen (Banks, 1991; Grummer, 1991). However, this practice is limited for economic reasons and because the resultant milkfat is more susceptible to oxidative rancidity. Also, some technical difficulties are involved in preparing encapsulated lipids that truly are protected from rumen action (Grummer, 1991). Concentration of fat in milk can be reduced by feeding diets low in roughage (e.g., by replacing forage with grains), and protein concentration has been shown to increase with low roughage diets (Sutton, 1989). However, milk yield usually is reduced also when low roughage diets are fed (Sutton, 1989), making this feeding option somewhat unattractive. Changing milkfat composition or concentration through genetic selection is another possible means of improving the fatty acid profile of milkfat. However, without economic incentives for change in milk composition, significant changes in fat content of milk will not occur (Gibson, 1989, 1991). Also, the rates of change through typical genetic improvement programs are slow, on the order of a small percentage per year for a characteristic such as milk yield (Gibson, 1991). More rapid changes

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could come about through the use of genetic engineering technology. For example, a gene that turns off or reduces the amount of acetyl CoA carboxylase could be introduced into dairy cattle to decrease fat production within the mammary gland. Much basic research remains to be done before genetic engineering can be a practical tool for changing milk composition (Bremel er a / . , 1989). Changing milkfat concentration and composition via changes in husbandry practices, especially feeding regimens, definitely has potential but each approach described also has drawbacks. More research in this area will be important. Adoption of any of these techniques, once scientific data prove them to be useful, ultimately depends on economic incentives for farmers to make changes.

6. Food Technology Approaches. Removal of fat from milk to be used in production of low-fat or nonfat dairy foods is accomplished readily by centrifugation. Of course this technology has been in routine practice for a long time, so fat removal from milk will not be discussed further here. Several technologies for modifying milkfat composition are now available or under study. Milkfat fractionation can be used to create liquid and hard fractions of milkfat with varying chemical compositions and physical properties and, therefore, different food applications. Fractionation can be achieved by melt crystallization, distillation, supercritical fluid extraction, or solvent fractionation (Boudreau and Arul, 1991a). The nutritional properties of various milkfat fractions are, for the most part, unexplored at present. Interesterification of milkfat can be carried out to change the triglyceride composition without changing the fatty acid composition, or milkfat-vegetable oil mixtures can be interesterified (Frede, 1991). Drawbacks to interesterification of milkfat include the loss of butter flavor and some undesirable physical properties such as poor mouthfeel (Frede, 1991). In addition, evidence is lacking that this procedure would result in nutritional benefit. Fractionation and interesterification technologies are likely to find application in creating specialty food ingredients, perhaps including a milkfat tailored for special nutritional purposes. With respect to milkfat-vegetable oil mixtures, for the dairy industry to exploit the nutritional and functional benefits that could be achieved with simple mixtures of milkfat and other fat sources, as has been done in the case of a few butter-margarine blends, would seem prudent. Nutrition research on the effects of adding modest amounts of unhydrogenated vegetable or fish oils to milkfat might reveal that a new product without nutritional “negatives” can be created for consumers who like the sensory properties of milkfat but avoid it for health reasons. Several methods for removal of cholesterol from milk and dairy foods have been developed, including steam stripping, supercritical fluid ex-

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traction, adsorption, and enzymatic conversion of cholesterol to innocuous compounds (Bradley, 1989; Boudreau and Arul, 1991b). Although consumers are interested in cholesterol-reduced or cholesterol-free foods, which may generate a marketing advantage for such products, the nutritional benefits for full-fat dairy foods are not clear because dairy foods do not make major contributions to dietary cholesterol. C. SUMMARY Inclusion of some full-fat dairy foods in typical varied diets is not likely to have the serum cholesterol-raising effects attributed to experimental diets unusually high in butter. Replacing milkfat with PUFA-rich oils may have an adverse effect on risk of certain cancers and gallstones. Omission of dairy foods, the main dietary calcium sources, from diets may increase the risk of osteoporosis. Moreover, expecting consumers to choose only low-fat or nonfat dairy products is not realistic because they like the taste, texture, and aroma of milkfat. Collectively, evidence suggests a need for balance in fatty acid intake and, therefore, variety in dietary fat sources. Moderation in fat intake also is important. Fat is energy dense and high-fat diets may contribute more to overweight than diets lower in fat but with the same energy content. Diets too restricted in fat (below -25 en%) may make meeting recommended intakes of vitamins and minerals difficult, can cause energy intakes to drop to undesirably low levels, and can have adverse effects on serum HDL cholesterol levels. Full-fat dairy foods can fit into diets that meet currently recommended fat guidelines, particularly for people with daily energy intakes of 2500 kcal (10.46 MJ) or more. To meet the dual goals of balance and moderation, a variety of dairy foods-from nonfat milk and yogurt, to low-fat milks and frozen desserts, to full-fat milks and cheeses-are already available to consumers. In addition, numerous technologies are under development to alter milkfat concentration or composition on the farm or at the food processing plant. VI. CONCLUSIONS AND RESEARCH NEEDS

Several conclusions about nutrient intakes from dairy foods and the health implications of milkfat in United States diets can be drawn. Contrary to popular belief, dairy foods (including butter) are not major contributors to dietary cholesterol and rank well behind meats, fish, and poultry and fats and oils as contributors to total fat. Intakes of dairy products and other foods vary widely among consumers, so a consider-

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able range in the contribution of specific food categories to fat intakes is seen. Dairy foods contribute important amounts of SFAs to United States diets, especially among women and children. Dairy foods also make key contributions to calcium, phosphorus, magnesium, zinc, riboflavin, vitamin B I Z ,vitamin A, vitamin D, protein, and other essential nutrients in United States diets. Fat-reduced dairy foods generally contain amounts of these nutrients similar to those in full-fat dairy foods, although many consumers prefer the taste, texture, and aroma of full-fat products. Much of the negative image of milkfat or full-fat dairy foods is derived from clinical or animal studies in which single fat sources (usually butter) constitute half to all the dietary fat, or from weak epidemiological data (international correlation studies). Therefore, the impact of milkfat from different dairy products in varied diets is not well defined. The adverse effects of dairy foods on CHD risk and the adverse effects of polyunsaturated vegetable oils on risk of certain cancers both are likely to be overestimated. Questions can and should be raised about how other fatty acids and diet components such as fiber or fruits and vegetables modify the cholesterolemic effects of butter. Some evidence from human and animal studies suggests that the impact of whole milk and perhaps of other dairy foods on serum lipids is not what would be predicted by their fat content and composition. A hypocholesterolemic factor in milk has been hypothesized but not yet identified. The benefits of fat-modified diets to cancer risk and risk of other chronic diseases (other than CHD) are undefined at present. The strongest evidence for a fat-cancer association is found for colon cancer. In addition to fat, dairy foods contain calcium and vitamin D, which have been shown to be protective against colon cancer. Therefore, even if an adverse effect of total fat on cancer risk is confirmed, full-fat dairy foods may not have the adverse effect predicted by their fat content alone. Epidemiological studies do not show any consistent effect of milkfat or dairy foods on cancer risk. Diets reduced in fat from current levels should be helpful as part of a long-term strategy for weight control, Also, potential adverse effects may occur if typical Western consumers switch to diets too low in fat (i.e., below -25 en%). One of the outcomes of adopting very-low-fat diets is that energy and food intakes can drop to levels that make it difficult to obtain essential nutrients. Omission of fat per se does not cause lower vitamin and mineral intakes; indeed, for most nutrients, nutrient density obviously increases as fat is removed. Rather, when consumers delete entire food groups (such as dairy or meats) or restrict total food intake unnecessarily, meeting recommended nutrient intake levels becomes difficult. Also, food choices are much more limited when trying

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TABLE XX FUTURE RESEARCH NEEDS AND STUDY DESIGN CONSIDERATIONS~

Research goal Improve databases on fatty acids in milkfat, other fat sources, and foods

Determine actual contributions of dairy products and other fat sources to intakes of fat and specific fatty acids in the United States Assess changes in consumption of specific fatty acids and other nutrients when standard-fat dairy foods are replaced by other foods Determine balance of fatty acids with most desirable impact on blood lipoproteins and apolipoproteins

Determine the effects of butter and other sources of milkfat on serum lipid profiles when fed at typical levels in mixed-fat diets Conduct a controlled human study to investigate the observed discordant effects of fat from whole milk rather than butter; other standard-fat dairy foods should be studied also ~~

Considerations Use modern chromatographic methods; many currently available analyses are outdated and ignore long-chain PUFAs, trans fatty-acid isomers, short-chain fatty acids Breakdown of data according to age, sex, geographic region, ethnicity of interest

Two age groups of special importance are teenagers and the elderly Focus should be on fatlfatty acid intakes reasonably achievable with selfselected diets; also of interest is determining balance of fatty acids with most desirable impact on LDL modification and thrombosis; however, at present. methods development and establishment of biological significance of in vitrolex vivo assays is more important in these research areas Dose-response relationship should be established; also, interactions with other fatslfatty acids (e.g., fish oils) should be assessed Control of food and nutrient intakes, as has been done in animal studies, is critical

~

~

Obtained in part from California Dairy Research Foundation Milkfat Expert Panel meeting, Burlingame, CA, October 13, 1992.

to meet dietary fat guidelines and to restrict energy intake at the same time. More effort should be made to highlight physical activity (rather than restriction in energy intake) as part of a long-term weight control strategy. This chapter highlights the need for further research designed to answer practical questions about milkfat in the diet. Is eating a diet very high in butter the same as eating a diet with several servings per day of a

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variety of full-fat dairy foods? What interactions take place in multisource fat diets to modify effects of single fats on serum cholesterol levels, thrombotic tendency, LDL modification, cancer risk, and so on? What are the long-term implications if United States consumers adopt very-low-fat and low-energy diets? Several critical research needs and important study design considerations are summarized in Table XX and in a recent summary of a meeting of a panel of experts (Berner, 1993). Answers to these and other questions will lay the groundwork for dietary recommendations based on practical considerations and not on exaggerated experimental situations. ACKNOWLEDGMENTS Financial support for this project was provided by the California Dairy Research Foundation. Drs. Barbara Schneeman and Denise Ney supplied important scientific input and helpful suggestions for improving this document. Drs. Joe O’Donnell and Emerita Alcantara gave helpful guidance during the preparation of this review.

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Walstra, P., and Jenness, R. (1984). “Dairy Chemistry and Physics.” John Wiley & Sons, New York. Wardlaw, G. M., and Snook, J. T. (1990).Effect of diets high in butter, corn oil, or higholeic acid sunflower oil on serum lipids and apolipoproteins in men. A m . J . Clin. Nuir. 51,815-821. Wargovich, M. J., Eng, V. W., Newmark, H. L., and Bruce, W. R. (1983). Calcium ameliorates the toxic effect of deoxycholic acid on colonic epithelium. Carcinogenesis 4, 1205-1207. Welsch, C. W., House, J. L., Herr, B. L., Eliasberg, S. J., and Welsch, M. A. (1990). Enhancement of mammary carcinogenesis by high levels of dietary fat: A phenomenon dependent on ad libitum feeding. J. Natl. Cancer h i . 82, 1615-1620. West, D. W., Slattery, M. L., Robison, L. M., Schuman, K. L., Ford, M. H., Mahoney, A. W., Lyon, J. L., and Sorenson, A. W. (1989).Dietary intake and colon cancer: Sex and anatomic site-specific associations. A m . J . Epidem. 130(5), 883-894. Willett, W. C. (1990). Epidemiologic studies of diet and cancer. Med. Oncol. Tumor Pharmacother. 7(2/3), 93-97. Willett, W. C., Stampfer, M. J., Colditz, G. A., Rosner, B. A., Hennekens, C. H., and Speizer, F. E. (1987).Dietary fat and the risk of breast cancer. New Engl. J . Med. 316, 22-28. Willett, W. C.. Stampfer, M. J., Colditz, G. A., Rosner, B. A., and Speizer, F. E. (1990). Relation of meat, fat, and fiber intake to the risk of colon cancer in a prospective study among women. New Engl. J . Med. 323, 1664-1672. Witschi, J. C., Capper, A. L., and Ellison, R. C. (1990). Sources of fat, fatty acids, and cholesterol in the diets of adolescents. J . Am. Diet. Assoc. 90(10), 1429-1431. Woollett, L. A., Spady, D. K., and Dietschy, J. M. (1992).Saturated and unsaturated fatty acids independently regulate low density lipoprotein receptor activity and production rate. J . Lipid Res. 33,77-88. Yamamoto, A. (1991).Regression of atherosclerosis in humans by lowering serum cholesterol. Aihero. 89, 1-10. Yamamoto, I., Sugano, M., and Wada, M. (1971).Hypocholesterolaemic effect of animal and plant fats in rats. Afhero. W , 171-184. Yanagi, S., Yamashita, M., Tsuyuki, M., Morimoto, J., Haga, S., and Imai, S. (1992).Milk cream does not enhance 2,7-dimethylbenz[a]anthracene-inducedmammary tumorigenesis. Cancer Lett. 61, 141-145. Yla-Herttuala, S.,Rosenfeld, M. E., Parthasarathy, S., Glass, C. K., Sigal, E., Witztum, J. L., and Steinberg, D. (1990).Colocalization of 15-lipoxygenase mRNA and protein with epitopes of oxidized low density lipoprotein in macrophage-rich areas of atherosclerotic lesions. Proc. Nail. Acad. Sci. U . S . A . 87,6959-6963. Zeisel, S. H., Char, D., and Sheard, N. F. (1986). Choline, phosphatidylcholine, and sphingomyelin in human and bovine milk and infant formulas. J . Nutr. 116,50-58. Zhao, L. P., Kushi, L. H., Klein, R. D., and Prentice, R. L. (1991).Quantitative review of studies of dietary fat and rat colon carcinoma. Nutr. Cancer 15, 169-177. Zock, P. L., and Katan, M. B. (1992). Hydrogenation alternatives: Effects of trans fatty acids and stearic acid versus linoleic acid on serum lipids and lipoproteins in humans. J. Lipid Res. 33,399-410.

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ADVANCES IN FOOD AND NUTRITION RESEARCH, VOL. 37

BIOCHEMISTRY OF CARDIOLIPIN: SENSITIVITY TO DIETARY FATTY ACIDS ALVIN BERGER AND J. BRUCE GERMAN Departmeni of Food Science and Technology University of California, Davis Davis, California 95616

M. ERIC GERSHWIN Division of RheumaiologylAllergy and Clinical Immunology University of California, Davis Medical School Davis, California 95616

I. Introduction 11. Discovery of Polyglycerophospholipids 111. Abundance of Pol yglycerophospholipids A. bis(Monoacy1glycero)phosphate B. Phosphatidylglycerol C. Cardiolipin IV. Pathways of Polyglycerophospholipid Synthesis A. bis(Monoacy1glycero)phosphate B. Phosphatidylglycerol and Cardiolipin C. Acyl-Specific Synthesis of Cardiolipin V. Intracellular Location of Polyglycerophospholipid Synthesis A. bis(Monoacylg1ycero)phosphate B. Cardiolipin and Phosphatidylglycerol VI. Conformation of Cardiolipin in Biomembranes A. Asymmetric Distribution B. Interaction with Cholesterol and Steroidogenesis C. Space-Filling Models of Cardiolipin D. Hex I1 Conformation of Cardiolipin E. Role of Cardiolipin as an Ionophore VII. Degradation of Polyglycerophospholipids A. bis(Monoacylg1ycero)phosphate B. Phosphatidylglycerol and Cardiolipin VIII. Oxidation of Cardiolipin IX. Association of Enzymes with Cardiolipin A. Possible Role of Cardiolipin in Mitochondria1 Protein Importation and Translocation B. Enzyme Associations X. Cardiolipin Acyl Composition A. Uniqueness of Cardiolipin Acyl Composition 259 Copyright 8 1993 by Academic Press. Inc. All rights of reproduction in any form reserved.

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XI.

XII. XIII.

XIV. XV.

B. Positional Distribution of Fatty Acids C. Interspecific Differences in Cardiolipin Acyl Composition D. Interorgan Differences in Cardiolipin Acyl Composition E. Influence of Diet on Cardiolipin Acyl Composition F. Acyl Changes in Cardiolipin Influence of Diet and Other Factors on Cardiolipin Content A. Temperature B. Hormones C. Diet D. Exercise E. Hyperthyroidism F. Ethanol G. Ischemia H. Malignancy Possible Role of Cardiolipin in Resistance to Ethanol-Induced Membrane Disordering Immunologic Activity of Cardiolipin A. Discovery of Anti-Cardiolipin Antibodies B. Anti-Cardiolipin Antibodies and Disease C. Binding of Anti-Cardiolipin Antibodies D. Noncardiolipin Antigens Directed Against Anti-Cardiolipin Antibodies E. Molecular Nature of the Reactive Epitope Chemical Synthesis of Acyl-Specific Cardiolipin Derivatives Chromatographic Separation of Cardiolipin References

I.

INTRODUCTION

Diphosphatidyl glycerol (cardiolipin) is a highly conserved phospholipid component of the mitochondrial membrane. The physiological role and nutritional sensitivity of this phospholipid class historically have been overlooked or poorly understood. Some research has indicated that this phospholipid is not only responsible for a great many biochemical and physiological functions but, far from being resistant to dietary modification, proves to be one of the phospholipid classes most sensitive to dietary fatty acids (Berger et al., 1992). For example, the fatty acid composition of its four acyl chains has been shown to vary from almost exclusively monounsaturated fatty acids (perhaps important in the newborn) to a predominance of fatty acids with two or more double bonds, including up to 30% 22:6n-3, all due to variations in diet (Berger el al., 1992). We are unaware of a phospholipid class that exhibits such profound sensitivity to diet. The evolving understanding of the essential structural role of cardiolipin in mitochondria1function, however, has not yet addressed acyl chain effects. This role has focused intense interest on this class of lipid and its interrelationship with nutritional status and dietary modification.

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Cardiolipin (CL) biochemistry and chemistry are reviewed by Ioannou and Golding (1979) and by Hostetler (1982), who reviewed all three polyglycerophospholipids. Historical accounts of the discovery and chemistry of this lipid class can be obtained in these two reviews. Daum (1985) later reviewed the mitochondrial lipid composition of microorganisms, plants, and animals and the function, synthesis, and degradation of mitochondrial lipids. The physiological effects of changes in the amounts and acyl composition of phosphatidyl glycerol (PG) and CL in microbes (Ohki et al., 1984; Mendoza and Farias, 1988; Ratledge and Wilkinson, 1988) and plants (Lynch and Thompson, 1984, 1988; Quinn, 1988) also have been reviewed. This chapter will update the nutritional, immunologic, and lipidprotein interaction aspects of CL, with emphasis on mammalian systems. PG and bis(monoacylg1ycero)phosphate [bis(MAG)P]will be considered briefly with respect to CL synthesis. II. DISCOVERY OF POLYGLYCEROPHOSPHOLIPIDS

The polyglycerophospholipids include cardiolipin [diphosphatidyl1’-glycerol-3’-phosphonoglycerol, or sn-1,2-diacylglycerol-3-phosphono(1”,2”diacyl) glycerol], phosphatidylglycerol (sn-I ,2-diacylglycerol-3phosphono-1’-glycerol), and bis(monoacylg1ycero)phosphate [lysobisphosphatidate, or sn-(monoacyl)glycero1-l-phosphono-l’(monoacyl) glycerol]. Considering its unique structure, its importance in mitochondrial function, and its clinical utility as an index of various pathological states by generating anticardiolipin antibodies, the discovery and molecular characterization of CL have been relatively recent events. CL was discovered in 1942 in beef heart by Pangborn (Pangborn, 1942). The correct structure was proposed in 1956-1957 and confirmed by chemical synthesis in 1965-1966. In 1966, CL was found to be positive in the serological test for syphilis. PG was isolated from alga in 1958 by Benson and Maruo (1958); the structure confirmed in 1964 (Benson and Maruo, 1989). bis(MAG)P was discovered in 1967 using silicic acid columns with ch1oroform:methanol (Hostetler, 1982). 111.

ABUNDANCE OF POLYGLYCEROPHOSPHOLIPIDS

CL is best considered one of the members of a family of polyglycerophospholipids. These molecules share common structures or biosynthe-

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tic routes and help explain both the function and the relative sensitivity of CL to nutritional modulation. Whereas many of the polyglycerophospholipids historically have been considered solely precursors of CL, new information about their unique abundance and activities in particular cells or organelles would argue against this oversimplification. Detailed information on the precise functions of these compounds, however, is lacking. A. bis(MONOACYLGLYCER0)PHOSPHATE bis(MAG)P is synthesized in lysosomes and found only in mammalian cells. bis(MAG)P represents less than 1% of phospholipids, except in alveolar lung macrophages, in which levels are as high as 14-18%. Very high levels of bis(MAG)P are found (up to 14%) in Niemann-Pick disease, as well as in sea-blue histocyte syndrome, neurovisceral lipidosis, and juvenile dystonic lipidosis. In Niemann-Pick disease, an increase in the number of tissue lysosomes is speculated to lead to increased bis(MAG)P. However, in diseases such as Tay-Sachs that are characterized by an increased number of lysosomes, no increase in bis(MAG)P is detected. Cationic amphiphilic drugs also are known to increase bis(MAG)P (Hostetler, 1982).

B. PHOSPHATIDYLGLYCEROL 1 . Microorganisms PG is the sole phospholipid of certain gram-positive bacteria and bluegreen algae. In Escherichia coli, PG and CL represent 22 mol% of the total phospholipid. Normal growth has an absolute requirement for I-2% PG (Raetz and Dowhan, 1990). Some of the PG present in microorganisms may be derivatized, by glucosamination of either of the free sn-2’ or -3‘ hydroxy groups and acylation of the 2’ position of the free glycerol moiety of PG, forming a triacylated PG derivative (Hostetler, 1982). 2. Plants PG is highly abundant in plants and, thus, makes a small but significant dietary contribution to most human diets. In plant chloroplasts, PG represents 20-30% of the phospholipids, thus rivaling phosphatidyl choline (PC) in animals with respect to abundance. In fruit, roots, and tubers, PG represents 2-8% of total polyglycerophospholipids. PG is present in many intracellular sites except in the potato, in which it is confined to the

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inner mitochondria1 membrane at 5% of total (Daum, 1985; Douce et al., 1987; Hostetler, 1982). 3. Animals

In animals, PG is ubiquitous at levels of 0.4-1% in mitochondria and 1% in liver microsomes, where it probably exists as a CL precursor. Only in bovine heart (11%) (Comte et al., 1976) and sheep adrenal cortex (7%) (Getz et al., 1968) mitochondria has the phospholipid fraction been reported to contain increased levels of PG. PG is also an important component of pulmonary surfactant, representing 7-11% of total phospholipids (Possmayer, 1988). PC and PG in surfactant predominantly have saturated acyl chains (50% 16 :0, 20-40% 18 : ln-9, and 3-5% 18 : 2n-6); the main molecular species is the dipalmitoyl moiety. Because of “kinks” at the double bonds of PC molecules with unsaturated residues, these molecules cannot be compressed without collapse to the same extent as dipalmitoyl PC and therefore are less effective in reducing surface tension, which is necessary to stabilize the alveoli and prevent alveolar collapse (Possmayer, 1988). Unsaturated PG molecules are also less stable. Pathways leading to the synthesis of the dipalmitoyl moiety have been proposed and investigated (Infante and Huszagh, 1987; Possmayer, 1988). The acidic phospholipids PG and phosphatidyl inositol (PI) are not, however, important for these physical and biological properties of the surfactant (Esko and Raetz, 1980; Beppu et al., 1983; Hallman et al., 1985). C. CARDIOLIPIN 1 . Microorganisms

In yeast species, levels of CL generally range from 12% (Saccharomyces cereuisiae) to 25% (Tetrahymena bovina) (Daum, 1985). The thermophilic strain Candick sloofjii, however, contains no CL (and therefore has no cytochrome c oxidase activity; see Section IX,B,l) (Arthur and Watson, 1976). Bacterial levels are also variable; log growth phase bacteria contain more PG and less CL than stationary organisms (Hostetler, 1982; Daum, 1985).

2. Plants The abundance of CL has not been studied extensively in plants but its overall abundance argues for more study of its role in nutrition and plant function. CL represents 10-22% of total phospholipid in plants such as

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corn and lettuce (16%), clover (lo%), alga (14%), and parsnip root (22%) (Hostetler, 1982). In castor bean endosperm, CL represents only 5% (Ohmori and Mitsuhiro, 1974).

3 . Animals

Cardiolipin generally is considered to vary in direct proportion to mitochondrial content. Although some intriguing exceptions to this trend have been observed, the direct implications are not yet understood. CL is high in rat and human heart (9-18%), since cardiac muscle is rich in mitochondria (Simon and Rouser, 1969; Poorthuis et al., 1976; Daum, 19851, low in rat brain (0.2-2%) (however, 11% in guinea pig brain mitochondria; see Daum, 1985), 12-17% in rat liver, 1% in rat macrophage alveolar cells, 7% in rat lung, 9-20% in rat kidney, 2-7% in rat skeletal muscle, and 2-3% in rat spleen (Rouser et al., 1969; Poorthuis et al., 1976; Daum, 1985). Red blood cells lack CL since they lack mitochondria. CL levels in various cell lines are 4-18% (Daum, 1985). Several tumor lines may contain 50-70% more CL relative to total mitochondria1 phospholipids than controls (Hostetler, 1982) (Sections X,F,l; X1,H). This observation and the potential for cardiolipin composition to relate to tumor function is likely to lead to an increase in research focus in this area.

IV. PATHWAYS OF POLYGLYCEROPHOSPHOLIPID SYNTHESIS

A. bis(MONOACYLGLYCER0)PHOSPHATE The synthesis pathway of bis(MAG)P is not yet well known. This molecule is formed from PG, lysoPG, and CL in uitro and in uiuo (Hostetler, 1982). Both PG and CL consist of 3-phosphonoglycerol, whereas bis(MAG)P consists of 1-phosphonoglycerol.Therefore, molecular rearrangement must occur. Somerharju and Renkonen (1980) demonstrated that sn-(monoacyl)glycero-3-[32P]phosphono-sn-rac-glyceroland sn(monoacyl)glycero-3-[32P]phosphono-~n-l’-glycerpol were converted in BHK cells to bis(monoacylglycero)[32P] phosphate with substantial amounts of sn-glycero-3-phosphate in the early phase. However, on prolonged incubation for 20 hr, the glycerol residues assumed primarily the sn-glycero-1-phosphate configuration. These results are consistent with PG being converted to bis(MAG)P by acyl transfer followed by unknown rearrangement (Hostetler, 1982).

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B. PHOSPHATIDYLGLYCEROLAND CARDIOLIPIN Detailed historical accounts of the synthesis of PG and CL are available (Hostetler, 1982; Mudd et al., 1987). The reactions leading to the synthesis of PG and CL in mammals, called the Kennedy Pathway, follow. Responsible enzymes, when known, are shown in brackets. 1. glucose or pyruvatw (DHAP).(via glycolysis or gluconeogenesis, depending on the tissue) 2a. DHAP+a glycerophosphate (GP) [glycerol 3-P dehydrogenasel 2b. glycerol + P,*aGP [glycerol kinase in liver] 3a. aGP + acyl donor-, 1-acyl glycerol 3-P (lysophosphatidate) [glycerol phosphate acyltransferase; EC 2.3.1.151 3b. DHAP + acyl donor-1-acyl DHAP; 1-acyl DHAP + NADPH1-acylglycerol 3-P + NADP [dihydroxyacetonephosphate acyltransferase].

In the mitochondria, where CL is synthesized, only the glycerol phosphate acyltransferase reaction (3a) seems to occur; both acylations (3a,b) occur in microsomes (Brindley and Sturton, 1982). 4a. 1-acyl glycerol 3-P + acyl donor-,diacylglycerol (DAG) 3-P (phosphatidate) [lysophosphatidateacyltransferase] 4b. DAG + P,+DAG-3P [DAG kinase] 5 . DAG 3-P + CTP-*CDP-DAG + PPi [phosphatidatecytidyltmsferase] The committed step in the biosynthesis of acidic phospholipids is Step 5 . In isolated rat liver mitochondria, the initial rate of PG synthesis is known to depend on the acyl composition of CDP-DAG. Dilauryl, dimyristoyl, dioleoyl, and dilinoleoyl CDP-DAGs are preferred substrates over CDP-DAGs with long-chain saturated fatty acids, presumably because of the enhanced solubility of the former (Hostetler et d.,1975). In lung, evidence suggests that a reversal of CDP-DAG inositol transferase produces CDP-DAG for PG synthesis by the reaction PI + CMP-CDP-DAG + myoinositol (Hallman and Gluck, 1980; Bleasdale and Wallis, 1981). In mammalian tissues, this enzyme is more active in microsomes than in mitochondria (Daum, 1985). 6. CDP-DAG + aGP-phosphoglycerol phosphate (PGP) [PGP synthetase] 7. PGP-PG' [PGPase; EC 3.1.3.271 8. PG + CDP-DAG-,CL2 [CL synthetase]

' A tetracylated derivative of PG,bis(DAG)phosphate has been isolated from cultured baby hamster kidney cells (see Hostetler, 1982). * In one species of bacteria, triacyl derivatives of CL are formed (5-7%).

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CL feedback inhibits CL synthetase. Two PG molecules may come together to form CL and glycerol in bacteria, but not in mammals (Hostetler, 1982). In mammalian mitochondria, CL synthesis requires divalent cations such as C02+, Mn2+, or Mg2+, but is inhibited by Zn2+, Fe2+, Cu2+, Hg2+,CaZ+,and nonionic detergents (Hostetler et al., 1975). Solubilized CL synthetase from rat liver and pig heart similarly requires Co2+, MnZ+,or Mg2+ (and the reaction product CL), and is inhibited by Ca2+, BaZ+,Hg2+,Cu2+,and Ni2+ (McMurray and Jarvis, 1980). C. ACYL-SPECIFIC SYNTHESIS OF CARDIOLIPIN CL contains up to 84 mol% 18 :2n-6. This spectacular apparent selectivity has been the subject of some investigation. Do biochemical events in the biosynthesis of cardiolipin exhibit this level of preference for linoleic acid? This question has not been answered, possibly because of the actual lack of high specificity in overall synthesis. Also, however, none of the enzymes of direct synthesis of CL, let alone possible retailoring enzymes, have been purified to date for such analyses. In in uitro studies, when CL is reacted with phospholipase A2 (PLA2) and reacylated with fatty acyl CoA substrates and acyl transferases, no preference was seen for linoleoyl CoA. Reaction frequency was stearoyl CoA>oleoyl CoA>>linoleoyl CoA (Bard et al., 1972; Eichburg, 1974). Similarly, CDP-dilinoleoyl glycerol was not especially preferred in the de nouo synthesis of CL compared with other CDP species (Hostetler, 1982). Such differences in phospholipid acyl specificity observed in uiuo and in uitro are not uncommon. For example, eicosapentaenoate (20 :52-3) is incorporated very minimally into the PI pool in uiuo (Von Schacky and Weber, 1985; Herold and Kinsella, 1986; Mori et al., 1987; Holub et al., 1988), but is incorporated readily into the PI pool of platelet and muscle phospholipid in uitro (Von Schacky and Weber, 1985; Croset and Lagarde, 1986; Yerram and Spector, 1989). In uiuo, CL containing predominantly linoleoyl chains may be achieved by selective acylation of deacylated precursors (Infante, 1984), more rapid turnover (i.e., deacylation-reacylation) of linoleoyl esters of CL compared with other fatty acids (Hostetler, 1982), and access to a polyunsaturated fatty acid mitochondria1 pool rich in linoleate (Reitz et al., 1969; Lynch and Thompson, 1984; Possmayer, 1988). Infante (1984) proposed a pathway for CL synthesis in which acyl specificity is not achieved by producing an acyl specific CDP-DAG, but by a separate acyl-specific proposed pathway in which fatty acids would

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be incorporated selectively into the deacylated precursors (glycerophosphoryl glycerol and phosphatidyl glyceroglycerophosphate). In these pathways, PI, PG, and CL do not share any common acylated intermediates such as DAG or CDP-DAG, so acyl-specific DAG or CDP-DAG is required for each phospholipid. Also, the pathways allow for a rational understanding of how a precursor molecule such as PG could have a fatty acyl composition different from that of the final product CL. The proposed pathway follows. 1. PG for storage is synthesized via PGPase, as described in Step 7 of the Kennedy pathway 2. PG (storage) + CMP+CDP-glycerol + DAG 3. CDP-glycerol + crGP+glycerophosphoryl gylcerol (GPG) [CDP glycerol :sn-3 glycerol phosphate glycerol transferase or GPG synthetase] 4. GPG + acyl donor+2-acyl GPG 5 . 2-acyl GPG + acyl donor+PG (acyl specific) 6. PG (acyl specific) + CDP-glycerol (from Step 2)+phosphatidyl glyceroglycerophosphate (PGGP) + CMP 7. PGGP + acyl donor+2’-acyl PGGP 8. 2’-acyl PGGP + acyl donor+diphosphatidylglycerol or CL (acyl specific)

V. INTRACELLULAR LOCATION OF POLYGLYCEROPHOSPHOLIPID SY NTHESlS A.

bis(MONOACYLGLYCER0)PHOSPHATE

Polyglycerophospholipids are not found in plasma lipoproteins (LP), red blood cells (RBC), or urine. Hence, these molecules can be incorporated into membranes only at the point of synthesis. To synthesize bis(MAG)P, CL, PG, and IysoPG probably are applied to the lysosome by autophagy (digestion of the cell constituents by enzymes of the same cell). Two IysoPG molecules could form bis(MAG)P after rearrangement (see Section IV,A; Hostetler, 1982). B. CARDIOLIPIN AND PHOSPHATIDYLGLYCEROL None of the mammalian enzymes involved in polyglycerophospholipid synthesis have been purified. However, in E. coli, PG phosphate

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synthase has been purified and PGPase has been purified partially (see Hostetler, 1982). The major intracellular source of synthesis for most phospholipids is the endoplasmic reticulum (ER) in animals (Voelker, 1985). In the lung and liver, the two tissues examined most closely, PG is synthesized in the mitochondria and the ER, and the former predominates (Hallman and Gluck, 1980; Mavis and Vang, 1981; Harding et al., 1983). Low concentrations of microsomal PI in the lung can inhibit the synthesis of PG in the microsome (ER), presumably by not providing a source of CDP-DAG for PG synthesis in the CDP-DAG inositol transferase reaction (PI + CMP-CDP-DAG + myoinositol; Hallman and Gluck, 1980; Bleasdale and Wallis, 1981). Bleasdale and co-workers (1981) also suggested that high CMP levels may stimulate PG synthesis in the ER by promoting formation of CDP-DAG (Quirk et al., 1980). In plants, PG is synthesized in mitochondria, microsomes, and the chloroplast envelope (Mudd et al., 1987). Most reports indicate that CL is synthesized almost exclusively in the inner mitochondrial membrane in plants (e.g., cauliflower, potato) and animals, from which the synthetic enzymes have been solubilized and partially purified (Hostetler, 1982; Vance, 1985). In potato tuber (Meunier and Mazliak, 1972; McCarty et al., 1973) and some microorganisms (Hallermayer and Neupert, 1974; Bednarz-Prashad and Mize, 1978; Bottema and Parks, 1980), the outer mitochondrial membrane contains amounts of CL that cannot be simply dismissed as contamination. In animals, until very recently, very small amounts of CL were reported in the outer mitochondrial membrane in the heart and in other organelle membranes. Microsomes, lysosomes, Golgi, and plasma membranes are reported to contain approximately 1% CL; the nuclear membrane is reported to contain 4% CL. Hovius et al. (1990) since reported that the outer mitochondrial rnembrane from rat liver contains 23% of the total mitochondrial CL, which. did not originate from inner membrane contamination and therefore is a true component of the outer membrane. The inner membrane was found to be virtually devoid of PI and phosphatidyl serine (PS). Mitochondria were isolated by a combination of differential and Percoll gradient centrifugation, resulting in a highly pure and intact preparation, as assessed by marker enzyme analysis, latency of cytochrome c oxidase, respiratory control index, and electron microscopy. Two different methods were compared for the separation of inner and outer membranes. In the swellshrink-sonicate procedure, glycerol was included, resulting in the isolation of one outer membrane and two inner membrane fractions of high purity. Using digitonin, a highly selective and gradual solubilization of the outer membrane was accomplished.

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In summary, PG is synthesized in the ER and the inner mitochondrial membrane and the latter predominates (Hostetler, 1982; Daum, 1985). Nonmitochondrial PG may be translocated to the mitochondria for CL synthesis; PG from the ER and mitochondria may be translocated to the lysosome for bis(MAG)P synthesis. PC, PI, sphingomyelin (SM), and sterols also are synthesized predominantly in the ER in eurkaryotes and similarly must be translocated to the mitochondria (Daum, 1985). The mechanism by which this translocation from the ER to the mitochondria occurs is not known. Theories of intracellular lipid transport include (1) transfer of PL by PL exchange proteins (Wirtz and Zilversmit, 1968, 1970), (2) vesicular mediated transport, and (3) target organelle modification of transported lipid (Voelker, 1985). In support of Theory 1, phospholipid exchange proteins have been discovered that transport all phospholipids including CL (Parkes and Thompson, 1970; Daum and Paltauf, 1984). In refutation of Theory 2, no clear demonstration of vesicle traffic from the ER and Golgi to the mitochondria has been done, the possibility remains for the transport of PG from the ER to the mitochondria to direct CL synthesis. In support of Theory 3, CDP-DAG has appreciable solubility and could diffuse in a nonfacilitated manner into the mitochondria to direct synthesis of CL in the mitochondria. This mechanism would not account for the acyl specificity of CL according to the Kennedy pathway but is consistent with the pathways for acyl specificity proposed by Infante (1984), in which acyl specificity is not achieved by producing an acyl specific CDP-DAG but by a separate acyl-specific pathway in which fatty acids are incorporated selectively into the deacylated precursors (see Section IV,C). Studies with 32P-labeledphospholipid and CDP-DAG indicate that the transfer of phospholipid from the ER to the inner mitochondrial membrane is a slow process requiring several hours. The limiting step seems to be translocation from the outer to the inner membrane. The transfer of phospholipid between the microsomes and the outer membrane is complete within 20 min (McMurray and Dawson, 1969; Jungalwala and Dawson, 1970; Blok et al., 1971; Wojtczak e f a l . , 1971; Eggens et al., 1979; Stuhne-Sekalec and Stanacev, 1979a,b; Stuhne-Sekalec et al., 1979). The suggestions that the ER must be in close proximity to the inner mitochondrial membrane or that disruption or detachment of the outer membrane is necessary for lipid to be translocated from the ER to the inner membrane (Sauner and Levy, 1971; Wojtczak et al., 1971) are not supported by these data. Once PG is translocated from the ER to the mitochondria, it must be translocated in a very precise manner to the inner mitochondria1 membrane. Based on the report by Hovius (1990), some of this CL would

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seem to be translocated from the inner membrane to the outer membrane of the mitochondria. Proposed mechanisms for intramitochondrial transfer of lipids include: (1) transfer of lipids by soluble proteins of the intermembrane space; (2) transfer without protein involvement; and (3) transfer via contact sites between the outer and inner mitochondrial membrane (see Daum, 1985; Simbeni et al., 1990). The first hypothesis is not supported since, to date, no soluble mitocondrial phospholipid transfer proteins have been discovered (Blok et al., 1971). The second hypothesis is unlikely since transfer of a lipophilic particle from a membrane compartment to a soluble space and back to a lipophilic area would be difficult without the aid of protein catalysis (Daum, 1983, but this process is not impossible (Baranska and Wojtczak, 1984). In support of the final hypothesis, contact sites between the two membranes have been observed in rat liver mitochondrial sections. Daum et al. (1982) and Van Venetie and Verkleij (1982) found that contact between the membranes is induced by Ca2+, Mn2+, or Mg2+, and therefore speculated that nonbilayer structures could be involved in this process. The finding that, on perfusion of rat liver with calcium acetate, transport of cytoplasmically synthesized PC from the outer to the inner mitochondrial membrane is accelerated further supports this hypothesis (Ruigrok et al., 1972). VI.

CONFORMATION OF CARDlOLlPlN IN BIOMEMBRANES

A. ASYMMETRIC DISTRIBUTION Mitochondria consist of four subfractions: matrix, inner membrane, intermembrane space, and outer membrane. Of CL in the mitochondrion, 60% is found to lie on the matrix side of the inner mitochondrial membrane (Cheneval et al., 1985), where most enzymes for phospholipid synthesis are located (Voelker, 1985). Earlier estimates were 75-90% (Nilsson and Dallner, 1977; Krebs et al., 1979; Harb et al., 1981). Daum (1985) speculated that CL may be distributed asymmetrically because it is associated with inner mitochondrial membrane proteins that are distributed asymmetrically or because inner mitochondrial membrane phospholipids seem to be arranged according to charge (e.g., negatively charged CL and PI are located preferentially on the matrix side of the inner mitochondrial membrane). Krebs et al. (1979) suggested that CL therefore may play a role in the binding of matrix Ca2+ and in changing the permeability properties of the membrane, since calcium is known to induce a bilayer to hex I1 conformation change in CL (see Section V1,D).

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For new synthesis of CL to occur, CL may be translocated entirely to the cytoplasmic side of the membrane, analogous to the process known to occur for phosphatidyl ethanolamine (PE) and PC (Voelker, 1985). Proteins may participate in this translocation (Yeagle, 1989).

