Low Density Lipoproteins

Low Density Lipoproteins

Ed£ted by

Charles E. Day The Upjohn Company Kalamazoo, Michigan

and

Robert S. Levy Department of Biochemistry University of Louisville School of Medicine Louisville, Kentucky

PLENUM PRESS· NEW YORK AND LONDON

Library of Congress Cataloging in Publication Data

Main entry under title:

Low density lipoproteins.

Includes bibliographical references and index. 1. Lipoproteins. 2. Lipoproteins-Analysis. 3. Metabolism, Disorders of. 4. Arte·

riosclerosis. I. Day, Charles E. II. Levy, Robert Sigmund, 1921- [DNLM: 1. Lipoproteins, LDL. 2. Arteriosclerosis-Metabolism. 3. Arteriosclerosis­Prevention and control. 4. Hyperlipemia. QU85 L912] QP552.L5L68 599'.01 '9245 76-25840 ISBN-13: 978-1-4684-2252-8

ISBN-13: 978-1-4684-2252-8 001: 1 0.1 007/978-1-4684-2250-4

© 1976 Plenum Press, New York

e-ISBN-13: 978-1-4684-2250-4

Softcover reprint of the hardcover 1 st edition 1976

A Division of Plenum Publishing Corporation 227 West 17th Street, New York, N. Y. 10011

All rights reserved

No part of this book may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission from the Publisher

Contributors

FrankP. Bell, Department of Pathology, McMaster University, Hamilton, Ontario, Canada

Thomas R. Blohm, Merrell-National Laboratories, Division of Richard­son-Merrell, Inc., Cincinnati, Ohio

G. D. Calvert, Department of Clinical Chemistry, Queen Elizabeth Hospi­tal, Birmingham, England

German Camejo, Laboratorio de Lipoproteinas, Centro de Biofisica y Bioquimica, Instituto Venezolano de Investigaciones Cientificas, Caracas, Venezuela

Charles E. Day, The Up john Company, Kalamazoo, Michigan Shlomo Eisenberg, Lipid Research Laboratory, Department of Medicine B,

Hadassah University Hospital, Jerusalem, Israel Waldo R. Fisher, J. Hillis Miller Health Center, Department of Medicine,

University of Florida, Gainesville, Florida Santibrata Ghosh, Department of Biochemistry, Northwestern University

Medical and Dental Schools, Chicago, Illinois Herbert]. Kayden, Department of Medicine, New York University School

of Medicine, New York Gerhard M. Kostner, Institute of Medical Biochemistry, University of

Graz, Graz, Austria Peter Laggner, Institut fUr Rontgenfeinstrukturforschung, Oster­

reichische Akademie der Wissenschaften und Forschungszentrum, Graz, Austria

Diana M. Lee, The Cardiovascular Research Program, Oklahoma Medi­cal Research Foundation, Oklahoma City, Oklahoma

Alfredo Lopez-S, Department of Medicine, Louisiana State University School of Medicine, New Orleans, Louisiana

v

VI Contributors

Josef R. Patsch, Medical Department, University of Innsbruck, Inns­bruck, Austria (Present Address: Division of Atherosclerosis and Lipoprotein Research, Baylor College of Medicine-The Methodist Hospital, Houston, Texas)

L. Fred Roensch, Merrell-National Laboratories, Division of Richardson­Merrell, Inc., Cincinnati, Ohio

o. A. Schjeide, Department of Biological Sciences, Northern Illinois Uni­versity, DeKalb, Illinois

Preface

Low density lipoproteins (LDL) are pathophysiologically important be­cause of their central role in the disease atherosclerosis and because atherosclerosis is the leading cause of death in developed countries. Many researchers believe that a more detailed knowledge of the struc­ture, function, and metabolism of LDL may eventually lead to a means to control atherosclerosis. For this reason a fairly large research effort has gone into the investigation of LDL over the past few years. The purpose of this book is to collect and summarize in one place most of the pub­lished information on LDL through 1975. To this end more than 1500 references are cited in the papers that make up this volume.