B. INTERACTION WITH CHOLESTEROL AND STEROIDOGENESIS Cholesterol is known to have a classical ordering effect in membranes. Using fluorescence anistrophy, prainarate as a probe, and 31Pnuclear magnetic resonance (NMR), CL was found to bind to cholesterol in a 1 :4 molar ratio (i.e., 1 mol cholesterol per CL acyl chain). High levels of CL in the inner mitochondrial membrane were found not to inhibit cholesterol uptake (Gallay and Vincent, 1986). CL is known to be exceptionally steroidogenic. In contrast, hydrogenated CL has no effect on steroidogenesis. Hence, polyunsaturated fatty acids (PUFAs) in the inner mitochondrial membrane may be required for steroidogenesis. Additionally, the head group of acidic phospholipids such as CL also may be important (Igarashi and Kimura, 1986). C. SPACE-FILLING MODELS OF CARDIOLIPIN Space-filling models of CL indicate that the polar head group has a maximum surface area of 1600 nm2 and that the four hydrocarbon chains are packed tightly in an area of 800-900 nm2. The polar head group of CL is believed to be below the water interface; the four fatty acid residues then account for the surface area of CL at the interface (Rainer et al., 1979; Ries, 1979, Houle et al., 1982). Mead and colleagues (1986) speculated that the shape of CL is suitable for the small radius of curvature of the inner mitochondrial membrane, which is extremely invaginated. D.

HEX I1 CONFORMATION OF CARDIOLIPIN

The hex I1 form is a nonbilayer arrangement that consists of a hydrocarbon matrix surrounded by hexagonally packed aqueous cylinders of 2-nm diameter. Three important questions must be answered about the presence of hex I1 phase lipids in bilayers. ( 1 ) What is the driving force for formation? (2) How is bilayer stability maintained in the presence of hex I1 lipids? (3) What are the advantages of these transient bilayer instabilities? A full discussion of these points is beyond the scope of this chapter. Briefly, however, one advantage of the hex I1 comformation

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might be that phospholipids can interact better with water by lessening the interaction with water in the tightly packed columns. Increased unsaturation increases the surface area each head group is forced to occupy in the lamellar (bilayer) phase. This unfavorable interaction would be exacerbated in the case of highly unsaturated phospholipids such as PE and CL, if they existed in a bilayer with water. An advantage of transient destabilization of the lipid bilayer structure is that membrane fusion (e.g., receptor mediated endocytosis and intracellular vesicular transport) is encouraged, since two membranes can come in closer proximity, with the removal of water between the two membranes (Lindblom and Rilfors, 1989; Yeagle, 1989). Phospholipids with both small head groups, such as PE (which is wedge-shaped so it fits into hex 11), and large head groups, such as CL (in the presence of Ca2+)(Rand and Sengupta, 1972), adopt the hex I1 phase. Monogalactosyl DAG also adopts this phase. As discussed, CL may adopt this form because the hex I1 form is favored with increasing unsaturation and CL is the most unsaturated mitochondria1 phospholipid. After isolation and hydration, the following additional conditions promote hex I1 formation: higher temperature; the presence of DAG (which decreases the transition time from bilayer to hex 11) (Yeagle, 1989); small radius of curvature, as in CL (a close packing of lipid head groups occurs in the hex I1 conformation); low pH or high salt concentration (Seddon et al., 1983); the presence of divalent cations such as Ca2+ (Rand and Sengupta, 1972; De Kruijff et d., 1982; Vasilenko et al., 1982); hydrophobic peptides such as gramicidin (Killian et al., 1986);presence of the cationic local anesthetics such as dibucaine and chlorpromazine (Cullis et al., 1978); mellitin (Batenburg et al., 1987); and basic water-soluble cytochrome c (De Kruijff and Cullis, 1980a). Transition from fluid bilayer La to inverted hex I1 is induced by lowering pH to below 2.8 or by increasing NaCl concentration to greater than 1.6 Mat pH 7 (Seddon et al., 1983). Macdonald and Seelig (1987) found that calcium (1 .O M ) did not have a destabilizing effect on CL bilayers (i.e., formation of hex I1 and isotropic phases) when CL was mixed with sufficiently large proportions of 1palmitoyl-Zoleoyl PC (1 :9 M/M ratio of CL :PC). The induction of the hex I1 phase of CL by calcium is known to be blocked by poly L-lysine (De Kruijff and Cullis, 1980b), adriamycin (Goormaghtigh et al., 1982), and cytochrome c oxidase at Ca2+/CLmolar ratios of 1-10 (Verheul et d.,1981) (see see Section IX,B,l). Prior to and during the formation of the hex I1 phase, two closely opposed bilayers have been postulated to fuse with one another. The bilayer stabili-

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zation by cytochrome c oxidase therefore may be caused by the repulsive forces generated between bilayers by the large hydrophilic region of the protein, which creates a fusion barrier between the vesicles (Rietveld el af., 1987). Alternatively, the membrane-spanning part of cytochrome c oxidase may experience an extensive hydrophobic interaction with the acyl chains of CL. Glycophorin also inhibits hex I1 formation in membranes containing CL (Tomita and Marchesi, 1975; Taraschi et af., 1983). Sankaran (1989) investigated salt-induced bilayer-to-inverted hex I1 phase transitions in CLs with different acyl chain compositions using ”P-NMR spectroscopy. Tetramyristoyl CL was found to undergo a thermotropic lamellar gel-to-lamellar liquid-crystalline phase transition at 33-35°C. This lipid exhibited an axially symmetric ”P-NMR spectrum corresponding to a lamellar phase at all NaCl concentrations between 0 and 6 M. In the case of tetraoleoyl CL, formation of a hex I1 phase was induced by salt concentrations of 3.5 M or greater. These observations, in addition to earlier findings that bovine heart CL aqueous dispersions adopt a hex I1 phase at salt concentrations of 1.5 M or greater, indicate that increasing unsaturation and length of the acyl chains favor formation of the hex I1 phase by CLs. E. ROLE OF CARDIOLIPIN AS AN IONOPHORE Mitochondria1 divalent cation transport is enhanced when hex I1 phase lipid is present, which might be a role for CL (De Kruijff et af., 1981). [See Ioannou and Golding (1979) for a complete discussion.] Van Venetie and Verkleij (1982) found that contact between the membranes is induced by Ca2+,Mn2+,or Mg2+,and therefore speculated that nonbilayer structures could be involved in this process. The finding that, on perfusion of rat liver with calcium acetate, transport of cytoplasmically synthesized PC from the outer to the inner mitochondria1 membrane is accelerated, further supports this hypothesis (Ruigrok et af., 1972).

Phospholipids, by virtue of their amphiphilic properties, can interact in nonpolar media forming “inverted” structures (micelles) that presumably have a hydrophilic core and might act as diffusional carriers (ionophores) of electrolytes across low dielectric constant media or lipid membranes. Accatino (1988) measured the Na+ ionophoretic capability of various purified phospholipids by (1) measuring 22Na+partitioning into the organic phase (chloroform) of a two-phase system and (2) directly measuring the translocation of 22Na+across a bulk chloroform phase separating two aqueous phases in a Pressman cell. All phospholipids tested, except PC, showed ionophoretic capability for Na+ at micromo-

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lar concentrations. CL and PS were the most efficient Na+ carriers, comparable with monensin, an established Na+ ionophore. Cholic acid (a hydroxylated bile acid), sodium dodecyl sulfate (SDS), and Triton X-100, which can induce and stabilize inverted structures in lipid membranes, were able to increase 5- to 8-fold the phospholipid-mediated Na+ transport. Interaction of CL with Na+ in the chloroform phase followed a rectangular hyperbolic function with an apparent K d within the physiological Na+ concentration range (16.9 5.1 mM).

*

VII.

DEGRADATION OF POLYGLYCEROPHOSPHOLIPIDS

An important aspect of cardiolipin abundance and specificity actually may lie in its relative degradation and turnover. This aspect is perhaps even less well described than the biosynthesis of the molecule. Nevertheless, our increasing understanding of how membrane composition is regulated in the case of other phospholipids argues that the degradation step may be very critical. Thus, the lack of information on cardiolipin is discouraging. A. bis(MONOACYLGLYCER0)PHOSPHATE Mitochondria can be degraded by lysosomal hydrolases. bis(MAG)P is resistant to phospholipases because of its unusual stereochemistry. This molecule is degraded by lysosomal phosphodiesterases to lysoPG and MAG (Hostetler, 1982).

B. PHOSPHATIDYLGLYCEROL AND CARDIOLIPIN Little is known about the degradation of PG and CL in mammalian systems. PG and CL can be attacked by mitochondria1 PLA2 (Section IX,B,9), phospholipase C (PLC), and phospholipase D (PLD). About 2% of PG is converted to DAG 3-P (phosphatidate) and glycerol via PLD (Hostetler 1982). Hostetler (1982) reported that CL is hydrolyzed slowly or not at all by most PLCs. In our laboratory, we have demonstrated that CL is cleaved readily by PLC from Bacillus cereus (Sigma #P-7147, St Louis, Missouri) in the presence of appropriate amounts of SDS in uitro. The turnover of CL is reported to be less rapid than that of other phospholipid classes, at least in liver (McMurray and Dawson, 1969). Mammalian CL has a low incorporation rate for phosphorus (Gurr et al., 1965) and GPGPG has a very low turnover rate compared with other phospholipids (McMurray and Dawson, 1969). In contrast, labeled fatty acids are incorporated rapidly into rat liver CL (Taylor et al., 1967). The

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half-lives of phosphate, glycerol, linoleate, and nonessential fatty acid components of CL in rat liver were found to be 10.4, 5.2, 2.2, and 3.1 days, respectively (Landriscina et al., 1976). Brain CL is reported to be very stable (Cuzner et al., 1966). VIII.

OXIDATION OF CARDIOLIPIN

The abundance of PUFAs, location in the inner mitochondrial membrane (in which electron transfer is ongoing), and the potential for active oxygen species production argue that CL could be a major target for oxidative deterioration. We have reported previously the dramatic incorporation of the highly unsaturated fatty acid 22: 6 n-3 in the CL of fish oil-fed mice; this molecule readily is autoxidized in uitro (Berger et al., 1992). Similarly, fish oil-derived alterations in mitochondrial function may be associated directly with CL as the primary target phospholipid (Malis et al., 1990). Finally, Cullis and Hope (1982) compared the number of double bonds in phospholipids of different tissues and found that more unsaturation occurred in more active membranes such as the inner mitochondrial membrane and nerve synapses. Thus, by virtue of its composition, sensitivity to highly unsaturated dietary fatty acids, and location, CL may be the most sensitive phospholipid to oxidation in uiuo. In addition to obvious alterations in structural properties as a membrane phospholipid, oxidation also could lead to the rapid generation of electrophilic decomposition products. Peroxidized CL could cause damage to associated key inner mitochondrial proteins and other proteins (Nielsen, 1978). Perhaps the fact that tocopherol is very high in the mitochondria- 1000 mol PUFA/mol tocopherol-is not coincidental (Buttriss and Diplock, 1988) (see Sections IX,XI,G). The significant potential for autoxidation of CL even has been recognized to compromise standardization of enzyme-linked immunosorbent assays (ELISA) for anti-CL detection (see Section XIII), (Krasnopol’skii et al., 1986). IX. ASSOCIATION OF ENZYMES WITH CARDIOLIPIN

A. POSSIBLE ROLE OF CARDIOLIPIN IN MITOCHONDRIAL PROTEIN IMPORTATION AND TRANSLOCATION The vast majority of mitochondrial proteins is encoded on nuclear DNA, although some proteins are encoded on mitochondrial DNA. Proteins encoded by nuclear DNA have their mRNA translated on free

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polysomes; the polypeptides are imported into the mitochondria posttranslationally. Proteins imported into the mitochondria then must be translocated to the proper membrane site within the mitochondria. [For reviews of mitochondrial protein importation, see Hart1 et al. (19891, Reithmeier (1983, and Roise (1988).] The exact mechanism of translocation, however, remains elusive. Important additional roles for membrane lipids have been suggested repeatedly, both on theoretical grounds and on the basis of experiments with model systems, but no direct evidence had been obtained (De Vrije et al., 1988). Ou et al. (1988) found that, in uitro, synthesized precursors of several mitochondrial proteins (P-450 SCC, adrenodoxin, and malate dehydrogenase) bound to liposomes prepared from mitochondrial phospholipids but not to those from microsomal phospholipids. When liposomes were prepared from various pure phospholipids, adrenodoxin precursor bound only to liposomes that contained CL. The mature forms of adrenodoxin and malate dehydrogenase did not bind to the liposomes. The binding of the precursors was dependent on the concentration of CL in the liposomes. Liposomes containing various CL derivatives with modified polar head groups showed very different binding affinity for adrenodoxin precursor, suggesting the importance of the structure of the polar head of the CL molecule. Two or three positively charged amino acid residues in the extension peptide of P-45O(SCC) precursor were replaced by neutral amino acid residues by site-directed mutagenesis. The mutated P-45O(SCC) precursors did not bind to the liposomes containing CL. These results indicate that mitochondrial protein precursors have specific affinity for CL, and that this affinity is caused by the interaction between the extension peptides of the precursors and the polar head of the CL molecule. CL also is known to bind to the precursor apo cytochrome c more strongly than to cytochrome c (Rietveld et al., 1983). Nesmeyanova and Bogdanov (1989) theorized that, in bacteria, positively charged hydrophobic regions of secretory proteins are inserted into the membrane bilayer where they interact with the unsaturated fatty acids of CL. This insertion is promoted by the fact that unsaturated chains allow for increased mobility. The secretory protein-CL interaction then leads to conversion of CL to PG via ATP-dependent PLD; unfolding of the secretory protein is initiated. Because of the neutralized charge of PG, the protein-PG complex acquires the ability to move to the hydrophobic region. Flip-flop is initiated by release of free energy by exclusion of the orderly bound water from the surface of both the protein and the phospholipid. PG then moves across the membrane, carrying the bound peptide with it. Subsequently, the secretory protein is released into the cytoplasm.

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In yeast, a presequence of 25 amino acids is known to be required for import of cytochrome c oxidase subunit IV into mitochondria. Goormaghtigh et al. (1989) investigated the structure and orientation of this 25-mer peptide in a lipid bilayer by infrared attenuated total reflection spectroscopy. These investigators demonstrated that incubation of the peptide with dioleoyl PC and dioleoyl PC-CL liposomes led to an increase in a-helical content compared with p structure. Polarization measurements indicated that the amphipathic helical segment was inserted parallel to the lipid acyl chains in CL liposomes.

B. ENZYME ASSOCIATIONS The inner membrane of the mitochondrion is a very selective permeable barrier, in contrast with the outer membrane, which has poreforming proteins similar to bacterial porins (Roos et al., 1982). In the inner mitochondrial membrane, 12 functionally different carriers are found (LaNoue and Schoolwerth, 1979), some of which are associated with CL. Such associations were recognized first by Richardson (1964). These workers found that CL was effective at restoring the original protein conformation of lipid-depleted inner mitochondrial membranes. In the sections to follow, a brief overview of the function and structure of specific proteins known to be associated with CL is provided. 1.

Cytochrome c Oxidase and Precursor Proteins; Na+IK+ ATPasg

Cytochrome c oxidase (EC 1.9.3.1) is the last of the three protonpumping assemblies of the respiratory chain at which electrons are transferred from cytochrome c to oxygen to form water. The protein contains two heme A groups and two Cu ions. Cytochrome c oxidase is encoded on mitochondrial DNA; subunit I1 is translated on mitochondrial ribosomes that are associated with the inner surface of the inner mitochondrial membrane. Cytochrome c oxidase probably is inserted cotranslationally into the inner mitochondrial membrane (Reithmeier, 1985). The association of CL and cytochrome c oxidase was reported first in 1970 (Awasthi et al., 1970, 1971). Now, cytochrome c oxidase is known to bind 2-3 molecules of CL very tightly, which is essential for activity of the enzyme (Chuang and Crane, 1973; Robinson et al., 1980; Fry and Green, 1980, 1981; Vik et al., 1981; Robinson, 1982a,b; Marsh, 1983; Dale and Robinson, 1988). For example, Robinson et al. (1980) showed that the addition of CL was essential to restore full activity to cytoCyt c oxidase is also called cyt oxidase and ferrocytochrorne c :O2 oxidoreductase.

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chrome c oxidase that had been delipidated with Triton X-100. The subunit of cytochrome c oxidase that binds CL is not known (Cheneval and Carafoli, 1988). Other phospholipids are associated with cytochrome c oxidase, but with lower affinity, or the association is not essential for activity (Robinson et al., 1980). The negative charges of CL may not be important in the tight binding of cytochrome c oxidase to CL (Robinson, 1982a) or in the weaker interaction of cytochrome c oxidase and CL in enzyme-phospholipid mixtures (Knowles et al., 1981). Studies using electron spin resonance spectroscopy (Knowles et al., 1981) and differential scanning calorimetry (Semin et al., 1984; Rigell et al., 1985) revealed that the acyl chains of CL may interact hydrophobically with the membrane-spanning part of cytochrome c oxidase. In this manner, cytochrome c oxidase may influence directly and perturb up to 55 CL molecules and 80-100 dimyristoyl PC molecules (i.e., CL forms a shell around the protein, which is in good agreement with a boundary layer model). Lee (1983) suggested that, in mixtures of phospholipids and proteins, the binding of the four acyl chains of CL to protein will be favored over the binding of only two acyl chains of other phospholipids to protein. Powell et al. (1987) then examined binding of CL derivatives to cytochrome c oxidase. The binding of CL derivatives from strongest to weakest was: CL-monolyso CL 1 pentaacyl CL (an extra fatty acid attached to C 2’) >dimethyl CL (methylation of PO4 groups) >>PC (zwitterionic). The same order of affinity of CL derivatives was established for Na+/K+ ATPase in the dogfish shark Squalus acanthias. (Esmann et al., 1988). Hence, the associations of CL with Na+/K+ ATPase and cytochrome c oxidase do not have just electrostatic origin since dimethyl CL had greater affinity than the more negatively charged PC or hydrophobic origin (relative to the number of fatty acid^).^ Dale and Robinson (1988) synthesized CL derivatives with fatty acyl substitutions at both of the 2 positions (2‘ and 2”) of CL and examined the activities of cytochrome c oxidase. Both activation parameters, the apparent dissociation constant and the maximum change in molecular activity of cytochrome c oxidase, were found to depend on the chain length of the tails, with less dependence on the degree of saturation. Natural CL (92% 18:2n-6, 8% 18: ln-9), and CL disubstituted with 18 : ln-9 (47% 18 : 2n-6, 52% 18 : ln-9) were equally effective at stimulating cytochrome c oxidase activity, with an apparent dissociation constant of approximately I p M when incubated in 0.3% Triton X-100 and assayed in lauryl maltoside. CL disubstituted with 6 :0, however, was For an interesting parallel discussion of the effects of derivatization of PC on the association with P-hydroxybutyrate, see the review by Daum (1985).

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found to be a poorer activator, with an apparent dissociation constant of 6.8 p M and a maximum change in molecular activity that was 50% of that achieved with natural CL. DilysoCL, with complete removal of two of the fatty acid tails, showed negligible stimulation of cytochrome c oxidase activity. In this investigation, 18 : ln-9 and 18 : ln-7 apparently were lumped together; the activation by monolysoCL was not examined. Semin et al. (1984) and Koppenol and Margoliash (1982) suggested that the binding of CL to cytochrome c oxidase may help orient cytochrome c oxidase and the dipolar cytochrome c in a way that reduces the activation energy of the reaction between these two. In this interaction of proteins, CL would be organized in a bilayer arrangement, since CL in the hex I1 phase does not associate with cytochrome c oxidase and because cytochrome c oxidase inhibits calcium-induced hex I1 formation at Ca*+/CLmolar ratios of 1-10 (Rietveld et al., 1987). 2 . FIFoATPase’

CL also is associated with and necessary for the activity of FIFO ATPase (Santiago et al., 1973; Vik et al., 1981). The FI subunit is located on the matrix of the inner membrane, and consists of five polypeptide chains. The normal role of the F, subunit is to catalyze the synthesis of ATP (solubilized F1 has ATPase activity). The Fo subunit spans the inner membrane, consists of four polypeptide chains, and probably forms the transmembrane pore for protons. The overall reaction catalyzed by this enzyme is ADP3- + Pt- f* ATP- + HzO. (see Section X,E,l) (Stryer, 1988). This enzyme also binds PS (Brown and Cunningham, 1982). The degree of chain length and unsaturation of the fatty acids plays a role in modulating enzyme activity (Bruni et al., 1975; Pitotti et al., 1980). The enzyme also is inhibited by cholesterol in uitro, presumably because of a condensing effect of cholesterol (Pitotti et al., 1980). 3. ADPIATP Carrier Protein6

The inner mitochondria1 membrane contains three to four protein carriers, depending on the tissue: the ADP/ATP carrier protein; the uncoupling protein or thermogenin, which resembles the ADP/ATP carrier protein; the dicarboxylate carrier protein; and the phosphate carrier

FIFOATPase is also called ATP synthase H+ ATPase oligomycin-sensitive ATPase. ADP/ATP carrier protein is also known as ATP-ADP translocase or adenine (nucleotide) translocase or adenine (nucleotide) carrier.

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protein. Based on comparison of the primary structure, the phosphate carrier protein, the ADP/ATP carrier protein, and the uncoupling protein all are thought to originate from a common ancestor (Aquila et al., 1987; Runswick et al., 1987). The ADPlATP carrier protein allows ATP and ADP to transverse the inner mitochondrial membrane by catalyzing the reaction: cytosolic ADP + mitochondrial ATP mitochondrial ADP + cytosolic ATP. Approximately 6 CL molecules are bound to the isolated ADP/ATP carrier protein (Beyer and Klingenberg, 1985). The binding of CL to the ADP/ATP carrier protein requires SDS denaturation and heat treatment (Cheneval and Carafoli, 1988). The binding of CL to this protein represents the highest specific phospholipid, binding discovered to date. The ADP/ATP carrier protein constitutes 9- 10% of the total mitochondrial membrane protein and is located on the outer layer of the inner membrane. The large abundance as well as the relatively low molecular weight of this dimeric intramembrane protein (67,000), implies that it contributes strongly to the total lipid-protein interaction in the inner mitochondrial membrane (Mende et al., 1983; Beyer and Klingenberg, --f

1985).

The mitochondrial creatine kinase (see Section IX,B ,5) ultimately must accept ATP from this ADPlATP carrier protein. Since both proteins are associated with CL, a close functional link between the three molecules must exist. Perhaps CL somehow couples the kinase to the carrier (Cheneval and Carafoli, 1988). The two molecules do not seem to interact physically, however (Cheneval et al., 1985). The activity of the ADP/ATP carrier protein is not absolutely dependent on CL interaction (Bradford, 1976). Cheneval and Carafoli (1988) proposed that the mitochondrial creatine kinase molecule itself could swing on its membrane-anchoring N terminus to bring the ATP-binding site of creatine kinase into contact with the ATP-delivering end of the ADP/ATP carrier protein during its lateral movement on the outer side of the inner membrane. CL thus would have a structural interaction with the ADP/ ATP carrier protein and functional interaction in the binding of creatine kinase. 4 . Phosphate Carrier Protein7

The phosphate carrier protein is located on the inner mitochondrial membrane and carries out the coupled transport of H2P04- and H+ (or Phosphate carrier protein is also known as phosphate transport protein, phosphate translocator,or phosphate/hydroxylexchanger.

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OH-) (Ligeti and Fonyo, 1987). The mechanism of transport of this protein involves interactions with lysine residues on the surface of the inner mitochondrial membrane and sulfhydryl groups of the protein (Genchi et a f . , 1988). CL is required for full activity of the reconstituted enzyme and for preventing irreversible inactivation of the protein during its extraction from the mitochondria with Triton X-100or Triton X-114 (Kadenbach et al. 1982; Aquila et al., 1987). 5. Creatine Kinase and Sequence Homologies of Other Proteins

Phosphocreatine is converted to creatine via creatine kinase (EC 2.7.3.2) in muscle, releasing ATP. The mitochondrial isoenzyme of creatine kinase was discovered in 1964 (Jacobs et a f . , 1964). Lipskaya (1980) found that excess cytochrome c oxidase inhibited binding of creatine kinase to the beef heart mitochondrial membrane, suggesting that creatine kinase binds to phospholipids. Cheneval et al. (1985) determined that creatine kinase binds to CL on the outer side of the inner mitochondrial membrane, where the enzyme is known to be located (Scholte et al., 1973). Schlame and Augustin (1985) found that a CL-specific phospholipase released the enzyme from the mitochondria, thus further supporting the association of CL and creatine kinase. The interaction of CL and the enzyme was thought to be ionic since the rebinding of solubilized creatine kinase to mitochondria and to CLcontaining vesicles was inhibited on increasing the ionic strength of the incubation medium with KCL or NaCl (Lipskaya et al., 1980). In a later experiment, Cheneval and Carafoli (1988) purified mitochondrial creatine kinase to homogeneity from rat heart mitochondria and identified the primary structure of the CL-binding domain; they also identified an ATPbinding site. The CL-binding domain was identified as a 25-amino-acid N terminus, abundant in the basic residues lysine, histidine, arginine, glutamine, and asparagine, and containing one contiguous region of three basic residues (Arg-Lys-His) that was thought to interact with the two negatively charged phosphate groups of CL. Chemical modification of the basic amino acids lysine and arginine abolished binding of CL to creatine kinase. CL did not interact with the cytosolic isoform of the creatine kinase, which shares no N-terminal amino acid sequence homology with the mitochondrial isoform. Cheneval and Carafoli (1988) examined sequence homologies among other CL-associated proteins and found that subunit I of cytochrome c oxidase had 53% homology with the CL-binding domain of creatine kinase, and subunit V, in a stretch of 23 amino acids, had 9 identical residues; three of these were basic. Hence, subunits I and V are implicated

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in the binding of CL. The C terminus of the ADP/ATP carrier protein (see Section IX,B ,3) shares sequence homology with the binding region of creatine kinase (e.g., lysine residues; Bogner et al., 1982). However, since SDS and heat treatment of the ADP/ATP carrier protein were necessary for association with CL, the ADP/ATP carrier protein is believed to interact with CL at a hydrophobic binding region that does not share sequence homology with creatine kinase (Cheneval and Carafoli, 1988). Cytochrome c oxidase also is thought to interact with CL through hydrophobic interactions (see Section IX,B,l). 6 . Carnitine/Acyl Carnitine Translocase, and Carnitine Palmitoyl-Transferase8

Interest in the essentiality of carnitine, the role of carnitine in diseased states, consequences of carnitine palmitoyl-transferase(CPT) deficiency, and consumption of megadoses of carnitine by health food faddists has resumed. For example, during ketotic states in which acetyl CoA carboxylase is inactive and less malonyl CoA is generated, CPT sensitivity to malonyl CoA inhibition is diminished to prevent further increases of ketone bodies, which are generated from fatty acids that enter the mitochondria. Fatty acids are activated on the outer mitochondrial membrane. The acyl group then is transferred from the sulfur atom of CoA to the hydroxyl group of carnitine to form acyl carnitine. This reaction is catalyzed by CPT I. Acyl carnitine translocase then shuttles acyl carnitine across the inner mitochondrial membrane and returns carnitine to the cytosolic side in exchange for another acyl carnitine. The acyl group is transferred back to CoA on the matrix side of the membrane by CPT 11. Some debate over whether CPT I and I1 are separate enzymes has been generated. CPT activity is located on both outer and inner sides of the mitochondrial inner membrane (Brady and Brady, 1987). In peroxisomes, carnitine octanoyl transferase is a separate enzyme with higher affinity for shorter chain fatty acids. Noel and Pande (1986) investigated the phospholipid requirement for optimal solubilization of carnitine/acyl carnitine translocase from the inner membrane vesicles of rat liver mitochondria and for reconstitution of carnitine/acyl carnitine translocase activity in liposomes. CL was the best phospholipid for solubilization of carnitinelacyl carnitine translocase at the octylglucoside solubilization step and was more effective than Carnitine palmitoyl-transferase previously was known as carnitine acyl transferase (CAT).

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other anionic phospholipids for reconstitution. Similar results were obtained by Noel et al. (1985) when CL was added to egg yolk PC liposomes. Carnitine/acyl carnitine translocase activity was diminished when adriamycin was used to bind CL. Fiol and Bieber (1984) purified CPT from beef heart mitochondria. The purified enzyme has a molecular weight of 67,000 and binds 18.9 mol phospholipids/mol enzyme. The bound phospholipids were shown to be CL, PC, and PE (three unidentified minor spots were detected also). Although these researchers did not perform a quantitative analysis, CL was the major component of the phospholipids. Noel and Pande (unpublished; cited in Noel and Pande, 1986) found that CPT activity was stimulated by CL only when the reaction was followed in the direction of palmitoyl carnitine. Since CL is associated with CPT and the carnitine/acyl carnitine translocase, Noel and Pande (1986) suggested that CL may provide adhesion sites for CPT and carnitine/acyl carnitine translocase within the membrane and facilitate, by electrostatic interaction, the flow of acyl carnitines from the outer to the inner side of the membrane. Brady and Brady (1987) found that addition of adriamycin to both intact rat liver and heart mitochondria (outer CPT) and inverted submitochondrial vesicles (inner CPT) depressed CPT in the forward direction of the reaction (palmitoyl carnitine formation), but inner CPT activity was more sensitive to the inhibitor. Adriamycin depressed the outer CPT reverse reaction (palmitoyl CoA formation) to 40% of control, but had no effect on the inner CPT reverse reaction. Addition of CL (0.25-1.0 mg/ assay) increased activity of the outer CPT forward reaction of both control and galactosamine-treated rats, but did not affect inner CPT activity. The results suggest that outer CPT and inner CPT may be influenced differently by perturbants that affect lipids of the membrane. 7. a-Glycerol-Phosphate Dehydrogenase a-Glycerol-phosphate dehydrogenase (a-GPDH; EC 1.1.99.5) is located on the inner mitochondrial membrane. Isolated a-GPDH from pig brain (Cottingham and Ragan, 1980) and rat liver (Garrib and McMurray, 1986) contains CL. In isolated liver mitochondria from hyperthyroid rats, the kinetics of the reaction catalyzed by a-GPDH were affected by adriamycin (see Section IX,B, 10). The effect of adriamycin was dependent on the electron acceptor. CL was thought to influence electron acceptor binding sites (hydrophobic quinone binding sites and hydrophilic sites) on both leaflets of the inner mitochondria1 membrane, since adriamycin penetrates into both leaflets (Beleznai and Jancsik, 1989).

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8. Phospholipase A2 Intracellular PLAz plays an important role in liberating arachidonate, eicosapentaenoate, and other important eicosanoid precursors from phospholipids (Dennis, 1987). The enzyme is also important for membrane retailoring and removal of peroxidized fatty acids (Van Kuijk et al., 1987). Conflicting data exist concerning the location of PLAz in the mitochondria (Wait, 1969; Nachbaur et al., 1972; Severina and Evtodienko, 1981; Zurini er al., 1981). The function of PLAz in subcellular organelles has not been elucidated fully (Van den Bosch, 1982). Reers and Pfeiffer (1987) found that PLAz extracted from the acetone powder of previously frozen rat liver mitochondria was inhibited strongly compared with the activity present before acetone powder preparation. Activity was recovered substantially on partial purification of the enzyme by gel filtration chromatography. Inhibitor activity was eluted into the void volume of the column when void volume fractions were subjected to Folch extraction; the inhibitor activity was present in the chloroform layer. Structural studies supported identification of the inhibitor as monolysoCL. Under the assay conditions employed, one molecule of the inhibitor per 5000 substrate molecules or 40 nM on a nominal concentration basis was Is0 for the mitochondrial enzyme. MonolysoCL was similarly effective against pancreatic and snake venom PLAz. MonolysoCL and dilysoCL prepared enzymatically from bovine heart CL were less potent than the material arising from rat liver CL by factors of 10- and 30-fold, respectively, yet were still highly potent compared with the other known inhibitors of this enzyme. Differences in acyl group composition, the degree of acyl group oxidation, or structural isomerism between the sn-1 and sn-2 positions of the lyso compounds may have accounted for the difference in potency between the materials derived from rat liver and bovine heart. In related publications, these authors reported that monolysoCL, at an inhibitor to substrate ratio of 1 : 1000, reduced PLAz activity by 50% (Reers and Pfeiffer, 1985; De Winter, 1987). Other researchers also have reported that hydrolysis products inhibit PLAz activity in uitro (De Winter et al., 1982). According to Lenting et al. (19881, however, these products are unlikely to act as inhibitors in uiuo where concentrations are lower. Lenting (1988) reported that the PLAz hydrolysis of trace amounts of 1-acyl-2-[l-'4C]linoleoyl PE, in membranes constructed of mitochondrial PE and PC in proportions similar to those of mitochondrial membranes (0.6 molar ratio), was stimulated by the addition of CL and PG. Other negatively charged phospholipids (P1,PS) did not have this effect. The authors speculated that the stimulation may be caused by a favorable

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interaction with the negatively charged phosphates of these molecules or by a change in conformation from and initial bilayer configuration of the PE-PC mixture to hex 11, after CL and PG addition, as detected by "P-NMR. To test whether the effects of CL and PG were caused by structural rearrangement, the authors added CL (in the presence of Cazf) and PG to membranes only containing PE. Under these conditions, both the initial PE and the added CL organized in a hex I1 phase (PG forms bilayers, but the possibility that the small amounts of PG added to the incubation mixture induced conformational changes in PE was considered unlikely (Rand and Sengupta, 1972; Cullis et al., 1978). Under these conditions, CL and PG still enhanced hydrolysis (PI and PS apparently were not tested in this experiment). In the presence of Triton X-100, in which the phospholipids exist in a mixed micellar conformation, the stimulation of CL and PG was less pronounced (see Section V1,D). 9. Protein Kinase C

Rodriguez-Paris et al. (1989) used dansyl PS (DPS) as a fluorescence probe to study interactions with protein kinase C (PKC) and phospholipid vesicles. DPS fluorescence (520 nm) was enhanced by PKC (excited at 285 nm), the fluorescence energy transfer indicative of a close association of PKC with DPS vesicles. Homogeneous vesicles containing 2 p M DPS plus 2 p M CL and 10 pA4 CaCI2 incubated with type 111 isozyme PKC showed enhanced excitation relative to incubation with PS alone (180% increase in fluorescense, relative to 120%). In heterogeneous vesicles containing PKC incubated with 2 pA4 DPS and 10pM CaCh and 8 p M CL, fluorescense was decreased (120% to 60% with CL present). CL may compete with PS for interaction with PKC. As the concentration of CL in vesicles was increased from 0.025 to 0.15 (PI : PE, 1.0 : 0.251, the apparent K , for PI decreased from 173 to 96 p M . 10. Glutamate Dehydrogenase CL and PS inhibit glutamate dehydrogenase (GDH) by binding to the enzyme (Julliard and Gauthernon, 1972; Dodd, 1973; Godinot, 1973; Nemat-Gorgani and Dodd, 1977; Pour-Rahimi and Nemat-Gorgani, 1987; Couee and Tipton, 1989). CouCe and Tipton (1989) reported that the inhibition of mitochondria1 GDH by CL, but not PS, was reversed by Lleucine (e.g., 5 mM leucine relieved 75% of CL inhibition). The concentration of CL necessary to give 50% inhibition was variable, ranging from 2 to 40 pg/ml (15-70 2-40 pg/ml for PS). PC had no effect on enzyme

286

ALVIN BERGER et al.

activity up to 55 p M ; hence, the inhibition by PS and CL is not simply nonspecific action. Interestingly, purified GDH from ox liver and brain revealed no bound phospholipid using an assay that could detect 5 nmol phosphate/mg protein. Nevertheless, in intact mitochondria, GDH appeared to be inhibited strongly (Dodd, 1973). Godinot (1973) found that the presence of glutamate increased the association of GDH with CL. 1 1 . DNA Polymerases

CL, PI, and phosphatidate have been shown to inhibit mitochondrial DNA polymerase y in uitro when preincubated with the enzymes in the absence of template primer (Yoshida et al., 1989). The inhibition of DNA polymerase y by CL was nearly competitive with template primer. CL also inhibited the reactions of a and /3 eukaryotic DNA polymerases and terminal deoxynucleotidyl transferase. CL is thought to interact with DNA polymerase y at the hydrophobic region since 0.05% Triton X-100 during preincubation reversed the inhibition. DNA polymerase y has been implicated in mitochondrial DNA replication; hence, CL and other phospholipids may be involved in mitochondrial DNA replication. In E. coli, CL is known to interact with a nucleotide binding site of a DNA protein that could be important in regulating the cell cycle (Sekimizu and Kornberg, 1988). 12. Other Enzymes Complex I (NADH ubiquinone oxidoreductase) (Heron et al., 1977, 1979; Ragan, 1978; Earley and Ragan, 1980; Fry and Green, 1981; Poore and Ragan, 1982) and complex I11 (bcl complex) (Yu et al., 1979; Fry and Green, 1981; Shimomura and Ozawa, 1981, 1982) bind CL, PC, and PE. Isolated ATP translocator, succinate cytochrome c reductase (Fleischer et al., 1977; Yu et al., 1978; Sondergaard, 1979), mitochondrial cytochrome P-450 (Hall et al., 1979; Lambeth, 1981), succinate oxidase (Lenaz et al., 1971), NADH oxidase (Lenaz et al., 1971), pig liver monoamine oxidase (Oreland and Olivecrona, 1971; Ekstedt et al., 1975), GDH ( Julliard and Gauthernon, 1972), a-protein (a delipidated bovine serum a-lipoprotein) (Fleischer et al., 1967a,b),rat liver lysosomal lipase (Kariya and Kaplan, 1973), and y-globulin (Marinetti and Pettit, 1968) also are known to bind to or be activated by CL. The cationic polypeptide polymyxin B was found to require anionic phospholipids such as CL or PG to bind and destroy gram-negative bacte-

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rial membranes (Teuber and Miller, 1977). Nicotinamide dinucleotide transhydrogenase activity in rat liver mitochondria was found to be inhibited by CL (Kelker and Pullman, 1979). Shirai et al. (1988) found that human serum carboxylesterase (EC 3.1.1.1) hydrolyzed tributyrin (40 nmol/mg proteidhr), but scarcely hydrolyzed triolein (<0.2 nmol/mg proteidhr), unless phospholipids were present (2.5 nmol/mg protein/hr with CL; 450-980 nmol/mg/hr with PS and PI). The rates of hydrolysis of monoolein, diolein, and triolein by carboxylesterase in the absence and presence of 100 pg/ml CL were 3.9, 0.5, and 0.2 nmol/mg/hr and 2.0, 0.6, and 4.0 nmol/mg/hr, respectively. Thus, on addition of CL, triolein hydrolysis was enhanced but tributyrin hydrolysis was decreased reciprocally. Triton X-100(0.1%) and NaCl (1.0 M ) decreased triolein hydrolysis but did not decrease tributyrin hydrolysis. Mercaptoethanol decreased triolein hydrolysis but not tributyrin hydrolysis. These results suggest that CL modifies the interaction of carboxylesterase with substrates in a way that facilitates interaction of the enzyme with a hydrophobic substrate, and that disulfide bonding might be involved in the substrate recognition site.

13. Adriamycin Adriamycin (doxorubicin) is used to treat leukemia and solid tumors. However, a negative consequence of this drug is cardiotoxicity, which may be a result of the formation of a CL-adriamycin complex (approximately 2 mol adriamycin/mol CL) that can lead to impaired enzyme function (Goormaghtigh et al. 1987; Hasinoff and Davey, 1988; DupouCCzanne et al., 1989). The planar anthracycline moiety of adriamycin intercalates between DNA base pairs, and the sugar moiety fits into the DNA large groove. Like DNA, CL also contains phosphodiester-linked ionized phosphate groups that are separated by three carbon atoms. Therefore, CL is a target for adriamycin binding (Schwartz, 1988). Whether the binding of adriamycin to CL itself is membrane damaging or whether the association simply might concentrate the drug in the mitochondria long enough for another type of mechanism, such as free radical damage, to occur is not clear (Weinstein, 1987). Hasinoff et al. (1989) found that the adriamycin-induced inactivation of cytochrome c oxidase is strongly dependent on the presence of Fe3+ and Cu2+,present in phosphate and Tris buffers. No adriamycin-induced inactivation of cytochrome c oxidase occurred in the presence of ethylene diamine tetraacetic acid (EDTA) or in phosphate buffers purified on a cation exchange column to remove trace metals. CL partially protects

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against cytochrome c oxidase inactivation, presumably by binding to the cytochrome c oxidase, whereas catalase or superoxide dismutase partially protects by scavenging damaging reactive oxygen species generated by a Fe3+-adriamycin-enzyme complex. A positive consequence of the adriamycin-CL association, however, is that adriamycin can be useful as a probe in CL-protein binding assays and in determining the asymmetric position of CL on the inner mitochondrial membrane (Krebs et al., 1979; Goormaghtigh et al., 1980, 1986; Cheneval et al., 1983, 1985; Daum, 1985; Cheneval and Carafoli, 1988). Delgado et al. (1989) exploited this association by administering adriamycin intraperitoneally in encapsulated liposomes containing CL, PC, cholesterol, and stearylamine to patients with advanced ovarian cancer. Liposome-encapsulated adriamycin was well tolerated. Mayhew et al. (1987) determined the effects of non-drug-containing liposomes of different compositions and sizes on the proliferation of nine cancer-derived cell lines and one normal cultured human cell line. Stearylamine- and CL-containing liposomes were toxic (IDSO)at 200 p M liposoma1 lipid concentrations or less, whereas PG-and PS-containing liposomes were toxic in the range 130-3000 pM. DipalmitoylPC liposomes were not toxic at 3000-4000 pM. In general, small liposomes were more toxic than large ones. The results indicate wide variations in toxicity of non-drug-containing liposomes to cultured human cells. The potential for nonspecific toxicity caused by the liposomes themselves should be considered carefully if human administration of drug-containing liposomes is to be done. Malatesta and Andreoni (1989) studied the molecular interaction between CL vesicles and two representative anthracyclines, daunomycin and 5-iminodaunomycin, by laser time-resolved fluorescence. The fluorescence lifetime of daunomycin is 1.03 nsec. For a CL : anthracycline ratio of 0.3-5, a longer lived transient (1.91-1.49 nsec) was present, originating from the excitation of daunomycin bound on a single phosphate group of CL. For a ratio of 0.3, two lifetimes were observed, the second one being due, partially, to free daunomycin and bound drug molecules embedded in the lipid bilayer. The fastest decaying species was present at a ratio of 0.5-2.0 and identified as two adjacent, stacked daunomycin molecules bound onto the two phosphate groups of CL. In the case of 5-iminodaunomycin, a less cardiotoxic analog, exponential decay was never observed and a fast-decaying component, approximately 0.2 nsec, was.already present at low ratios of CL to drug and vanished for ratios greater than 0.5. The constancy of the lifetimes of the longer lived species may originate from the reorientation of the bound drug from the hydrophilic to the lipid domain.

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289

CARDIOLIPIN ACYL COMPOSITION

Although few studies have been undertaken to determine the role of nutritionally based fatty acid composition on CL function, various reports have developed a database from which implications can be drawn. Acyl changes in phospholipids are known to influence membrane fluidity (Lynch and Thompson, 1984, 1988; Greene and Selivonchick, 1987) and to alter membrane-associated enzymes or receptor activities (Lynch and Thompson, 1984; Neuringer et al., 1988; Zuniga et al., 1989). Investigating the changes possible and coupling these possible changes with mechanistically based alterations in specific functions is, thus, important. Considering the overall homogeneity and relative constancy of composition of most phospholipids, changes in CL composition are genuinely remarkable. CL acyl composition is affected by age (Innis and Clandinin, 1981; Keranen et al., 1982), alcohol ingestion (Waring et al., 1981; Cunningham et al., 1982; Cunningham and Spach, 1987; Ellingson et al., 1988), hyperthyroidism (Landriscina et al., 1976; Hoch et al., 1980; Ruggiero et al., 1984), disease state (Dyatlovitskaya et al., 1973a; Morton et al., 1976), and dietary modification (Divakaran and Venkataraman, 1977; Hsu and Kummerow, 1977; Ishinaga et al., 1982; Wolff et al., 1985a,b, 1988; Wolff, 1988). CL acyl composition also differs among species (Charnock et al., 1986; Swanson and Kinsella, 1986; Yamaoka et al., 1988; Berger and German, 1990), within species, and between organs (Wolff et al., 1985b). A. UNIQUENESS OF CARDIOLIPIN ACYL COMPOSITION With relatively few exceptions, phospholipids exhibit a pattern of fatty acyl distribution in which a saturated fatty acid is esterified at the sn-1 position and an unsaturated fatty acid is esterified at the sn-2 position. The fatty acyl composition of CL is unique in containing up to 84% 18 :2n-6 in mice (Berger and German, 1990) and rats (Yamaoka et al., 1988); 18 : ln-7, the elongation product of 16 : ln-7 (Reichwald-Hacker et al., 1979), in equal or greater abundance than 18 : ln-9 (8-13%) (Wolff et al., 1985b; Berger and German, 1990), small amounts of the elongation products 20 : 3n-3 (0.8%) and 20 :2n-6 in mice (Berger and German, 1990) and rats (2-3%); and A5,11,14 20: 3 (Wolff, 1988). In contrast, 18 :2n-6 does not predominate in bis(MAG)P and PG. The composition of rat liver mitochondria1 PG is 12% 16 :0, 14% 18 :0, and approximately 20% each 18 : ln-9, 18 :2n-6, and 20: 0 (Gray, 1964). Hence, the fatty acyl composition is widely different among the po-

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lyglycerophospholipids. This result is interesting considering the interrelationships of the compounds (see Section IV,C). In bacteria, PG and CL have a more similar fatty acid composition (Hostetler, 1982).

B. POSITIONAL DISTRIBUTION OF FATTY ACIDS Wolff (1985b) reacted CL with PLAz to find the positional acyl specificity of CL. In the mitochondria of rat heart, liver, and kidney, the 2 and 2’ position consisted of 16: 0 at 1.4-3.5%, 18 :0 at 0.4-0.7%, 18 : ln-9 at 3.7-6.2%, 18 : ln-7 at 15-26%, and 18 :2n-6 at 62-78%. In the 1 and 1’ position, 18 : 2n-6 occupied 85-89% and was, therefore, more abundant and less variable than in the 2 and 2’ position. Overall octadecenoate was 9-10 times more abundant at the 2 and 2’ position than at the 1 and 1’ position. Teng and Smith (1985) attempted to define the molecular species of CL using reversed-phase higher performance liquid chromatography (HPLC) and, in some cases, thin layer chromatography (TLC), melting behavior, and infrared absorption spectra. Single molecular species rarely were resolved. 18 :2n-6 was identified in all peaks. Molecular species tentatively identified included trilinoleoyl-palmitoleoyl, dilinolenoyllinoleoyl-stereoyl, and trilinoleoyl-linolenoyl CL. C. INTERSPECIFIC DIFFERENCES IN CARDIOLIPIN ACYL COMPOSITION Enormous interspecific differences exist in the acyl composition of CL. The fatty acyl composition of bovine heart CL is 1% 16: 0, 1.5% 16: ln-7, 1% 18:0, 4% 18: ln-9,7, 87% 18: 2n-6, and 5.5% 18:3n-3. In contrast, E. coli CL contains 32% 16: 0, 1% 18 : 0, 38% 18 : ln-9,7, no 18:2n-6, and 29% cyclopropane fatty acids (Avanti Polar Lipids, Pelham, Alabama). Other bacterial CLs contain 40-50% 16:O (Mendoza and Farias, 1988; Ratledge and Wilkinson, 1988) or up 84% 14: 0 (e.g., the spirochete Treponema pallidum, which causes syphilis in humans) (Krasnopol’skii et al., 1986). The total amount of saturated fatty acid residues is as high as 98% in some bacteria (Vasilenko et al., 1982). Yeast CL contains negligible amounts of 18 :2n-6 and 42% 16 : ln-7 (see Ioannou and Golding, 1979). If any metabolic functions of CL require 18:2n-6 specifically, these functions apparently are not operating in bacteria.

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29 1

D. INTERORGAN DIFFERENCES IN CARDIOLIPIN ACYL COMPOSITION Very little research has been directed specifically to determine the basis of interorgan or intertissue variations in phospholipids in general. Thus, CL is not unusual in the lack of data on variations possible and mechanisms proposed to explain them. In the rat brain, lung, and testes, 18:2n-6 constitutes 8-15% of the total fatty acids in CL whereas, in other tissues, 16 :0 and 18 :2n-6 account for >60% (see Hostetler, 1982). In 21-day-old Wistar rats (probably fed standard rat chow), heart CL contained more 22:6n-3 and less 18:2n-6 than liver and kidney CL (Wolff et al., 1985b). The unique location and synthesis of polyglycerophospholipids may make them a useful basis for investigating fatty acid targeting to specific tissues. E. INFLUENCE OF DIET ON CARDIOLIPIN ACYL COMPOSITION

I . Polyunsaturated Fatty Acids Berger and German (1990) demonstrated that dietary 20 : 3n-3 was incorporated selectively into the CL pool of various organs at a level of 4.1-8.2% after only 2 wk feeding of 2% fat diets to mice. Yamaoka et al. (1988) found that, in rats fed diets containing 16.8% fish oil, 22 : 6n-3, 18: ln-7, and 18: ln-9 increased and 18:2n-6 decreased (from 84 to 24-44%) in heart CL. In studies by Charnock et al. (1986) and Swanson and Kinsella (1986), fish oil feeding similarly increased 22 : 6n-3 and diminished 18 : 2n-6 in the heart CL pool. Roblee and Clandinin (1984) showed that the 18: 20-6 content of CL was elevated significantly with low-fat feeding (15% calories as fat) compared with high-fat feeding (40% as fat) in rat heart mitochondria. These acyl changes in CL may affect the activity of enzymes associated with CL (Table I). For example, when 22:6n-3 content increased and 18: 2n-6 decreased in rat heart CL, the activity of cytochrome c oxidase was decreased by 50% and the oxygen consumption rate of rat heart mitochondria decreased (Yamaoka et al., 1988). Fish oil feeding, however, increased activity of FIFo ATPase, which also requires CL for its activity (Santiago et al., 1973; Vik et a f . , 1981) (see Section IX,B). The ADP/O ratio was not changed, which indicates that uncoupling of electron transport did not occur; in essential fatty acid (EFA) deficiency, such uncoupling is known to occur (Divakaran and Venkataraman,

N

8

TABLE I META-ANALYSIS OF THE SELECTIVITY OF DIETARY FATTY ACIDS FOR INCORPORATION INTO CARDIOLIPINa

Organ

Species

Heart Heart Heart Heart Heart Heart Heart Heart Heart Heart Heart Heart Heart Heart Heart Heart Heart Heart Heart Heart Heart Heart Heart

Rat Rat Rat Mouse Rat Rat Rat Rat Rat Rat Chick Chick Rat Rat Rat Mouse Rat Rat Rat Rat Mouse Rat Rat

Length of diet (mo) 1.00 4.00 2.50

1.so

1.50 1.00 1.00 1.OO 2.50

0.30 0.80 0.80 I .so 4.00 I .00 1.50 12.00 5.00 2.75 0.75 0.50

0.75 2.75

Lipid source Mixture Olive Mixture Olive Rapeseed Rapeseed Rapeseed Rapeseed Rapeseed Rapeseed Rapeseed Sunflower Corn Corn Corn Safflower Sunflower Sunflower Safflower Coconut Safflower Coconut Soybean

Fatty acid precursor 18:ln-9 18:ln-9 18: In-9 18:ln-9 22: In-9 22: 1n-9 22: In-9 22: In-9 22: In-9 22: In-9 22: In-9 18:2n-6 18:2n-6 18:2n-6 18:2n-6 18:2n-6 18:2n-6 18:2n-6 18:2n-6 18:2n-6 18:211-6 Ik2n-6 18:3n-3

Fatty acid ratio” 0.81

1.48 2.97 3.98 0.20 0.43 0.50 0.65 1.19 I .27 1.51 4.91 5.92 6.% 7.09 8.58 10.38 10.72 12.10 25.40 27.16 49.63 0.0

Reference 6 6 6 6

4 4 4 4 4 4 4

e

0

e e e e 9

e 8

e e

n

Javouhey (1990) Kramer (1980) Hey ( 1990) Berger er al. (1992) Hsu and Kummerow (1977) Blomstrand and Svensson (1974) Blomstrand and Svensson (1974) Innis and Clandinin (1981) Blomstrand and Svensson (1983) Blomstrand and Svensson (1974) Renner er al. (1979) Renner et al. (1979) Hsu and Kummerow (1977) Kramer (1980) Yamaoka ef al. (1988) Berger et al. (1992) Charnock et al. (1986) Astorg (1991) Hey (1983) Swanson and Kinsella (1986) Berger and German (1990) Swanson and Kinsella (1986) Hey (1983)

Heart Heart Heart Heart Heart Heart Heart Heart Heart Heart Heart Heart Heart Heart Liver Liver Liver Liver Liver Liver Liver Liver Liver Liver Liver Liver Liver

Rat Rat Rat Rat Mouse Mouse Rat Rat Rat Rat Rat Rat Rat Mouse Rat Mouse Rat Rat Mouse Rat Mouse Rat Rat Mouse Mouse Rat Mouse

1.OO 4.00 5.00 1.OO 1S O

0.50 0.75 3.00 0.75 0.75 I .00 I .OO 12.00 1S O

2.50 1.50

0.30 5.00 1S O 2.75 0.50 2.75 5.00 0.60 0.50 0.30 1S O

Soybean Soybean Mixture Mixture Linseed Linolenate Menhaden Cod liver Menhaden Menhaden Mixture Sardine Tuna Menhaden Mixture Olive Corn Sunflower Sflower Safflower Safflower Soybean Linseed Linseed Linolenate Sardine Menhaden

18:3n-3 18:3n-3 18:3n-3 18:3n-3 18:3n-3 18:3n-3 Hn-3 in FO' En-3 in FO Zn-3 in FO Hn-3 in FO Hn-3 in FO En-3 in FO Zn-3 in FO En-3 in FO 18:ln-9 18:ln-9 18:2n-6 18:2n-6 18:2n-6 18:2n-6 18:2n-6 18:3n-3 18:3n-3 18:3n-3 18:3n-3 Zn-3 in FO Hn-3 in FO

0.94 0.95 O.% 1.87 2.72 10.00 0.91

n

1.06

R

1.08 2.36 5.36 5.68 9.12 17.81

n R R R R R

1.64

6 6

2.67 6.92 8.16 8.49 9.12 26.7 0.0 0.99 3.73 5.90 1.48 4.02

R

n R R R

n

9

e e

e

e

n R

n R R R

lnnis and Clandinin (1981) Kramer (1980) Astorg (1991) Javouhey (1990) Berger er a/. (1992) Berger and German (1990) Swanson and Kinsella (1986) Gudbjarnason (1978) Swanson and Kinsella (1986) Swanson and Kinsella (1986) Javouhey (1990) Yamaoka et a / . (1988) Charnock et a / . (1986) Berger et a/. (1992) Hey (1990) Berger et a/. (1992) Yamaoka er a/. (1988) Astorg (1991) Berger et a/. (1992) WY( 1983) Berger (1990) Hey (1983) Astorg (1991) Berger er a/. (1992) Berger (1990) Yamaoka (1988) Berger er a/. (1992)

Reprinted with permission from Berger et a / . (1992). Calculated as the ratio of the mol%, wt%, or area% of 18: In-9 (6), 22: ln-9 (&, 18:2n-6 (e), or 22:6n-3 (R) in cardiolipin divided by the respective mol%, wt%, or area% of the designated precursor fatty acid. a

h)

%

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ALVIN BERGER

et

al,

1977). Mitochondria1 PC and PE also are known to be necessary for electron transport but are not associated specifically with cytochrome c oxidase. From this study, one cannot disprove that changes in cytochrome c oxidase activity were caused by changes in the amount of the enzyme or that steps other than cytochrome c oxidase were rate limiting during respiration. Haeffner and Privett (1975) similarly found that replacement of 18 :2n6 with other PUFAs in total phospholipid of rats resulted in decreased cytochrome c oxidase activity and a presumed compensatory increase in FIFOATPase activity. Administration of an arachidonate-rich diet increased hepatic cytochrome c oxidase activity. Roblee and Clandinin (1984) found that feeding low-fat diets (15% of calories versus 40% of calories) or diets with a lower PUFA/SFA ratio (0.25 versus 2.0, adjusted with beef tallow and soybean oil) resulted in higher rates of ATP-32Pi exchange by the mitochondrial ATPase.

2 . Essential Fatty Acid Deficiency Stancliff et al. (1969) examined acyl changes in CL and PC from rat liver mitochondria after EFA deficiency. These researchers found no changes in respiration or oxidative phosphorylation. One can speculate that the 18 : 2n-6 content of CL would be depleted during extended EFA deficiency. EFA deficiency is known to cause the uncoupling of oxidative phosphorylation in rat liver mitochondria (Divakaran and Venkataraman, 1977). Rafael et al. (1984) found that cytochrome c oxidase activity of rat heart mitochondria (which is associated with CL) was not altered by an EFA deficiency induced by feeding hydrogenated coconut oil. However, whether the fatty acyl composition of mitochondrial phospholipids changed under the feeding regimen employed was not determined. Microbial mutants have been used extensively to study the effects of EFA deficiency on mitochondrial functioning. For a review, see Daum (1985). Wolff (1988) fed 21-day-old rats either laboratory chow for 7 or 20 days (control group); 0.07% fat for 1 , 2, 3, 7, or 66 days; or the low-fat diet for 7 days and then switched to the chow diet for 1 , 2, 5, or 9 days. In liver mitochondrial phospholipids, 18 :2n-6, 20 :4n-6, and 22 :6n-3 decreased abruptly during the first 3 days of fat deficiency and remained stable until day 7. In contrast, in CL, 18:2n-6 decreased continuously from 79% (day 1) to 33% (day 7), reaching a value of 19.6% on day 66. When deficient rats were transferred to an equilibrated diet, the fatty acid profile of phospholipids was identical to that of control rats within 2 days. A considerably longer period (9 days) was necessary for CL to reach a level

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analogous to that of control rats. The decrease in 18 : 2n-6 in CL was compensated for by increases in 16: ln-7, 18 : ln-9, and 18 : ln-7, which accounted for 18.0%, 17.7%, and 17.9%, respectively, on day 7. The proportion of 18 : ln-7 relative to total octadecenoic acids was decreased from 75% to 50% in CL with fat deficiency. After 66 days of fat deficiency, 18:2n-7 reached a level of 4.3% in CL and 2% in other phospholipids; 20 : 3n-9 reached a level of only 0.8% in CL compared with 13% in other phospholipids; and 20: 3n-7 (up to 0.7%) and 5,11,14eicosatrienoate levels increased slightly in CL and total mitochondrial phospholipids. Elimination of markers of essential fat deficiency (18 :2n7, 20 : 3n-9) was slow for all mitochondria1 phospholipids. The increases in 18 :2n-7 (e.g., 2.6% 18 : 2n-7 in rat liver CL after 3 mo EFA deficiency; Lemarchal and Munsch, 1965), 20: 3n-7 (Klenk and Tschope, 1963; Spence, 1971; Sprecher and Lee, 1971) and 5,11,14-eicosatrienoate (Schmitz et al., 1977) with EFA deficiency have been reported previously. 5,11,14-Eicosatrienoateis the A5 desaturation product of 20 : 2n-6, which is present in CL (Ullman and Sprecher, 1971; Sprecher and Lee, 1975). The presence of 18:2n-7 is surprising because this fatty acid must be formed via A8 desaturation (A1 1 18 : b A 8 , 11 18 :2); evidence suggests that this desaturase does not occur in animals (Klenk and Mohrhauer, 1960; Rivers et al., 1975; Sprecher and Lee, 1975; Dhopeshwarkar and Subramanian, 1976; Jurenka et al., 1988) or in plants (Takagi, 1982; Lie Ken Jie, 1987). In rat testes, some evidence exists of a A8 desaturase (Albert and Coniglio, 1977). 20 : 3n-7 (A7,10,13 20 : 3) would be formed by A7 desaturation of the precursor 20:2n-7 (A10,13). This enzyme also converts 16 : 0+16 : In-7. 3. Saturated and Trans Fatty Acids and Rapeseed Oil

The cardiotoxicity of rapeseed oil (10% erucate; 22 : ln-9) was demonstrated first by Roine (1960). In subsequent studies, rats fed rapeseed oil developed foci of macrophage infiltration in heart, skeletal muscle, and adrenals (Abdellatif and Vles, 1970; Houtsmuller et al., 1970). Triacylglycerol content of heart mitochondria increased and of phospholipid decreased with the consequence of impaired j3 oxidation of other mitochondrial fatty acids (see Blomstrand and Svensson, 1974, 1983; Hsu and Kummerow, 1977). The demonstration that ATP synthesis was lowered after feeding 22: ln-9 (Houtsmuller et al., 1970) and that CL was necessary for the activity of FIFOATPase (Santiago et al., 1973) led to dietary investigations examining specific phospholipids (e.g., CL) (Blomstrand and Svensson, 1974; Renner et al., 1979; Innis and Clandinin, 1981).

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Blomstrand and Svensson (1974) fed rats diets containing 40 cal% rapeseed oil/peanut oil to yield 1.4, 2.6, or 9.8% 22 : ln-9. After feeding 9.8% 22 : ln-9, heart CL incorporated 12.4% 22 : ln-9, compared with approximately 5% for PC and PE. Hence, the authors concluded that CL has a special affinity for 22 : ln-9. However, when rats were fed only 1.4 or 2.6% 22 : ln-9 for 28 days, no selective incorporation of 22 : ln-9 into CL was seen compared with PC and PE. Rocquelin (1981) fed rats 15% by wt sunflower or low- or high-22 : ln-9 rapeseed oil and also found incorporation of 18 : 1, 20 : 1, and 22 : 1 into CL after rapeseed oil feeding. Innis and Clandinin (1981) fed rats 20% rapeseed oil or soybean oil in a cross-over design. These investigators did not observe the preferential increase in 22: ln-9 in CL relative to PE and PC, but pointed out that these changes may not have been fully evident because of the ageassociated increase in 18 :2n-6 from weaning to 5 wk that they observed (see Section X,D). When rats were switched from soybean oil to rapeseed oil for 10 days, 18 :2n-6 content decreased from approximately 30 to 20%; when crossed back to soybean oil for 10 days, 18 : 2n-6 increased back to control levels, thus illustrating the dynamic nature of CL acyl changes. Compared with PC and PE, turnover of fatty acids in CL was slower. Hsu and Kummerow (1977) fed rats 22% corn oil, rapeseed oil (containing 49.1% 22 : ln-9), or hydrogenated fats. Rapeseed and hydrogenated fat feeding lowered heart mitochondrial oxygen uptake, lowered respiratory control ratios, decreased ADP/02 ratios and lowered the rate of ATP synthesis. The addition of erucyl and elaidyl carnitine derivatives to heart mitochondrial preparations similarly lowered oxygen consumption compared with oleoyl derivatives. After 6 wk feeding, heart CL content of 16 :0 increased from 1.2% (corn oil) to 5.1% with rapeseed oil, cis 18 : ln-9 (probably including 18 : ln-7) increased from 10.4 to 24.1%, 22 : ln-9 increased only slightly from 0.4 to 2.2%, and 18 :2n-6 decreased from 72.3 to 55.9% (no statistics provided). In summary, whether cis 22 : ln-9 has special affinity for CL is unclear since this fatty acid was found to be incorporated selectively into CL only when animals were fed very high levels (9.8 cal% or approximately 4.4 wt% 22 : ln-9) (Blomstrand and Svensson, 1974). Also, whether lowered heart mitochondrial oxygen uptake, respiratory control ratios, ADP/Op ratios, and rate of ATP synthesis with rapeseed feeding are due to decreased 18 :2n-6 content of CL (Hsu and Kummerow, 1977), incorporation of 22 : ln-9 into CL, or factors not related to CL is not clear. Trans fatty acids, unlike PUFAs (see Section X,F), are not rapidly incorporated into CL (Blomstrand and Svensson, 1974; Sohal and

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Bridges, 1977; Wolff et al., 1988). In a study feeding 10% trielaidate (tri 22: ln-9t) to rats for 1 mo, the order of incorporation of 18 : I t and 16: I t into heart and kidney was PI > PE > PC >> CL, indicating that CL is most resistant and PI apparently has no discrimination to trans fatty acid incorporation (Wolff et al., 1988).

F. ACYL CHANGES IN CARDIOLIPIN I.

With Development

Keranen (1982) compared the fatty acyl composition of term, 1-day, and 4-day fetal (mixed sexes) and adult (3-6 month-old females) rat liver mitochondrial and microsomal phospholipids. With increasing age, a decrease in the monounsaturated fatty acids 18 : ln-9 and 16: ln-7 and an increase in 18 : 2n-6 were seen. 18 : ln-7 and 18 : ln-9 apparently were not distinguished in this investigation. Bruce (1974) similarly found that the relative amount of 18 :2n-6 increased during the prenatal period, reaching maximum values 2 mo after birth. 2 . With Ethanol Administration

In rats fed an ethanol liquid diet for 31 days providing 36% of calories as ethanol or isocaloric amounts of maltose-dextrin (controls), the 18 :2n-6 content of CL decreased from 51.3 to 42.8%. Ethanol did not change the amount of mitochondrial phospholipid (see Section XI) (Cunningham et al., 1982). Waring (1981) similarly found 18 : 2n-6 to be decreased from 72.1 to 54.3% with ethanol administration. Accompanying this decrease, 16 :0 increased from 4.1 to 7.3%, 18 :0 increased from 2.9 to 6.8%, and 18 : ln-9 increased from 18.9 to 29.1%. Changes in CL acyl composition were more pronounced than for mitochondrial PC and PE. Ellingson (1988) also reported a decrease in 18 :2n-6 and an increase in 18: ln-9 in CL with ethanol consumption. This increased saturation of CL with alcohol administration may explain why mitochondria from chronic alcoholic rats are more resistant to uncoupling by ethanol at physiological temperature (Rottenberg et al., 1980). 3. With Hyperthyroidism

In rats made hyperthyroid with injection of triiodothyronine, Ruggiero (1984) found large increases in 16 : 0 from 11.5 to 33.0% and 18 :0 from 7.3 to 16.5% and a decrease in 18 :2n-6 from 52.5 to 20.1% in liver CL. Of the phospholipids examined, CL was the one most affected by hyper-

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thyroidism. Hence, either a doubling of hepatic synthesis of 16:O and 18 :0 or less synthesis of 18 :2n-6 took place proportionately less 18 :2n-6 was available since hyperthyroidism increased the amount of hepatic CL (see Section X1,E). In PC and PE, hyperthyroidism led to increases in 20 :4n-6. In hypothyroidism, other investigators have found a decrease in 20 :4n-6 in PC and PE and found that the acyl composition of CL was unchanged (Landriscina et al., 1976; Hoch et al., 1980). Since the quantity of phospholipid in hepatic microsomal membranes was decreased with hyperthyroidism, the authors speculated that thyroid hormones might stimulate phospholipid transfer from microsomes to mitochondria, possibly acting thru phospholipid transfer proteins. Since liver microsomal phospholipid fatty acid composition was not affected by hyperthyroid state, the authors speculated that a particular PUFA pool may be accessible only to the mitochondrial membranes, probably through an acylationdeacylation process, the metabolism of which is subjected to thyroid hormone regulation (Reitz et al., 1969) (see Section IV,C). Also of interest was the strong decrease in the cholesterol to phospholipid ratio with hyperthyroidism which generally indicates decreased membrane fluidity. Cytochrome c oxidase (Wolff and Wolff, 1964), ADP/ATP carrier protein (Babior et al., 1973), and Na+/K+ ATPase (Ismail and Edelman, 1970) all are influenced by thyroid hormones. All are known to be associated with CL (Beyer and Klingenberg, 1985; Powell et al., 1987; Rietveld et al., 1987; Esmann et al., 1988) (see Section IX). Therefore, the effects of hyperthyroidism on these enzymes may be mediated via acyl changes in CL since, at least in the case of cytochrome c oxidase, Yamaoka et al. (1988) demonstrated that a fish-oil-induceddecrease in CL 18 : 2n-6 from 84 to 24-44% was associated with a 50% decrease in the activity of cytochrome c oxidase in rats. The oxygen consumption rate of rat heart mitochondria decreased as well. Mitochondria from hyperthyroid rats also show decreased oxygen consumption when succinate, NADH, palmitate, or malate was used as a substrate (Ruggiero et al., 1984). 4.

With Malignancy

Tumor cells may exhibit lower levels of mitochondrial 18:2n-6 and higher levels of 18 : ln-9 and 16 :0 than normal cells (Dyatlovitskaya et al., 1973a; Morton et al., 1976). No pronounced acyl differences in CL were found in Jensen sarcoma (Dyatlovitskaya et al., 1969) or in nephroma (Dyatlovitskaya et al., 1973b). The usual distribution of saturated and unsaturated fatty acids on the sn-1 and sn-2 positions, respectively,

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does not apply in hepatoma cells, presumably because of altered or absent acyltransferases (Haldar et al., 1979). 5.

With External Temperature and Membrane Fluidity

Acyl changes in phospholipids are known to influence membrane fluidity. For example, in cold-adapted plants and fish, an enhancement of unsaturated fatty acids is seen in phospholipids (Lynch and Thompson, 1984, 1988; Greene and Selivonchick, 1987). Active membranes such as the inner mitochondrial membrane nerve synapses and the retinal photoreceptor membranes also are known to have more unsaturated phospholipids for enhanced fluidity (Cullis and Hope, 1982; Birkle and Bazan, 1987; Rotstein and Aveldano, 1988). CL has been suggested to fluidize the inner mitochondrial membrane, based on spin-labeling studies (Stuhne-Sekalec and Stanacev, 1977), and to have an essential role in the integrity of the mitochondrial membrane, since the membrane collapses after PLA2 treatment (Awasthi et al., 1969). In in uitro studies, the phase-transition temperatures of various acyl-specific PGs and CLs have been determined by direct scanning calorimetry to be 62.2"C for tetrapalmitoyl CL, 47.6"C for tetramyristoyl CL, 403°C for dipalmitoyl PG, and 25°C for dimyristoyl PG. Hence, the crystalline-to-liquid crystalline transition temperature is increased by increasing chain length of the saturated fatty acid and by changing the head group (Nagamachi et al., 1985). The effects of increased unsaturation on phase transition temperature were not determined. In uiuo, cold acclimatization of carp (Wodtke, 1981) and goldfish (van den Thillart and Modderkolk, 1978) increased the quantity of unsaturated fatty acid in PC as expected; however, the quantity of unsaturated fatty acid in CL decreased. The acyl changes in CL were thought to reflect temperature-dependent modifications in the dynamic surface shape of integral membrane proteins (Daum, 1985). In plants, the acyl composition of PG is known to affect the thermal stability of the photosynthetic apparatus and to predispose some plants to chill sensitivity (Lynch, 1984; Sekiya, 1987; Murata, 1983). Plants containing 16 :0/16 :0 and 16 :0116 : In-13t as the major molecular species have been found to be less chill resistant. Note that, in plants, both prokaryotic and eukaryotic pathways of PG synthesis are used (Roughan, 1987) and that plants with the prokaryotic pathway may have up to 50% disaturated PG (e.g., dipalmitoyl PG) in chloroplasts (Quinn, 1988). Also note that plant PG is unique in containing 16: ln-13t at the sn-2 position (Mudd et al., 1987).

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XI.

INFLUENCE OF DIET AND OTHER FACTORS ON CARDlOLlPlN CONTENT

A. TEMPERATURE We are not aware of temperature-induced changes in CL content in mammals. However, in Tetrahymena exposed to high temperature, the quantity of CL increased, ultimately leading to cell death (Ohki et al., 1984). In Neurospora, a temperature shift from 37" to 15°C resulted in decreased mitochondrial PC and CL, but increased PC (Aaronson et al., 1982).

B. HORMONES Synthesis of polyglycerophospholipids may be controlled hormonally since, in castrated rats, a 48% decrease in PG synthesis was noted in the prostate; levels were restored with testosterone. Other hormones implicated include cortisol (alveolar cells), prolactin (lung), and CAMP(Hostetler, 1982). C. DIET In liver microsomes, the quantity of CL (approximately 1%) was not affected by EFA deficiency in rats (Christon et al., 1988). Blomstrand and Svensson (1974) fed peanut oil or rapeseed oil to rats and found no difference in the amount of heart CL. Innis and Clandinin (198 1) reported a similar finding with rapeseed oil feeding. In contrast, Rocquelin (1981) fed rats 15% by wt sunflower or low- or high-erucate rapeseed oil and found that the amount of heart CL increased from 3.3 mglg tissue to 4.4 (averaged for the two rapeseed types). The authors speculated that their results may differ from those in other publications because of the differing quantities of erucate used in different feeding studies. Roblee and Clandinin (1984) fed weanling rats diets containing 15 or 40% calories as fat, with PUFA/SFA ratios of 2.0 or 0.25 (adjusted with beef tallow and soybean oil). These investigators also found that the heart mitochondrial CL content was not affected significantly by total fat or the PUFA/SFA ratio. D. EXERCISE Rocquelin (1981) found that treadmill training of rats significantly increased the CL content of the heart. Earlier, Prioreschi and Peterman

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(1967) found that rat heart CL was decreased 75% and 18 : 2n-6 content almost abolished after only 4 min forced restraint. Longer periods of restraint (up to 24 hr) did not decrease the amount of CL further. With rats immobilized for 24 hr and then allowed to rest for up to 72 hr, CL and 18 : 2n-6 levels returned to normal; after 96 hr rest, levels of CL and 18 :2n-6 were higher than normal. E. HYPERTHYROIDISM In rats made hyperthyroid with injection of triiodothyronine, Ruggiero (1984) found an increased mol% of CL from 13.4 to 18.0. F. ETHANOL Ethanol has not been shown to induce changes in rats in the amounts of mitochondrial CL (Cunningham et al., 1982; Ellingson et al., 19881, whereas mitoplast hepatic PE decreased from 42.0 to 38.0 mol% and PC increased from 45.4 to 49.2%.

G. ISCHEMIA Malis and Bonventre (1988) exposed mitochondria to calcium and reactive oxygen species to simulate ischemic conditions. These researchers found that FIFo ATPase activity and ADP/ATP carrier protein activities decreased to 45 and 35% of control values, respectively. The authors suggest that calcium activates PLA2 and that reactive oxygen species increase permeability of the inner and outer mitochondrial membranes. Thus, activated PLA2, which is located predominantly in the outer membrane, now readily can permeate the inner membrane gaining access to and causing persistent degradation of the electron transport chain (NADH CoQ reductase), FIFo ATPase, and ADP/ATP carrier protein. Further, during ischemia, CoA, which is a substrate for lysophospholipid acyltransferase, forms disulfide dimers, thus preventing reacylation of lysophospholipids. The PLA2-generated cytosolic free fatty acids then may act as detergents, further contributing to membrane damage. Dibucaine, a PLA2 inhibitor, was shown to preserve uncoupled respiration at control levels. Okayasu er al. (1985) found that ischemic rat hepatic mitoplasts had 60% less CL than normal control mitoplasts and a reduced 18 : 2n-6 content. LysoPC, PE, CL, and free fatty acids increased, indicative of increased PLA activity. The specific activity of cytochrome c oxidase was unchanged.

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CL is known to be a substrate for PLA2 (Hostetler, 1982; see Section VI and to be associated with and necessary for maximal activity of FIFO ATPase (Vik et al., 1981), ADP/ATP carrier protein (Beyer and Klingenberg, 1985), and mitochondrial PLA2(Lenting et al., 1988). Hence, these reports demonstrate that, during ischemia, electron transport may be impaired because of damage to CL. H. MALIGNANCY In several tumor lines, the actual mitochondrial content (e.g., pg) of total phospholipids may be 50-70% higher than in controls (Hostetler, 1982). In hepatoma cells and Jensen sarcoma subcellular particles, the phospholipid pattern of different subcellular organelles (microsomes, mitochondria, nuclei, and plasma membrane) resembles that of the whole tissue (Bergelson et al., 1968, 1970). This “chemical dedifferentiation” is hypothesized to be more pronounced in highly malignant, poorly differentiated cells (Ioannou and Golding, 1979). Composition of CL, specifically in malignant cells, has not been reported, nor has the effect of diet in altering tumor specific mitochondria. XII.

POSSIBLE ROLE OF CARDlOLlPlN IN RESISTANCE TO ETHANOL-INDUCED MEMBRANE DISORDERING

Ethanol is known to have disordering or fluidizing effects on biological membranes (reviewed by Taraschi and Rubin, 1985; Hoek and Taraschi, 1988) (see also Section X,F,5). Rats that chronically consume ethanol develop membranes that are resistant to the disordering effects of ethanol in uitro. Ellingson et al. (1988) investigated the molecular basis of this tolerance in the inner mitochondrial membrane from control and ethanolfed animals as follows. Either intact mitoplast membranes or vesicles of recombined extracted mitoplast phospholipids (separated by HPLC) were labeled with a spin-labeled fatty acid or phospholipid probe; the order parameter, S, was determined with electron spin resonance. Recombined bilayer vesicles were prepared by combining HPLC-separated mitoplast phospholipid in their naturally occurring molar ratios of 46% PC, 42% PE, 2% PI+PS, and 10% CL. To identify the phospholipid component responsible for membrane tolerance, bilayers were made by recombining all individual HPLC-separated phospholipids from control or ethanol-fed rats in these naturally occurring molar ratios except that, in each preparation, one different phospholipid class was omitted and the

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missing phospholipid from the control preparation was replaced by the corresponding one from ethanol-fed rats. Recombined membrane vesicles of phospholipids from control animals were disordered by 50-100 mM ethanol when PC (46%) or PE (42%) from the ethanol-fed preparation was substituted for its counterpart from controls. In contrast, when CL (10%) from ethanol-fed animals was substituted into control vesicles, bilayers were rendered resistant to disordering by ethanol. As little as 2.5% CL from ethanol-fed rats combined with 7.5% CL from control rats conferred tolerance to reconstituted bilayers made from phospholipids of either rat mitoplasts or bovine tissues. Reconstituted mitoplasts constituted solely of CL from ethanoltreated animals also conferred tolerance to ethanol disordering. These authors also demonstrated that PI in liver microsomes (Taraschi et al., 1986) and PS from synaptosomal membranes, a membrane that contains 10% PS (Hoek and Taraschi, 1988), of ethanol-treated rats conferred tolerance to those membranes. When ethanol-adapted animals are withdrawn from the ethanolcontaining diet, both the membrane tolerance and the presence of the modified PI or CL disappear in parallel over a period of 2-4 days. Reacquisition of membrane tolerance when ethanol was reintroduced into the diet took a much shorter time (5-15 days) than the initial development of the adaptive response. Whether fatty acyl or head group modifications of these anionic phospholipids are responsible for inducing membrane tolerance is not known at this time. The anionic character of the phospholipids suggests that the interactions (hydrogen bonding or ionic interactions) in the polar region of the membrane are important. Nevertheless, conceiving of head group changes that could occur that would not compromise the inherent structure of the phospholipid is difficult. This suggestion argues for a molecular species, that is, a fatty acid chain mechanism, to account for this effect. Since ethanol is known to decrease the 18:2n-6 content of CL, acyl chain modifications may be important. Remodeling acyl chains within or between different phospholipids would not require net synthesis of particular fatty acids and, thus, could proceed rapidly within the membrane. These changes in the membrane lipids could alter the properties of enzymes bound within it significantly. For example, the acyl side chains in the hydrophobic core of the membrane can affect the distance between interacting membrane components and thereby influence the frequency of those interactions (Hoek and Taraschi, 1988). Ethanolinduced acyl changes in CL also may affect the activity (Yamaoka et al., 1988) and binding affinity (Powell et al., 1987; Esmann et al., 1988) of

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mitochondria1 enzymes associated specifically with CL, especially by its remodeling in the presence of the membrane perturbing actions of ethanol. XIII. IMMUNOLOGIC ACTIVITY OF CARDlOLlPlN

A. DISCOVERY OF ANTI-CARDIOLIPIN ANTIBODIES Anti-cardiolipin antibodies (aCL) probably were reported first in 1906 when Wassermann described a complement fixation test to detect reagin in the sera of syphilitic patients (Hazeltine et al., 1988). In 1941, Pangborn showed that the antigen bound by reagin was an acidic phospholipid obtained by alcohol extraction of ox heart muscle, subsequently named CL (Pangborn, 1942). Today, aCLs are detected using solid phase immunoassays and by their effect on prolonging phospholipid-dependent clotting tests. This latter phenomenon is termed the lupus anticoagulant (LA) (see Section XIII,B,2). aCLs are IgG and IgM immunoglobulins. B. ANTI-CARDIOLIPIN ANTIBODIES AND DISEASE 1 . Thrombosis

The position that autoantibodies are inconsequential and useful only as markers of disease is no longer widely held. The circumstantial evidence that antibodies are responsible for clinical disease is powerful, yet the direct evidence implicating autoantibodies in the immunopathogenesis of clinical disease is meager (Harley and Gaither, 1988). Speculating on the possible effects of substantially altering CL composition on the development and expression of the various pathologies now linked to aCLs is intriguing. Unfortunately, no experiments to support or refute these possibilities have been done. This chapter reviews many of the autoantibodies and antigens found in systemic lupus erythematosus (SLE), the clinical consequences associated with these antibodies, and a historical account of the tests used for detection of the various antibodies. Anti-dsDNA is the harbinger of renal disease, particularly when coexistent with evidence of serum complement consumption. Anti-dsDNA is very specific and useful in diagnosis of SLE, whereas anti-ssDNA is nonspecific. The predominant theory of renal injury is that preformed immune complexes deposit directly in the glomerulus, that DNA is trapped and bound in situ by the anti-DNA

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antibody, or that anti-DNA binds directly to the glomerular basement membrane. Interest in aCL has been sparked because of the association of these antibodies with spontaneous venous and arterial thrombo~is,~ immune thrombocytopenia (decreased number of platelets), recurrent fetal loss, chorea, livedo reticularis, occasionally neurological manifestations, and thrombotic infarctions of the skin and the GI tract (in Degos’ disease). Other proposed mechanisms of thrombosis include alterations in prekallikrein, antithrombin 111, and protein C, as well as functional defects in fibrinolysis and platelet function (Hughes et al., 1986; McNeil et al., 1988; Danao and Camara, 1989).

2. Systemic Lupus Erythematosus Raised levels of serum aCL have been reported most commonly in patients with SLE (for reviews, see Harris et al., 1983, 1985, 1987a,b,c, 1988a,b; Loizou, 1985; Hughes, 1986, 1989; Bingley and Hoffbrand, 1987; Harris, 1987, 1988, 1989; Harris and Hughes, 1987; Lockshin, 1987; Lockshin et al., 1987; Sontheimer, 1987; Boey, 1988; Harley and Gaither, 1988; Laskin, 1988; Schwartz, 1988; Danao and Camara, 1989; Parke, 1989; Mackworth-Young, 1990) and other autoimmune disorders. SLE is a disease characterized by fever, rash, arthritis, hemolytic anemia hemorrhages in skin and mucous membranes, inflammation of the pericardium, and, in serious cases, involvement of the kidney and central nervous system (Smolen and Zielinski, 1987; Vaarala et al., 1987; Pascual-Salcedo et al., 1988). Mouse models that develop an SLE-like syndrome include MRL/MpJ-Ipr; NZB/NZW F1; and BALB/c (Gershwin, 1978; Kotzin and Palmer, 1987; Kotzin and Palmer, 1988). Conflicting reports have been made of the aforementioned clinical associations of aCL in patients with SLE. Kalunian et al. (1988) examined serum samples and clinical and laboratory data from 85 consecutive outpatients with SLE and 40 control subjects. The presence of aCL was documented in 42.4% of patients with SLE. These antibodies were associated significantly with thrombosis, fetal loss, and thrombocytopenia, but not with other manifestations. Fluctuations in the levels of aCL made it difficult for the authors to interpret a single negative result. Thus, positive aCL test results are not predictive for other clinical manifestations of SLE, including activity and severity of disease. Generally, in SLE, aCL have been associated with thrombosis. However, chlorpromazine-induced lupus anticoagulant and aCL levels appear not to be associated with an increased incidence of thrombosis (Canoso and de Oliveira, 1988).