The A, B, C apolipoprotein classification system was adopted for use throughout this work. In addition to the A, B, C, and "D" families of apolipoproteins, apoE is used to designate the "arginine-rich" apolipo­protein. This classification system is used because it is far less cumber­some than other proposed classification schemes for apolipoproteins.

In reviewing the extant information on LDL, one becomes painfully aware of the major gaps that exist in our knowledge about this lipopro­tein. The most obvious lack of information is on the apolipoprotein of LDL (apoB). Even the number of peptides constituting apoB is un­known. Estimates range from one to many. Although the apoA and apoC families of peptides have been sequenced, no sequence informa­tion is available for apoB. Elucidation of the structure and properties of apoB is a major problem to be confronted in future studies.

In the past two years much has been learned about LDL catabolism primarily from tissue culture studies. The current view is that LDL is picked up and degraded by peripheral cells such as fibroblasts and smooth muscle cells. However, since these cells are unable to degrade the

vii

Vlll Priface

sterol nucleus, it is postulated that a transport system must exist for carrying cholesterol back to the liver where it can be degraded and excreted as bile acids. This "reverse cholesterol transport" hypothesis of LDL catabolism needs to be investigated more extensively.

Since high LDL levels defmitely contribute to premature atherosclerosis, better means to control the atherogenicity of LDL are urgendy needed. Such control may take the form of either an absolute reduction in LDL levels or a modification in the structure of LDL. Cau­tion should be exercised in reducing LDL levels since such reduction might contribute to increased deposition in peripheral tissues, including arteries. As discussed in the last chapter of this book, bile acid seques­trants probably are the best agents to reduce LDL concentrations phar­macologically. As evidenced by decreased cardiovascular mortality, these agents also appear to inhibit or reverse atherosclerosis. Thus, control of LDL levels may be clinically quite significant. Definitely, better and more varied means of LDL control are needed.

We feel that these are a few of the major gaps in our knowledge of LDL that should be explored in the future. Ideally, a method to effec­tively control atherosclerosis will evolve from such studies. It is our de­sire that this volume will be useful to the many investigators who un­doubtedly will be involved in this quest. We hope that this compilation of information on LDL will contribute in some small way to the eventual conquest of the atherosclerotic scourge of mankind.

Kalamazoo, Michigan Louisville, Kentucky

Charles E. Day Robert S. Levy

Acknowledgments

We express our sincere thanks to the authors of each chapter whose work made this book possible. It was a pleasure working with everyone. Our appreciation is also extended to our secretaries, Judy Woods, Cindy Shattuck, Audrey Bos, and Lora Abell, who spent many hours working on all facets of this project. Also, we wish to thank The Up john Company (CED) and the University of Louisville School of Medicine (RSL) for granting us the time to complete this volume on low density lipoproteins.

A few authors in this volume have reproduced figures published previously. We gratefully acknowledge the following sources (and their publications) for permission to reproduce this material: Growth Publish­ing Company, Inc. (Growth); Professor G. A. Kerkut (Comparative Bio­chemistry and Physiology); The Rockefeller University (Journal of General Physiology and Journal rif Clinical Investigation); ASP Biological and Medical Press B. V. (Atherosclerosis and Biochimica Biophysica Acta), Springer-Verlag, New York, Inc. (Immunogenetics), Academic Press, Inc. (Advances in Lipid Research, Analytical Biochemistry, and Chemistry and Molecular Biology of the Intercellular Matrix), The Biochemical Society (The Biochemical Journal), American Chemical Society (Journal of the American Chemical Society), Dr. DeWitt S. Goodman, Dr. E. Shafrir, and Dr. M. Burstein.

ix

C. E.D. R. E. S.

Contents

I. Isolation and Structure

Chapter 1 Isolation and Characterization of Low Density Lipoproteins Diana M. Lee

I. Introduction ......................................... 3 A. The Origin and Defmition of the Term "Low Density

Lipoproteins" ..................................... 3 B. Nomenclature Based on Protein Moieties ............ 4