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3. N o Well-Dejned Disease

Several groups of patients with raised aCL levels do not have any other well-defined disease, but do have clinical features associated with these raised antibodies. Exner and Koutts (1988) found that patients being treated with oral anticoagulant therapy (warfarin), especially those being treated for thrombotic episodes, had aCL levels greater than 4 SD above normal. In this study, borderline DNA binding studies, with some positive aCL results, suggested autoimmune etiology in the minority of these cases. LA was strongly detectable in only one such patient. The aCLs were predominantly IgG (14/ 17) and did not appear to compromise the conditions of patients on anticoagulant therapy. Hence, raised aCLs may be a highly significant marker for an acquired prothrombotic state. Mackworth-Young et al. (1989) described the clinical, hematological, and serological features of patients with raised aCL levels who did not fulfill criteria for the diagnosis of SLE, including 20 patients with raised IgG aCLs, 12 with raised IgM aCLs, and 6 with raised levels of both isotypes. Of 19 patients, 9 had raised antinuclear antibody levels. Also, 7 patients had a history of venous thrombosis and 5 had definite or presumed arterial thrombosis, for example, stroke. Of the 15 female patients who underwent pregnancy, 12 experienced fetal loss, with up to 8 abortions each (mean, 3.6). Of the patients, 6 individuals had thrombocytopenia and 4 others had migraines. Other clinical features included livedo reticularis, cardiac and neuropsychiatric disorders, arthralgias, and Raynaud’s phenomenon. These findings confirm that the clinical features of individuals with what may be called the “primary aPL syndrome” are similar to those in patients with other diagnoses who have raised aPLs. The findings indicate that the aPL syndrome may be related to SLE and other autoimmune diseases but that, although it frequently overlaps with these disorders, the aPL syndrome also exists as a distinct entity. As mentioned, Kalunian et al. (1988) examined serum samples and clinical and laboratory data from 85 consecutive outpatients with SLE and 40 control subjects. Most intriguing is that the authors documented aCL in 7.5% of control subjects, who apparently did not have thrombotic episodes, unlike their SLE counterparts with raised aCL. aCL, as well as a plethora of other autoantibodies also have been reported in 52% of a healthy elderly population of 32 males and 32 females examined (Manoussakis, et ul., 1987). 4 . Other Disorders

Other diseases in which aCLs have been detected include Sneddon’s syndrome (Montalban et al., 1988), Guillain-Barre syndrome, progres-

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sive systemic sclerosis (Bamberga et al., 1987), Wiskott-Aldrich syndrome (Pascual-Salcedo et al., I988), Lyme disease (Mackworth-Young et al., 1988), and AIDS (Canoso et al., 1987). aCLs also are seen in persons with malaria, in which both biologically false positive tests for syphilis and anti-DNA are frequent; high gallium uptake (Drane et al., 1988); cerebral infarction and ischemia, with (Asherson et al., 1989; Kushner and Simonian, 1989) and without showing SLE (Briley et al., 1989; Coull et al., 1987); rheumatoid arthritis (49% of 90 patients examined) (Keane et al., 1987; Klemp et al., 1988); ischaemic heart disease (IgG and IgM aCLs); and tuberculosis (IgG only) (Klemp et al., 1988); and in women with clinically unexplained recurrent abortions (Norberg et al., 1987; Unander et al., 1987; Greenspoon and Buchanan, 1989) and pre-eclampsia (a toxic condition late in pregnancy associated with increased blood pressure, edema, weight gain, headache, and visual disturbances) (Scott, 1987). Reports have been made that “lowering aCL levels in women with these antibodies and fetal loss, may result in live births (Lubbe et al., 1984; Branch et al., 1985). As mentioned, aCLs also are found in persons with syphilis (Costello and Green, 1988). Costello and Green (1988) found that, of 22 SLE serum samples tested and 47 syphilitic sera samples tested, antibody affinity was four- to fivefold lower in the SLE sera than in the syphilitic sera. All 47 syphilitic sera reacted with CL, phosphatidate, andlor PS. C. BINDING OF ANTI-CARDIOLIPIN ANTIBODIES 1 . Platelet

Colaco and Male (1985) suggested that aCL binds to acidic phospholipids on platelet membranes, promoting aggregation, activation, and degranulation and eventually leading to thrombocytopenia (hemorrhage). Hazeltine et al. (1988) found aCLs to be associated positively with lower mean platelet counts, C3 and C4 levels, and positive Coombs tests.

2. Endothelial Cells Vismara (1988) found that absorption with CL liposomes partially inhibited endothelial cell binding of anti-endothelial cell antibody (detected in lupus sera). Further, affinity purified aCLs reacted with intact human endothelial cells; the binding was not via Fc receptors since blocking with rabbit IgG did not affect the endothelial cell reactivity. Thus, aCLs may lead to thrombotic episodes by disruption of endothelial cells, which produce the platelet anti-aggregatory eicosanoid product, PGIz . This

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claim is not supported by the work of Hasselaar et al. (1988), who examined 23 antiphospholipid antibody positive SLE sera, 4 aPL negative sera, and 17 control sera for agonist-induced endothelial PGI2 and platelet TXA2 production. Depending on the agonist used, 4-19% of the SLE sera inhibited PG12 release and 4-28% enhanced PG12 release. The TXA2/PG12ratio was imbalanced in some patients, but did not correlate with a history of thrombosis. Thus, according to the authors, the thrombosis associated with aPL cannot be explained by effects on endothelial and platelet prostanoid synthesis. 3. Red Blood Cell

Studies with RBC eluates and absorption studies using fixed RBCs suggested that some aCLs may act as anti-RBC antibodies, similarly directed at membrane phospholipid epitopes (Hazeltine et al., 1988). Hammond et al. (1989) showed that, in sera from patients with SLE, aCL levels (both IgG and IgM subclasses) were correlated inversely with complement receptor type I (which clears immune complexes). CL is known to activate the first component of complement C1 (Peitsch et al., 1988). IgM aCL, but not IgG aCL, levels were correlated positively with RBC C4d and C3d levels. Anti-ds DNA antibodies were not correlated with CRl, C4d, or C3d numbers. The authors suggest that CL or a closely related antigen may bind to the RBC and be involved directly in the mechanism for reduction of RBC CRI expression in SLE patients. Recall that RBCs contain no mitochondria and therefore no CL. 4 . Prothrombin Complex and Lupus Anticoagulant Levels

LA is an immunoglobulin with affinity for negatively charged phospholipids. LA binds to phospholipids in the prothrombin complex (factors Xa and V), leading to neutralization of anticoagulation and thrombosis (Shapiro and Thiagarajan, 1982; Feinstein, 1985; Lechner and PabingerFasching, 1985; Vermylen et al. 1986). McNeil (1988) investigated the relationship between aCLs and the LA activity of plasma in 14 patients, including 7 with SLE, 6 without SLE, and 1 with chlorpromazine-induced LA. Plasma of these patients exhibited both LA activity and high levels of aCL. Of these patients, 7 had a history of thrombosis and 7 did not, despite high antibody levels. Plasma aCL was determined with an ELISA; LA activity was determined with kaolin clotting time (KCT) and activated partial thromboplastin time (APTT). No correlation existed between baseline aCL levels and parameters of LA activity (KCT or APTT), in contrast to previous reports.

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However, a concurrent reduction in both LA and aCL levels over 24 hr during incubation with CL was seen in all patients. The rate of reduction of both parameters was highly correlated (r = 0.99; p < 0.001). The relative reduction of LA activity versus aCL level varied between patients, and may represent different affinities for phospholipid in thromboplastin than in solid phase. Thus, despite the lack of concordance between LA and aCL in many patients, the two activities can be removed concurrently in uitro, suggesting similar binding specificities of the antibodies. The incomplete concordance could be explained by varying affinities for different structural presentations of the lipid antigen. Kushner and Simonian (1989) studied 23 patients suffering from cerebral ischemia who also had laboratory evidence of either LA or abnormal aCL. Of the patients studied, 4 had lupus or a lupus-like illness, 3 had drug-induced lupus, and 16 had no overt evidence of collagen-vascular disease. Cerebral ischemic events were multiple in 71% of the patients; 2 patients presented with multi-infarct dementia. Recognized cerebrovascular disease risk factors were present in 57% of the patients. The partial thromboplastin time was prolonged in only 35% of the patients. LA was identified in 15 of 21 patients tested; elevated aCL titer was identified in 10 of 12 patients tested. Simultaneous assays for LA and aCL were discordant in 8 of 10 patients tested. LA- and aCL-associated brain ischemia is often recurrent, but other risk factors for cerebrovascular disease are often present. The laboratory findings in such patients may display considerable heterogeneity.

5 . Splenic T and B Lymphocytes Tiwari et al. (1988) fed mice fat diets (20% by wt) containing low (4% total calories) and high (22% total calories) levels of 18 : 2n-6. These investigators found lower levels of 20 :4n-6 and 22 :6n-3 in SPH, PS, and PE (no change in PC) in normal (MRL/MpJ-+/+) mice T lymphocyte membranes compared with autoimmune MRL/MpJ-Ipr mice. In B cells of normal mice, 20 :4n-6 levels were higher in PC and lower in PE (no differences in PS and SPH); 22 : 6n-3 levels were lower in PE compared with autoimmune mice. These authors did not examine CL composition. Diet had a minor effect on 18 : 2n-6 content of B and T lymphocyte phospholipids, which varied with the phospholipid examined. The magnitude with which dietary fats are known to alter membrane composition is underscored in this study, since animals normally receive varied linoleate levels in the diet and are able to regulate the levels of 18:2n-6 incorporated into membranes, desaturated, and elongated to arachidonate. Future studies must examine whether alteration of the phospho-

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lipid fatty acid composition of lymphocytes contributes to differences in the rate of progression of age-related changes in autoimmune disorders, since alteration of the lymphocyte membrane may influence immune responsiveness (Schroit and Gallily, 1979; Gum, 1983; Johnston, 1988). Misra et al. (1989) found that, in mononucleosis, affinity purified IgM aCLs reacted with the cell membrane of. transformed lymphocytes (as shown with indirect immunofluorescence), but not with resting lymphocytes. These authors speculated that transformed lymphocytes may be the antigenic epitope in mononucleosis and other disorders in which aCLs are detected. Future researchers should ascertain whether the reactive epitope is, in fact, CL on the lymphocyte membrane, and determine the CL acyl composition of the lymphocyte. D. NONCARDIOLIPIN ANTIGENS DIRECTED AGAINST ANTICARDIOLIPIN ANTIBODIES 1 . Lipopolysaccharide

Vaarala et al. (1988) found that, in gram-negative infections in which aCLs are detected, lipopolysaccharide (isolated from Salmonella minnesota strain Re595) effectively inhibited the binding of CL to IgG aCL. This inhibition was not observed in gram-positive infections, syphilis, and SLE. Hence, in gram-negative infections and possibly in other disorders, CL may not be the primary inducing antigen.

2 . Other Phospholipids Green (1972) was able to abolish Rh expression by butanol extraction, and regenerate antigen expression with different phospholipids. PC and PE were more effective at antigen regeneration than PS and CL. Thus, Green noted that the primary antigenic determinant may not be CL. The data of Hazeltine (1988) support the hypothesis that antibodies directed at RBC epitopes are capable of cross-reacting with CL directly. Inoue and Nojima (1969) examined the reactivities of various CL analogs to sera (isolated from rabbits immunized with the different analogs) using the VDLR microglocculation test. The results of these investigators suggested that a free /3 hydroxyl group and two phosphodiester bonds of CL were important in the immunological reaction of CL as an antigen. Specifically, the degree of clumping was as follows: tetrapalmitoyl CL (disodium salt; uncharged CL = beef heart CL (disodium salt) > deoxyCL (uncharged CL with 1’,3’-propanediolgroup instead of 1’,3’glycerol) = 0-benzyl CL (uncharged CL with 0-benzyl attached to C-2’) > derivatives with only one phosphodiester group.

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These authors did not set out to describe the binding affinity of different molecular species of CL to aCL. However, they found equal binding of the disodium salts of tetrapalmitoyl CL and beef heart CL. These molecules have a dramatically different acyl composition since bovine and ox heart CL contain only 1% 16: 0 and 7 2 4 7 % 18 : 2n-6 (Avanti Polar Lipids Catalogue; Gray, 1964). At least in their crude assay, acyl composition was unimportant. Brown et al. (1989) reported that, of 45 lupus patients that were LA positive, 6 reacted only to human cadaver heart tissue CL as the antigen and not to bovine CL. Of these patients, 88% had antibodies to human CL, whereas only 75% had antibodies to bovine CL. Hence, fatty acyl composition of the antigen may affect binding to aCL. Unfortunately, the authors did not examine the fatty acyl composition of the tissues used and did not quantify oxidation of the fatty acids of the human cadaver heart CL. Since the composition of CL varies widely with diet, one cannot estimate which fatty acid may be responsible for differences between the two CL sources. Gharavi et al. (1987) used quantitative isotype specific ELISA to determine the distribution of immunoglobulin isotypes and phospholipid specificities of aCL in 40 patients with one or more of the following aPL associated clinical complications: thrombosis, fetal loss, and thrombocytopenia. Of these 40 patients, 12 had IgG, IgM, and IgA aCL; 10 patients had IgG and IgM, 5 patients had IgG and IgA, and 3 patients had IgM and IgA aCL. No statistical association was found between any single isotype or any group of isotypes and thrombosis, fetal loss, or thrombocytopenia. The presence of IgG aCL in 36 of the 40 patients suggests that this isotype may be most important in determining clinical complications, but 4 patients without IgG aCL also appeared susceptible to thrombosis, fetal loss, and thrombocytopenia. IgG, IgM, and IgA aCL bound the negatively charged PL, PS, and PI, but not the zwitterionic PC. No significant difference between binding to CL and binding to other negatively charged phospholipids was found, suggesting that the specificity of these antibodies is for negatively charged phospholipids in general rather than for CL in particular. Staub et al. (1989) determined that most patients with LA activity have coincident antibodies to a group of negatively charged phospholipids. These researchers suggested that LA and aCL tests detect antibodies with overlapping specificities. Some discordance between the two assays has been described, however. One patient presenting with severe thrombotic disease (recurrent deep vein thrombosis, pulmonary embolism, inferior venocaval obstruction, myocardial infarction, and digital gangrene) showed strong LA activity. An ELISA showed no binding to the negatively charged phospholipids CL, PS, and phosphatidic acid, but binding

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to zwitterionic PE was demonstrated. Inhibition studies and affinity purification confirmed this finding. Interestingly, the serum did not bind to the KCT reagent when used as antigen in an ELISA. The pathogenic significance of anti-PE antibodies and their relationship to LA remains to be clarified. Further studies of the occurrence of anti-PE antibodies in patients with LA activity who have negative aCL tests are suggested. E. MOLECULAR NATURE OF THE REACTIVE EPITOPE In SLE, antibodies are produced predominantly to native dsDNA, in which case the reactive epitope is believed to be the helical structure of the molecule; to denatured ssDNA, in which case the reactive epitope is believed to be the individual bases; or to both molecules, in which case the reactive epitope is believed to be the sugar-phosphate group common to both ss and dsDNA (Cohen et al., 1971; Bourdage and Voss, 1988). In general, the immunochemical cross-reaction between DNA and CL leads most investigators to speculate that the arrays of phosphodiester groups on the nucleic acid backbone and in phospholipid micelles are the reactive epitopes (Schwartz, 1988; Voss, 1988). In fact, both molecules contain phosphodiester-linked phosphate groups that are separated by three carbon atoms. Ben et al. (1988) investigated the structural features of the interaction between DNA and anti-DNA in competition experiments with low molecular weight synthetic compounds. Two correctly spaced chemical components, a substituted aromatic ring system and a negatively charged acidic residue, were found to be required for the binding of most antiDNA antibodies to their respective antigens. These chemical elements are combined in the structure of several anionic dyes, including some certified food colors. The dyes were found to compete efficiently for lupus DNA. Therefore, this family of compounds may serve as a basis for the development of a new approach to drug therapy in SLE. Rauch et al. (1984) injected BALB/c mice intraperitoneally with a solution of the monoclonal autoantibody H102, which binds both DNA and CL suspended in Staphylococcus aureus (Cowan strain) cultured fluid, incubated with a liposomal preparation of CL, and emulsified with Freund’s adjuvant or saline. No adjuvant was used for subsequent immunizations. Mice injected with CL without the adjuvant showed low DNA binding and no significant CL binding. Mice given only the adjuvant without CL showed little or no binding to CL and DNA. However, mice given CL and the adjuvant produced both anti-DNA (antibodies) and aCL. This result suggests that CL and DNA share an epitope that is both antigenic and immunogenic, but whether the anti-DNA antibodies of

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SLE really originate from this kind of “antigenic mimicry” remains unknown (Schwartz, 1988). In competitive assays, large excesses of CL were needed to compete for single and double stranded serum anti-DNA antibodies, suggesting that only a minor population of anti-DNA antibodies binds to both DNA and phospholipid (Edberg and Taylor, 1986; Eilat et al., 1986). In competitive radioimmunoassays (RIAs), the ability of several phospholipid micelles to compete with denatured DNA for hybridoma autoantibody binding was as follows: CL=phosphatidate=denatured DNA >> PG; PS, PC, and PE failed to inhibit the reaction at the highest level tested, which was 832x more than that required for 50% inhibition by CL (Lafer et al., 1981). For other hybridoma antibodies, denatured DNA was up to 80 times more effective as a competitor than CL. For a base-specific autoantibody with a marked preference for a guanine- or hypoxanthine-containing determinant, no level of any phospholipid inhibited denatured DNA binding. Phosphatidate and PG may react as well as CL because both have a series of repeating phosphate groups on a micellar surface. In contrast, when PC, PE, and PS are presented as a micelle, the positively charged groups of these molecules exposed to the aqueous solvent may interfere with the binding of the phosphate groups with the antibody. These observations also support the notion that the reactive epitope is the phosphodiester linkage. In this experiment, phospholipid micelles were prepared by drying the lipids under NZand slowly adding saline citrate. Rauch and co-workers (Janoff and Rauch, 1986; Rauch et al., 1986) tested the structural specificity of different polymorphic forms of PE for structural specificity against 1 1 isolated hybridoma anticoagulants. The polymorphic forms of PE used were (1) dipalmitoyl PE, which forms a bilayer at 37°C; (2) dioleoyl PE; (3) monooleoyl PE at pH<8.5, which exists as a bilayer; (4) bovine brain PE; ( 5 ) egg PE, which adapts a hex I1 phase under 37°C; (6) egg PE derived from phospholipase C cleavage of egg PC [abbreviated egg PE(PC)], which forms a bilayer at 37°C and (7) a hex I1 phase at 43°C; (8) monomyristoyl PE; and (9) monopalmitoyl PE, both of which form micelles at 37°C. Phase behavior of phospholipids were determined with 3’P-NMR. Note that, under the experimental conditions employed, the phase preference of egg PE is not entirely predictable since egg PE has a loosely defined bilayer to hex I1 transition that occurs between 25 and 35°C. The hybridoma antibodies tested were of the IgM class and were obtained by fusing peripheral blood lymphocytes from venous blood of 13 patients with lupus with an IgG producing GM 4672 human lymphoblastoid cell line. Hybridoma supernatants were tested for the production of LA using a modified APTT assay. LAs are

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aPLs that presumably bind to phospholipids of the prothrombin activator complex phospholipid, thus prolonging normal clotting time in in v i m coagulation assays (measured as increased APTT). Of 67 hybridoma IgM supernatants examined, all with varying immunoglobulin concentrations, 53 gave APTT values below or equal to that of the GM 4672 negative control. An antibody was defined as having anticoagulant activity only if its APTT exceeded the AFTT of the GM 4672 by 5 SD. Of the antibodies tested, 14 met this criteria and 11 were available in sufficient quantity. To clarify, a strong inhibition of the binding of hybridoma LAs to phospholipids of the prothrombin activator complex (i.e., a decrease in clotting time and decreased APTT) by different exogenous phospholipids is indicative of the binding affinity of different exogenous phospholipid sources for LA. Over the concentration range of 2-100 nmol phosphorus, only egg PE (hex 11) inhibited anticoagulant activity (APTT) of a representative hybridoma LA, whereas egg PE(PC) and PE (bilayer) did not inhibit APTT over this range. Overall, hex I1 lipid [bovine brain PE, egg PE, dioleoyl PE, egg PE (PC) at 43"CI inhibited anticoagulant activity whereas bilayer lipid [dipalmitoyl PE, egg PE(PC) at 37"CI did not. Monooleoyl PE at pH<8.5 exists as a bilayer and inhibited lupus anticoagulation activity, although not in the hex I1 phase, so the LA epitope can distinguish between diacyl and monoacyl PE because of increased motional freedom for the molecule or a difference in the orientation of the shielding tensor due to changes in the conformation of either the glycerol backbone or the PE head group. XIV. CHEMICAL SYNTHESIS OF ACYL-SPECIFIC CARD10LIPIN DERIVATIVES

Very little information exists on the antigenic and protein-binding affinities of CL derivatives with different acyl chains. CL derivatives with oxidized fatty acids (Nielsen, 1978; Krasnopol'skii et al., 1986), extra fatty acids (Esmann et al., 1988), and other modifications (Inoue and Nojima, 1969; Esmann et al., 1988) have been synthesized for these purposes, as well as for the study of phase-transition temperature (Nagamachi et al., 1985). Houle et al. (1982) describes procedures for purifying CL. Not surprisingly, few studies are devoted to the chemical synthesis of acyl specific CL. The method developed by Stanacev et al. (1973), modified by Nagamachi et al. (1985), and reviewed by Ioannou and Golding (1979) involves reacting acyl specific PGs with PLD. Briefly, 30 mg dried PG is dissolved in 100 ml diethylether. To this substrate, 60 mg PLD

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(dissolved in 100 ml 100 mM acetate buffer, pH 5.6, containing 10 mM EDTA) is added. The incubation proceeds for 4-12 hr at 28°C. After the reaction, the diethylether is evaporated and the reaction mixture is partitioned between chloroform and water. The lower chloroform layer is collected and evaporated. The phospholipids are separated by TLC and eluted from the silica. CL is purified in a solvent system of chloroformmethanol-water (65 :25 :4 v/v/v) or with hexane :diethyl ether (95 :5 v/v) and SI silica columns (Analytichem International; 100 mg). Fatty acid homology is checked by gas chromatography (GC). Approximately 95% purity of the phospholipid is expected (Nagamachi et al., 1985). Greater purity may be obtained by DEAE-cellulose column chromatography, as described by Stanacev et al. (1973). Acyl specific PG molecules may be purchased from various manufacturers. Sigma (St. Louis, Missouri) supplies dimyristoyl, distearoyl, dioleoyl, and dipalmitoyl moieties; Avanti Polar Lipids (Pelham, Alabama) supplies dilauroyl, dipentadecanoyl, dilinoleoyl, and 1palmitoyl-2-oleoyl moieties. In this way, tetramyristoyl, tetrapalmitoyl, dimyristoyl-dipalmitoyl CL, and so on can be obtained. Other CL molecular species can be obtained via PLD-transphosphatidylation reactions by reacting appropriate PC species (Sigma) with glycerol (Dawson, 1967; Yang et al., 1967). The postulated two-step reaction mechanism is (1) PC + PLD + phosphatidyl-PLD + choline and (2) phosphatidylPLD + glycerol +PG + PLD. According to Mishina et al. (1987), these few known methods for synthesizing acyl specific CL molecules are characterized by serious deficiencies such as multistage nature, low yield of the desired compounds, poor reproducibility of the individual stages, and the inadequate accessibility and lability of the intermediates in the synthesis. These authors synthesized acyl specific CL by reacting PC with PLD to yield acyl specific phosphatidate. The phosphatidate is then reacted with 2-0-tertbutyl dimethylsilglycerol in the presence of 2,4,6-triisopropylbenzene sulfonyl chloride. Moschidis (1988) reported the synthesis of the phosphono analog of 1”,2”CL, namely, 1,2-dipalmitoyloxypropyl-3-(2’-hydroxypropyl-3’)-( dipalmitoyl glycero1)biphosphonate.This compound was prepared by the condensation of l12-dipalmitoylglycerol with 1,2-dipalmitoyloxypropyl3-(2’-hydroxypropyl-3’)biphosphonatecatalyzed by triisopropylbenzene sulfonyl chloride in pyridine. The final product was characterized by elemental analyses, phosphonophosphorus determinations, TLC, and infrared spectroscopy. The silicic acid column chromatographic behavior of the phosphono analog of CL was studied also. New photoreactive analogus of CL have been synthesized chemically.

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Photoreactive aryl azido acyl groups were placed at two different locations within the CL molecule: at the sn-2 position of the sn-2 glycerol of CL; at the sn-2 position of the sn-3 phosphatidyl group; or at both locations to provide a dual-labeled analog. Thus, three different CL analogs, distinguished by the positions of the aryl azido acyl groups, were prepared. Two different aryl azido acyl groups were employed in these syntheses and the site of acylation was identified stereospecificially using several phospholipids of known specificity for CL. Acylation of CL with the symmetrical anhydride of either aryl azido acyl fatty acid analog, 2-(N-4-azido-2-nitrophenyl)p-alanineor 12-(N-4-azido-2-nitrophenyl)aminododecanoic acid, provided l-(sn-3-phosphatidyl)-2-(acyl aryl azido)-3-( sn-3-phosphatidyI)-sn-glycerol.Acylation of monolysoCL [ l-(sn-3-phosphatidy1)-3-(1-acyl-2-lyso-glycero(3)phospho)-sn-glycerol]provided two products. l-(sn-3-phosphatidyl)-3-(l-acyl-2-(acyl aryl azido)-giycero(3)phospho)-sn-glycerol and the doubly labeled l-(sn-3-phosphatidyl)-2(acyl aryl azido)-3-(1-acyl-2-(acyl aryl azido)glycero-(3)phospho)-snglycerol. These are the first reported photoreactive analogs for CL. The analogs were positive effectors for cytochrome P-450(sec). As shown by SDS-PAGE, they labeled the single subunit of cytochrome P-450sec and the smallest subunits of cytochrome c oxidase from beef heart (Fowler et al., 1988). Kuppe et af. (1987) reported the syntheses of two new radioactive probes derived from CL and PC. These probes are derivatives of natural lipids and contain an amine-specific benzaldehyde in the head-group region. This functional group allows a choice of timing of the reaction (e.g., after equilibration and detergent removal) because an irreversible covalent bond is formed only on the addition of reducing agent. These probes, as well as a benzaldehyde analog of phosphatidic acid and a water-soluble benzaldehyde reagent, were attached covalently to bovine heart cytochrome c oxidase. After reconstitution into vesicles, the lipid-. benzaldehyde probes selectively incorporated into the smaller polypeptides of the enzyme, whereas the remaining subunits (I-IV) exhibited little incorporation of label. The accessibility of amine groups labeled under the conditions used here was independent of the structural and charge differences between the benzaldehyde probes. This result suggests that all three lipid probes react with polypeptides of the cytochrome c oxidase complex at general contact sites for membrane phospholipids. A water soluble benzaldehyde reagent predominantly labeled subunits IV, Va, and Vb and polypeptides of VII-VIII. A comparison of these results facilitates a more refined view of the disposition of polypeptides of cytochrome c oxidase with respect to the lipid and aqueous phases.

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Kuz’mina et al. (1988) described synthesis of CL analogs containing an ionophore residue in the fatty acid moiety. The ionophore dibenzo-18crown-6 has been incorporated into the second position of the glycerol residue by acylating mono- and dilysoCL with a modified fatty acid anhydride. Lyso derivatives of CL have been prepared by enzymatic hydrolysis of beef heart CL by snake (Naja naja oxiana) venom PLA2. Parinandi et al. (1988) report commercial preparations of bovine CL in chloroform solution contain substantial amounts of oxidation products. These oxidized derivatives, characterized by the presence of varying amounts of hydroperoxides and conjugated dienes, can be separated from unoxidized CL by normal phase HPLC using UV detection. When purified CL is subjected to autoxidation in aqueous media, oxidation products with similar HPLC properties are produced. Storage of CL in chloroform induces both autoxidation and hydrolysis, whereas storage in ethanol and other solvents does not. Chloroform is not recommended for the long-term storage of CL.

XV. CHROMATOGRAPHIC SEPARATION OF CARDIOLIPIN

One of the hurdles to intensive studies of CL has been its routine analysis. Newer methodologies promise to add great insight to its functions. In simple separation, we have found standardization of methods essential to reproducible analysis of CL species. Many solvent systems are available for the separation of phospholipids by TLC. In our laboratory, we use the procedure of Holub and Skeaff (1987) since all major phospholipid classes are separable and CL migrates clear of all other phospholipids with a Rf value of approximately 0.73. Phospholipids are separated into classes by TLC using a chloroform :methanol :acetic acid : water (50.0 : 37.5 :3.5 : 2.0, v/v/v/v) solvent system and 10 x 10 cm HPTLC precoated Silica Gel 60 plates (Merck, Darmstadt). Waring (1981) describes a solvent system to separate CL and other phospholipids with silicic acid columns (Sigma SIL-LC 325 mesh). Phospholipids are eluted with 4 column volumes of chloroform, and CL is eluted with 4 column volumes each of chloroform :acetone 2 : 1 ; acetone : methanol 20 : 1 ; and chloroform : methanol 14 : 1. Powell et al. (1987) described a method to separate dilysoCL. (MonolysoCL was synthesized by PLAz attack on CL.) Extracted lipids were placed in 2.8 x 20 cm columns with silica gel (Merck 60, 70-230 mesh) and dilysoCL was eluted with 500 ml each of ch1oroform:methano1 : water (100 : 15 : 1, 65 :30 :3, and 65 : 35 :5 v/v/v). DilysoCL was iso-

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lated from pooled fractions with TLC using a solvent system of acetone :chloroform : methanol :acetate : water (40 : 30 : 10 : 10 :5 ) . ACKNOWLEDGMENTS This review was supported in part by awards from the International Life Sciences Institute, the California Committee on Cancer Research, and the Clinical Nutrition Research Unit at the University of California at Davis.

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ADVANCES IN FOOD AND NUTRITION RESEARCH, VOL. 37

DISEASES AND DISORDERS OF MUSCLE A. M. PEARSON Department of Animal Sciences Oregon State University Corvallis, Oregon 9733I

RONALD B. YOUNG Department of Biological Sciences University of Alabama in Huntsville Huntsville, Alabama 35899

I. Introduction 11. Disorders and Diseases of Muscle A. Clinical Symptoms B. Methods of Study C. Muscular Dystrophies D. Inflammatory Myopathies E. Atrophy and Hypertrophy F. Neuropathies G. Normal and Abnormal Fatigue H. Aging and Effects of Exercise I. Muscular Dysgenesis J. Myoclonus 111. Disorders of Energy Metabolism A. Glycolytic Disorders B. Acquired Diseases of Glycolysis C. Disorders of Mitochondrial Oxidation D. Disorders of Lipid Metabolism E. Myotonic Syndromes IV. Diseases of the Connective Tissues A. Nutritional Diseases B. Genetic Diseases C. Acquired Disorders 339 Copyright 0 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

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A. M. PEARSON AND RONALD B. YOUNG V. Research Needs A. Biopsies B. Fiber Typing and Computerized Tomography C. Nuclear Magnetic Resonance and Nuclear Magnetic Imaging D. Measurement of Protein Turnover and Related Techniques E. Use of Muscle Cell Cultures F. Aging and Effects of Exercise G. Energy Metabolism H. Connective Tissue Diseases and Disorders I. Gene Mapping Studies VI. Summary References

I.

INTRODUCTION

Muscle and the supporting connective tissue system may be subject to numerous disorders and diseases that are important both in humans and in animals. These abnormalities fall into three broad classes: (1) disorders or diseases of the muscular system, (2) disorders of the sarcoplasmic proteins, and (3) disorders or diseases of the connective tissues. The disorders or diseases that fall into each of these classes are reviewed briefly by pointing out their characteristic manifestation and symptoms, as well as their basic causes and treatments, if known. Preceding the discussion of the different disorders or diseases is a review of some of the general clinical signs of muscular disorders. Then some of the methods commonly used to study muscle abnormalities are discussed, in addition to techniques for evaluating and diagnosing muscle and supporting connective tissue abnormalities. This discussion is not intended to replace diagnostic prognosis practiced by medical specialists, but to give researchers some understanding of such techniques as a supporting means of research. Appreciation of some of the currently used diagnostic techniques can be helpful in gaining a better understanding of normal functions as well as in recognizing some of the abnormalities or diseases that are found in muscle and connective tissues. An understanding of some of these disorders or abnormalities could lead to their use as models in studying changes in muscle that are related to meat quality, such as cross-linking of collagen and meat tenderness or toughness. Other disorders are of interest because they are caused by nutritional deficiencies. Some of these nutritional diseases also can be used as models to study factors related to meat quality.

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II. DISORDERS AND DISEASES OF MUSCLE

Edwards and Jones (1983) reviewed the diseases of skeletal muscle, discussing the history of recognition of abnormal conditions in muscle and the clinical symptoms of muscle abnormalities or diseases, and subsequently focusing on the methods commonly used to study muscle disorders.

A. CLINICAL SYMPTOMS Muscle disorders have many different manifestations. The most obvious symptom of a muscle disease or disorder is muscular weakness, that is, the inability to produce the normal amount of force. The manifestations of muscular disorders generally can be classified into three types: (1) muscular weaknesses in which the afflicted individual is unable to perform normal body muscular functions, such as sitting, standing, walking, or climbing; (2) weakness in which the suffering individual has normal muscular strength after resting but, on minimal exercising, suffers from excessive or premature fatigue; and (3) muscle disorders that are characterized by disturbance of normal muscular functions, such as spasticity, but in which muscular strength or fatigue is not involved (Edwards and Jones, 1983). The last group of disorders also involves those in which the muscles lack the ability to relax after contraction (myotonia), in which the individual suffers abnormally severe muscle cramps, and in which episodes of muscular weakness occur (Edwards and Jones, 1983). Some individuals also suffer from muscle aches and pains that are dimcult to diagnose. These individuals are characterized as psychopathic or hysterical, but Edwards and Jones (1983) have indicated that this label frequently is unjustified because of a failure to recognize the basic cause of such aches and pains, which merely reflects ignorance about the origin of such problems. 1. Muscle Weakness

The amount of force generated by any muscle is proportional to the muscle cross-sectional area occupied by the contractile proteins, according to Edward and Jones (1983). As a result, muscle hypertrophy will increase the capability of a muscle to generate force, whereas muscle atrophy will decrease force generation. Growth in length, on the other hand, does not alter the ability to generate force. This condition was discussed in terms of the effects of use and disuse by Pearson (1990).

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Muscle weakness almost always is caused by a loss of contractile tissue, except in the case of a few rare muscle diseases. Loss of contractile tissue (muscle area) commonly occurs in one of two ways: ( 1 ) by destruction or damage to the contractile tissue (such conditions commonly are classified as destructive myopathies) and (2) by shrinkage and atrophy of the contractile area without any losses in the number of muscle cells (atrophic myopathies) (Edward and Jones, 1983). In destructive myopathies, the replacement of muscle fibers by fat and connective tissue may give the impression of no change in contractile tissue area since muscle bulk is not altered, a condition called pseudohypertrophy. In atrophic myopathies, the weakness is the result of wasted or thin muscles with a loss in myofibrillar cross-sectional area without any loss in muscle cell numbers. 2. Muscle Fatigue

According to Edwards and Jones (1983) muscle fatigue can be defined as the failure to achieve or maintain the expected force or power output. Thus, an individual with a decrease in muscle bulk cannot maintain the same absolute force as a normal subject, which will be manifested by a decrease in maximum force or more rapid tiring. However, some patients that have normal amounts of muscular area become easily fatigued. Edwards and Jones (1983) classified individuals who have normal myofibrillar mass but become easily fatigued into two main groups: (1) those suffering from myasthenic symptoms, that is, having a defect at the neuromuscular junction that causes a neuromuscular block during attempted sustained contraction, and (2) those having a defect in muscle energy metabolism which results in unusually rapid depletion of glycogen stores, and thus, causes premature fatigue. However, abnormal muscle metabolism does not always cause rapid fatigue since individuals with hypothyroidism, who have a marked decrease in muscle ATP turnover, can sustain isometric contractions for appreciably longer periods of time than normal individuals (Wiles et al. 1979). 3. Myoglobinuriu

The presence of myoglobin in the urine is indicative of muscle destruction and may cause renal damage (Edwards and Jones, 1983). This condition frequently arises from physical damage to muscle, as can occur in crushing injuries. According to Edwards and Jones (1983), myoglobinuria often is observed in clinical practice as a result of active myositis (inflammation of the voluntary muscles) or rhabdomyoma (an active tu-

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mor in muscle cells). The condition also is observed more rarely as a symptom of some metabolic muscle disease, such as carnitine palmitoyltransferase deficiency or myophosphorylase deficiency. Regardless of the cause, myoglobinuria is serious because it indicates the degradation of muscle. Unless alleviated, this degradation can lead to serious consequences. 4 . Unusual Muscle Function

The two most common forms of unusual muscle function are myotonia (tonic muscle spasms) and spasticity. Spasticity is a disorder of the central nervous system. Myotonia involves the inability of muscle to relax completely following contraction. This condition may be of congenital origin (myotonia congentia) or be a form of dystrophy (myotonia dystrophy). The physiological bases for the two forms of myotonia are probably different, although they are not well understood (Edwards and Jones, 1983). B. METHODS OF STUDY The various procedures that are helpful in diagnosing and studying muscle diseases include (1) history and clinical examination, (2) blood biochemistry studies, (3) electromyography, (4) muscle biopsies, ( 5 ) nuclear magnetic resonance, (6) measurement of cross-sectional area by ultrasound, computerized tomography, or both, (7) function tests, (8) provocation tests involving exercise and fasting, and (9) studies on protein turnover. All these procedures are reviewed briefly next. For a more extensive review, see Edwards and Jones (1983). 1 . Clinical Examination

The symptoms of any muscle disease should be observed and recorded carefully. Sometimes the information obtained may suggest that the problem is of genetic origin. Brooke (1977) and Dubowitz (1978) have described the procedures that can help in the diagnosis. Physical examination may aid in detection of defects in performance and may permit assignment to the muscle groups that are involved. 2. Blood Biochemistry

Evaluation of plasma electrolytes, thyroid hormones and thyroid stimulating hormone activities, differential blood cell counts, erythrocyte

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sedimentation rate, plasma protein and immunoglobulin levels, and serum creatine kinase (CK) activity should be determined (Edwards and Jones, 1983). If the patient has any history of pigmenturia, urine myoglobin should be measured. A more sensitive test is measuring myoglobin in the blood serum, which could detect muscular dystrophy even in the absence of myoglobinuria (Kagen et al., 1980). Hypo- and hyperkalemia may be associated with episodes of flaccid paralysis, as discussed in greater detail by Edwards and Jones (1983), whereas altered thyroid function also can cause marked changes in muscle function. If blood calcium concentrations are normal, bone disease (osteomalacia) can be eliminated as a cause of muscular weakness. A high erythrocyte sedimentation rate would be indicative of inflammatory diseases. Plasma CK activity is among the most useful blood biochemistry measures, since this enzyme is released from muscle cells under a variety of conditions. High activity is indicative of an active disease (Pennington, 1980). In the case of severe muscle disease, CK activity may be as much as 2- to 3-fold higher than normal. Although CK levels often are used in diagnosis of Duchenne muscular dystrophy, marginally higher levels of CK activity must be interpreted with care.

3. Electromyography Electromyography is a useful tool in differentiating diseases of myopathic origin from those of neuropathic origin. Myopathic diseases are characterized by an increased number of small polyphasic action potentials that can be contrasted with the giant action potentials associated with nerve sprouting and fiber grouping that occurs in neurogenic atrophy and reinnervation. Nevertheless, clinical changes in electromyography are difficult to quantify. Power-spectrum analysis has been used to study muscle fatigue (Gross et al., 1979), but also gives quantitative information about interference patterns. Electromyographic recordings in conjunction with nerve stimulation can be used in diagnosis of myasthenia. Restoration of the normal responses can be achieved by administration of cholinesterase inhibitors, which overides the abnormal decremental responses. Neuromuscular blocking can be detected by recordings from two muscle fibers innervated by the same motor unit, where absence or delayed firing indicates blocking, and can be useful in diagnosis of myasthenia (Stalberg et al., 1974) or abnormalities found in muscular dystrophy (Stalberg et a1. 1972).

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4 . Muscle Biopsies

Muscle biopsies are extremely helpful in the diagnosis of muscle diseases and have provided much of the raw material used in the characterization of abnormalities. Samples commonly are removed from large proximal muscles, such as the quadriceps or deltoid, either through a skin incision or with the percutaneous biopsy needle. Histochemical and biochemical tests then can be carried out. Clinical applications using the needle biopsy technique for detecting pathological or biochemical abnormalities in muscle are reviewed by Edwards et al. (1980). In clinical practice, histochemical analysis usually has the first priority and is followed by electron microscopic examination and, finally, by specific enzyme analyses for both the glycolytic and the oxidative pathway. Measurement of metabolites and electrolytes also may be carried out if the quantity of sample permits. Samples used for histological examination commonly are stained with hematoxylin-eosin to allow assessment of the muscle architecture and to reveal inflammatory responses. Examination should focus on fiber-typing to ascertain whether preferential involvement of one fiber type is occurring; this determination usually is accomplished best by staining with myosin-ATPase and certain oxidative stains. Specific enzymatic abnormalities can be detected using histochemical stains, for example, myophosphorylase deficiency in McArdle’s disease can be diagnosed by staining for phosphorylase activity (Edwards et al., 1980).