II. Isolation Methods ..................................... 6 A. Electrophoretic Technique ......................... 6 B. Chemical Fractionation ............................ 6 C. Immunochemical Fractionation ..................... 10 D. Chromatographic Techniques ...................... 11 E. Ultracentrifugation ................................ 11

III. Preservation of LDL .................................. 17 IV. Assessment of Purity .................................. 17

A. Analytical Ultracentrifugation ...................... 18 B. Zonal Electrophoresis .............................. 18 C. Immunology ...................................... 19

V. Immunochemical and Chemical Properties of LDL and LP-B ................................................ 19 A. Immunochemical Properties ....................... 19 B. Lipid and Protein Composition ..................... 21 C. Amino Acid Composition .......................... 26 D. Studies of the Protein Moieties ..................... 26

xi

Xli Contents

E. Composition and Concentration of Apolipoproteins in LDL Subfractions ................................. 29

VI. Physicochemical Properties of LDL and LP-B with an Interpretation of Molecular Species .................... 31

VII. Conclusions .......................................... 37 References

Chapter 2 Physicochemical Characterization of Low Density Lipoproteins Peter Laggner

38

I. Introduction ......................................... 49 II. Hydrodynamic Analysis. Compositional and Structural

Heterogeneity ........................................ 50 III. Morphology .......................................... 53

A. Electron Microscopy ............................... 53 B. Small-Angle X-Ray Scattering ...................... 54

IV. Internal Structure and Molecular Interactions ........... 56 A. The Organization and Physical State of Lipids ....... 56 B. The Disposition and Conformation of Protein ....... 60

V. Summary and Conclusions ............................. 62 References ........................................... 66

II. Metabolism Chapter 3 Mechanisms of Formation of Low Density Lipoproteins: Metabolic Pathways and Their Regulation Shlomo Eisenberg

I. Introduction ......................................... 73 II. Composition and Structure ............................ 74

III. Metabolic Conversion of Lipoproteins .................. 76 A. Human .. ..... ................................... 76 B. Rat .............................................. 77

IV. Mechanism of Formation of Intermediate Lipoproteins .. 79 V. LDL Synthesis: An Integrated Scheme and Possible

Regulatory Mechanisms ............................... 82 A. General Considerations ............................ 82

Contents Xlll

B. Synthesis of Triglyceride-Rich Lipoproteins .......... 83 C. Formation of Intermediate Lipoproteins ............ 84 D. Clearance of Intermediate Lipoproteins without

Conversion to LDL ................................ 85 E. Conversion of Intermediate Lipoproteins to LDL .... 85 F. Removal of LDL from Circulation .................. 86

VI. Conclusions .......................................... 86 References ........................................... 87

Chapter 4 Interactions of Lipoproteins with Cells in Culture L. Fred Roensch and Thomas R. Blohm

I. Introduction ......................................... 93 II. Interactions of Lipoproteins with Human Fibroblasts: The

LDL Receptor ........................................ 94 III. Interactions of Lipoproteins with Other Cell Types ...... 98 IV. Interactions of Lipoproteins with Smooth Muscle Cells: Cell

Proliferation ......................................... 104 References ........................................... 107

Chapter 5 Lipoprotein Lipid Exchange in Biological Systems Frank P. Bell

I. Introduction ......................................... III II. Phospholipid Exchange ............................... 112

A. Exchange between Lipoproteins .................... 112 B. Exchange between Lipoproteins and Cells, Membranes,

and Tissues ....................................... 114 III. Cholesterol Exchange ................................. 116

A. Exchange between Lipoproteins .................... 116 B. Exchange between Lipoproteins and Cells, Membranes,

and Tissues ....................................... 116 IV. Cholesteryl Ester Exchange ............................ 118 V. Triglyceride Exchange ................................ 118

VI. Unesterified Fatty Acid Exchange ...................... 118 VII. a-Tocopherol Exchange ............................... 119

VIII. Factors Modifying Lipid Exchange ..................... 120 A. Temperature ..................................... 120

XlV Contents

B. Organic Solvents ................................. . C. Surface Charge .................................. . D. Exchange Proteins ............................... . E. Lipoprotein Fatty Acid Composition ............... . F. Lecithin:Cholesterol Acyltransferase (LCAT) ........ .

IX. Physiological Significance of Lipid Exchange ........... .

120 120 120 121 121 121 123 126

X. Possible Mechanisms of Lipid Exchange ............... . References

Chapter 6 Effect of Exercise on Serum Lipids and Lipoproteins Alfredo Lopez-S

I. Exercise and Serum Cholesterol ........................ 135 II. Exercise and Serum Triglycerides ...................... 136

III. Exercise and Serum Phospholipids ..................... 138 IV. Exercise and Serum Lipoproteins ...................... 139 V. Exercise and Metabolism of Serum Lipids and Lipoproteins 142

VI. Conclusions .......................................... 145 References ........................................... 146

III. Aberrations of Metabolism

Chapter 7 The Hyper-/3- and Hyperpre-/3-lipoproteinemias Waldo R. Fisher

I. Introduction ......................................... 151 II. The Hyperlipoproteinemic Syndromes. . . .. .. .. .. . . . .. . . 153

A. Hyper-~-lipoproteinemia .......................... 154 B. Combined Hyperlipoproteinemia ................... 155 C. Hyperpre-J3-lipoproteinemia ....................... 156 D. Sporadic Hyperlipoproteinemia .................... 157

III. The Structure of LDL in Hyperlipoproteinemia ......... 158 IV. Metabolic Derangements in the Synthesis of LDL ........ 163

A. The Enzymatic Hydrolysis of VLDL Triglyceride .... 164 B. Kinetic Studies on VLDL Triglyceride Metabolism in

Hyperlipoproteinemia ............................. 167

Contents XV

C. Kinetic Studies on Apolipoprotein Metabolism in Hyperlipoproteinemia ............................. 169

V. Metabolic Derangements in the Catabolism of LDL ...... 173 VI. Variables in the Physiologic Control of Lipoprotein

Metabolism ........................................... 174 A. The Control of VLDL Synthesis .................... 175 B. The Conversion of VLDL to LDL .................. 177

VII. Pharmacologic Control of Lipoprotein Metabolism ....... 178 A. Nicotinic Acid .................................... 179 B. Clofibrate ........................................ 180

/ _- __ C. Cholestyramine ................................... 182

l~!!0 Conclusions .......................................... 183 References ........................................... 185

Chapter 8 Type III Hyperlipoproteinemia Josef R. Patsch

I. Definition and History ................................ 197 II. Plasma Lipids and Lipoproteins ........................ 198

A. Classes of Lipoproteins ............................ 198 B. Lipoproteins of Density 1.006-1.019 (LP-III,

Intermediate Density Lipoprotein, or IDL) . . . . . . . . . .. 200 C. VLDL (d 0.95-1.006, Sf2O-40Q) .................... 204 D. LDL (d 1.019-1.063, Sf 0-12) ....................... 208 E. Chylomicrons (d < 0.95, Sf> 400) .................. 208 F. HDL (d 1.063-1.210, F 1.21 0-9) .................... 209

III. Metabolic Defect ...................................... 210 IV. Diagnosis ............................................ 213 V. Clinical Features ...................................... 216

A. Age of Detection .................................. 216 B. Vascular Disease .................................. 216 C. Xanthomatosis .................................... 217 D. Other Clinical Features ............................ 217

VI. Treatment ........................................... 218 VII. Genetics ............................................. 219

VIII. Summary ............................................ 220 References ........................................... 221

XVI Contents

Chapter 9 Lp(a) Lipoproteins and the Genetic Polymorphisms of Lipoprotein B Gerhard M. Kostner

I. Introduction: Nomenclature and Abbreviations 229

II. Historical Development ............................... 231 III. Inherited Variants of Human Serum ~-Lipoproteins ..... 232