5 . Nuclear Magnetic Resonance The noninvasive technique nuclear magnetic resonance (NMR) has been shown to be useful for studying chemical changes in intact muscle during development of fatigue (Dawson et al., 1980). Chalovich et al. (1979) showed that NMR can be used to measure phosphodiesters in dystrophic muscle, indicating that this technique may prove valuable in following chemical composition and metabolism in both normal and diseased muscles. The development of larger magnets should permit greater resolution of various constituent components of muscle and make the NMR method more useful for in situ studies in muscle, as has been demonstrated by Cresshull et al. (1981) and Edwards et al. (1982). New instrumental adaptations no doubt will continue to result in further improvement in the NMR method. NMR ultimately may have application in following metabolites in muscle that are involved in meat quality.

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6 . Measurement of Muscle Cross-Sectional Area

As indicated earlier in this chapter, the area of a muscle in cross section determines its force-generating capacity as well as the intrinsic strength of the contractile elements. Although measurement of muscle cross-sectional area is difficult, muscle volume has been shown to be related to development of force (Edwards et al., 1979), leaving little doubt about their relationship. Two major procedures are used to determine muscle cross-sectional area: ( 1 ) ultrasound and (2) computerized tomography. a . Ultrasound. The imaging of muscle by ultrasound is based on the principle that reflection of sound waves occurs at tissue interfaces. The method has been used widely in measurement of fat and lean areas for selection and marketing of livestock (Lauprecht et al., 1957; Hazel and Kline, 1959; Price et al., 1960a,b; Stouffer et al., 1961). Measurement of muscle area by ultrasonics also has been used to assess the influence of strength-training programs in humans (Ikai and Fukunaga, 1968,1970; Dons et al., 1979) and to study the wasting of muscle that occurs from inactivity due to a fracture of the lower limb (Young et al., 1979). One of the problems with ultrasound is that the method does not always differentiate between muscle and fat since it recognizes only the tissue interfaces and does not identify the type of tissue responsible. Thus, recognizing fatty tissue replacement of muscle, as occurs in some of the dystrophies, is not always possible (Edwards and Jones, 1983). Nevertheless, ultrasound is still useful for studying muscle; its accuracy is in part dependent on the skill of the operator in interpretation. b. Computerized Tomography. This relatively new technique can be used to visualize the cross-sectional area of muscle and to give information on its composition. The procedure distinguishes differences in density between muscle and fatty tissues. The usefulness of this technique in recognition of replacement of muscle with fat is illustrated in Fig. 1 , where partial to almost complete replacement of muscle by fatty tissue occurs. This method also has been shown to be useful in diagnosing unilateral swelling of the calf muscle in humans, a condition that subsequently was found to be caused by an infiltrating lipoma (Fete11 et al., 1978). No doubt computerized tomography can be used to visualize the lean-fat ratio in meat animals.

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7 . Tests of Muscle Function Duchenne (1867) first reviewed the physiology of motion and discussed the functions of most human muscles. Since that time, the response to electrical stimulation has been investigated using a number of peripheral muscles with readily accessible motor nerves (Merton, 1954; Stephens and Taylor, 1972; Burke et al., 1974; McComas et al., 1977). Small distal muscles also have been used to study neuromuscular function, such as myasthenia (Desmedt and Boronstein, 1976), myotonia (Wiles and Edward, 1977), and Duchenne muscular dystrophy (Desmedt et al., 1968). However, use of the distal muscles suffers from two major shortcomings, according to Edwards and Jones (1983): ( 1 ) the muscle temperature must be controlled carefully or it may not be typical of other muscle groups and (2) distal muscles are not typical in the early stages of muscle disease, since muscle disorders usually occur in proximal muscles only during the late stages of diseases. Edwards and Jones (1983) recommended the quadriceps muscle for testing muscle function for the following reasons. (1) It is a proximal muscle group that often is affected severely by muscle disorders. (2) The group is load bearing and, thus, is important for maintenance of mobility. (3) The quadriceps muscle group is relatively free of major blood vessels, so biopsies can be taken and used simultaneously for histochemical, chemical, and microscopic examination. Simultaneous studies permit the coordination of functional tests with the results of the biopsies. Assessment of muscle function commonly includes objective measurement of the force at maximum voluntary contraction. Isometric contraction of the quadriceps muscle can be measured conveniently with the muscle testing chair (Tornvall, 1963). The commercially available Cybex@machine described by Thorstensson et al. (1976) can be used for isokinetic contractions to determine the force-velocity characteristics of the quadriceps muscle, which then must be related to the fiber-type composition as determined by biopsy. Edwards and Jones (1983) concluded that normal individuals can generate a force (kg) at the ankle during maximum isometric contraction that equals about 75% of their body weight in kg, with the lower limit of the normal (3rd percentile) being 50% of their body weight. Those individuals generating less than 50% of their body weight have difficulty performing tasks such as rising from a chair or walking up stairs. On the other hand, walking on level ground requires only low force generation by the quadriceps and often can be continued even in the presence of severe muscle wasting. Percutaneous electrical stimulation-induced contraction of the quadriceps can be achieved by stimulating the nerve terminals in the motor end

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FIG. 1. Computerized tomography of a West Indian male suffering from limb-girdle muscular dystrophy. (Top) Cross-section of midthigh region showing partial replacement of quadriceps and almost complete replacement of posterior thigh (hamstring) muscles with fatty tissue. (Borrom) Cross-section through area of maximum calf circumference showing hypertrophy of anterior tibia1 muscles, preservation of normal gastrocnemius, and nearly complete replacement of soleus muscles by fatty tissue. Similar infiltration of fatty tissue in muscle of meat animals also has been observed occasionally. Reprinted with permission from Edwards and Jones (1983).

plate region. Use of large pad electrodes activates 30-50% of the whole muscle and yields useful information with little discomfort to the patient. This procedure is almost free of risk and has been used for adults (Edwards et al., 1977) and children (Hoskins et al., 1976). Electrical stimulation gives an objective measurement of muscle function that is uncomplicated by emotional or psychological problems; therefore this technique is a valuable tool for determining the causes of muscular fatigue. 8. Provocation Tests

Exercise and fasting tests commonly are used to impose muscular stress and to help diagnose muscle disorders. Exercise as an adjunct for

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diagnosis of problems associated with cardiac and pulmonary disorders commonly is used by medical practitioners and specialists. Jones et al. (1975) outlined the use of similar techniques to identify metabolic myopathies. Brooke et al. (1979) and Carroll et al. (1979a) used exercise to classify patients into three categories: (1) those with psychogenic symptoms who have a relatively normal exercise response; (2) those with metabolic myopathies who show signs of muscle damage, that is, a raised CK level, on exercising; and (3) those with mitochondrial abnormalities, which may be manifested by accumulation of abnormally high levels of blood lactate. Blood lactate levels for an individual suffering from a defect in mitochondrial metabolism, with the accumulation of blood lactate during exercising and the slowness of recovering, is shown in Fig. 2. Fasting alone or in conjunction with exercise provides additional muscular stress that may be helpful in diagnosing metabolic abnormalities. Individuals with fatty acid metabolism defects are affected more severely during fasting, since the body relies on fat and ketone bodies as a major substrate. Patients suffering from defects in glycolytic pathways usually are not affected, and exercise performance actually may improve during fasting (Carroll et al., 1979b).

m

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zi:

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FIG. 2. Comparison of blood plasma lactate concentrations of normal subject (work done, l%W at 70% of maximum) with that of a patient with a defect in mitochondrial pyruvate metabolism (work done, 65W at 59% of maximum) during rest, exercise, and recovery periods. Note that the individual with a defect in mitochondrial pyruvate metabolism ( 0 ) showed a faster and much greater rise in plasma lactate concentrations and recovered more slowly than the normal individual (m). Reprinted with permission from Edwards er al. (1981).

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9. Studies on Protein Turnover

Changes in total body protein turnover have been shown to be useful for diagnosing myopathies. Nitrogen balance studies have been shown to be related closely to deterioration or recovery of quadriceps strength in a patient with polymyositis (Edwards et al., 1979). Thus, protein turnover can be used to follow development of or recovery from various muscle disorders. Excretion of creatinine is used as an index of muscle mass. Graystone (1968) concluded that about 1 g creatinine is quantitatively excreted in the urine per 20 g muscle. Administration of [''Nlcreatine by mouth has been used to measure muscle mass by following the labeled creatine in biopsy samples (Reeds et al. 1978). Whole body potassium also can be used to estimate muscle mass (Pfau, 1965), but appears to suffer from the fact that fatty tissue also contains some potassium as a component of each adipose tissue cell (Lawrie and Pomeroy, 1963; Flear et al., 1965; Gillett et al., 1965,1967). Protein turnover is an important response that is used in nutritional studies. Factors that influence the rate of turnover have a profound effect on efficiency and determine the ultimate amount of muscle or lean tissue deposited in both farm animals and humans. Myofibrillar protein catabolism also can be estimated by urinary excretion of 3-methylhistidine, which is derived from breakdown of actin and myosin and is excreted in that form. To determine its catabolic rate, muscle mass must be estimated, which frequently is done by expressing the ratio of urinary 3-methylhistidine to creatinine. Intracellular free 3methylhistidine in muscle is correlated with the rate of urinary creatinine excretion, according to Rennie et al. (1980). However, Millward et al. (1980) criticized its use based on the fact that some of the 3-methylhistidine that is excreted arises from non-skeletal muscle sources, that is, from smooth and cardiac muscle. In the case of wasting muscle, the quantitative contributions of non-skeletal muscle sources could become proportionately larger, thus increasing the error (Edwards and Jones, 1983). For additional information on measuring protein turnover in muscle, a detailed review by Waterlow et al. (1978) is recommended. C. MUSCULAR DYSTROPHIES Muscular dystrophy in the true sense refers to the group of inherited diseases that are manifested primarily as progressive degeneration of skeletal muscle. This rather diverse group of genetic muscular disorders is grouped according to such criteria as the pattern by which the defect is inherited, the clinical distribution of symptoms among the different mus-

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cle groups in the body, and the clinical severity of the symptoms. The major classes of muscular dystrophy are presented here; however, the reader is referred to Dubowitz and Brooke (1973) for an especially detailed description of some of the pathological changes in each of these diseases as detected through the use of muscle biopsies.

I. Duchenne and Becker’s Muscular Dystrophy The most severe form of human muscular dystrophy is Duchenne muscular dystrophy (DMD), which was first described by Duchenne (1867). This condition results from defective expression of the muscle protein dystrophin. This type of dystrophy is inherited as an X-linked recessive trait; females act as carriers and expression occurring in approximately 1 in 3500 males. Spontaneous mutations take place with a relatively high frequency in DMD, however, and approximately one-third of the cases diagnosed are in patients who do not have a previous family history of the disease. The onset of the disease usually corresponds with the time at which the child is first learning to walk. Clinically, the disease is first noticeable as difficulty running or as a slight waddling gate. The muscles of the lower leg initially exhibit a significant level of hypertrophy. The hypertrophy gives way to muscle atrophy after a short period of time. For this reason, DMD is sometimes referred to as pseudohypertrophic muscular dystrophy. Once the symptoms of DMD have started, the disease is irreversibly and steadily progressive; the ability to walk unaided is lost by 8-12 years of age. The muscle weakness usually affects the lower limbs before it affects the upper portion of the body; muscles in the proximal portions of the limbs generally are affected more severely than muscles in the distal portions of the limbs. The rate of progression of the disease may be accelerated if the child has been immobilized for periods of time with other illnesses; conversely, maintenance of a higher level of physical activity seems to delay the ultimate destructive force of the dystrophy. Most of the victims die from the disease during their teenage years (frequently from pneumonia), although a few patients have lived into their twenties. The histological and morphological characteristics of muscle fibers in DMD have been studied in detail; however, no pathological alterations that are unique enough to diagnose all cases of the disease unequivocally are apparent. Although cardiac and skeletal muscles have many biochemica!, histological, and dorphological characteristics in common, the effects of DMD are manifested almost exclusively in skeletal muscle. Among the effects of the disease in skeletal muscle are changes in fiber

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size and diameter, but the changes in fiber size are due mostly to an increased variability in the distribution of fiber sizes rather than to a consistent increase or decrease. If any consistent pattern of changes in fiber sizes is present at all, it is that the fibers tend to be slightly larger than normal in younger patients and slightly smaller than normal in older patients. This situation is, perhaps, logical in view of the diminished level of activity in the more advanced stages of the disease, whereas the increased fiber diameter in younger patients may reflect the pseudohypertrophic characteristics mentioned earlier. The size of neither of the major fiber types seems to be affected preferentially by the disease, but an increase in the proportion of fibers that are not precisely defined as fast or slow does occur, and dystrophic muscle tends to have a higher proportion of slow fibers than normal muscle. This observation likely results from changes in expression of myosin isoforms as the muscle fibers are subjected to abnormal levels of muscular activity in patients with the disease. In summary, the most consistent changes in DMD muscle biopsies include degeneration, significant increases in endomysial connective tissue (and, in later stages of the disease, a generalized increase in total connective tissue), replacement of muscle with fat, variation in fiber size, and a slight increase in centrally located fiber nuclei because of the regeneration of new muscle fibers (Dubowitz and Brooke, 1973). The clinical characteristics and distribution of muscle weakness in Becker’s muscular dystrophy (BMD) are similar to those of DMD discussed in the preceding paragraphs. BMD is inherited as an X-linked recessive characteristic; the site of the chromosomal mutation in BMD is localized with the DMD gene (i.e., dystrophin) on the X chromosome (Kunkel, 1986). These two diseases will be discussed together in this section. The primary difference expressed at the clinical level between DMD and BMD is the age at which loss of ambulatory ability takes place. Whereas DMD patients are no longer ambulatory by approximately 12 years of age, patients with BMD retain their freedom of movement into the late teenage years and on into adult life. Thus, BMD is a milder form of DMD. The few differences that are apparent in muscle biopsies from DMD patients are even more inconsistent in biopsies from BMD patients. Although DMD is an X-linked recessive trait and, therefore, normally expressed only in males, a small number of cases also have been documented in females. In all cases, these female patients have been found to carry chromosome translocations involving variable autosomal sites, but always with a breakpoint within band Xp21 of the X chromosome. In these female patients, the normal X chromosome is preferentially inacti-

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vated, which presumably silences their one normal gene for dystrophin and thereby subjects the patient to the effects of the aberrant gene on the translocated chromosome. The Xp21 region has been identified as the site of the DMD lesion. These very unusual translocations in females have provided the key to cloning, sequencing, and ultimately identifying the exact nature of the gene responsible for the genetic defect. Using this information, Ray et al. (1985) cloned the region spanning the translocation breakpoint in one such patient. A sequence derived from the Xchromosomal portion of the clone detected a restriction fragment length polymorphism (RFLP) that was linked closely to the DMD gene and indicated the presence of chromosomal deletions in DMD patients. Burmeister and Lehrach (1986) determined the restriction map of a major fraction of the region of the X chromosome in which the DMD gene is located. This physical map covered two regions of three million base pairs (approximately 2% of the X chromosome). The size of the DMD gene was estimated to be a minimum of 600 kb and the direction of transcription was determined to be from the centromere toward the telomere (Burmeister and Lehrach, 1986). As indicated by Monaco et al. (1986), “a large size for the DMD locus is consistent with the high frequency of mutation observed in the disease, the heterogeneity of translocation breakpoints in Xp21 which give rise to DMD or BMD in females, the 5% recombination of restriction fragment length polymorphisms (RFLPs) from the DXS164 locus with DMD and BMD mutations, segregating in families, the large size of deletions detected in DMD males, and the heterogeneity of deletion breakpoints” (Moser, 1984; Monaco ef al., 1985; Ray et al., 1985; Boyd and Buckle, 1986; Kunkel, 1986). The sequence of the cDNA of the DMD locus has been completed (Burghes et al., 1987; Koenig ef al., 1987a,b), as has the predicted amino acid sequence of the protein, dystrophin, whose absence is responsible for the degenerative characteristics of DMD (Hoffman et al., 1987b). Hoffman et al. (1987a) also isolated and characterized not only the cDNA from human muscle, but also the corresponding gene product from mice. Sequence analyses show that the DMD gene is approximately 90% homologous between the two species, and that 0.001-0.01% of total RNA in skeletal and cardiac muscle RNA consists of the dystrophin gene transcript. Transcripts also were detectable in tissue extracts of newborn mouse leg muscle, combined uterus and placenta, newborn heart, and adult heart. From these initial observations about the molecular biology of the gene responsible for DMD and BMD, information about dystrophin and its gene has accumulated rapidly. The dystrophin gene is nearly 10 times larger than the next largest gene that has been characterized, and occu-

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pies about 1% of the human X chromosome (Hoffman and Schwartz, 1991). The dystrophin gene contains approximately 2,500,000 nucleotides, but only 0.5% of the gene is composed of actual exon coding information for the dystrophin protein product. The remaining 99.5% of the dystrophin gene consists of introns, which are removed when the primary transcript is processed into the final translatable mRNA. This coding information for dystrophin is contained in 70 exons averaging approximately 200 nucleotides in size; these 70 exons are spliced together to produce a mRNA of 14,000 bases. Accordingly, the mRNA is translated into a large protein which contains approximately 3600 amino acids. The large size of the dystrophin gene seems to explain the unusually high mutation rate-the gene is a large target for random mutational events (Hoffman and Schwartz, 1991). The mutations in DMD and BMD consist of deletions or duplications of various sizes that occur within the Xp21 region. The deletions that cause DMD are frequent and quite large, but not large enough to cause the visible appearance of a gross chromosomal aberration at Xp2 1 during cytogenetic analysis. Dystrophin has been characterized in detail, and appears to contain four primary structural domains (Leger et al., 1991). Two dystrophin molecules are thought to self-associate to form antiparallel homodimers; the homodimers further self-associated into a higher order network beneath the plasma membrane, where the protein is essential to muscle cell function. The sequence of dystrophin has marked homologies with spectrin and a-actinin, both of which are cytoskeletal proteins (Davison and Critchley, 1988; Koenig ef al. , 1988). Biochemical and immunohistochemical studies have localized dystrophin to the internal face of the muscle plasma membrane (Hoffman and Schwartz, 1991). Analysis of sequence data suggests that this large protein interacts with a number of other highly conserved structural proteins in muscle, and that it serves a specific structural role in muscle tissue (Hoffman et al., 1987b,c,d). The protein is present in skeletal muscle as a large oligomeric complex that contains four glycoproteins of 156, 50, 43, and 35 kDa and another protein of 59 kDa (Ohlendieck and Campbell, 1991). Some of the residues of the N-terminal domain of dystrophin interact with actin. This region is the one that displays sequence homology with a chick a-actinin sequence (Davison and Critchley, 1988);the greatest similarity is localized within the N-terminal region of the proteins. Since this region of a-actinin may be involved in anchoring actin to a variety of intracellular structures, the sequence similarity between these two proteins is consistent with the interpretation that dystrophin also is involved in actin binding (Davison and Critchley, 1988). The long central domain of dystrophin has different discontinuous protease-sensitive

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areas. Dystrophin molecules have the same capacity for self-association in oligomeric structures as do other cytoskeletal proteins. The C-terminal domain of dystrophin is arranged close to the cytoplasmic internal face of the plasma membrane (Knudson et al., 1988), and interacts with the membrane integral glycoproteins. Different isoforms of dystrophin or dystrophin-related proteins appear to be expressed either exclusively or coexpressed in different muscle tissues or in other tissues such as brain and liver. These protein isoforms are translated from different mRNAs, and are generated by direct transcription of genes located on at least two different chromosomes (X and 6) or through alternative splicing. Alternative splicing has been reported to occur in the C-terminal region of the skeletal, brain, and smooth muscle isoforms (McAndrew et al., 1991); two different promoter regions apparently are utilized to generate the isoforms that are expressed in muscle cell types and in neuronal cell types (Chelly ef al., 1991). The precise function of dystrophin in muscle cells is not known. However, Hoffman and Schwartz (1991) describe a hypothesis in which dystrophin increases the stability of the muscle fiber plasma membrane. Since muscle fibers produce substantial mechanical force during contraction and show dramatic changes in cross-sectional area, these forces might be expected to cause stress on the plasma membranes of normal dystrophin-containing fibers. Dystrophin may afford some protection against damage from these forces, since most muscle fibers in the absence of dystrophin seem to sustain transient holes in the plasma membrane. The biochemical consequences of these membrane tears are massive efflux of muscle enzymes out of the muscle fibers and movement of substantial amounts calcium ions into the fiber. The influx of calcium ions is the likely mediator of necrosis in dystrophin-deficient muscle fibers (Hoffman and Schwartz, 1991). A complete lack of dystrophin is responsible for the symptoms of DMD; variable changes in the dystrophin protein molecule are observed in the milder allelic form of BMD. Voit et al. ( 1 9 9 1 ) studied the distribution of the dystrophin protein in 95 patients with DMD or BMD using immunofluorescent and Western blotting techniques. Dystrophin assessment revealed abnormal abundance or distribution in all 95 patients with DMD or BMD. Only trace amounts of dystrophin were detected in 29% of the DMD patients and complete absence of dystrophin was found in 71%. In two females with DMD but with normal karyotype, single dystrophin-positive fibers were found among more than 90% negative fibers. Of 26 patients with BMD, 73% had a dystrophin molecule of abnormal molecular weight (Voit et af., 1991). Thus, clearly all patients with DMD and BMD have a mutation in their dystrophin gene that

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greatly diminishes or eliminates expression of dystrophin. At least three distinct types of mutations account for the reduced expression, including deletions, in which segments of the gene are lost; duplications, in which segments of the gene are duplicated; and point mutations, in which single nucleotides are changed (Hoffman and Schwartz, 1991). Approximately 65% of DMD patients have deletion mutations, approximately 7% have duplications, and the remaining 30% are presumed to have point mutations. The size of a deletion may not necessarily determine the severity of the dystrophin reduction. For example, a relatively small deletion that results in a shift in the protein synthetic reading frame may lead to the placement of a termination codon early in the transcript. In this instance, a shortened or truncated polypeptide would be produced. Such individuals would exhibit the severe symptoms associated with DMD because of the absence of a functional protein product. Conversely, due to the enormous size of the dystrophin molecule, a relatively large deletion that has no new termination codons in the resulting reading frame would result in a shorter, but semi-functional, dystrophin polypeptide. These individuals might be expected to exhibit milder symptoms of dystrophy, such as those associated with BMD (Hoffman and Schwartz, 1991). The mutation need not even be within the exon or intron regions of the dystrophin structural gene, since a mutation within the promoter region of the dystrophin gene can lead to depressed production of dystrophin and, therefore, to the symptoms of BMD (Bushby et al., 1991). Deletions in DMD patients also may be distributed unevenly along the large DMD gene, resulting in “hot spots” for mutations in the central portion of the gene (Pizzuti et al., 1991). In this study, Pizzuti et al. (1991) reported that most of the hot spot corresponds to a single long intron (number 44) in which more than 30% of deletions have at least one boundary and the 5’ end of at least one deletion fell within a transposon-like sequence that also is present in normal individuals. Improper splicing of mRNA transcripts that results in exon skipping also may lead to certain types of DMD (Matsuo et al., 1991). In an initial effort to understand the mechanism that leads to aberrant formation of the dystrophin gene in humans, Hu et al. (1991) investigated three tandem duplications in DMD that had been shown in each case to have a subset of duplicated dystrophin genes. The origin of these duplications was traced to the single X chromosome of the maternal grandfathers, suggesting that an intrachromosomal event (i.e., unequal sister chromatid exchange) was involved in the formation of these duplications. Hu et al. (1991) cloned and analyzed the sequences of the duplication junctions and the normal DNA sequence that corresponded to both ends of the duplicated regions. The sizes of the duplications were 130 kb,

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approximately 300 kb, and 35-80 kb. One of these duplications was created by homologous recombination between two repetitive elements (Alu sequences); the other two were created by recombination between unrelated nonhomologous sequences. In the latter cases, the preferred cleavage sites of the eukaryotic type I and I1 DNA topoisomerases were found at the junctions of the duplications, suggesting a possible role of these enzymes in the chromatid exchange events (Hu et al., 1991). Additional comparisons of the dystrophin amino acid sequence to databases has revealed other information about its possible function. For example, homology alignments and domain recognition pattern analysis have identified highly significant correlations between dystrophin and other calcium regulating proteins (Gurusinghe et al., 1991). A major portion of the dystrophin sequence contains repeating units of approximately 100 amino acid residues; these repeating units exhibit significant homology to the muscle protein troponin I. Troponin I is known to interact with troponin C (the calcium regulatory protein in skeletal and cardiac muscles) and calmodulin. The regions of highest homology were characterized by patterns of high localization of charged amino acids and, thus, could represent possible calmodulin or troponin C surfaceaccessible binding sites. Since subcellular localization studies indicated that dystrophin is associated with the triadic junction, Gurusinghe et al. (1991) suggested that dystrophin could be involved in controlling intracellular calcium homeostasis. Although clearly a deficiency in dystrophin is the underlying biochemical defect in DMD (Hoffman et al., 1988,1989), no direct temporal correlation between dystrophin deficiency in muscle and the progressive and lethal clinical features of DMD has been determined (Hoffman and Schwartz, 1991). The dystrophin deficiency is present from fetal life onward and does not change with age; however, the clinical symptoms of the disorder are progressive with age. DMD seems to be initiated by dystrophin deficiency in muscle, but the progressive muscle wasting appears to result from other factors that compound the problems caused by dystrophin deficiency. Some of these factors include the gradual loss of regenerating capacity of muscle because of a diminishing pool of myogenic stem cells, to excessive proliferation of fibrotic tissue, or the involvement of other dystrophin deficient tissues such as vascular smooth muscle or neurons (Hoffman et al., 1988; Miyatake et al., 1990). Regardless of the detailed mechanism, much evidence seems to focus on a central role of an increased concentration of calcium ions in the cytoplasm (Knudson et al., 1988), possibly as a consequence of a leaky muscle cell membrane, as a key component in the progressive deterioration of skeletal muscle. Additional indirect evidence that a deficiency of dystro-

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phin does not itself directly cause the symptoms of DMD is apparent from studying dystrophin deficiency in other species. For example, dystrophin-deficient mice and cats (Bulfield et al., 1984; Hoffman et al., 1987b; Carpenter et al., 1989) exhibit a clinical disease that is markedly different from that of dystrophin-deficient humans. Dystrophin deficiency in mice and cats exhibits muscular hypertrophy rather than the muscle atrophy and weakness found in human dystrophy (Hoffman and Schwartz, 1991). In summary, rapid progress has been made toward identifying the defect in DMD. However, a great deal of new information is still needed to understand how the genetic defect at the DNA level leads to the observed physiological characteristics of the disease. At present, no therapies are available to slow the course of DMD or BMD. However, at least two “replacement therapies” have been attempted. These two strategies are based either on the possibility of transplanting genetically normal myoblasts into dystrophic individuals or transfering a normal gene for dystrophin into affected individuals, followed by expression of the transferred gene into a normal protein product. Love and co-workers (Acsadi et al., 1991; Dickson et al., 1991; Dunckley et al., 1992) demonstrated expression of a 12,000-bp, full-length human dystrophin cDNA gene and a 6300-bp BMD-like gene in cultured cells and in uiuo. When human dystrophin expression plasmids were injected intramuscularly into dystrophin-deficient mdx mice, the human dystrophin proteins were present in the cytoplasm and sarcolemma of approximately 1% of the muscle fibers (Acsadi et al., 1991). More importantly, muscle fibers expressing human dystrophin contained an increased proportion of peripheral nuclei, suggesting an improvement in one of the cellular symptoms of dystrophin deficiency. Thus, the primary limitation observed at this stage seems to be the low percentage of muscle fibers that will take up the plasmid; a higher percentage will be necessary to obtain improvements in the clinical symptoms of the disease. In a second innovative approach toward alleviating the symptoms of DMD, Huard et al. (1992) transplanted myoblasts from immunocompatible donors into the muscles of four DMD patients in the advanced stages of the disease. Although no immunosuppressive treatments were used, none of the patients exhibited any clinical signs of rejection. Muscle biopsies of one of the injected muscles in the four patients revealed that 80, 75, 25, and 0%, respectively, of the muscle fibers showed some degree of dystrophin immunostaining. A few months subsequent to muscle injection, one patient had a 143% increase in strength during wrist extension and two others exhibit a 41% and a 51% increase in strength (Huard et al., 1992). In summary, injection of myoblasts may show promise for localized improvements;

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however, obvious limitations for clinical improvements in DMD and BMD patients exist.

2 . Limb Girdle Muscular Dystrophy Limb girdle muscular dystrophy is inherited as an autosomal recessive disease, and primarily affects the muscles that are associated with the pelvic girdle and the scapulo-humeral region of the body. The proximal muscles of the limbs are affected more severely than the distal muscles; this phenomenon is the basis for naming the disease. Weakness can be concentrated in either the shoulder region or the pelvic region of the body, although the frequency of occurrence of the disorder is higher for the pelvic girdle region than for the upper body. In fact, both regions of the body usually are affected, but the severity of the dystrophy is concentrated in one region or the other. Limb girdle dystrophy, as an isolated disease, is sometimes difficult to distinguish from BMD because of similarities in the distribution of weakness. The age at which onset of limb girdle muscular dystrophy is first apparent is extremely variable; the earliest onset is in early childhood and the possibility of occurrence exists well into adult life. Although the course of the disease is usually slowly progressive, periods of rapid progression can be followed by periods of relative stability. In comparison to Duchenne dystrophy and BMD, the symptoms and progression are relatively mild; a nearly normal life-span may be expected in many instances. The biochemical and molecular biological analyses of limb girdle dystrophy are nearly nonexistent compared with DMD and BMD, but a small amount of information is available about the histochemical aspects of the disease. Among the most distinctive histological characteristics is the presence of fibers with very large diameters (Dubowitz and Brooke, 1973). The degree of fiber hypertrophy can be pronounced, with diameters of up to 250 p m (normal muscle fiber diameter is approximately 50 pm, depending on the fiber type). This information, coupled with accurate family histories, is useful in diagnosis. 3. Myotonic Dystrophy (Steinert’s Disease)

Myotonic dystrophy is manifested clinically primarily as muscle weakness or stiffness, as well as the inability of a muscle to relax following a strong contraction. This condition usually is exacerbated by cold weather. The age of onset of myotonic dystrophy is highly variable; perhaps the majority of cases become apparent during adult life. The disease

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also may be manifested in young children or infants; a large degree of variability exists in the severity of the disease within the same family. Once the disease is initiated, however, the primary manifestations include a slowly progressive weakness coupled with increasing levels of myotonia. The distal muscles of the limbs tend to be affected more severely than the hips or shoulders. The relentless progression of the disease leads to a rather complete handicap late in adult life. Several physical manifestations of myotonic dystrophy lead to a characteristic appearance of individuals who are affected. Some of these characteristics include frontal balding, weakness of the facial muscles, drooping of the eyelids, wasting of the muscles required for chewing, and wasting of the muscles associated with the temple on the face. Some of the other clinical problems associated with myotonic dystrophy are cataracts, atrophy of the gonads, abnormalities in the endocrine system, heart defects, and low plasma levels of IgG (Dubowitz and Brooke, 1973). Among the histological changes apparent in myotonic dystrophy are a disparity between the sizes of different fiber types: red fiber types are abnormally small and white fiber types are abnormally large (Dubowitz and Brooke, 1973). This disparity becomes less apparent in patients who have had myotonic dystrophy for longer periods of time or who are affected most severely by the disease. Additionally, the number of small angular fibers increases with progression of the disease; this characteristic is reminiscent of the appearance of muscle fibers following denervation. Another histological feature of muscle in myotonic dystrophy is a major increase in the number of muscle nuclei located in the interior of the muscle fiber rather than at the periphery (Dubowitz and Brooke, 1973). In muscle biopsies from patients with advanced stages of the disease, for example, nuclei appear almost randomly scattered through the interior of the muscle fiber rather than beneath the sarcolemma. Myotonic dystrophy is inherited as an autosomal dominant trait located on chromosome 19. The incidence of myotonic dystrophy is estimated to be approximately 13.5 per 100,000 births, but the actual incidence is presumably higher because of the variable expression of the gene for the disease and the variable age of onset. Unlike DMD, the frequency of new mutations is exceedingly low; virtually all cases of myotonic dystrophy have documented instances in family pedigrees. Through the use of clinical and genotypical analysis of large families in which the disease is present, a successful linkage analysis of the approximate location of the defective gene on chromosome 19 has been carried out. The apolipoprotein C-I1 locus has been found to be linked closely to the myotonic dystrophy locus (Meredith et al., 1985; Pericak-Vance et al., 1986). This information has been used to screen RFLPs on chromo-

DISEASES AND DISORDERS OF MUSCLE

36 I

some 19 to produce a probe that can be employed successfully for the prenatal diagnosis of myotonic dystrophy (Bartlett et al., 1987). Through the use of 14 polymorphic markers on the long arm of human chromosome 19, Harley e f al. (1992) localized the gene more precisely to a region between 19q13.2 and 19q13.3. This position corresponds to a region within approximately two million base pairs of the genes for apolipoprotein C-I1 and muscle creatine kinase. A human genomic clone that detects novel restriction fragments specific to individuals with myotonic dystrophy was isolated (Harley et al., 1992). A two-allele EcoRI polymorphism is observed in normal individuals, but in most affected individuals one of the normal alleles is replaced by a larger fragment that varies in length both between unrelated affected individuals and within families. This consistency has been confirmed further by the isolation of an expressed sequence from this region that detects a DNA fragment that is larger in affected individuals (Buxton er al., 1992). The unstable nature of this region may explain the characteristic variation in severity and age of onset of the disease; increases in size of this region through successive generations parallel the increasing severity of the disease (Buxton e? al., 1992; Harley et al., 1992). Finally, Aslanidis et a f . (1992) cloned this essential 19q13.3 region (including proximal and distal marker genes) and studied it through the use of subsequent subclones and restriction maps. Four probes were isolated that are all situated within approximately 10 kb of genomic DNA; these researchers detect an unstable genomic segment in myotonic dystrophy patients. The sequence tends to increase in size only in affected individuals and is situated adjacent to a transcribed region (Aslanidis et al., 1992). Although the actual gene involved in the defect has not been identified, the tools are available and likely will lead to its identification in a short period of time. 4 . Ocular Myopathies

The last group of the muscular dystrophies is associated initially with the function of the eye muscles. These diseases can be grouped generally into three different categories (Dubowitz and Brooke, 1973). The first category of ocular myopathies initially is manifested clinically by drooping of the eyelids, followed by atrophy of the other facial muscles and the muscles of the limbs. The oculopharyngeal dystrophies include the same symptoms, but difficulty swallowing is also a component of the disease. The third group of myopathies includes the opthalmoplegias. In this group, the wasting or weakness of the eye muscles is only a small component of a more complex disease process that also includes stunted

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growth, mental retardation, deafness, retinitis pigmentosa, motor neuron damage, and cardiac conduction defects (Dubowitz and Brooke, 1973). The time of onset of these disorders can vary from early childhood to early adolescence; frequently the disorders are inherited as autosomal dominant characteristics. As expected from their relatively rare occurrence, even less is known about these myopathies than about the major muscular dystrophies discussed earlier in this section. D. INFLAMMATORY MYOPATHIES Polymyositis and dermatomyositis are the most commonly encountered of the acute muscle diseases (Edwards and Jones, 1983). Both can be severe enough to produce myoglobinuria and may lead to urinary failure. Muscle loss can result in weakness and even immobilization. If the respiratory muscles become involved, life-threatening respiratory failure may occur. Both diseases are characterized by muscular weakness and, less frequently, by tender and wasted muscles. Early biopsies show marked variation in muscle fiber size, increased lysosomal enzyme activity, and infiltration by macrophages. A cross section of muscle from a patient suffering from polymyositis illustrates the marked variation in fiber sizes, infiltration with macrophages, and some nuclei located in the central portions of the fibers (Fig. 3). In advanced cases of the disease, the amount of destruction may be severe and resemble dystrophic muscle (Edwards and Jones, 1983). Polymyositis appears to be caused by a heterogeneous group of conditions, whereas dermatomyositis appears to be more homogeneous. Both diseases seem to be the result of autoimmune reactions against muscle, and are probably cell mediated (Rose and Walton, 1966; Pearson, 1976). Lymphocytes from humans and animals suffering from polymyositis are cytotoxic toward muscle cell cultures, according to Johnson et al. (1972). The factors responsible for initiating the autoimmune reaction have not been identified but probably include virus infections, drugs, and neoplasia (Edwards and Jones, 1983). Immunosuppressive drugs and corticosteroids are used for treatment. However, large doses of corticosteroids can cause muscle wasting, so the immunosuppressive effects of these chemicals must be balanced against this deleterious influence (Edwards ef al., 1979). To make a decision about the effects of steroids in treatment of the disease, quantitative information about changes in residual muscle strength because of the natural course of the disease and those originating from corticosteroid therapy is helpful (Edwards and Jones, 1983).

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363

FIG. 3. Photomicrograph showing cross-section of quadriceps muscle or patient with polymyositis. Note marked variation in the size of the fibers, the internal nuclei, and the infiltration with macrophages. Reprinted with permission from Edwards and Jones (1983).

E. ATROPHY AND HYPERTROPHY Immobilization and inactivity are well established to result in muscle atrophy, whereas increased activity that is associated with strength training results in muscle hypertrophy. These effects were discussed in considerable detail by Pearson (1990) in a review of the effects of exercise; resistive exercises were concluded to increase both muscle mass and strength . 1 . Atrophic Myopathies In muscle atrophy, typically a reduction in the cross-sectional area of the individual muscle fibers occurs. Although a loss of contractile material occurs in both oxidative (red) and glycolytic (white) fibers, the proportion of the loss is usually larger in the white fibers. However, the difference in the amount of atrophy in the fiber types is nonspecific and probably of little diagnostic significance (Edwards and Jones, 1983). On the other hand, atrophy of the glycolytic fibers commonly is observed in

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individuals suffering from hypothyroidism (McKeran et al., 1975; Young and Munro, 1980), osteomalacia (Dastur et al., 1975; Young et al., 1978), and a number of other conditions including chronic alcoholism, prolonged steroid therapy, rheumatoid arthritis, and muscle wasting occurring as a result of a carcinoma or malnutrition (Edwards and Jones, 1983).

Atrophied fibers are smaller and more angular in shape, but they exhibit no evidence of abnormal lysosomal or inflammatory responses. The greater amount of atrophy in white fibers is believed to be the result of inactivity. Similar cases of atrophy in white fibers also have been observed in the gluteus and quadriceps muscles of patients immobilized by total hip replacement surgery (Sirca and Susec-Michieli, 1980), but patients immobilized by knee injuries or lower limb fractures have been reported to have red fiber atrophy in the quadriceps muscle instead (Haggmark and Eriksson, 1979). On the other hand, Grimby et al. (1976) have reported that patients suffering from traumatic cervical cord transection have a complete absence of red (oxidative) fibers. Successful treatment of the causative disorder results in recovery of muscle strength and is paralleled by the growth and return to normal size of the previously red or white atrophied fibers (Young et al., 1978). Alteration of protein turnover rates may be involved in muscle atrophy but the mechanism is not understood. This disorder could involve either faster degradation or decreased rates of protein synthesis. Edwards and Jones (1983) have discussed the evidence for both mechanisms and the possible role of various hormones in protein turnover, but the evidence does not justify conclusions about which of the two mechanisms is involved in muscle atrophy and how the condition could be prevented. Although fast-contracting (white) fibers most often suffer from atrophy, red (slow-contracting)fibers are also susceptible, which further complicates diagnosis.

F. NEUROPATHIES The maintenance of an intact, healthy, and active nerve supply is essential to normal development and function in muscle. Dysfunction or damage to either the central motoneurons or the peripheral motor axons will cause pathological changes in muscle. Loss of an individual axon or motoneuron will result in the atrophy of all myofibers of the same type that depend on that particular motor unit for their nerve supply. Reinnervation of denervated fibers can occur by collateral sprouting of nearby motor axons and result in reestablishment of the nerve supply. Sprouting by nearby motor units generally will result in regeneration of the same

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365

fiber type, since most of the nearby fibers tend to be of the same type. However, reinnervation may be by a different type of motoneuron, which would result in a change in fiber type. Once a myofiber is renervated, it will recover from atrophy, although the fiber type may be changed if reinnervation occurs from an axon of a different fiber type. For more information on these changes, see a discussion by Pearson and Young (1989) on muscle fiber types relative to reinnervation. G. NORMAL AND ABNORMAL FATIGUE Fatigue is a normal occurrence that commonly is encountered following vigorous exercise. The phenomenon is a natural one. Mosso (1918) concluded that fatigue is central in origin, that is, the central nervous system tends to reduce its motor drive before any evidence of failure by the contractile apparatus of muscle is detectable. Merton (1954) demonstrated, by studies using the adductor pollicis muscle, that fatigue could occur as a result of failure in the muscle per se. Stephens and Taylor (1972) showed that fatigue may occur at the neuromuscular junction, failure resulted in a loss of force in both stimulated and voluntary contraction. One of the consequences of normal fatigue is the slowing of muscle relaxation, which has been shown in both humans (Edwards et al., 1972) and animals (Feng, 1931). Edwards et al. (1972) proposed that slowing of the relaxation rate may reflect the rate of crossbridge turnover. On the other hand, Dawson et al. (1980) did not find any change in ATP turnover rates on fatigue in frog muscle and suggested that a decreased rate of calcium reuptake by the sarcoplasmic reticulum (SR) is responsible. Regardless of the exact mechanism, researchers agree that the slowing of muscle relaxation on fatigue is associated with a reduced muscle phosphagen [ATP + creatine phosphate (CP)] level (Edwards and Jones, 1983). Evidence for this view is found in the fact that the recovery of the relaxation rate parallels resynthesis of CP. After heavy or ischemic exercise, the shape of the force-frequency curve changes, that is, the amount of force generated at lower frequencies of stimulation is depressed relative to the force at higher frequencies. This phenomenon is demonstrated in Fig. 4 by the tracings of force generated by fresh and fatigued quadriceps muscle stimulated at different frequencies. Full recovery may require several hours and has been observed to take as long as 24 hr (Edwards and Jones, 1983). This type of fatigue may be associated with damage to the cell membranes and require protein synthesis for their repair. Other difficulties in measuring fatigue are discussed by Edwards and Jones (1983).