A. The Ag System ................................... 232 B. Relationship of the Ag System to Lp(a) Lipoproteins 234 C. Nature of the Ag Antigen ......................... 234 D. Other Polymorphic Systems of Human Serum

~-Lipoproteins .................................... 235 IV. Lp(a) Lipoproteins .................................... 236

A. Genetics of Lp(a) Lipoproteins ..................... 236 B. Possibility of Heterogeneity of the Lp(a) Factor: The

Lp(x) Antigen .................................... 238 C. Methods for Demonstration of Lp(a) Lipoproteins ... 238 D. Isolation and Purification of Lp(a) Lipoproteins ...... 243 E. Chemical and Physicochemical Properties of Lp(a)

Lipoproteins ...................................... 245 F. Distribution of Lp(a) Polypeptides among Lipoproteins

of Different Density Classes ........................ 254 G. Lp(a) Lipoprotein Values in the Normal Population .. 254 H. Lp(a) Lipoproteins in Species Other than Man. . . . .... 255 I. Relationship of Lp(a) to Histocompatibility Antigens 256 J. Lp(a) Lipoproteins, Hyperlipoproteinemia, and

Atherosclerosis .................................... 257 K. Lp(a) Lipoproteins and Liver Diseases .............. 262

V. Summary and Conclusions ............................. 263 References ........................................... 265

Chapter 10 Abetalipoproteinemia Herbert]. Kayden

I. Introduction ......................................... 271 II. The Lipoproteins and Apoproteins ..................... 272

A. HDL and Its Apoproteins .......................... 272 B. Lipoproteins with Density Less than 1.063 g/ml ...... 275

III. LCA T Activity ........................................ 276

Contents xvii

IV. Genetic Aspects ....................................... 277 V. Conclusions .......................................... 277

References ........................................... 278

IV. Comparative Biology

Chapter 11 Mammalian Low Density Lipoproteins G. D. Calvert

I. Introduction ......................................... 281 II. General Studies ....................................... 282

III. Rats ................................................. 284 A. Normolipidemia: Properties of LDL ................ 284 B. LDL Metabolism .................................. 286 C. Hypolipidemia: Orotic Acid and Essential Fatty Acid

Deficiency ........................................ 287 D. Hyperlipidemia ................................... 288 E. Hypolipidemia: CPIB, Tomatine, Glucagon ......... 288

IV. Guinea Pigs .......................................... 289 A. Normolipidemia: Properties of LDL ................ 289 B. Hyperlipidemia ................................... 290 C. LDL Metabolism .................................. 292

V. Rabbits .............................................. 293 A. Normolipidemia: Properties of LDL ................ 293 B. Hyperlipidemia ................................... 293 C. Physiological Changes ............................. 294

VI. Ungulates ............................................ 294 A. Cattle ............................................ 294 B. Sheep ............................................ 295 C. Swine ............................................ 296 D. Horses ........................................... 298

VII. Marine Mammals ..................................... 298 A. Killer Whales ..................................... 298 B. Dolphins ......................................... 299 C. Sea Lion, Walrus, and Harbor Seal .................. 299

VIII. Dogs ................................................ 300 A. Normolipidemia .................................. 300 B. Hyperlipidemia ................................... 301

XVlll Contents

C. LDL Metabolism .................................. 302 D. Bile Duct Obstruction ............................. 302

IX. Cats ................................................. 302 X. Nonhuman Primates .................................. 303

A. General Considerations ............................ 303 B. Family Cercopithecoidea (Old World Monkeys) ...... 303 C. Family Pongidae, Genus Pan (Chimpanzee) .......... 306 D. Family Cebidae (New World Monkeys) .............. 306

XI. Conclusion ........................................... 308 References .................................. '......... 309

Chapter 12 Low Density Lipoproteins of Nonmammalian Vertebrates o. A. Schjeide

I. Introduction ......................................... 321 II. Occurrence and Characteristics of "Normal" LDL of Fish,