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A. M. PEARSON AND RONALD B. YOUNG

A

B

FIG. 4. Tracings of force generated by percutaneous stimulation of quadriceps muscle at different frequencies. Stimulation used five impulses beginning at 1 Hz for 2 sec and successively at 10, 20, 50, and 100 Hz. (A) Fresh rested muscle. (B)Fatigued muscle. Same muscle used in A after 20-min step-test and 10-min rest. Note that maximum tetanic force is nearly the same in the rested and fatigued muscle, but the force generated at low frequencies is much reduced in the fatigued muscle. Adapted with permission from Edwards and Jones (1983).

1 . Myasthenia Gravis

The symptoms of myasthenia gravis are muscle weakness and the ease of becoming fatigued. The muscles also may be partially or completely paralyzed without any evidence of atrophy or sensory disturbance. The onset may be rapid or insidious and occur at any age, but is most common during the second decade of life. Its incidence is from 1 : 10,000 to 1 :50,000; twice as many females as males are affected. Although all muscles of the body can be involved, most commonly the disorder affects those of the face, lips, tongue, throat, and neck. The droopy eyelids seen in some individuals gives them a characteristic appearance. Individuals affected often have difficulty speaking and swallowing; if the respiratory muscles are affected, the disease may prove fatal. After resting, the muscles sometimes regain their normal strength, but weakness or paralysis quickly develops after brief activity. The disease can be diagnosed by its characteristic clinical electromyography (EMG) tracing, by a rapid and sometimes dramatic improvement in muscle strength following injection of a cholinesterase inhibitor (edrophonium chloride), or by an increased sensitivity to curare. Myasthenic muscle shows abnormal decrements of force and action potential amplitude during repetitive nerve stimulation (Desmedt and Borenstein, 1976). Normal muscle stimulated at 20 and 30 Hz will maintain normal force and action potential amplitude for at least 30 sec, but this is not true with myasthenic muscle. Desmedt (1973) described the responses of normal and myasthenic muscle to brief stimulation at 3 Hz for six consecutive impulses; the fifth action potential was compared to the first. No decre-

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367

ment is seen in normal muscle but a marked decrement is apparent in myasthenia. The beneficial effects of cholinesterase inhibitors show that the myasthenic muscle defect is at the neuromuscular junction. However, the question becomes whether the lesion occurs at a pre- or postsynaptic site. Dahlback et al. (1961) demonstrated that the abnormality is associated with a reduced quantity of acetylcholine being released at the presynaptic membrane. Sufficient similarities exist between myasthenia and partial blockage by curare to suggest that blood components similar to curare may be responsible for the myasthenic symptoms, a suggestion that is reinforced by the results of Simpson (1978), who described the existence of a neonatal form of myasthenia. Also, Ito et af. (1978) showed that myasthenic end plates have a reduced sensitivity to acetylcholine. However, evidence suggests the involvement of the postsynaptic receptor sites, and is supported by two facts: (1) the curare-like action of the antibodies blocks the binding to existing binding sites and (2) a reduction is seen in the number of binding sites. Conclusive evidence suggests that myasthenia gravis is a disease of the autoimmune system. The factors responsible for its development are elusive and problematic, as they are for other autoimmune diseases. However, virus infections and emotional stress are believed to be contributors. Myasthenia does not appear to have any genetic basis (Jacob et al., 1968), but may be associated with the human histocompatibility antigen HLA-8 (Simpson, 1978). This association would suggest that predisposing factors place some persons at a greater risk than others. Studies on myasthenia gravis demonstrated that this autoimmune disease results from the loss of T and B cell tolerance to acetylcholine receptors (Lindstrom et af., 1988; Steinman and Mantegazza, 1990). Therapy with antibodies to V regions of the acetylcholine receptor has been successful in treatment of paralytic disease in experimental allergic encephalomyelitis (Steinman and Mantegazza, 1990). Although antibodies to the V region of the acetylcholine receptor or vaccination against these receptors per se may prove to be effective in treating myasthenia gravis in humans, Steinman and Mantegazza (1990) indicated that more information is needed on ( 1) the acetylcholine receptor fragments that are immunogenic in myasthenia gravis, (2) major histocompatibility complex molecules that bind these fragments, and (3) T cell receptors that recognize the acetylcholine receptor-major histocompatibility complexes. Once such information is available, highly specific approaches for treatment of myasthenia gravis should be feasible in humans. Thymectomy may be helpful in treating myasthenia if used early in the course of the disease when the T cells are involved in antibody produc-

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tion, but is ineffective when production of antibodies moves to extrathymic sites (Edwards and Jones, 1983). The most effective long-term treatment involves use of anticholinesterase inhibitors to relieve the symptoms of the disease. Research is concentrating on long-lasting preparations that give relief and do not have any toxic side effects. Longrange goals are the development of procedures for controlling the underlying autoimmune response that causes the disease.

H. AGING AND EFFECTS OF EXERCISE Aging of humans is characterized by loss of the functional reserve capacity, which is the difference between basal and maximal function. Thus, the effects of aging are determined by maximally stressing the muscles and relating performance to maximal performance, often taken to be that of individuals in the 20-30 year age range. The changes in functional reserve capacity and its changes with aging in exercised and sedentary individuals was the subject of one symposium (Holloszy , 1987). The symposium examines the relationship between exercise, functional reserve capacity, and life-span in humans and other animals, that is, rats. Astrand et al. (1973) and Robinson et al. (1975) indicated a fairly uniform decline in maximum oxygen uptake capacity (VO,,,) with age. This measurement, which represents the capacity of the cardiovascular system to provide oxygen to the working muscles and their ability to use the oxygen on delivery, appears to decline at a fairly uniform rate with age for men: 0.40-0.50 ml/kg-'/min-'/yr-'. In women, the decline is slower, amounting to 0.20-0.35 ml/kg-'/min-'/yr-'. Buskirk and Hogdson (1987) concluded an overall indication that active individuals show a slower decline than sedentary individuals, but results are not uniform. These authors suggest that the discrepancies in VOzmaxover the entire age range may be caused by a curvilinear relationship; active individuals show a slow decline throughout life whereas sedentary persons may decline at a faster rate during their 20s and 30s and more slowly thereafter. Hagberg (1987) concluded that exercise may slow the rate of decline in VO2max with age to 5% per decade, in contrast to the 10% per decade found for sedentary individuals. Older persons (men and women over 60 years of age) appear to be able to minimize the reduction in VOzmaxthat occurs with age by maintaining high levels of physical activity. Gerstenblith et al. (1987) found that, in highly motivated but not athletically trained subjects, the only cardiovascular parameter to increase with age was systolic blood pressure. One reason for the age difference in cardio-

369

DISEASES AND DISORDERS OF MUSCLE

vascular response to exercise may be the diminished responsiveness to P-adrenergic stimulation. Ehsani (1987) pointed out that VOZmaxand left ventricular function decrease with age, whereas vigorous endurance training results in an increase in VOZmaxin the elderly. The failure of exercise to prevent deterioration of left ventricular function was suggested to reflect insufficient training stimulus rather than the inability of the heart to adapt to training in elderly subjects. Lakatta and Spurgeon (1987) presented evidence showing that the age-related decreases in myocardia function of the rat could be reversed by exercise. Finally, Holloszy and Smith (1987) suggested that exercise can improve survival in rats by countering the deleterious effects of a sedentary life combined with overeating. The relationship between voluntary exercise and survival in rats is shown in Fig. 5 . Thus, evidence suggests that vigorous exercise may be beneficial in maintaining and prolonging the functional reserve capacity of cardiac and perhaps even of skeletal and smooth muscle.

. \

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20 22 24 Age (months)

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FIG. 5. Relationship between voluntary running and survival in rats. Note that the figures shown at each time period are the number of survivors. The animals decreased their running with age. Reprinted with permission from Holloszy and Smith (1987).

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A. M. PEARSON AND RONALD B. YOUNG

I. MUSCULAR DYSGENESIS Although pregnant women and other animals are well aware of the movement of the fetus during development, most are not acquainted with the importance of fetal movement in muscular development, as is illustrated by muscular dysgenesis in the mouse. Evidence from studies on the dysgenic mouse has shown that fetal muscle does not develop normally without the stimulation of exercise (fetal movement). Muscular dysgenesis provides a model for studying both muscular development (Powell, 1990) and excitation-contraction coupling (Adams and Beam, 1990a,b). Muscular dysgenesis in the mouse is a lethal embryonic hereditary mutant that is expressed by disruption in skeletal muscle development, in contrast to the muscular dystrophies in which the onset of the condition occurs only in postnatal muscle (Pai, 1965a,b; Platzer, 1979). Gross abnormalities manifested in the newborns include a frozen fetal position, a small lower jaw in all animals, a cleft palate in most, and a severe reduction in muscle mass; individual muscle groups often are virtually indistinguishable from each other (Powell, 1990). The responsible gene is inherited as a homozygous recessive gene in which both parents contribute to the condition (mdg/mdg). Respiratory failure at birth, because of the lack of contraction of the intercostal muscles, is responsible for death (Adams and Beam, 1990; Powell, 1990). Although skeletal muscle does not develop normally in dysgenic animals, neither cardiac nor smooth muscle is affected (Pai, 1965b). The earliest signs of abnormal myogenesis, which are apparent in the intercostal muscles, occur at the 13th day of gestation (Platzer and Gluecksohn-Waelsch, 1972). Although many of the early myotubes develop into primitive myofibrils with apparently normal sarcomeric organization, the fibrillar organization deteriorates as the SR becomes swollen and the Z bands become irregular, indistinct, or wider than normal. Other abnormalities of dysgenic muscle cells include centrally located nuclei, nuclear inclusions, retraction clots, and areas of coagulated myofilaments, which are indicative of attempts to contract. Addition of spinal cord cells to dysgenic muscle in cell culture results in restoration (rescue) of contraction (Koenig et al., 1982; Rieger et al., 1987) and argues for involvement of the nervous system in muscular dysgenesis. However, addition of fibroblasts to the dysgenic muscle cells in culture also restores contraction (Courbin et al., 1989) and suggests that other normal cell types are capable of rescue, providing evidence against neuronal involvement. Other research has demonstrated that the mutant (mdg/mdg) muscle is

DISEASES AND DISORDERS OF MUSCLE

37 1

not capable of excitation-contraction (E-C) coupling that is characteristic of normal muscle (Powell and Fambrough, 1973). The failure of E-C coupling appears to be due to the absence of the a1 subunit for the skeletal muscle receptor of the dihydropyridine (DHP) calcium channel modifiers (Leung et al., 1987; Tanabe et al., 1988; Knudson et al., 1989). The DHP receptor is theorized to function in E-C coupling of normal skeletal muscle by serving as the voltage sensor that triggers calcium release from the SR and thus initiates contraction, as outlined by Adams and Beam (1990b). The a1subunit of the DHP receptor also appears to function as the ion channel that is responsible for slowly activating the DHPsensitive calcium current in normal muscle, but in dysgenic muscle this receptor is incapable of acting as a voltage sensor so the muscle fails to contract (Klaus et al., 1983). Muscular dysgenesis can be used as a model for determining the role of various factors on muscle development (Powell, 1990). Similarly, dysgenic muscle already has been and can continue to be used to understand further how E-C coupling takes place in normal and abnormal muscle (Chaudhari and Beam, 1989; Adams and Beam, 1990a). Although nearly all the research on muscular dysgenesis to date has been carried out with the dysgenic mouse, Kieny et al. (1988) have described muscular dysgenesis in an apparently lethal mutant for the chick-the crooked neck dwarf. These two mutants may serve as models for studying muscle development and answer the following questions posed by Powell (1990): (1) What is the role of membrane activity and contraction per se in development of muscle? (2) What is the role of muscle inactivity (lack of contraction) on basal lamina production, tendinous structure, reduction of polyneuronal innervation, and neuronal cell death? (3) What is the role of the slow calcium current or the presence of the diphosphopyridine receptor in morphogenesis of the t tubule and triad system and on the sarcofibrillar organization of muscle? and (4) What effect do nuclear domains for the DHP receptor have on myotube membranes? Similarly, Adams and Beam (1990a) raised several intriguing questions concerning the role of E-C coupling and the DHP receptors in normal and dysgenic muscle that, when answered, will help clarify the role of the DHP receptors of E-C coupling in normal muscle.

J. MYOCLONUS Myoclonus is an imprecise clinical term used to describe a brief shocklike jerky muscle movement of involuntary origin and is a symptom of impaired muscle function that may be associated with neurological dysfunction. This disorder differs from clonus, which typically occurs in

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decerebrated experimental animals or in humans with upper spinal cord or brain stem lesions, only because the latter condition is manifested by sustained rhythmical muscle contraction whereas contraction is not sustained in myoclonus (Stein and Lee, 1988). Both disorders, however, imply synchronized firing of motor neurons, are sensitive to peripheral sensory input, and may occur in seemingly normal humans and animals, commonly during sleep. Spontaneous myoclonus has been observed only in Polled Hereford cattle, the photosensitive baboon, and the spastic mouse (Snodgrass, 1990). Myoclonus has been reported to occur as a result of anoxic brain injury of humans. A limited number of patients have been shown to respond to treatment with the serotonin precursor 5-hydroxytryptophan (Snodgrass, 1990). Although benzodiazepines are often helpful in treatment of myoclonus, their beneficial effects decline with chronic administration, according to Snodgrass (1990). I . Epilepsy Progressive myoclonus epilepsy refers to a group of diseases characterized by a jerky movement generated by involuntary muscular contractions. Generalized epileptic seizures are manifested in their early stages by jerking of the head and neck and are followed by excessive salivation, loss of consciousness, and major disturbances in blood pressure and respiration. Snodgrass (1990) reviewed the treatment of epilepsy and other form of myoclonus with y-aminobutyric acid (GABA) agonists and some GABA uptake blockers which, when properly administered, may have anticonvulsant effects. Although stimulation of GABAA(y-aminobutyric acid type A) receptors may either stimulate or prevent myoclonus, the result may depend on which GABA receptors are involved. The inheritance of the different types of epilepsy has not been elucidated fully, although the GABA receptors may be involved in some forms of this condition. Epilepsy in humans is well known to occur in certain families and, hence, may have a genetic basis. Additional studies will be required to resolve the genetic basis of epilepsy in humans.

2. The Spastic Mouse The spastic mouse is a mutant in which a motor disorder is inherited as an autosomal recessive trait. The spastic condition is manifested by tremors, myoclonic episodes, and a disturbance in the righting response. Becker (1990) demonstrated that the mutant has a substantial deficit in

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the postsynaptic glycine receptor channels, which results in a dramatic reduction of glycinergic synaptic inhibition. The glycine receptor protein appears to be unaffected, suggesting that the spastic mutation is caused by a regulatory rather than a structural defect. Studies summarized by Becker (1990) suggest that GABAA receptormediated inhibition alleviates symptoms of the spastic condition. The cloning of the cDNA for the glycine receptor subunits may help elucidate the molecular basis by which the spastic gene causes the glycine receptor deficit. Although the symptoms in the spastic mouse are similar to those observed in startle disease of humans (Heller and Hallett, 1982), the relationship of the spastic mouse to this disease in humans needs to be investigated from the perspective of receptor dysfunction.

3. lnherited Myoclonus in Hereford Cattle Inherited congenital myoclonus occurs in Polled Hereford calves and is characterized by hyperesthesia and myoclonic jerks of the skeletal musculature that develop both spontaneously and in response to sensory stimuli. The condition is inherited as an autosomal recessive trait (Healy et al., 19851, and shows no pathological lesions in the central nervous system on examination with either the light or electron microscope (Harper et al., 1986). Symptoms of the disorder suggest a failure of spinal inhibition and, according to Gundlach (1990), show a marked similarity to those of subconvulsive strychnine poisoning (strychnine is a high affinity antagonist of the action of glycine at the synapse). Biochemical studies summarized by Gundlach (1990) showed a marked deficit in the [3H]strychnine binding sites in brain stem and spinal cord membranes from myoclonic calves compared with normal calves, reflecting a decrease in the number of inhibitory glycine receptors in the calves suffering from myoclonus. Thus, the major clinical signs of the myoclonic disorder can be explained by the deficiency of inhibitory glycine receptors in the brain stem and spinal cord of the affected animals. The characteristics of this disease are similar to those of the spastic mouse. The myoclonic calves may provide a useful model for studying the plasticity of various components in other neurotransmitter systems in the absence of the spinal inhibitory system, and to study further the regulation of expression of the neurotransmitter receptor protein, which is only one of a large family of ion-channel receptors. Gundlach (1990) suggested that such studies could lead to a DNA-based diagnostic test to identify the heterozygote, which could help prevent this defect in the susceptible cattle population.

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111.

DISORDERS OF ENERGY METABOLISM

Several defects in energy metabolism are of genetic origin, including defects in the glycolytic pathway, defects in the mitochondrial enzymes involved in both pyruvate and fatty acid metabolism, and defects in the cytochrome components of the electron transport system. All these pathways are involved with the secondary supply of energy following depletion of the primary reserve supply of CP. To date, no known defects in the actomyosin ATPase or the muscle creatine kinase reactions exist. Patients with metabolic energy defects are generally of normal or near normal muscle strength but have limited endurance. If the defect occurs in the glycolytic pathway, contraction of exercised muscle is often painful. In contrast, patients with mitochondrial disorders have a limited exercise capacity that is associated with high blood lactate concentrations, as though their muscles were ischemic.

A. GLYCOLYTIC DISORDERS Diseases of carbohydrate metabolism, which have been classified into different types, are summarized in Table I. These glycogen storage diseases cause muscle weaknesses, but the cause is not clear. However, the weakness may be caused by mechanical damage to the contractile mechanism resulting from accumulation of abnormal and less soluble forms of glycogen. 1.

Von Gierke’s Disease (Type 1 Glycogenesis)

Von Gierke’s disease is caused by a deficiency of the enzyme glucose6-phosphatase that catalyzes conversion of ~-glucose-6-phosphateto Dglucose plus phosphate. This reaction is very important in the liver since it delivers free glucose to the blood stream. The disease affects liver, kidney, and skeletal muscle, with clinical symptoms of hepatomegaly, growth retardation, hypoglycemia, ketosis, hyperlipemia, and lactate acidosis (Table I). Von Gierke’s disease also is known as glycogen storage disease and consists of at least four different manifestations of the condition, called glycogen storage disease type la, type lb, type lc, and type Id, depending on the exact cause of the disorder (Burchell, 1990). Glycogen-6phosphatase (Gd-Pase) has the unique feature of being localized so its active site is located inside the lumen of the endoplasmic reticulum (ER), whereas all other enzymes known to be involved in gluconeogenesis and glycogenolysis are found elsewhere in the cell. Genetic evidence demon-

TABLE I DISEASES OF GLYCOLYSIS, ENZYMATIC DEFICIENCIES, TISSUES AFFECTED, A N D CLINICAL SYMPTOMS~

Disease

Enzyme deficiency

Tissues affected

Clinical features

Von Gierke’s disease (Type I)

Glucose-Gphosphatase

Liver, kidney, skeletal muscle

Hepatomegaly, growth retardation, hypoglycemia ketosis, hyperlipemia, lactic acidosis

Pompe’s disease (Type 2) Infancy

Acid (lysosomal)-l,4- and - 1 6 glucosidase

All tissues

Cardiomegaly, extreme weakness, death within 1st year of life Proximal myopathy of variable severity, respiratory failure Hepatosplenomegaly, fasting hypoglycemia, weakness

Skeletal muscle and liver

Adults Con-Forbes disease (Type 3)

Andersen’s disease (Type 4) (amylopectinosis)

McArdle’s disease (Type 5 ) (phosphorylase deficiency) Hers’ disease (Type 6) Tarui’s disease (Type 7) (phosphofructokinase deficiency) a

Debrancher system results in abnormal glycogenphosphorylase-limit dextrin Branching system results in abnormal glycogen-longer peripheral chains, fewer branching points Glycogen phosphorylase

Liver, muscle. red blood cells, fibroblasts

Glycogen phosphorylase

Liver, white blood cells; skeletal muscle normal Skeletal muscle, red but not white blood cells

Phosphofructokinase

Adapted with permission from Edwards and Jones (1983).

Liver, kidney,white blood cells, muscle, central nervous system, spleen

Hepatosplenomegaly, cirrhosis of liver

Skeletal muscle

Cramps after exertion, myoglobinuria Hepatomegaly, hypoglycemia, lactic acidosis Cramps after exertion, my oglobinuria

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strates that G-6-Pase is composed of at least five different protein components; the active site of the catalytic subunit of G-6-Pase is associated with a regulatory Ca2+stabilizing protein (SP). In addition, at least three transport proteins, T I , T2, and T3, actively transport glucosedphosphate, phosphate (and pyrophosphate), and glucose, respectively, across the ER membrane, as explained by Burchell(l990). Type la glycogen storage disease was first described by Cori and Cori (19521, who showed that the condition was caused by the absence of G-6-Pase in its classical form. The only reliable method for currently diagnosing type la disease is assaying for Gd-Pase in liver samples (Burchell, 1990), which makes it possible to detect the condition in early childhood and clinically maintain normoglycemia and reduce other metabolic abnormalities so that affected children can survive longer. The surviving children, however, often develop adenomas and, subsequently, hepatomas (Huijing, 1975; Howell and Williams, 1982), whereas others may develop renal problems and succumb to renal failure. Type lb glycogen storage disease has symptoms similar to those of classical type la glycogen storage disease. In patients with type lb glycogen storage disease, the activity of Gd-Pase is completely normal in fully disrupted microsomes but is abnormal in intact microsomes and in uiuo (Huijing, 1975; Howell and Williams, 1982). Type l b glycogen storage disease has been demonstrated to be associated with a deficiency in the TI transport protein which, in normal tissue, transports glucosedphosphate across the ER membrane (Nordlie and Sukalski, 1986). Care must be taken in diagnosis of type lb glycogen storage disease, since defects in T2 and T3 also will lead to abnormal G-6-Pase activity in both intact microsomes and in uiuo. Type lc glycogen storage disease is caused by a deficiency of T2, which is the microsomal phosphate/pyrophosphate transport protein. Some patients with this condition have impaired insulin release in response to glucose whereas others do not (Nordlie e f al., 1983; Burchell et al., 1987). The different clinical features of type lc glycogen storage disease can be explained by the fact that some patients have a defect in T2 not only in liver and kidney (Burchell and Waddell, 1990) but also in the pancreatic islets (Waddell and Burchell, 1988), whereas others have the defect only in liver and kidney. Type Id glycogen storage disease is not tested for by most laboratories since no direct enzymatic assay is available for T3 transport capacity. Although the deficiency appears to be caused by decreased T3 transport capacity, identifying the disorder is difficult without a satisfactory assay for the T3 protein. The sudden infant death syndrome or crib death may be associated with abnormally high hepatic glycogen levels and be related to G-6-Pase

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deficiency (Burchell, 1990). Emery et al. (1988) proposed that many genetic deficiencies that have the potential to cause severe hypoglycemia may be involved in sudden and unexpected death, yet any clear relationship between glycogen storage disease and the sudden infant death syndrome has not been established to date (Burchell 1990). 2. Pompe’s Disease (Type 2 )

Pompe’s disease is caused by a deficiency of the lysosomal enzymes a-l,4-glucosidase and a-l,6-glucosidase, and is characterized by a large accumulation of glycogen in skeletal muscle. The symptoms of the disease are cardiomegaly and extreme muscle weakness. Normally, the condition is present at birth and results in severe muscle hypotonia. Death usually occurs from respiratory failure within the first year of life. Similar symptoms have been observed in adults (Angelini and Engel, 1973; DiMauro et al., 1987a,b). 3. Cori-Forbes Disease (Type 3) Failure to have adequate amounts of debranching enzyme leads to accumulation of large amounts of structurally abnormal glycogen, which characterizes Cori-Forbes disease. In the absence of debranching enzyme, myophosphorylase removes glycosyl units attached by 1,4 linkages until it reaches a branch point that cannot be cleaved. This reaction leaves a limit dextrin, which can be identified by the red color produced by reaction with iodine. Cori-Forbes disease affects muscle, liver, red blood cells, and fibroblasts. Clinical symptoms of the disease include hepatosplenomegaly , muscle weakness, and fasting hypoglycemia. 4 . Andersen’s Disease (Type 4 )

Andersen’s disease of glycolysis is caused by a deficiency in branching enzyme, which results in storage of large amounts of abnormal glycogen with longer peripheral chains and fewer branch points. The affected tissues include muscle, liver, and some other tissues (Table I). The primary clinical symptoms include hepatosplenomegaly and cirrhosis of the liver. If untreated, the condition usually results in death. 5 . McArdle’s Disease (Type 5 )

McArdle’s disease was the first to be recognized as a myopathy caused by a defect in glycolysis (McArdle, 1951). The disorder is caused

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by a deficiency in the adult form of myophosphorylase. Patients suffering from McArdle’s disease have normal strength on resting but exercise causes muscle pain. Continued exercise at high rates leads to very painful contractures that can block local circulation and result in severe ischemic damage, with myoglobinuria and ultimate kidney damage. Exercise at low levels may result in diminishing pain so exercise ultimately may cause no discomfort. However, high rates of exercise result in anaerobic conditions, so the absence of myophosphorylase limits the ability to perform. Only about half the ATP flux is available. The impaired ATP supply alters the excitability of the muscle membranes, so the contracture developing during fatigue is electrically silent. This condition may be analogous to contractures in rigor mortis. Although McArdle’s disease is caused by a genetically based enzyme defect, phosphorylase activity can be demonstrated in cultured cells from patients suffering from the disease (Roelofs et al., 1972). The defect in myophosphorylase activity has been suggested to be due to the presence of a fetal form of myokinase that normally is lost during childhood (DiMauro et al., 1978b). 6 . Hers’ Disease (Type 6 )

Glycogen phosphorylase is also the enzyme that is deficient in Hers’ disease, but the tissues affected and the symptoms are different (Table I). In contrast to McArdle’s disease, in which only skeletal muscle is affected, Hers’ disease manifests skeletal muscle that is normal, whereas the liver and white blood cells become target tissues. Symptoms include hepatomegaly, hypoglycemia, and lactic acidosis, none of which are clinical signs of McArdle’s disease (Table I).

7. Tauri’s Disease (Type 7) Phosphofructokinase (PFK) is deficient in Tauri’s disease. Patients with the disease suffer from painful contracture on heavy exercise and recover during light exercise; in this way, the condition is similar to McArdle’s disease. The improvement during light exercise is not due to utilization of blood glucose, as occurs in McArdle’s disease, since the deficiency in PFK precludes the utilization of glycogen and pentose sugars. Therefore, the patients are dependent on free fatty acids in the blood as a source of energy. Ketogenic diets may be beneficial in treatment of the disease (DiMauro and Eastwood, 1977). Tauri’s disease affects muscle in a way similar to McArdle’s disease, resulting in cramps after exercising and, in severe cases, causing muscle

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damage and myoglobinuria with kidney damage. The disorder also affects the red blood cells but does not affect the white blood cells (Edwards and Jones, 1983). 8. Glycolysis and Meat Quality

The rate of glycolysis is well established to be involved in pale, soft, and exudative (PSE) pork and the related conditions of dark, firm, and dry (DFD) pork and the porcine stress syndrome (PSS) in pigs (Pearson and Young, 1989). The various glycogen diseases may be involved in the etiology of the PSE, DFD, and PSS conditions. Studying the expression and symptoms of the different glycolytic disorders in humans may provide leads about the basic causes of these three problems in the quality of pig muscle as meat.

B. ACQUIRED DISEASES OF GLYCOLYSIS Edwards and Jones (1983) reviewed other diseases of glycolysis that affect muscle metabolism. These disorders are classified as acquired and include alcoholism, which results in a striking reduction in glycolysis. Impaired lactate metabolism in ischemic forearm exercise also has been observed in the alcoholic myopathy. Histochemical studies of muscle from patients suffering from hypothyroidism have shown them to have a reduction in phosphorylase activity, which indicates impairment of glycolysis. This condition may, in part, be a reflection of the predominance of red (oxidative)fibers and their lower content of phosphorylase. Also, a reduction in the contribution of anaerobic glycolysis to overall energy exchange during ischemic contraction occurs in hypothyroidism that is reversed after several months of thyroid treatment. A deficiency in the enzyme acid maltase occurs in hypothyroidism as well; treatment of the deficiency results in an increase toward normal (Hurwitz et al., 1970). C. DISORDERS OF MITOCHONDRIAL OXIDATION Several disorders involving mitochondrial oxidative metabolism are reviewed by Edwards and Jones (1983). A number of different cases were described. The first involved a young woman with a hypermetabolic condition but normal thyroid function (Luft et al., 1962). The principal abnormality on biopsying her muscle was an abnormal mitochondria1 structure and some evidence of partially uncoupled mitochondrial metabolism. Only one other case of this condition has been confirmed

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(Schotland et al., 1976), indicating the disorder is rare. Although abnormalities of mitochondrial structure are not uncommon, no regularly appearing relationship between structure and abnormal function is known. Although the red fibers stained with trichrome often have a characteristic ragged appearance, this condition appears to be due to subsarcolemmal aggregation of mitochondria around the periphery of the fibers and is associated with muscle weakness in childhood. Most often, this condition occurs in specific muscular dystrophies (Dubowitz and Brooke, 1973).

Morgan-Hughes et al. (1977) identified a patient suffering from muscle cytochrome b deficiency. The principal clinical symptoms were exercise intolerance and persistent lactic acidosis. Other cases of cytochrome and mitochondrial enzyme deficiencies were described by Land and Clark (1979), who pointed out the difficulties in assessing normal and abnormal function because of complicating factors such as the influence of activity, bed rest, and training. Despite these difficulties, Willems et al. (1977) were able to identify a patient with a marked decrease in cytochrome c activity, whereas Gohil et al. (1960) found a young man with exercise intolerance who had the same mitochondrial enzyme defect. These authors also described a more severe and fatal case of cytochrome c deficiency in a baby. Pyruvate dehydrogenase (PDH) deficiency in brain and fibroblasts has been studied extensively by Blass (1980). Patients exhibit ataxia and tend to exhibit lactic acidemia, which can be reduced to some extent by placing them on a high-fat diet. At present, whether PDH deficiency is manifested in muscle and other tissues is not known. Defects in mitochondrial function have been produced experimentally in animals using uncoupling agents and chloramphenicol, which inhibit mitochondrial protein synthesis (Patterson and Klenerman, 1979). I . Mitochondria and Meat Quality

Buege and Marsh (1975) first proposed that release of Ca2+ions by the mitochondria of prerigor muscle under the influence of cold was responsible for the phenomenon of cold shortening and cold-induced toughness in meat. Later Cornforth et al. (1980) verified the fact that holding prerigor muscle at cold temperatures (OOC) resulted in the release of Ca" by the mitochondria and also demonstrated that the SR was involved in cold shortening through its inability to bind the excess Ca2+ released by the mitochondria. The disorders of mitochondrial metabolism in humans may provide a model for studying the effects of the mitochondrial enzymes in the release of Ca2' ions by both the mitochondria and SR, and

DISEASES AND DISORDERS OF MUSCLE

38 1

their possible relationship to cold shortening or toughening in meat science. D. DISORDERS OF LIPID METABOLISM Involvement of disorders in lipid metabolism was first recognized by Bradley et a f . (1969,1972) and by Engel and Siekert (1972) on observing excessive lipid accumulation in muscle biopsies. The symptoms of lipid metabolism disorders are muscle weakness, exercise intolerance, and muscle stiffness and pain, and sometimes are accompanied by myoglobinuria. At times when free fatty acids are being used as the main substrates, for example, during prolonged submaximal exercise or fasting, the symptoms may become more severe. Although fat accumulation around the muscle fibers also occurs in Duchenne muscular dystrophy, disorders of fat metabolism result in the uniform distribution of fat droplets within the fibers. The two major lipid metabolism disorders in muscle are carnitine deficiency (Engel and Angelini, 1973) and carnitine palmitoyl-transferase (CPT) deficiency (DiMauro and DiMauro, 1973). Carnitine and carnitine palmitoyl-transferase function in the transfer of free fatty acids into the mitochondria, where they undergo /3-oxidation. Two transferases (CPTI and CPTII) are believed to be located on the inner mitochondria1 membrane. 1 . Carnitine Dejiciency

Synthesis of carnitine occurs in the liver. Then the molecule is transported by the blood to other tissues, including muscle, where active uptake takes place (Willner et al., 1978). The disorders of fat metabolism associated with carnitine deficiency may be caused by (1) failure of the liver to synthesize carnitine or (2) failure of carnitine uptake by the muscles. Both conditions can take place in humans, and have similar clinical symptoms. However, the site of the disease can be ascertained by measuring blood serum and muscle carnitine concentrations. Normal serum carnitine levels and low muscle concentrations indicate the disorder is in muscle carnitine uptake (Angelini et al., 1976), whereas low serum and low muscle concentrations suggest the defect is in synthesis. Carnitine deficiency is treated by giving oral carnitine. Such treatment has improved the exercise tolerance in patients with hepatic involvement. Dietary carnitine therapy also has resulted in some clinical improvement in patients with the defect in muscle uptake (Angelini et a f . , 1976), although muscle concentrations did not increase measurably.

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Several acquired partial carnitine deficiencies exist, including low muscle levels in patients receiving regular dialysis for renal failure and in malnutrition-associated liver disease. The latter condition is more likely to develop if the diet is low in lysine or methionine. Sepsis also has been reported to cause carnitine deficiency. Fatty degeneration of the myocardium precipitated by diptheria toxin also may involve a localized deficiency in carnitine. Carrier and Berthillier (1980) followed urinary excretion of carnitine during growth and development of human subjects and found that the level was low at birth, increased during the first few weeks of life, dropped again during the next 3 years, reached adult levels by 10 years of age, and remained stable throughout life.

2. Carnitine Palmitoyl-Transferase Dejkiency CPT deficiency has been identified in 22 patients only 2 of which were female (Edwards and Jones, 1983), suggesting the disease is more prevalent in males. The dominant symptoms of CPT deficiency are recurrent pain and myoglobinuria, which may be caused by prolonged exercising or fasting. Muscle weakness is not commonly encountered except when it is accompanied by pain and myoglobinuria. Muscle carnitine concentrations may be raised by CPT deficiency. Although lipid accumulates, this phenomenon is less marked than in carnitine deficiency. The symptoms of the disease generally are preceded by a marked rise in CK activity, which is indicative of muscle damage. The requirement for carnitine and CPT in the translocation of longchain fatty acids into the mitochondria may be alleviated by treating patients with medium-chain triglycerides. A high carbohydrate-low fat diet often adequately causes significant improvement and reduces the pain and associated myoglobinuria. A broad distinction can be made between patients lacking CPT and those suffering from carnitine deficiency. The latter exhibit muscle weakness, whereas the CPT deficient group have normal muscle strength after resting. However, fasting or exercise tends to cause muscle destruction, which is accompanied by high blood serum levels of CK and myoglobinuria. 3 . Relationship of Carnitine to Meat Quality

Although carnitine plays a key role in fat metabolism, the molecule is not required in the diet under normal conditions. However, carnitine deficiency can occur as a result of failure of the liver to synthesize carnitine or the inability of the muscles to take up carnitine. In both cases,

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carnitine is given by mouth and has been shown to be effective even in persons having a defect in muscle carnitine uptake. Thus, addition of carnitine to the diet can be useful in cases of pathological carnitine deficiency. Although meat is an excellent source of dietary carnitine, larger amounts of carnitine normally are needed only in cases of pathological deficiency (Willner, 1978). Since carnitine is involved in lipid metabolism, carnitine deficiency may serve as a useful model for studying the role of different fatty acids in meat quality, for example, in animal models with induced carnitine deficiency, in which one actually could study different fatty acids and their effects.

E. MYOTONIC SYNDROMES Numerous myotonic conditions in muscle are associated with slow or delayed relaxation following contraction. Hypothyroidism also results in slow relaxation, as occurs in fatigued muscle, but myotonia differs because the failure to relax is accompanied by continued electrical activity.

I . Myotonia Congenita (Thomsen’s Disease) Thomsen’s disease was discussed by Edward and Jones (1983). Unlike most other muscle diseases in which muscle is replaced with fat, myotonia congenita results in true muscle hypertrophy. Myotonic stiffness frequently affects the limb muscles and may be aggravated by cold. Diagnosis of myotonic contractions can be accomplished by direct percussion of the muscle; the thenar muscles of the thumb or the tongue muscles frequently are used to demonstrate the disorder. No evidence exists of persistent muscle weakness, although tetanic contractions may have a tendency to result in undue fatigue. A condition similar to myotonia congenita was described by Bryant (1979) in a certain strain of goats. Brown and Harvey (1939) showed that slow relaxation of the muscles in the myotonic goat is associated with continuing electrical activity. These researchers also demonstrated that myotonia produced by percussion was still evident after nerve transection and curarization. These facts suggest that the disorder occurs in the muscle rather than in the central nervous system. Although now myotonia is thought to originate in the muscle per se, McComas (1977) implied that the myotonic discharges may involve the motoneuron. Since most of the latter observations were with dystrophic mice, the exact role of the central nervous system is still not clear. In the case of the myotonic goats, the myotonic fibers have a higher

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than normal membrane resistance. Replacement of the external chloride with an impermeable anion in normal goat muscle raises the membrane resistance and induces myotonic behavior. Thus, the primary factor in development of myotonia in the goat appears to be an increase in membrane resistance as a consequence of chloride anion. Treatment of myotonia congenita is generally with substances that reduce muscle excitability. Quinine, phenytoin, and procainamide all have been used, but phenytoin generally is preferred.

2 . Paramyotonia Congenita Paramyotonia congenita has symptoms similar to those of myotonia congenita. Unlike those of myotonia congenita, however, the myotonic discharges become worse with repeated contractions. Symptoms frequently occur only at cold temperatures. Studies have not been made on the muscle membrane properties, but the condition is probably a variant of hypokalemic periodic paralysis, which is discussed later in this chapter. If so, the defect probably is not related to the chloride anion, as is the case for myotonia congenita, but likely is caused by the effects of Na' and K+ ions on membrane conductance. Paramyotonia congenita is linked to an autosomal dominant gene. In contrast, a recessive variant in addition to an autosomal dominant condition may exist in myotonia congenita (Edwards and Jones, 1983). 3. Periodic Paralysis The two main types of familial periodic paralysis are (1) hypokalemic and (2) hyperkalemic. Although their symptoms are similar, consisting of periodic attacks of muscle weakness, the conditions have entirely different causes. a. Hypokalemic Periodic Paralysis. Hypokalemic periodic paralysis consists of periodic attacks of muscle weakness that generally first occur during the second decade of life. This condition was described by Edwards and Jones (1983), and is precipitated by eating high carbohydrate meals after strenuous exercise; attacks often occur at night. The attack can last up to 24 hr. In the case of severe onset, most of the skeletal muscles may be involved in flaccid paralysis which, in some cases, may impair speech and cause coughing. Insulin will provoke attacks. Regardless of the cause, the attacks can be alleviated by administration of 10-15 g potassium chloride or citrate orally. Attacks also may be reduced by mild exercising of the affected muscles. Muscle strength is fairly normal between attacks, but repeated attacks can lead to vacuoles

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in the muscles that tend to persist, with loss of myofibrillar material and increasing muscle weakness. The weakness accompanying attacks of hypokalemic periodic paralysis characteristically is accompanied or preceded by a fall in serum K+ levels, which may be as low as 1.5 mM. The degree of weakness is related to the low content of K+ in the serum, but often persists after serum levels return to normal. Thus, K+ levels in the muscle actually may be increased during attacks. Studies with rats maintained on a low-K+ diet have been used as a model. These studies indicate that the surface membranes are depolarized to the extent that Naf-conductance channels become depolarized and the muscle becomes refractory. Treatment of hypokalemic periodic paralysis can be achieved by potassium supplementation and restriction of Na+ intake and by avoidance of insulin, carbohydrates, and heavy exercise. Mild acidosis may be useful; the increased H+ ion concentration helps block the sodium channels. Edwards and Jones (1983) suggested that acetazolamide may prove valuable in treatment because it is a carbonic anhydrase inhibitor and K+ diuretic. b. Hyperkalemic Periodic Paralysis. Hyperkalemic periodic paralysis also was described by Edwards and Jones (1983). The onset of this condition usually occurs in the first decade of life, and is provoked by K+ and relieved by glucose and insulin, in contrast to hypokalemic periodic paralysis. During attacks, serum K+ usually rises and K+ moves out of the muscle. Muscle Na+, on the other hand, increases as muscle K+ decreases. Vacuolization of the muscle fibers occurs that is similar to that seen in hypokalemia. Although the increase in serum K + and the decrease in intracellular K+ may reduce the resting membrane potential, this is unlikely to be the complete explanation of depolarization. The weakness also often occurs before serum K+ begins to increase. Thus, hyperkalemia may be a consequence and not the cause of muscle depolarization. Although the cause of depolarization is not known, Creutzfeldt e f al. (1963) suggested that an increase in the intracellular level of Na' may be responsible for depolarization. Since the attacks are periodic, not only must an increased resting conductance be present, but some mechanism must induce the changes. A small rise in serum K+ may be the stimulus, either because of diurnal variation, ingestion of K+, or muscle activity, and may trigger a further increase in membrane sodium conductance, depolarization of the muscle membrane, and movement of K+ out of the muscle. On muscle membrane depolarization, the membranes become hyperexcitable and paralysis develops. Management and treatment of hyperkalemia is the exact opposite for

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that of hypokalemia. In other words, high carbohydrate intake, glucose, and insulin all are helpful in minimizing attacks, probably by aiding in K + uptake by the muscles. Acetazolamide is effective on long-term management of hyperkalemic periodic paralysis just as it is for hypokalemic periodic paralysis. The P-adrenergic stimulant salbutamol also has been reported to be an effective treatment for hyperkalemia (Wang et al., 1978).

4 . Malignant Hyperexia (Hyperthermia)

Malignant hyperexia appears to be similar to malignant hyperthermia in the pig and seems to be involved in PSS, discussed in some detail by Pearson and Young (1989). The condition in humans is encountered during anesthesia of apparently healthy individuals undergoing routine surgery and is often fatal. This disorder was described first by Denborough and Love11 (1960): anesthesia induced muscle stiffness that was accompanied by a rapid rise in body temperature and increased plasma K + and lactate levels. The disorder results in cardiac failure and death in about 50% of the cases. Myoglobinuria and renal failure are also other consequences of the disorder. The factors precipitating the onset of malignant hyperexia are the use of halothane as a general anesthetic or suxamethonium as a muscle relaxant. The major defect responsible for malignant hyperexia appears to be in the lack of control of intracellular Ca2+concentrations (Britt, 1979). Failure to control Ca2+ levels properly results in rapid E-C coupling and accounts for the rigidity of the affected muscles and the rapid rise in body temperature. The increase in serum K+ levels and blood plasma lactate are a consequence of the rapid E-C and may contribute to the problem further. The condition appears to be inherited as an autosomal dominant trait. Reportedly, the incidence is twice as high in males as in females, but. this relationship merely may reflect the greater incidence of traumatic accidents requiring surgery in boys and young men (Ellis and Halsall, 1980). Diagnosis can be made using an in uitro test for propensity to contract in the presence of caffeine (Moulds and Denborough, 1974; Ellis e f al., 1978).

Management of malignant hyperexia susceptible patients requires monitoring body temperature and avoiding stress and other factors that may contribute to its development. If hyperexia occurs, intravenous administration of dantrolene will serve as an antidote, although strenuous efforts must be made to alleviate any rise in body temperature and to reduce the increase in plasma lactate and K+ concentrations (Edwards

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and Jones, 1983). Dantrolene is a peripheral muscle relaxant and is thought to act by controlling the release of excess Ca2+,thus preventing continuous cycling of muscle excitation and contraction (Nelson, 1978). IV. DISEASES OF THE CONNECTIVE TISSUES

The numerous diseases of the connective tissues include those of nutritional, genetic, inflammatory, degenerative, and hormonal origin. These groups are reviewed briefly; a few specific diseases are discussed, including not only those involving collagen but also those of elastin and those of the mucopolysaccharides. A. NUTRITIONAL DISEASES The best known of the nutritional diseases of connective tissues include scurvy and certain other diseases that are caused by consumption of lathyrogens. 1 . Scurvy

Scurvy is a disease of the connective tissues that is characterized by fragility of the blood vessels and other collagen-containing tissues. The disorder is caused by a dietary deficiency in ascorbic acid (vitamin C), although humans, other primates, and guinea pigs are the only vertebrates that require ascorbic acid in the diet. Scurvy was known as the scourge of seas by early mariners and explorers, who largely subsisted on salted cured meats and had few or no vegetables and fruits in their diets. An excellent book by Carpenter (1986), describing scurvy and the curing of the disease by adding fruits and vegetables and finally vitamin C to the diet, provides an interesting account of scurvy that culminated in the discovery of ascorbic acid and virtual elimination of this dreaded disease. The symptoms of scurvy include muscular weakness, anemia, frequent hemorrhaging, spongy bleeding gums with loss of teeth, skin lesions, reddening of the extremities because of the accumulation of blood on the surface of the muscles, and brawny induration. The hemorrhages are associated with blood vessel fragility as a consequence of the inability to hydroxylate collagen, since ascorbic acid is needed to maintain lysyland prolyl-hydroxylase in the reduced state so that these enzymes can catalyze hydroxylation. Thus, unless ascorbic acid is present, an abnormal collagen low in hydroxylysine and hydroxyproline is produced

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that lacks structural strength (Bailey and Etherington, 1980). For more discussion on the role of ascorbic acid in hydroxylation, see Pearson and Young (1989). Treatment of scurvy is by supplying ascorbic acid, which results in a speedy recovery. Citrus fruits and juices, potatoes, fresh vegetables, and fruits are excellent dietary sources of ascorbic acid. Scurvy is encountered rarely today, and then usually because of some metabolic error. 2. Lathyrism

Lathyrism is a disease of the connective tissues that is of nutritional origin. Although first observed on feeding animals lathyrogens, which are constituents of lupines or chickpeas, the disorder since has been studied widely to help understand the mechanism involved in hydroxylation of collagen. Experimental lathyrism can be produced by administering /3-aminoproprionitrile (BAPN) or penicillamine, or by making animals copper deficient (Bailey and Etherington, 1980). Excessive zinc intake also can act as an antagonist of copper and cause lathyrism. All these substances produce poorly cross-linked collagen that lacks structural strength (Pearson and Young, 1989). The use of lathyrogens to induce lathyrism is, today, primarily an experimental technique and seldom is observed naturally. However, the technique may be useful for studying the relationship between crosslinking and meat tenderness. 3. Starvation and Protein Dejiciency At the onset of starvation, a loss in body weight occurs that first uses the energy reserves in the form of glycogen and fat. As starvation proceeds, maintenance of vital processes mobilizes the body proteins. Studies with mice during starvation have shown an increase in urinary excretion of hydroxyproline (Bell, 1969), although little change occurs in hydroxyproline excretion by rats on a protein deficient diet (Dawson and Milne, 1978). Nevertheless, a continued synthesis and deposition of the connective tissue proteins appears to occur during early starvation, although a significant loss of muscle protein occurs also (McClain and Wylie, 1977; Dawson and Milne, 1978). This result supports the concept that nutritional deprivation may spare the connective tissues preferentially, except in the case of severe undernutrition. Love et al. (1976) demonstrated that extra collagen is laid down in the skin and myocommata of cod fish during periods of nutritional stress. However, the accumulation of connective tissues then disappears during

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a period when food is plentiful. These facts suggest that the connective tissue proteins are less affected by nutritional stress than the other tissue proteins, a characteristic that may be related to their functions in muscles. Degradation of the muscle proteins during starvation is not limited to the connective tissue proteins; the myofibrillar and sarcoplasmic proteins are affected also. This condition was discussed by Millward et al. (19801, who pointed out that the time of the onset of protein degradation during starvation may be variable but the rate ultimately increases dramatically. Marked changes occur in the lysosomal enzyme system during starvation; a large increase in lysosomal enzyme activity apparently occurs because of the greater fragility of the lysosomal membranes. Lysosomes become more abundant and tend to be localized in the I-band region. Nevertheless, muscle fiber fragments are not commonly found in the autophagic vacuoles. However, alterations in the sarcotubular system, especially at the I band, appear to occur, with some fiber splitting at the periphery. Similar changes have been observed in cardiac muscle of starved animals. Nonmuscle cells may invade muscle per se during starvation and may be responsible for the enhanced degradation of muscle fibers, since the invading cells are rich in hydrolases. The cells usually have enhanced cathepsin D and acid phosphatase activity and also may contain appreciable amounts of other acid hydrolases. Good evidence exists that the calcium-activated proteinase plays an important role in extracellular digestion of the myofilaments.

B. GENETIC DISEASES Several diseases of the connective tissues have a genetic basis, including dermatosparaxis in cattle and sheep, and the Ehlers-Danlos syndrome, the Marfan syndrome, osteogenesis imperfecta, homocystinuria, and alcaptonuria in humans, which are discussed briefly here. 1 . Dermatosparaxis

Dermatosparaxis is a recessively transmitted genetic disorder that occurs in cattle and sheep. The genetic defect is the result of the failure of procollagen peptidase to cleave the N-terminal region from procollagen (Bailey and Etherington, 1980). Dermatosparactic collagen is, thus, characterized as a disorganized collagen that results in long fine microfibrils in which lateral aggregation is defective. This structure results in the inhibition of intermolecular cross-links and prevents production of the

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normal thick cylindrical collagen fibers of high tensile strength. A significant proportion of the collagen of dermatosparactic animals consists of type I al(1) and a2(I) chains containing the N-terminal region that is characteristic of procollagen. Therefore, the skin and bones of the affected calves and lambs do not have enough tensile strength to allow them to stand and nurse, so they usually die. Morris et al. (1975) reported that the N terminus of procollagen is cleaved off before the C terminus, whereas other workers (Goldberg et al., 1975; Haswood et al., 1975; Davidson et al., 1977) identified intermediate forms of the molecule. This discrepancy led Morris et al. (1975) and Davidson et al. (1977) to propose that two different endopeptidases are involved in cleavage of the N and C termini. This processing is illustrated schematically in Fig. 6, which proposes that the C terminus of procollagen is cleaved off before the N terminus is removed. This scheme actually suggests, but does not prove, that two different endopeptidases may be involved in cleaving the procollagen terminal groups. This theory would help explain why dermatosparactic collagen still contains the N terminus but the C terminus has been cleaved from the procollagen molecule. Regardless of whether or not dermatosparactic collagen requires two different endopeptidases, Lenaers et al. (1971) showed that procollagen extracted from skins of animals suffering from dermatosparaxis is converted to collagen by treating with pepsin. Additional evidence for a defect in the endopeptidase system is found in the fact that Lapiere et al.

-/

4

NH2-terminal peptidase

I @

- - @ - - - - - - - _ - - - --. E7 5

N-terminal fragment

4

-

-

U

0 COOH-terminal peptidase

C-terminal fragment

FIG. 6. Proposed mechanism by which the NH2-terminal peptidase and the COOHterminal peptidase sequentially cleave off the ends of procollagen (top) to produce collagen (bottom). Taken from Bailey and Etherton (1980).

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(1971) isolated an enzyme from the tissues of normal calves that converted the procollagen from both dermatosparactic and normal calves to collagen. This result provides convincing evidence that dermatosparaxis is caused by a defect in the endopeptidase system that results in the incomplete conversion of procollagen to collagen.

2. Ehlers-Danlos Syndrome The Ehlers-Danlos syndrome (EDS) is a group of at least 10 different heterogeneous inherited disorders of collagen metabolism. These disorders are discussed by Krane (1984) and are characterized most commonly by joint hypermobility and skin hyperextensibility , as illustrated in Fig. 7A and 7B, respectively. Skin splitting and bruising is common; tissue friability occasionally is so extensive that major blood vessels or viscera may rupture and result in death. The expression of the symptoms, however, varies widely in different families, according to Beighton et al. (1969). EDS can be categorized into 10 different classes; the biochemical defect responsible for each type is shown in Table 11. The biochemical defects responsible for types I, 11, and 111 have not been identified. Nevertheless, types 1-111 are the most commonly encountered types of EDS and are characterized by joint hypermobility and hyperextensibility of the skin. Examination of affected relatives suggests that more than one type of mutation may be responsible for an apparently identical biochemical defect, which may arise from mutations in structural portions of the genes, regulatory portions of the genes, or genes encoding posttranslational modifying proteins. In EDS type IV, affected individuals bruise easily, apparently because of arterial fragility, which is a distinguishing feature of this form of the disease. Evidence suggests that type IV EDS, exhibits genetic heterogeneity; both autosomal dominant and autosomal recessive inheritance have been seen (Pope et al., 1977; Byers et al., 1981a). Minor trauma may produce extensive ecchymoses that result in thin pigmented scars. In severe cases of arterial fragility, a major artery may rupture and lead to sudden death. The disease also may lead to weakened connective tissue in the viscera and result in perforation of the viscus-most often of the colon or the uterus in pregnant women-and result in death. Type IV EDS is characterized by a biochemical defect in synthesis or secretion of type 111 collagen (Table 11). Byers et al. (1981b) verified the decreased synthesis of type 111 collagen and obtained quantitative evidence that the amount secreted was approximately the amount expected if only one allele of a single locus was abnormal. These results were confirmed

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by ultrastructural examination. The genetic defect observed in EDS type IV does not appear to be the same in all affected subjects. At least some subjects do not appear to be capable of producing any type I11 collagen (Pope et al., 1975), whereas most produce some, although the amount may be reduced to 10-30% of normal levels (Aumailley et al., 1980; Byers et al., 1981b). Although marked differences occur in the amino acid content of aorta from patients with EDS type IV and normal subjects, as shown in Table 111, the decreased content of 4-hydroxyproline and hydroxylysine can be attributed to the absence of type I11 collagen in EDS type IV. Thus, type IV EDS appears to be caused by a deficiency in producing type I11 collagen. Type V EDS can be distinguished from the other forms of this disease on the basis of an apparent X-linked inheritance and probably is caused by a defect in peptidyl lysyl oxidase (Table 11). The deficiency in peptidyl lysyl oxidase activity appears to be only 13-26% of control values. Type VI EDS probably is inherited as an autosomal recessive trait, although this has not been proven definitely (Krane, 1984). However, clearly type VI EDS shows a deficiency of hydroxylysine, especially in ocular tissue, which results in a decreased amount of cross-linking. The decrease in cross-linking seems to be associated with a lower level of peptidyl lysyl oxidase. Studies on type VI EDS, however, have revealed several concepts of collagen metabolism. (1) Secretion of collagen molecules from the cell (fibroblast) does not require hydroxylation of the lysine residues and glycosylation of the hydroxylysines. (2) Collagen molecules of the same type but located in different tissue can undergo separately controlled posttranslational modifications. (3) Urinary markers of collagen degradation can be derived from sources other than skin collagen. (4) The role of hydroxylysine in cross-linking has been emphasized by the decrease in hydroxylysine in type VI EDS (Krane, 1984). Type VII EDS is characterized by a condition known as arthrochalasis multiplex congenita, which is manifested by multiple joint subluxations, ligamentous tears, a peculiar scooped-out facies with epicanthal folds and hypertelorism, and a thin velvety skin. Patients accumulate incompletely processed collagen in the skin, probably pN collagen. The defect is caused by the absence or a low level of activity of procollagen endopeptidases. Although originally believed to be similar to dermatosparaxis, the condition is not the same. Skin changes in EDS type VII are much less severe than those in dermatosparaxis in animals. Other forms of EDS have been suggested, as shown in Table 11, although they are not as well characterized as types I through VII. These conditions include types VIII, IX, and X, which occur in periodontal disease, in skeletal and urinary tract dysplasia, and in fibronectin defi-

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FIG. 7. Photographs showing typical hypermobility of the thumb joint and hyperextensibility of skin from individual suffering from Ehlers-Danlos syndrome. (A) Thumb joint (probably type I) and (B) hyperextensibility of the skin (probably type I). Reprinted with permission from Krane (1984).

TABLE I1 CLASSIFICATION OF THE EHLERS-DANLOS SYNDROME"

Type

Name ~~

~~

I I1 I11 IV

Gravis Mitis Benign hypermobile Ecchymotic

V VI

X-linked Hydroxylysine deficient (ocular) Arthrochalasis multiplex congenita Periodontal disease Skeletal and urinary tract dysplasia Fibronectin deficient

VII VIII IX X a

Biochemical defect

~~

Reprinted with permission from Krane (1984).

Unknown Unknown Unknown Decreased type I11 collagen synthesis and/or secretion Peptidyl lysine oxidase defect (?) Peptidyl lysine hydroxylase defect Decreased procollagen N protease or resistant cleavage site Unknown Peptidyl lysine oxidase defect (?) Abnormal fibronectin (?)

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TABLE Ill AMINO ACID COMPOSITION OF COLLAGEN OBTAINED FROM AORTA OF PATIENT WITH EHLERS-DANLOS SYNDROME TYPE IV IN

COMPARISON TO NORMAL CONTROLS'

Residues/ lo00 Amino acid 4-H ydrox yproline

Aspartic acid Threonine Serine Glutamic acid Proline GIycine Alanine Cystine (half) Valine Methionine Isoleucine Leucine Tyrosine Pheny lalanine Hydroxylysine Histidine Lysine Arginine

EDS IV

Controls

10

30 56 29 34 76 91 236 131 13 89 3.6 25 53 18 24 3.3 14 32 35

47 26 22 56 93 226 168 6 I I7 2.2 21 76 22 37 0.5 17 36 23

Reprinted with permission from Krane (1984). using data from Pope ef al. (1975).

ciency, respectively. The last defect appears to be related to abnormal platelet aggregation. Nevertheless, further clarification of all three of these types is needed. 3. Osteogenesis Imperfecta

Another group of heterogeneous heritable disorders has been classified under osteogenesis imperfecta (01)because of the tendency of the bones to fracture. 01 frequently is associated with other abnormalities in the connective tissues (skin, teeth and sclerae) and, like other similar disorders, exhibits considerable heterogeneity. Sillence et al. (1979) grouped these disorders into four classes; two of them are inherited through an autosomal dominant mechanism (types I and IV) and the other two

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through an autosomal recessive mechanism (types I1 and HI), as outlined in Table IV (Krane, 1984). In type I 01, which is the best characterized of the four types of this collagen disorder (Table IV), Sykes et al. (1977) found that the ratio of type 111 to type I collagen in the skin of patients over the age of 20 was 0.26, compared with 0.14 for normal controls. These studies imply that decreased amounts of type I collagen in other tissues, such as skin, may lead to an increase in type I11 relative to type I collagen in skin. Additional evidence to support this concept was obtained by Krieg et al. (1981), who showed that the relative amount of type I procollagen synthesized by type I 01 cells was decreased. In the case of type I1 01, extreme bone fragility often leads to intrauterine or early infant death (Table IV). Again, skin collagen shows an increase of type 111 relative to type I collagen. Whether or not bone from type I1 0 1 patients contains type I11 collagen has not been investigated, although normal bone contains only type I collagen. No evidence exists for more than one set of structural genes for al(1) and a2(1) chains for type I collagen (Krane, 1984). The situation in type I 0 1 may be analogous to that of some individuals with type IV EDS, in which a genetically determined decrease in synthesis of a specific type of collagen has been shown to occur. Type I1 0 1 has been shown to be inherited as an autosomal recessive

TABLE IV SUGGESTED CLASSIFICATION SYSTEM FOR OSTEOGENESIS IMPERFECTA'

Type

Phenotype

Mode of inheritance

I

Disease moderate; early onset of fractures; not usually progressively deforming; sclerae blue; hearing impairment as young adults; no dentinogenesis imperfecta Onset in utero; often stillborn or death soon after birth; marked long bone bowing and severe skull involvement Progressively deforming with multiple fractures and kyphoscoliosis; normal sclerae; hearing loss infrequent; dentinogenesis imperfecta prominent Variable age of onset and variable deformity; sclerae pale blue or white; dentinogenesis imperfecta variable

Autosomal dominant

I1 I11

IV

a

Autosomal recessive Autosomal recessive

Autosomal dominant

Reprinted with permission from Krane (1984), after Sillence et al. (1979).

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trait and is characterized by extreme bone fragility leading to intrauterine or early infant death as a consequence of fractures (Sillence el al., 1979). Bones from lethal cases of type I1 01 contain more type V collagen than normal and also have some type I11 collagen, which may account for the decreased content of type I collagen. Trelstad et al. (1977) on analysis of several tissues from an infant with type I1 01 phenotype found a 1.5- to 2.5-fold increase in hydroxylation of lysine residues in the al(1) and a2(I) chains of bone collagen. This hydroxylation may be the result of hypocalcemia in urero, but the cause and effect is far from clear. Osteogenesis imperfecta includes several syndromes that appear to be caused by different defects of collagen biosynthesis. Study of this group of diseases may lead to understanding the structure of human collagen genes and control of their expression (Prockop, 1980). 4 . Marfan Syndrome

Marfan syndrome has been estimated to be present in 4-6 persons per 100,OOO of the population (Krane, 1984). The disorder is manifested by abnormalities of the eye, the blood vessels, and the skeleton, but since its expression is variable it is often difficult to diagnose. Pyeritz and McKusick (198 1) suggested that more than one mutation may be responsible for the diversity in expression of Marfan syndrome. The disease, however, appears to involve a genetic defect in the cross-linking of collagen. Boucek et al. (1981) found that patients suffering from Marfan syndrome had decreased levels of dihydroxylysinonorleucine relative to hydroxylysinonorleucine in the skin, compared with normal controls. Overall results seemed to indicate that adequate amounts of lysine and hydroxylysine aldehydes are formed, indicating that the defect is not in peptidyl lysyl oxidase activity (Krane, 1984). Rather, the defect appears to be associated with misalignment of the collagen molecules in the fibrils and formation of the wrong contacts with the side chains. Thus, Krane (1984) proposed that the defect may occur in the a2(I) chains. Also evidence suggests that some patients suffering from Marfan syndrome may have a problem in regulation of hyaluronate biosynthesis, resulting in excessive tissue accumulation of hyaluronic acid (Matalon and Dorfman, 1968). Cell-free extracts from fibroblasts originating from patients with Marfan syndrome have been demonstrated to have 3- to 10-fold greater hyaluronate synthetase activity than comparable extracts from normal individuals (Appel et al., 1979). Thus, the relationship between Marfan syndrome and its origin in the connective tissue is far from clear at this time, and needs further clarification. Nevertheless, the syndrome is clearly a disease of connective tissue and has a genetic basis.

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5 . Homocystinuria The term homocystinuria is used in reference to a group of metabolic disorders that are characterized by a deficiency of cystathionine P-synthase (Mudd et al., 1970). This form of the disease is expressed clinically by ectopia lentis (progressive downward displacement of lens of the eye), osteoporosis, occasional arachnodactyly (spider-like fingers and/or toes), and dilation and thrombosis in medium-sized arteries and veins (McKusick, 1972). These symptoms overlap some of those occurring in the Marfan syndrome. Homocystinuria appears usually to be inherited as an autosomal recessive trait. The enzymatic block in formation of cystathionine from homocysteine and serine, a reaction that normally is catalyzed by cystathionine P-synthase, results in accumulation of homocystine, homocysteine, and homocysteic acid and may be responsible for the connective tissue abnormalities and mental deficiencies that accompany this disease (Lehninger, 1975). D-Penicillamine, a compound closely related to homocysteine, has been shown to interfere with collagen cross-linking by binding to aldehydes and forming a stable thiazolidine ring structure (Deshmukh and Nimni, 1969). Siege1 (1977) found that D-penicillamine blocks the formation of polyfunctional cross-links in which allysine, but not hydroxyallysine, participates-even at low concentrations. Krane (1984) suggested that homocysteine may function in the same manner, thus preventing allysine polyfunctional cross-link formation. 6 . Alcaptonuria

Alcaptonuria is a rare hereditary disease characterized by the presence of homocysteic acid in the urine and subsequent development of degenerative joint disease (Krane, 1984). The disorder is accompanied by pigmentation of the connective tissues, particularly of cartilage. The metabolic defect is caused by a deficiency in the enzyme homogentisic acid oxidase, which catalyzes the conversion of homogentisic acid to maleylacetoacetic acid (LaDu, 1978). Homogentisic acid forms a polymer that is deposited in the connective tissues and, in some way, causes pigmentation and produces degenerative joint disease. Murray et al. (1977) proposed a different mechanism in which homogentisic acid may inhibit peptidyl lysine hydroxylase from forming hydroxylysine-derived intermolecular cross-links. However, no data are available on the levels of hydroxylysine or its cross-links in tissues from patients with alcaptonuria, so the former mechanism seems more plausible.

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7 . Epidermolysis Bullosa Epidermolysis bullosa describes a group of disorders in which blisters develop from trivial trauma to the skin. Different types have been defined on the basis of their site of development as well as clinical, biochemical, and genetic features. For example, a so-called dystrophic type of epidermal bullosa is inherited as a recessive trait and is characterized by subepidermal blisters (Krane, 1984). Patients suffering from this type of the disorder synthesize increased amounts of a structurally altered collagenase and can be treated with phenytoin (Bauer et al., 1980). Another type of the disease, known as epidermolysis bullosa simplex, is a dominant genetic trait. Although the disorder is heterogeneous, it appears to be associated with low activity of the enzyme galactosylhydroxylysine glucosyltransferase (GGT). Distinctly low levels of GGT in affected individuals suggests that this defect is in some way associated with the pathogenesis of blister formation. Krane (1984) theorized that attachment of epidermal cells to basement membrane collagens that are normally highly glycosylated may be involved in blistering. 8. Rheumatoid Arthritis and Polyarthritis

Development of rheumatoid arthritis can occur at almost any time, from a few months after birth through old age and senescence, although its frequency increases with age (Lamont-Havers, 1967; Vaughan, 1980). Rheumatoid arthritis in its classic form occurs in 1-2% of the population over 30 years of age (Lamont-Havers, 1967) but affects 8-11% of the population in their late 60s, according to Vaughan (1980). The predisposition to develop this disease is no doubt family related but the mode of inheritance has not been elucidated, perhaps because of its diversity in expression (Vaughan, 1980). However, the disorder does appear to be associated with an immunoglobulin (Ig) interaction with the immune system, as outlined by Katz (1980). Rheumatoid arthritis in humans shows some similarities to polyarthritis in swine, which is manifested by severe articular distortion, especially in the distal joints (Leader et al., 1967). In both human rheumatoid arthritis and polyarthritis of swine, the lesions consist of nonsuppurative granulomatous proliferation of the synovial tissue, pannus formation, lymphoid infiltration, intraarticular fibrous adhesions, and destruction of cartilage (Leader et al., 1967). Sikes et al. (1969) have identified two etiological agents, Erysipelothrix insidiosa and Mycoplasma hyorhinis, as the causative organisms involved in polyarthritis of swine. Shuman (1959) found that polyarthritis in pigs, which also often is called erysipe-

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las insidiosa, was a common basis for condemnation of hog carcasses by meat inspectors, accounting for 15.5% of all condemned carcasses. Since swine polyarthritis has lesions similar to rheumatoid arthritis in humans (Leader et al., 1967,1983), Collins and Goldie (1940) suggested that swine polyarthritis may be a good model for the study of rheumatoid arthritis in humans. Although swine polyarthritis is caused by bacterial invasion, the mechanism by which rheumatoid arthritis develops in humans is still not clear (Moll, 1983; Wallace, 1989). Treatment of rheumatoid arthritis may involve use of antiinflammatory drugs such as aspirin, indomethacin, ibuprofen, and naproxen (Robinson, 1982). These drugs, especially aspirin, have been used widely to alleviate the pain associated with rheumatoid arthritis (Lamont-Havers, 1967; Robinson, 1982). Moll (1983) pointed out that aspirin in small dosages relieves pain, but at higher dosages has antiinflammatory properties. However, aspirin often causes stomach upsets so some of the modified aspirin drugs may be prescribed instead (Moll, 1983). Miescher et al. (1988) discussed new approaches to the treatment of rheumatoid arthritis. These investigators indicated that the use of glucocorticoids often is limited by their side effects. Antimalarial drugs, D-penicillamine, and gold compounds also are among the promising new treatments; Dpencillamine and gold therapy are believed to act by mediating the effects of the disease on the immune system. Prevention or treatment of rheumatoid arthritis also may include exercise to keep the muscles, tendons, ligaments, and bones active and strong; avoidance of pounding or jarring exercises; cushioning of shock by wearing shock-absorbing shoes, and reducing one’s body weight (Wallace, 1989). 9. Aleutian Disease Aleutian disease occurs only in mink. The disorder gets its name from the fact that it originally was believed to occur only in the pelt color known as Aleutian, which is a recessive trait. Although, all mink are now known to be able to contract the disease, the Aleutian phenotype (aa) has a hereditary predisposition toward the disease (Leader et al., 1967, 1983). Aleutian disease is a chronic progressive infectious condition characterized by hypergammaglobulinemia; glomerulonephritis; marked plasmacytosis of spleen, liver, kidneys, lymph nodes, and bone marrow; and, frequently, periarteritis. In addition, these lesions usually are accompanied by fibrinoid changes in the connective tissues. Some evidence suggests that its onset may be precipitated by a virus that has been isolated from the urine and feces of affected animals. Also, a familial ten-

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A. M. PEARSON AND RONALD B. YOUNG

dency to develop Aleutian disease exists; the aa genotype makes affected animals less vigorous so they succumb to the effects of the disease more quickly (Leader et al., 1967,1983). This disease is of interest because it has some similarities to systemic lupus erythematosus. 10. Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) or lupus, as it is commonly known, is a chronic inflammatory disease of unknown cause that can affect virtually any part of the body, with development of distinct abnormalities of the immune system that cause the afflicted patients to produce antibodies against their own cells (Schur, 1983). These so-called antinuclear antibodies may be produced against DNA (deoxyribonucleic acid), the basic genetic material in the cells (Schur, 1983). SLE is difficult to diagnose because of polymorphism in the clinical symptoms (Tsokos and Balow, 1984). SLE results in morphologic changes in five groups of tissues: (1) acute necrotic and dystrophic changes in the connective tissues and the parenchyma; (2) subacute interstitial inflammation and vasculitis; (3) sclerotic changes; (4) immunomorphologic changes; and ( 5 ) specific changes in one or more of the following systems-kidney, cardiovascular, liver, and nervous (Laufer, 1967; Strukov, 1967; Wader, 1967;Aladjem, 1985). The most characteristic lesion observed in SLE is nephritis with diffuse thickening of the glomerular capillary walls, which frequently have electron-dense deposits along the basement membrane (Vasquez and McCarter, 1967). According to Vasquez and McCarter (1967), the glomerular lesions have been shown to contain y-globulin localized in the capillary walls, which may be bound to epidermal cell nuclei. SLE appears to have a genetic basis. At least a familial relationship appears to exist (Schur, 1983;Tsokos and Balow, 1984); some families exhibit a tendency or predisposition to the disease. Schur (1983)calculated that incidence of SLE among close relatives (parents, children, siblings, and first cousins) of a patient with the disease is 1 in 20, in contrast to 1 in 400 people in the United States as a whole. Women are more susceptible to lupus than men during the child-bearing years, with a frequency of 9 or 10 to 1. This frequency declines at older ages (Aladjem, 1985). Black women are more susceptible to lupus than white women, with an incidence about 3-fold higher (Aladjem, 1985). Leader et al. (1983) reviewed the differences and similarities of the symptoms and etiology of SLE in several different species that suffer from this malady. After weighing the evidence, these investigators con-

DISEASES AND DISORDERS OF MUSCLE

40 1

cluded that the mouse provides the best model for the disease in humans. Treatment of SLE was reviewed by Schur (1985), who pointed out that some drugs may do more harm than good. General guidelines given by Schur (1985) for treatment include (1) regular rest when the disease is active and increased physical activity when in remission or quiescent; (2) use of sunscreens by photosensitive patients; (3) aspirin and aspirinlike drugs to decrease pain; (4) the antimalarial drug, hydroxychloroquine, to help in case of severe joint or skin involvement; (5) cortisone drugs, to help in cases of severe organ involvement (however, cortisone may have hazardous side effects); (6) consulting a physician in case of fever; (7) having regular checkups, including blood and urine tests; and (8) calling a specialist whenever in doubt. As can be seen, many of these guidelines are not specific for SLE. However, many of the symptoms of lupus are also nonspecific, which makes diagnosis of this disease difficult and treatment even more tentative (Schur, 1985). C. ACQUIRED DISORDERS Some diseases of connective tissues appear to be environmental or acquired for some reason. These conditions include disorders resulting in abnormal calcification of both elastin and collagen, interstitial lung disease, and hepatic fibrosis. These diseases are not well understood but do not appear to have a genetic basis. 1 . Abnormal CalciJication

Disorders involving abnormal calcification of collagen and elastin in the soft tissues have been documented. Mineralization occurs primarily in mesenchymal tissue, especially in collagen and elastin, and somewhat less often in muscle (Yu and Blumenthal, 1967). Calcification of elastin and collagen is not uniform at different anatomical sites; elastin of the aorta and large arteries calcifies readily, whereas ligament calcifies less easily, and dermis, lung, and hollow viscera are calcified little, if at all. Large arteries of individuals over 35 years of age almost always exhibit some calcification of connective tissues. Nucleation centers apparently are formed in the connective tissues and serve as locations for calcification, where calcium and phosphate are deposited. Yu and Blumenthal (1967) suggested that elastin is more likely to become calcified than collagen; the latter fibers only become calcified when the process reaches massive proportions.

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2. Systemic Sclerosis

Systemic sclerosis or scleroderma is a disorder of unknown etiology that most frequently affects the skin and subcutaneous tissue. The disease often is associated with Raynaud’s phenomenon or gangrene. Scleroderma can be classified as systemic, in which it results in calcification of the internal organs, or as epidermal, in which the skin and epidermis are affected. In epidermal scleroderma, the skin is thin and atrophic and the dermal layer is bound to deeper connective tissues as a result of excessive collagen deposition. The disorder is characterized by distorted dilated capillaries and obliteration of the small arteries. The affected tissues appear to have an increased collagen content, apparently as a result of increased collagen synthesis by scleroderma fibroblasts (Krane, 1984). Systemic scleroderma affects the lungs, kidneys, gastrointestinal tract, and heart of affected individuals. Clinical features of scleroderma have been related to disorders of tryptophan metabolism. Urinary excretion of kynurenine is elevated in scleroderma, a condition that may be associated with decreased activity of the enzymes involved in the oxidation of this product of tryptophan breakdown. Plasma kynurenine levels were shown to be high in 7 of 15 patients suffering from scleroderma (Krane, 1 984). Shulman (1975) described another variant of scleroderma that is characterized by fibrosis of deep subcutaneous tissue and fascia and is associated with peripheral eosinophilia. Krane (1984) indicated that mononuclear cell infiltration in these lesions is common, as is the case in scleroderma, but clinical features such as those that occur in Raynaud’s disease are absent in this disorder. 3. Interstitial Lung Disease Pulmonary fibroses or interstitial lung disorders comprise a number of heterogeneous diseases that involve the lung parenchyma (Crystal et al., 1981). Fibrosis of the alveolar interstitium (the portion of the alveolar tissue between the epithelial and endothelial basement membranes) is the main histologic feature of this disorder. Most of these diseases are progressive and often fatal, resulting from the loss of alveolar capillary units and right-sided cardiac hypertrophy. Cor pulmonale, a syndrome resulting from failure of the right side of the heart, is quite often responsible for death from this disease. In approximately one-third of the patients suffering from interstitial lung disease, the cause can be linked to some specific environmental

DISEASES AND DISORDERS OF MUSCLE

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factor. The most common etiologic agents are chronic inhalation of dusts containing silica or silicates. Thus, these diseases occur most frequently in miners and asbestos workers. The initial event occurring in pulmonary fibroses is an inflammation of the alveolar interstitium and is characterized by the infiltration of lymphocytes and polymorphonuclear leukocytes, which are not normally present in the lungs. Eventually, the alveolar structures are replaced by cystic spaces separated by wide bands of collagenous tissue interspersed with chronic inflammatory cells (Krane, 1984). In normal lung tissue, type I11 collagen constitutes about one-third of the total lung parenchymal collagen; types IV and V collagen are present predominantly in the basement membranes. In interstitial lung disease, type I collagen predominates, resulting in a corresponding decrease in type I11 collagen. This group of diseases is diverse and, no doubt, caused by a number of different etiologic agents (Crystal et al., 1981). Bleobomycin therapy has been demonstrated to stimulate alveolitis. Research using cultured lung fibroblasts has shown that bleobomycin increases synthesis of type I collagen, which may help explain the disproportionate accumulation of type I collagen in interstitial lung diseases (Krane, 1984). 4 . Hepatic Fibrosis

An increase in the content of connective tissue in the liver is a rather common occurrence (Gay and Miller, 1978), and results in hepatic fibrosis. Depending on the distribution and amount of hepatic connective tissue and the cause of the disease, hepatic fibrosis may be of little clinical importance or a serious life-threatening disorder in the form of cirrhosis (Krane, 1984). Although numerous causes of hepatic fibrosis are known, liver injury is frequently a predisposing event. The major etiologies in hepatic fibrosis in the western world include alcoholism, nutritional diseases, and viral hepatitis. Other underlying causes include hemochromatosis (iron overload), Wilson’s disease (a heritable hepatolenticular degenerative disease secondary to a heritable disorder in copper metabolism), extrahepatic biliary obstruction, primary biliary cirrhosis, and chronic congestive heart failure. In developing countries, hepatic schistosomiasis is the major etiologic cause of this disease. Although all the hepatic diseases are accompanied by collagen deposition, the patterns of the connective tissue deposits vary widely depending on the specific disease. For example, alcohol-induced liver cirrhosis results in deposition of small connective tissue nodules (ca. 3 mm in diameter) and regular bands of septa1 connective tissue. In contrast, the so-called postnecrotic cirrhosis associated with viral hepatitis is charac-

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terized by different sized nodules (up to several cm in diameter) and irregularly arranged septa. Apparently, lipocytes are the precursors of parenchymal fibroblasts and secrete collagen during the early phases of hepatic fibrosis. Later, the parenchymal fibroblasts become the principal pathway for collagen deposition. Excessive collagen deposition results, and produces disorganized connective tissue, which leads to portal hypertension, decreased biliary secretion, and impairment of hepatic cell function. Although type I11 collagen constitutes 47% of collagen in normal livers, it decreases to only 18-34% of total collagen in hepatic fibrosis and constitutes a disproportionate amount of total collagen in cirrhotic tissue. Finding increased amounts of collagenase activity in liver as a consequence of experimentally induced fibrosis suggests that this enzyme may, in some way, reflect accumulation of collagen (Carter et al., 1982). Although treatment of cirrhosis is difficult, in the case of alcoholinduced cirrhosis good nutrition is essential and, coupled with abstinence, may result in reversal and ultimately a return to near normal hepatic function. Also, some evidence suggests that prostaglandins may not only suppress collagen synthesis but also modulate intracellular degradation. Krane et al. (1982) reviewed these mechanisms in regard to rheumatoid tissue, which also may have application to hepatic fibrosis. 5 . Carcinomas of Connective Tissue Cancer is frequently, but not always, a disease of connective tissues. The uncontrolled growth of connective tissue cells is encountered in skin carcinomas; in cancer of the stomach, intestines, and lower bowel; as well as in lung cancer, bone cancer, and several other manifestations of this diverse disease. The factors initiating and controlling growth are only beginning to be understood. Since this discussion is not intended to be comprehensive, it will center only on the relationship of fibronectin and laminin to this disease. Akiyama and Yamada (1983) discussed the role of fibronectin in this disease. This glycoprotein occurs in many organs and body tissues, frequently as a constituent of the basement membrane-a common site of cancer initiation. Fibronectin decreases in or is lost from malignant tumors as the invasion process progresses. However, three areas of uncertainty remain. (1) Whether fibronectin decreases in benign tumors is not known. (2) Whether metastases have less fibronectin than primary carcinomas is not clear. (3) Whether or not sarcosomas have decreased concentrations of fibronectin is not known (Akiyama and Yamada, 1983). Although men suffering from prostatic cancer may have elevated levels

DISEASES AND DISORDERS OF MUSCLE

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of urinary fibronectin, the significance of this observation is not yet known in light of the decrease or absence of fibronectin at the site of the tumor invasion. Since fibronectin and laminin are initially present at relatively high concentrations at cell-to-cell surfaces and function in intercellular communication (Carter and Hakomori, 1981; Rao er al., 1982), the question of whether or not they are involved in initiation of either benign or malignant tumors is not known. Further, the virtual absence of fibronectin in carcinomas could reflect a role in tumor growth and its disappearance could be the result of such a relationship (Ruosalahiti et al., 1981). Conversely, the absence of fibronectin in tumors may signal that the decrease is a defense mechanism against cancerous growth. Only when these questions are answered will a full understanding of the role of fibronectin in malignant tumor growth be possible. V.

RESEARCH NEEDS

Emphasis in this chapter on diseases and disorders of muscle has, from time to time, focused on using some of the disorders described as models for studying meat quality. Thus, readers should examine the entire chapter for its implications for meat quality. However, some of the diseases discussed are of nutritional origin and should be considered from the viewpoint of their implications on the health and well-being of humans and the need for understanding the basic mechanisms involved. A.

BIOPSIES

From the standpoint of understanding the growth and composition of muscle, investigations are needed that concentrate on using biopsies to follow such changes, which are important in elucidating not only basic mechanisms but also nutritional effects. Basic to such studies are the selection of representative muscles and their accessibility for biopsies, which has been discussed by Edwards and Jones (1983) for humans. Similar information is needed for both laboratory and farm animals. Only by use of properly selected and implemented muscle biopsies do the data become meaningful.