Amphibia, Reptiles, and Birds ......................... 322 A. General Comments ................................ 322 B. Flotation Studies of Lipoproteins and Lipid Analyses . 324 C. Electrophoretic Studies ............................ 329 D. Apoprotein Analyses .............................. 330

III. Effects of Estrogens and Other Hormones on Rates of Synthesis and Composition of LDL ..................... 333

IV. Correlation between LDL of Plasma and Liver Structure 335 V. Functional Roles of LDL and VLDL in Nonmammalian

Vertebrates .......................................... 337 VI. Manipulation of Light Lipoproteins in Nonmammalian

Vertebrates .......................................... 341 A. Cholesterol Feeding ............................... 341 B. Feeding of Specific Fatty Acids and Unique Lipids ... 342 C. Fasting or Overfeeding ............................ 342 D. Ionizing Radiation and Pesticides ................... 343 E. Temperature ..................................... 344

VII. Summary ............................................ 344 References ........................................... 345

Content5

v. Relationship to Atherosclerosis

Chapter 13 Interaction of Low Density Lipoproteins with Arterial Constituents: Its Relationship with Atherogenesis German Camejo

XIX

I. General Considerations ................................ 351 II. Irritative Components of LDL ......................... 352

III. Passage of LDL across the Endothelial Barrier ........... 353 IV. Arterial Wall Components that Interact with LDL ....... 354

A. Enzymes Acting upon LDL ........................ 354 B. Elastin .............................. : . . . . . . . . . . .. 355 C. Complex Macromolecules .......................... 356

V. Affinity of LCF for Serum LDL from Coronary Heart Disease Patients ....................................... 363 References ........................................... 366

Chapter 14 Interaction of Low Density Lipoproteins with Small and Large Molecules Santihrata Ghosh

I. Introduction II. Interaction with Small Ions and Molecules ............. .

A. Binding of Hydrogen Ions ........................ . B. Binding of Metal Ions ............................ . C. Binding of Free Fatty Acids (FF A) ................. . D. Interaction with ANS ............................. .

III. Interaction of LDL with Macromolecules in Solution .... . A. Components of the Extracellular Matrix of the Arterial

Wall ............................................ . B. Mucopolysaccharide-LDL Interaction .............. . C. Heparin-LDL Interaction ......................... . D. Dextran Sulfate-LDL Interaction .................. . E. Amylopectin Sulfate-LDL Interaction .............. . F. Collagen-LDL Interaction ........................ . G. Elastin-LDL Interaction .......................... . H. Interaction with Nonionic Polymers ................ .

371 372 372 376 378 381 383

383 387 388 390 391 396 397 397

xx Contents

IV. Interaction at the Tissue Level ......................... 398 A. Isolation of Complexes from Arterial Tissue ......... 398 B. Interaction of LDL in a Gel Matrix ................. 402

V. Nature of the Interaction between LDL and Polyanions .. 408 A. Ionic Aspects of the Interaction .................... 408 B. Specific Features of the Polysaccharides Affecting

Complex Formation ............................... 410 C. Nonionic Aspects of the Interaction ................. 411

References ....................................... 412

Chapter 15 Control of Low Density Lipoproteins and Atherosclerosis with the Bile Acid Sequestrants Colestipol, Cholestyramine, and Polidexide Charles E. Day

I. Introduction ......................................... 421 II. Colestipol ............................................ 423

A. Effect on Serum Cholestrol and Triglycerides ....... 423 B. Effect on Serum Lipoproteins ...................... 425 C. Modification of Cholesterol Metabolism ............. 426 D. Reduction of Cardiovascular Mortality .............. 427 E. Side Effects ....................................... 428

III. Cholestyramine ....................................... 428 A. Effect on Serum Lipids, Lipoproteins, and Cholesterol

Metabolism ....................................... 428 B. Effect on Atherosclerosis .......................... 429 C. Side Effects ....................................... 431

IV. Polidexide ........................................... 432 V. Conclusions ........................................ ... 432

References ........................................... 433

Index ...................................................... 439