B. FIBER TYPING AND COMPUTERIZED TOMOGRAPHY Other methods that may be used to follow changes in muscle during growth and development include fiber typing and assessment of changes

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in fiber types that occur (Pearson and Young, 1989). What factors are responsible for these changes and what are their effects on meat quality? Closely related areas include measurement of muscle areas and shapes that can be followed by computerized tomography (Edwards and Jones, 1983) and ultrasonic visualization (Stouffer et al., 1961). Not only is the relative size of a muscle important, but its shape also may have major implications for its performance. Further, questions should be asked about the relative needs for supporting connective tissue and the need for accompanying adipose tissue, not only from the standpoint of maximal performance but also for the best meat quality. C. NUCLEAR MAGNETIC RESONANCE AND NUCLEAR MAGNETIC IMAGING NMR and nuclear magnetic imaging (NMI) are techniques that have been used widely by the medical profession (Edwards and Jones, 1983). However, these related techniques should be applicable to meat studies, particularly to understanding the state of the water in muscle (Fennema, 1977; Garlid, 1979; Offer and Knight, 1988). Such studies should prove valuable in elucidating the mechanism(s) involved in water binding in meat and meat products. These issues are not only important physical aspects but also important economical aspects of meat. D. MEASUREMENT OF PROTEIN TURNOVER AND RELATED TECHNIQUES Other techniques that could be important to both meat quality and nutrition include protein turnover (Edwards and Jones, 1983), creatinine coefficient (Reeds et al., 1978), and whole body potassium (Pfau, 1965). Although these techniques can be used to follow changes in composition, many questions about their usefulness remain to be explored. For example, what factors control protein accretion and breakdown? What factors initiate a decrease in protein degradation and an increase in accretion? How can one control tissue growth and composition? Advances in methodology discussed in a book on growth regulation in farm animals (Pearson and Dutson, 1991) make exploration of many growth-related and compositional indices attractive in light of their potential for understanding the effects of growth on muscle proliferation and composition. Closely related to growth and composition of muscle is research on 3-methylhistidine as a measure of skeletal muscle breakdown. 3Methylhistidine is a urinary catabolite that was believed to be indicative

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of skeletal muscle breakdown (Rennie et al., 1980) until recently, since both actin and myosin contain 3-methylhistidine, which on degradation cannot be used for protein synthesis since the cell does not contain either the aminoacyl-RNA or the aminoacyl-tRNA synthase that recognizes 3-methylhistidine (Pearson and Young, 1989). Thus, this methylated amino acid cannot be reincorporated into protein, so it is quantitatively excreted in the urine and has been used as a measurement of myofibrillar protein degradation (Young and Munro, 1980). Harris and Milne (1980) indicated that 3-methylhistidine also is present in smooth muscle actin and myosin, so this metabolite may not be a good measure of skeletal muscle degradation. Thus, basic studies are needed to ascertain whether the amount of 3-methylhistidine derived from breakdown of smooth and cardiac muscle result in a significant error when using this methylated amino acid as an index of degradation of the myofibrillar proteins in skeletal muscle. E. USE OF MUSCLE CELL CULTURES Muscle cell culture is another basic technique that should be useful in studying factors that influence cell and tissue growth. Rowland (1988) provided evidence that muscular dystrophy, at least Becker’s and Duchenne, is associated with a sarcolemmal membrane dysfunction. Investigations need to concentrate on the mechanism of the dysfunction at the cellular level and the nature of the factors controlling its expression. Muscle cell culture should be a useful technique for elucidating the role of nutritional factors in growth. Use of this technique may be helpful in determining the mode by which the growth promoters and inhibitors exert their effects (Witkowski, 1986).

F. AGING AND EFFECTS OF EXERCISE Data from exercising rats, collected by Hollozy and Smith (1987), indicated that exercise can have a profound influence on longevity. More data are needed to determine whether exercise will alter the physical well-being and longevity of humans greatly, and, if so, what amount of activity is needed to achieve the desired affects? One may ask whether exercise is beneficial up to a certain point, but is harmful beyond that point. The effects of different training regimens on race horses running different length races is virtually unexplored, although it could be determined scientifically. At the same time, even less is known about the training of athletes and the effects of nutrition on their performance.

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G. ENERGY METABOLISM Exploration of some of the defects in energy metabolism, including the glycolytic disorders, may be related to the PSE muscle or the related phenomena of DFD and PSS muscle in pigs. The glycogen storage diseases in humans may provide a model for investigating the causes of these muscle problems in farm animals, especially in pigs and cattle. Similarly, the disorders of mitochondria1 oxidation in humans (Edwards and Jones, 1983) may aid in the investigation of PSE, PSS, and DFD pig meat. As mentioned earlier, disorders of mitochondrial metabolism may be useful in elucidating the exact mechanism for release of Ca2+ions by both the mitochondria and the SR in cold shortening and toughening of meat when exposed to low temperatures before the onset of rigor mortis. Also, as discussed earlier in this chapter, carnitine deficiency could be used as a model for studying the role of various iron-related factors in meat quality, including muscle color and firmness. Malignant hyperexia is another myotonic syndrome that already has been shown to be a useful test for the PSS condition in pigs (Monin et al., 1981). The application of the halothane test for malignant hyperexia in the occurrence of PSE, PSS, and DFD muscle in pig (Pearson and Young, 1989) has proven useful in identifying susceptible animals. This example of application of principles used in treatment of malignant hyperexia in humans to a condition in farm animals should lend encouragement to nutritionists and meat scientists to follow developments in diagnosis and treatment of the same or similar diseases made by the medical profession for applications in their fields.

H. CONNECTIVE TISSUE DISEASES AND DISORDERS Connective tissue diseases and disorders are of special interest in meat quality, especially tenderness. The use of lathyrogens in determining the role of collagen cross-linking in meat texture offers an opportunity to better understand meat tenderness. Since lathyrogens block cross-linking in collagen, one should be able to determine whether or not collagen cross-linking is a major contributor to meat tenderness. A number of other disorders in collagen metabolism (Bailey and Etherington, 1980; Krane, 1984) could also be useful in studying the role of collagen in meat tenderness. Some indirect evidence suggests that weaknesses of blood capillaries result in the escape of blood into the tissues and account for excessive bruising in chickens fed aflatoxins (Chen et al., 1984), causing large economic losses. Whether aflatoxins cause similar problems in pigs and cattle still is unknown. Polyarthritis is a common cause of condemnation of hog carcasses (Shuman, 1959); this disorder is related to

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changes from normal to abnormal collagen. Methods for preventing or treating this disorder in collagen metabolism are badly needed.

I. GENE MAPPING STUDIES Mapping of the human genome is already underway and has resulted in identification of the gene associated with Duchenne and Becker’s muscular dystrophy (Hoffman et af. 1988). Similar gene mapping work is needed for the meat producing species. Once the genes and the traits for which they are responsible are known, controlling various inherited diseases and manipulating meat quality traits such as tenderness should be possible. Although gene mapping programs are expensive, their pay-off in terms of potential control of desirable and undesirable traits holds great promise. VI. SUMMARY

Muscle may suffer from a number of diseases or disorders, some being fatal to humans and animals. Their management or treatment depends on correct diagnosis. Although no single method may be used to identify all diseases, recognition depends on the following diagnostic procedures: (1) history and clinical examination, (2) blood biochemistry, (3) electromyography, (4) muscle biopsy, ( 5 ) nuclear magnetic resonance, (6) measurement of muscle cross-sectional area, (7) tests of muscle function, (8) provocation tests, and (9) studies on protein turnover. One or all of these procedures may prove helpful in diagnosis, but even then identification of. the disorder may not be possible. Nevertheless, each of these procedures can provide useful information. Among the most common diseases in muscle are the muscular dystrophies, in which the newly identified muscle protein dystrophin is either absent or present at less than normal amounts in both Duchenne and Becker’s muscular dystrophy. Although the identification of dystrophin represents a major breakthrough, treatment has not progressed to the experimental stage. Other major diseases of muscle include the inflammatory myopathies and neuropathies. Atrophy and hypertrophy of muscle and the relationship of aging, exercise, and fatigue all add to our understanding of the behavior of normal and abnormal muscle. Some other interesting related diseases and disorders of muscle include myasthenia gravis, muscular dysgenesis, and myclonus. Disorders of energy metabolism include those caused by abnormal glycolysis (Von Gierke’s, Pompe’s, Con-Forbes, Andersen’s, McArdle’s, Hers’, and Tauri’s diseases) and by the acquired diseases of glycolysis

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(disorders of mitochondria1 oxidation). Still other diseases associated with abnormal energy metabolism include lipid-related disorders (carnitine and carnitine palmitoyl-transferase deficiencies) and myotonic syndromes (myotonia congenita, paramyotonia congenita, hypokalemic and hyperkalemic periodic paralysis, and malignant hyperexia). Diseases of the connective tissues discussed include those of nutritional origin (scurvy, lathyrism, starvation, and protein deficiency), the genetic diseases (dermatosparaxis, Ehlers-Danlos syndrome, osteogenesis imperfecta, Marfan syndrome, homocystinuria, alcaptonuria, epidermolysis bullosa, rheumatoid arthritis in humans, polyarthritis in swine, Aleutian disease of mink, and the several types of systemic lupus erythematosus) and the acquired diseases of connective tissues (abnormal calcification, systemic sclerosis, interstitial lung disease, hepatic fibrosis, and carcinomas of the connective tissues). Several of the diseases of connective tissues may prove to be useful models for determining the relationship of collagen to meat tenderness and its other physical properties. Several other promising models for studying the nutritionrelated disorders and the quality-related characteristics of meat are also reviewed.

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INDEX

A Accessory cells, in Peyer’s patches, 15 Acetazolamide, for hyperkalemic periodic paralysis, 386 Acidophilus milk, unfermented, 86, 101 aCLs, see Antibodies, anti-cardiolipin Acquired immunodeficiency syndrome gastrointestinal suppression, 34 nutritional therapies for gastrointestinal disease, 51-52 Acyl composition, cardiolipin diet effects, 291-297 interorgan differences, 291 interspecific differences, 290 related factors, 289 uniqueness, 289-290 Adherence, probiotic cultures in human intestine, 74-77, 120 ADPIATP carrier protein, cardiolipin association, 279-280 Adriamycin, cardiolipin association, 287-288 Aging effects on muscle function, 368-369 research needs, 407 AIDS, see Acquired immunodeficiency syndrome Alcaptonuria, 397 Aleutian disease, 399-400 Allergens, food characterization and identification, 40-42 detection and avoidance, 46 research needs, 54 Allergies, food, 25-26 Amino acid sequences, dystrophin gene, 357

y-Aminobutyric acid, agonist and uptake blockers, for epilepsy, 372 Andersen’s disease, 377 Animal husbandry, effect on milkfat composition modifications, 233-235 Animals cardiolipin, 264 dietary fat effects on cancer risk, 2 19-224 phosphatidylglycerol, 263 Antibiotics, p-lactam, in foods, 43 Antibodies anti-cardiolipin in absence of well-defined disease, 306 discovery, 304 endothelial cell binding, 307-308 lupus anticoagulant levels, 308-309 noncardiolipin antigens directed against, 310-312 platelet binding, 307 red blood cell binding, 308 splenic B and T cell binding, 309-3 10 in systemic lupus erythematosus, 305 in thrombosis. 304-305 in various diseases, 306-307 antinuclear, in systemic lupus erythematosus, 400 Antigens food, detection and avoidance, 46 orally presented, 22 uptake in gut via intestinal cells, 12-14 via Peyer’s patch, 14-15 Anti-inflammatory drugs, for rheumatoid arthritis. 399 425

426

INDEX

Anti-inflammatory effects, short-term fasting, 52 Antimicrobials, production by lactic acid bacteria, 77-78 Antinuclear antibodies, in systemic lupus erythematosus, 400 Apolipoproteins, serum butter effects, 166-169 dietary fat type and source effects, 154- 159 in MUFA-rich vs. low-fat diets, 174, I89 n-3 polyunsaturated fatty acid effects, 178-179 Arthrochalasis multiplex congenita, 392 ATPase FI Fo.association with cardiolipin, 279 Na+/K+,association with cardiolpin, 277-279 Atrophic myopathies, 363-364 Autoimmunity myasthenia gravis, 367 nutritional therapies, 52 research needs, 55 role of gut immune system, 33 suppression by oral tolerance induction, 50-51 Azoreductase, fecal, lactic culture effects, 99-101

B Bacterial agents gastroenteritis-producing, 35-37 ingested, immunostimulatory properties, 49-50 Bacterial diarrheal diseases, 23-24 Bacteriocin, production by lactic acid bacteria, 77-78 Becker’s muscular dystrophy, 351-359 Bifidobacteria effects on cancer-related fecal enzyme levels, 99-101 cancer suppression, 99-103 Candida-related diseases, 92 cholesterol reduction, 97-98 immune response, 109-110 fecal recovery, 72-74 role in lactose intolerance alleviation, 83-84

Biopsies, muscle. 345.405 B lymphocytes characterization, 8 splenic, aCLs binding. 309-310 Botulinum toxin, effects on gastrointestinal immunity. 35 Breast cancer, effects of lactic cultures, 106-107 Breast feeding, effects on gastrointestinal immune system, 45 Butter, effects on serum lipoproteins and apolipoproteins, 166-1 70 Butterfat, as source of dietary fat, 168- I69

C Calcification, abnormal, collagen and elastin, 401 Calcium ion, intracellular. in malignant hyperexia, 386-387 Cancer intestinal tract, 24-25 risk factors, milk- and dairy product-related animal studies, 219-223 breast cancer, 21 1-212 colon cancer, 212-213 research needs, 237 Cancer suppression, effects of lactic cultures, 99-107, 119-120 Candidal vaginitis, effects of Lacrobacill~sacidophilus cultures, 112-1 14 Carbohydrates, role in lactobacilli adherence, 76 Carboxylesterase, cardiolipin association, 287 Carcinomas, connective tissue, 404-405 Cardiolipin acyl changes, effects of development, 297 ethanol administration, 297 external temperature, 299 hyperthyroidism, 297-298 malignancy, 298-299 membrane fluidity, 299 acyl composition, 289-290 factors affecting, 289 interspecific differences, 290 uniqueness, 289-290

INDEX acyl-specific derivatives. chemical synthesis, 314-317 adriamycin association, 287-288 analogs. binding to aCLs, 310-312 in animals, 264 anti-cardiolipin antibodies in absence of well-defined disease, 306 discovery, 304 endothelial cell binding, 307-308 lupus anticoagulant levels. 308-309 noncardiolipin antigens directed against, 310-312 platelet binding. 307 red blood cell binding. 308 splenic B and T cell binding. 309-3 10 in systemic lupus erythematosus, 305 in thrombosis, 304-305 in various diseases, 306-307 cholesterol interaction. 271 chromatographic separation, 317-3 18 conformation in biomembranes. 270-274 degradation, 274-275 diet effects on acyl composition, 291-297 on quantity, 300 distribution, asymmetric. 270-274 enzyme associations ADP/ATP carrier protein, 279-280 carboxylesterase, 287 carnitine/acyl carnitine translocase, 282-283 carnitine palmitoyl-transferase. 282-283 cytochrome c oxidase, 277-279 DNA polymerase, 286 F, Fo ATPase , 279 glutamate dyhydrogenase. 285-286 a-glycerol-phosphate dehydrogenase. 283 NADH ubiquinone oxidoreductase, 286 Na+/K' ATPase. 277-279 phosphate carrier protein, 280-281 phospholipase A2, 284-285 protein kinase C, 285 various enzymes, 286-287 fatty acids, positional distribution, 290 hex I1 formation. 271-273

ionophore. 273-274 in microorganisms. 263 in mitochondria1 protein importation and translocation, 275-277 oxidation. 275 in plants, 263-264 reactive epitope. 312-314 space-filling models. 271 steroidogenicity. 271 synthesis acyl-specific. 266-267 intracellular location. 267-270 pathway, 265-266 Carnitine/acyl carnitine translocase. cardiolipin association. 282-283 Carnitine deficiency. effects in muscle, 381-382 Carnitine palmitoyl-transferase cardiolipin association, 282-283 deficiency effects in muscle, 382 Celiac disease, 29-30 Cell cultures, muscle, research needs, 407 Cell-mediated immunity, gut, 19-21 effects of lactic cultures. 107-1 10 CHD, see Coronary heart disease Chemicals, foodborne immunotoxicit y , 4 1-43 research needs, 54-55 Chemical synthesis, acyl-specific cardiolipin derivatives, 3 14-3 I 7 Cholera toxin, effects on gastrointestinal immunity, 35 Cholesterol cardiolipin interaction, 271 content in dairy products, 150-152 lactic acid bacteria effects, 92-98, 120 in milkfat. 150 serum levels, fat quality and dietary cholesterol effects, 160-161 Cholesteryl ester transfer protein, 193 Cholinesterase inhibitors, effects on myasthenic muscle, 367 Chromatography. cardiolipin separation, 3 17-3 I 8 Cirrhosis, etiology and treatment, 403-404 CL, see Cardiolipin Clinical examinations, for muscle diseases and disorders, 343

427

428 Collagen abnormal calcification, 401 deficient synthesis in Ehlers-Danlos syndrome, 392-393 dermatosparactic. 389-390 in hepatic fibrosis, 404 metabolism disorders, 391 Colon cancer, bifidobacteria effects. 105 Colostrum, effects on gastrointestinal immune system, 45 Common mucosal immune system, 21 Competitive colonization, by probiotic microbes. 91 Computerized tomography for muscle cross-sectional area, 346 research needs. 405-406 Connective tissue diseases, see also specific disease acquired disorders, 401-405 genetic, 389-401 nutritional. 387-389 research needs, 408-409 Constipation, effects of lactic cultures, 1 10-1 I 1 Contaminants, in foods, 41-43 Cori-Forbes disease, 377 Coronary heart disease genetic factors, 192-194 link with dietary fatty acids, 153-154 risk factors milk- and dairy product-related, 203-206 research needs. 237 Cortisol. effects on cardiolipin content, 300 Cow’s milk protein intolerance, 30-31 Creatine kinase, serum levels, 344 Creatinine, excretion index for muscle mass, 350 Crohn’s disease, 31-32 Cross sections, muscle, 346 Cyclic AMP, effects on cardiolipin content, 300 Cystathionine P-synthase deficiency, in homocystinuria, 397 Cytochrome b deficiency, in muscle, 380 Cytochrome c oxidase, cardiolipin association, 277-279 Cytokines, characterization, 8 Cytotoxicity, gut-associated, 19-21

INDEX

D Dairy products, see also Milkfat cholesterol content, 150-152 contributions to fathutrient intake, 140-143 diets meeting current recommendations available products, 229-232 modified products, 232-233 by animal husbandry, 233-235 by food technology, 235-236 effect on CHD risk, 203-206 effect on serum lipids animal studies, 200-203 human trials, 194-200 fat content, 150-152 fermented, immunostimulatory properties, 49-50 Dark, firm, and dry pork, see Pork Daunomycin, cardiolipin association, 288 Degenerativejoint disease, alcaptonuria-related, 397 Degradation, pol yglyerolphospholipids, 274-275 7-a-Dehydroxylase, fecal, lactic culture effects, 100 Delayed-type hypersensitivity reactions, gut-associated, 19-21, 29 Dermatomyositis, 362 Dermatosparaxis, 389-390 Development, effect on cardiolipin fatty acyl composition, 297 DFD pork, see Pork Diarrhea, effects of lactic cultures, 84-92 Diarrheal diseases bacterial, 23-24 research needs, 118-1 19 Diet effects on cardiolipin acyl composition, 291-297 quantity, 300 modification of gastrointestinal immunity autoimmunity suppression, 50-5 1 breast-feeding effects, 45 control of microbial flora, 47-50 food antigens and allergens, 46 hypoallergenic foods, 47 nutritional therapies, 51-52

429

INDEX total, role of milkfat CHD and cancer risks, 224-225 current fat recommendations available products, 229-232 modified products, 232-236 effect of weight control, 226-229 fat deficienc y-related diseases, 225-226 research needs, 237-238 Diethylstilbestrol, residues in food, 42-43 Dihydropyridine receptor, in muscular dysgenesis, 371 Diseases, muscle, see Muscle diseases and disorders Disorders, muscle, see Muscle diseases and disorders DNA polymerase, cardiolipin association, 286 Doxorubicin, cardiolipin association, 287-288 Drugs, immunotoxic, in foods, 43 Duchenne muscular dystrophy, 351-359 Duplications, dystrophin gene, 356-357 Dysgenesis, muscular, 370-371 Dy splasia skeletal, 392-393 urinary, 392-393 Dystrophin function in muscle cells, 355 gene, 353-357 isoforms, 355 structural domains, 354

E Ehlers-Danlos syndrome, 391-394 classification table, 393 Elastin, abnormal calcification. 401 Electrical stimulation, for muscle function, 347 Electrom yography for muscle diseases and disorders, 344 myasthenia gravis. 366-367 Endopeptidase system, in dermatosparaxis. 390-391 Endothelial cells. anti-cardiolipin antibody binding, 307-308 Energy metabolism disorders glycolysis-related acquired disorders, 379

glycolytic, 374-379 lipid metabolism, 381-383 mitochondria1 oxidation, 379-38 1 myotonic syndromes, 383-387 research needs, 408 Enteropathies, food-sensitive celiac disease, 29-30 cow’s milk protein intolerance. 30-31 soy protein, 3 1 Epidermolysis bullosa, 398 simplex, 398 Epilepsy, 372 Epithelial cells, gut, antigen uptake, 12-14 Epitope, cardiolipin, 312-314 Erysipelas insidiosa, 398-399 Erythromycin treatments, effect of bifidobacteria consumption, 84-86 Essential fatty acid deficiency, cardiolipin acyl changes, 294-295 Ethanol, effects on cardiolipin content, 301 fatty acyl composition, 297 Examinations, for muscle diseases and disorders, 343 Excitation-contraction coupling, in muscular dysgenesis, 371 Exercise effects on cardiolipin content, 300-301 muscle function, 368-369 research needs, 407 tests for muscle disorders, 348-349 Eye muscles, ocular myopathies, 361-362

F Familial periodic paralysis, 384-386 Fasting anti-inflammatory effect, 52 tests for muscle disorders, 348-349 Fat, dietary, see also Fatty acids butterfat comparison with other food fats, 168-169 content in dairy products, 150-152 disappearance data, 133-136 effects on cancer risk animal studies, 219-223

430

INDEX breast cancer, 21 1-212 colon cancer, 212-213 fat composition and source, 2 13-2 15,221-222

fat quantity, 210-213,220-221 milk and dairy products, 215-219 trace lipids, 223 coronary heart disease risk, 153-154 LDL modification, 171-181 serum lipoproteins and apolipoproteins, 154- I59 monounsaturated fatty acids, 171- 178 n-3 polyunsaturated fatty acids, 178- 179

saturated and unsaturated fatty acids, 159-170 thrombosis tendency, 181-185 intake data, 136-140 optimal balances for CHD protection, 206-208

response determinants genetic factors, 192-194 quantity and composition, 189-192 triglyceride structure, 185-188 Fatigue, muscular, 341-342.365-368 Fatty acids, see also Fat, dietary cardiolipin, positional distribution, 290 essential fatty acid deficiency. 294295

in milkfat, 144-146 n-3, 147-148

predominant, 144-146 trans isomers, 146-147 monounsaturated cis monounsaturates, 171-175 effects on serum lipoproteins and apolipoproteins, 159-178 trans fatty acids, 175-178 polyunsaturated effects on serum lipoproteins and apolipoproteins, 159-170 incorporation into cardiolipin, 29 1-294 in milk, 146 n-3, 178-179 saturated effects on serum lipoproteins and apolipoproteins, 159-170 incorporation into cardiolipin, 295-297

selectivity for incorporation into cardiolipin, 292-293 trans in fats and oils, 146-147 incorporation into cardiolipin, 295-297

Fibronectin deficiency in Ehlers-Danlos syndrome, 392-394

role in connective tissue carcinomas, 404-405

Fibrosis, hepatic, 403-404 Flora, microbial in gastrointestinal tract control, 47-50 distribution, 71 Food allergens characterization and identification, 40-42 detection and avoidance, 46 research needs, 54 Food allergies, characterization and classification, 25-26 Food antigens detection and avoidance, 46 research needs, 55 Food hypersensitivities classification, 26 delayed-type, 19-21, 29 nonreaginic, 28-29 reaginic, 27-28 Food intolerance, 25-26, see also Intolerance Foods, hypoallergenic, 47 Food technology, modification of milkfat composition, 235-236

G Gallstones, prevalence in men on low-cholesterol diets, 225 Gastrointestinal tract anatomy, 2-4 immune response modification by breast-feeding, 45 food antigens/allergens, 46 hypoallergenic foods, 47 microbial flora control, 50 nutritional therapies, 51-52 oral tolerance induction, 50-5 1 nutrition effects, 44-45 research needs, 53-56

43 1

INDEX role of bacterial agents, 35-37 foodborne chemicals, 41-43 parasitic agents, 39 viral agents, 38 immune system diseases autoimmunity, 32-33 food hypersensitivity, 25-26 food intolerance, 25-26 food-sensitive enteropathies, 29-3 1 immune complex-related, 32-33 inflammatory bowel disease, 3 1-32 intestinal cancer, 24-25 microbial infections, 23-24 nonreaginic hypersensitivities, 28-29 reaginic hypersensitivities, 27-28 immunosuppression, 33-34 microbial ecology, 71-72 nonspecific immune mechanisms, 4-6 specific immune response, 6-8 antigen uptake, 12-15 cell-mediated responses, 19-21 common rnucosal immune system, 21 gut-associated lymphoid tissue, 10-12 humoral responses, 15-19 immunocompetent cells, 8-10 nonspecific mechanisms, 4-6 oral tolerance, 22 phases, 7 stimulation of specific immunity, 2 1-22 Gene mapping, for muscle diseases and disorders, 409 Gene mutation, dystrophin gene, 355-356 Genes dystrophin, 353-357 Steinert’s disease, 361 Genetic diseases, in connective tissue, 389-401 Genetic factors. in response to dietary fat, 192-194 Gene transfer, pAMPl from Lactobacillus reuteri to Enterococcus faecalis in uiuo, 114-1 15 P-Glucuronidase, fecal. lactic culture effects, 99-10] Glutamate d yhydrogenase, cardiolipin association, 285-286

Gluten intolerance, 29-30 a-GI ycerol-phosphate dehydrogenase, cardiolipin association, 283 GIycogenesis type 1 Won Gierke’s disease), 374-377 type 2 (Pompe’s disease), 377 type 3 (Cori-Forbes disease), 377 type 4 (Andersen’s disease), 377 type 5 (McArdle’s disease), 377-378 type 6 (Hers’ disease), 378 type 7 (Tauri’s disease), 378-379 GI ycol ysis acquired diseases, 379 enzyme deficiencies, table, 375 relationship to meat quality, 379 Goodpasture’s syndrome, 33 Grave’s disease, 33 Growth promoters, residues in food, 42-43 Gut-associated lymphoid tissue, 10-12

H HDL, see High-density lipoprotein cholesterol Hematopoiesis, characterization, 8 Hepatic fibrosis, 403-404 Hers’ disease, 378 Hex I1 form, cardiolipin, 271-273 High-density lipoprotein cholesterol, effects of lactic acid cultures, 93 HIV infection, see Human immunodeficiency virus infection Homocystinuria, 397 Homogentisic acid oxidase deficiency, 397 Hormones, effects on cardiolipin content, 300 Host specificity, adherence of probiotic cultures, 75 Human immunodeficiency virus infection, 34 effects of hydrogen peroxide-producing Lactobacillus acidophilus, 114 enteric pathogens in, 34 Humoral immunity effects of lactic cultures, 107-1 10 gut, 15-19 Hyaluronate synthesis, in Marfan syndrome, 396 Hyperexia, malignant, 386-387,408

432

INDEX

Hyperkalemic periodic paralysis, 385-386 Hypersensitivities, food-related classification, 26 delayed-type, 19-21.29 nonreaginic, 28-29 reaginic, 27-28 Hyperthermia, malignant, 386-387 Hyperthyroidism cardiolipin content, 301 cardiolipin fatty acyl composition, 297-298 Hypertrophy, muscle, 363-364 Hypokalemic periodic paralysis, 384-385

I 5-Iminodaunomycin,cardiolipin association, 288 Immune response, gastrointestinal tract antigen uptake, 12-15 cell-mediated, 19-21 common mucosal immune system, 21 effect of foodborne chemicals, 41-43 microbes and microbial products, 35-40 gut-associated lymphoid tissue, 10-12 humoral responses, 15-19 immunocompetent cells, 8-10 modification food antigens/allergens, 46 hypoallergenic foods, 47 microbial flora control, 50 nutritional therapies, 5 1-52 oral tolerance induction, 50-51 role of breast-feeding, 45 nonspecific, 4-6 nonspecific mechanisms. 4-6 nutritional effects, 44-45 oral tolerance, 22 research needs, 53-56 specific, 6-8 stimulation of specific immunity, 21-22 Immune surveillance. in intestinal cancer, 24-25 Immune system common mucosal, 21 stimulation with lactic cultures, 107-110, 120 Immunoglobulins F, region, 16

kA adjuvants for gut responses, 22 deficiency, selective, 33-34 induction by oral immunization, 21-22 related hephropathy, 33, 35.40 secretory malnutrition effects, 44 structure and functions, 17-18 structure and functions, 16-17 IgE, functions, 18-19 IgG, functions, 19 IgM, functions, 19 isotypes. 16-17 properties. table. 17 structure, 15-16 Imrnunostimulation, ingested bacteria, 49-50 Immunosuppression gastrointestinal immune system, 33-34 nutritional therapies, 5 1-52 Immunotoxicity, ingested chemicals, 4 1-43 Inflammatory bowel disease, 31-32 Inflammatory myopathies, 362 Inherited myoclonus, 373 Interstitial lung disease, 402-403 Intestinal diseases, nutritional therapies, 51 Intestinal tract cancer, 24-25 Intestine. adherence of probiotic cultures, 74-77 Intolerance cow’s milk protein, 30-31 food, 25-26 gluten, 29-30 lactose, 79-84 research needs, I18 Ionophores, cardiolipin, 273-274 Irradiation side-effects, Lacrobacillus acidophilus effects, 90-91 Irritable bowel syndrome, unfermented acidophilus milk effects, 86 Ischemia, effects on cardiolipin content. 30 1-302

K Killer cells mononuclear, 10 natural, 10

433

INDEX

L 0-Lactam antibiotics. in foods, 43 Lactate levels, blood, 349 Lactic acid bacteria bacteriocin production, 77-78 cultures, health targets cancer suppression. 99-107 constipation, 110-1 1 I diarrhea, 84-92 immune system stimulation. 107-1 10 lactose digestion. 79-84 vaginitis. 1 1 1-1 14 fecal recovery, 72-74 health effects, 70 history. 69-70 research needs, 116-120 reviews. 70 safety issues, 114-1 15 strain selection. 115-1 16 Lactobacilli bacteriocin production, 77-78 effects on cancer-related fecal enzyme levels, 99-101 cancer suppression, 99-103 cholesterol reduction, 92-97 prevention of enterotoxigenic Escherichia coli-induced diarrhea. 86 Lactobacillus acidophilus effects on candidal vaginitis, 112-1 14 immune response, 108-109 irradiation side-effects, 90-91 fecal recovery, 72-74 hydrogen peroxide-producing, effects on HIV, 114 Lactose digestion, effects of lactic cultures, 79-84 intolerance, research needs, I18 Lamina propria, components, 10 Laminin. role in connective tissue carcinomas, 404-405 Lathyrism, 388 LDL, see Low-density lipoprotein cholesterol Left ventricular function, effects of age, 369 Leukocytes characterization, 8

distribution, 10 functions in specific immunity. 9 Limb girdle muscular dystrophy, 359 Lipid metabolism, disorders, 381-383 Lipopolysaccharide effects on gastrointestinal immunity, 35 inhibition of C L binding to IgG aCL. 310 Lipoproteins, serum butter effects, 166-169 dietary fat type and source effects, 154- 159 lactic acid cultures effects, 93-96 in MUFA-rich vs. lowfat diets, 174, I89 n-3 polyunsaturated fatty acid effects, 178- 179 Low-density lipoprotein cholesterol effects of lactic acid cultures, 93-96 modification, dietary fat effects, I7 1 18 1 Lung disease, interstitial, 402-403 Lupus anticoagulant, anti-cardiolipin antibody binding, 308-309 Lymphocytes, see also B lymphocytes, T lymphocytes intraepithelial, 10-1 2 in lamina propria, 10-12

-

M Maldigestion, lactose. 79-84 Malignancy acyl differences in cardiolipin, 298-299 effects on cardiolipin content, 302 Malignant hyperexia, 386-387.408 Malnutrition effects on gastrointestinal immunity, 44-45 research needs. 55 Marfan syndrome, 396 Mast cells, characterization. 10 Maximum oxygen uptake capacity, effects of age, 368-369 McArdle's disease, 377-378 M cells, function, 15 Meat quality, see also Pork carnitine relationship, 382-383 research needs. 406-407 role in mitochondria1 metabolism disorders, 380-381

434

INDEX

Membrane disordering, ethanol-induced, cardiolipin-related resistance, 302-304 Membrane fluidity ethanol-induced. cardiolipin-related resistance, 302-304 phospholipid acyl changes, 299 Memory, immune system. 6.8 3-Methylhistidine,research needs, 406-407 Microbial flora, gastrointestinal tract control, 47-50 distribution, 71 Microbial infections, gastrointestinal immune system, 23-24 Microorganisms cardiolipin, 263 phosphatidylglycerol, 262 Milkfat, see also Dairy products cholesterol, 150 composition n-3 fatty acids, 147-148 predominant fatty acids, 144-146 trans isomers, 146-147 consumption disappearance data, 133-136 intake data, 136-143 effect on serum lipids animal studies, 200-203 human trials. 194-200 overall impact on health, 224-229 phospholipids, 150 relationship to CHD risk, 203-206 research needs, 236-239 in total diet CHD and cancer risks, 224-225 products meeting current recommendations available products, 229-232 modified products, 232-236 research needs. 237-238 trace lipids, 150, 223 triglyceride structure, 148-149 Mitochondria cardiolipin content, ischemia effects, 301-302 membrane fluidity, cardiolipin effect, 299 metabolism, lactate indicator, 349 oxidative metabolism disorders, 379-381

protein importation and translocation. cardiolipin role. 275-277 bis(Monoacylg1ycero)phosphate degradation. 274-275 distribution. 262 synthesis. 262. 264 Mononuclear killer cells, characterization. 10 Monounsaturated fatty acids cis monounsaturates. 171-175 effects on serum lipoproteins and apolipoproteins. 159-170 trans fatty acids. 175-178 MUFAs, see Monounsaturated fatty acids Muscle area, cross-sectional. 346 Muscle diseases and disorders age effects. 368-369 atrophy, 363-364 clinical symptoms muscle fatigue. 342 muscle function, unusual. 343 muscle weakness, 341-342 myoglobinuria. 342-343 connective tissue diseases acquired disorders. 401-405 genetic diseases, 389-401 nutritional diseases. 387-389 diagnosis biopsies, 345 blood chemistry, 343-344 clinical examination, 343 electrical stimulation tests, 347-348 electromyography, 344 nuclear magnetic resonance. 345 protein turnover, 350 provocation tests, 348-349 exercise effects, 368-369 glycolytic disorders, 374-379 acquired diseases, 379 lipid metabolism disorders, 381-383 mitochondria1oxidation disorders. 379-381 myotonic syndromes, 383-387 hypertrophy, 363-364 inflammatory myopathies, 362 muscular dysgenesis, 370-371 myoclonus, 371-373 neuropathies, 364-365 research needs, 405-409

435

INDEX Muscle fatigue. 341-342 Muscle function electrical stimulation. 347-348 provocation tests. 348-349 related quadriceps. 348 unusual. 343 Muscle weakness. 341-342 in Duchenne and Becker's muscular dystrophies. 352 Muscular dysgenesis. 370-371 Muscular dystrophies Becker's. 35 1-359 Duchenne. 351-359 limb girdle. 359 myotonic. 359-361 ocular myopathies. 361 -362 Steinert's disease. 359-361 Myasthenia gravis. 33. 366-367 Myoclonus. 371-373 epilepsy. 372 inherited. in Hereford Cattle. 373 spastic mice. 372-373 spontaneous. 372 Myoglobinuria. 342-343 Myopathies atrophic. 363-364 inflammatory. 362 ocular. 361-362 Myositis. active. 342 Myotonia congenita. 383-384 Myotonic muscular dystrophy. 359-361

N NADH ubiquinone oxidoreductase. cardiolipin association. 286 Natural killer cells. characterization. 10 Neuropathies. muscular. 364-365 Nitroreductase. fecal. lactic culture effects. 99-10! Nuclear magnetic resonance. for muscle disease/disorders. 345. 406 Nutrition. effects on gastrointestinal immunity. 44-45 Nutritional diseases. of connective tissue. 387-389 Nutritional therapies AIDS. 51-52 immunosuppressed conditions. 5 1-52 intestinal diseases. 5 I

0 Obesity. low-fat diet effects. 226-229 Ocular myopathies. 361-362 Oral immunization. for IgA responses. 2 1-22 Oral tolerance induction. autoimmune therapy by. 50-5 I as specific response. 22 Osteogenesis imperfecta. 394-396 classification system. 395 Osteoporosis. prevalence in women on low-cholesterol diets. 225-226 Oxidation. cardiolipin. 275 Oxidative metabolism. mitochondria1 disorders. 379-381

P Pale. soft, and exudative pork. see Pork Paramyotonia congenita. 384 Parasites. gastroenteritis-producing. 39 Pathogens. microbial. control. 47-48 Pediatrics. effects of lactic cultures on diarrhea. 87-89 Penicillin. in foods. 43 Peptidyl lysyl hydroxylase defect. in Ehlers-Danlos syndrome, 392-393 Peptidyl lysyl oxidase defect. in Ehlers-Danlos syndrome. 392-393 Periodic paralysis hyperkalemic. 385-386 hypokalemic. 384-385 Periodontal disease. in Ehlers-Danlos syndrome. 392-393 Pesticide residues, in foods. 43 Peyer's patches accessory cells, 15 characterization. I5 components. 10 gut antigen uptake. 14-15 Phosphate carrier protein. cardiolipin association. 280-281 Phosphatidy lglycerol in animals. 263 degradation. 274-275 in microorganisms. 262 in plants. 262-263 synthesis

436

INDEX

intracellular location, 267-270 pathway, 265-266 Phospholipase A*. cardiolipin association, 284-285 Phospholipids, in milkfat, 150 Pigmenturia, 344 Plants acyl composition of phosphatidylglycerol, 299 cardiolipin, 263-264 phosphatidylglycerol, 262-263 Platelets, anti-cardiolipin antibody binding, 307 Polyarthritis. 398-399 Polychlorinated biphenyls, in foods, 43 Pol ygl ycerophospholipids abundance, 261-262 degradation, 274-275 discovery, 261 synthesis intracellular location, 267-270 pathways, 264-267 Polymyositis, 362 Polyunsaturated fatty acids, see Fatty acids, polyunsaturated Pompe's disease, 377 Porcine stress syndrome, 379,408 Pork, see also Meat quality dark, firm, and dry (DFD), 379,408 pale, soft, and exudative (PSE). 379, 408

Potassium supplements, for hypokalemic periodic paralysis, 385 Probes, radioactive, cardiolipin-derived, 3 16-3 17

Probiotic cultures adherence in human intestine, 74-77 ingestion, 48-49 lactic acid bacterial strains, 115-1 16 Procollagen endopeptidase, in Ehlers-Danlos syndrome, 392-393 Progressive myoclonus epilepsy, 372 Protein deficiency, effects on connective tissue, 388-389 Protein kinase C, cardiolipin association, 285

Protein turnover in atrophic muscle, 364 for myopathy diagnosis, 350 research needs, 406-407

PSE pork. see Pork Pyruvate dehydrogenase deficiency, in muscle, 380

Q Quadriceps muscle, testing, 347

R Radioactive probes, cardiolipin-derived, 3 16-3 I 7 Rapeseed oil, incorporation into cardiolipin, 295-297 Reactive epitope, cardiolipin, 312-314 Red blood cells, anti-cardiolipin antibody binding, 308 Replacement therapy, for Duchenne and Becker's muscular dystrophies, 358 Rhabdomyoma, 342-343 Rheumatoid arthritis, 398-399 Rice, polychlorinated biphenyl-contaminated, 43 Risk factors cancer animal studies. 219-223 breast cancer. 211-212 colon cancer, 212-213 milkfat relationship, 224-225 research needs, 237 coronary heart disease effects of milk and dairy products, 203-206

milkfat relationship, 224-225 research needs. 237

S Safety. lactic acid bacteria consumption, 114-1 15

Saturated fatty acids effects on serum lipoproteins and apolipoproteins. 159-170 incorporation into cardiolipin. 295-297 Scleroderma, 402 Scurvy. 387-388 sIgA. see IgA. secretory Sodium ion. ionophoretic capability of cardiolipin, 273-274 Soy protein enteropathy. 31

437

INDEX Spastic mice, 372-373 Species differences, cardiolipin acyl composition, 2% Staphylococcal enterotoxin, effects on gastrointestinal immunity, 35 Starvation, effects on connective tissue, 388-389 Steinert’s disease, 359-361 Steroidogenesis. cardiolipin, 27 1 Strain differences, lactic cultures, 80-82 Systemic lupus erythematosus anti-cardiolipin antibodies, 305 characterization, 400 genetic basis, 400 treatment, 401 Systemic sclerosis, 402

T Tauri’s disease. 378-379 Temperature, effects on cardiolipin content, 300 cardiolipin fatty acyl composition, 299 Testosterone, effects on cardiolipin content, 300 Thomsen’s disease, 383-384 Thrombosis anti-cardiolipin antibodies, 304-305 dietary fat effects, 181-185 Thymectomy treatment, for myasthenia gravis. 367-368 T lymphocytes characterization, 8

cytotoxic, gut-associated, 19-2 I effector functions, 8 regulatory functions, 8 splenic, aCLs binding, 309-310 Triglycerides acyl differences in cardiolipin, 298-299 in milkfat, 148-149 serum, effects of lactic acid cultures, 93-96 structure in dietary fats, 185-188 Tumor cells. cardiolipin content, 302 Tumor growth, effects of lactic cultures, 103-107. 119-120

U Ulcerative colitis, 31-32 Ultrasound, for muscle cross-sectional area, 346

V Vaginitis, effects of lactic cultures, 1 1 1-114 Viral agents, gastroenteritisproducing, 38 Viral myocarditis, 33 Vomitoxin, effects on gastrointestinal immunity, 35.40 Von Gierke’s disease, 374-377

W Weakness, muscle, 341-342 Weight control, relationship to low-fat diets, 226-229

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