Coronary Heart Disease and the Mucopolysaccharides (glycosaminoglycans) - Chondroitin Sulfate 0398029032, 9780398029036

Citation preview

ee AOS Paar

Nee oy

mine ale

ee 104"

a

aor

ss

CORONARY HEART DISEASE AND THE MUCOPOLYSACCHARIDES (GLYCOSAMINOGLYCANS)

PLATE

1

PLATE 2

PLATE 3

PLATE 4

PLATE 5

PLATE 6

“g

Mis

i

at)«|

PLATE 7

PLATE 8

PLATE 9

Plate 1: Upper left: Normal human adult coronary artery intima cells in organ culture. Lower left: Same cells saturated with lipids and lipoproteins from human blood serum. Sudan IV stain shows heavy intracellular fat deposits. Upper right: Normal human adult coronary artery intima cells saturated with human blood serum lipids and lipoproteins; coronary cells and extra-cellular tissues are shown saturated with lipids and lipoproteins. Sudan IV stain. Lower right: Human coronary intima cells saturated with human blood serum lipids and lipoproteins three days after treatment with one dose of CSA shows complete intra- and extra-cellular clearing of lipids and lipoproteins. Sudan IV stain. (The above four photographs are supplied through the courtesy of A. Whitley Branwood, Professor of Pathology, College of Physicians and Surgeons, Columbia University, New York.) Plate 2. Cross sections of coronary arteries from x-irradiated rats fed a cholesterolcontaining diet. Left: Heavy sudanophilic stain shows almost complete lipid obstruction of lumen and complete lipid infiltration of the artery. (Control). Right: Addition of 0.4% chondroitin sulfate A to the above diet reveals no lipid deposition, completely normal artery. (CSA-treated group). Plate 3. Left: Rat coronary artery containing atheromatous stained plaque after six weeks of hypervitaminosis D, atherogenic diet. Plaque characteristically invades arterial lumen. Right: Rat coronary artery showing complete absence of plaque formation after six weeks of hypervitaminosis D, atherogenic diet plus 1% chondroitin sulfate. A. Calcium and fibrous connective tissue deposits remain. Plate 4. Normal aorta of young, 2 1/2- to 3-year old squirrel monkey of South America. These control animals are unique in that when older and thus corresponding to middle-aged or elderly human males and females, they develop natural or spontaneous atheroarteriosclerosis virtually identical to the lesions and vascular disease of the coronary arteries and aorta of human patients. Note the normal, smooth, glistening surface of the aorta, free of any lesions as found in normal healthy children. Plate 5. Monkey aorta from a control fed a cholesterol, atherogenic diet for nine months plus receiving daily saline injections. Note the significant degree of atherosclerotic plaques and infiltrative athero-arteriosclerosis throughout the aorta. The heavy red or crimson color is produced by Oil-red-O stain to bring out the localization and extent of the atheroarteriosclerotic plaques and lesions. Plate 6. Marked arteriosclerotic disease in the aorta of a monkey receiving 20 mg of heparin injections daily plus a cholesterol atherogenic diet for nine months. It is interesting to note that heparin is a sister acid mucopolysaccharide to chondroitin4-sulfate (CSA) since it is an isomeric form or derivative of dermatan sulfate (CSB).

Plate 7. Photograph of an aorta taken from a monkey following nine months of subcutaneous injection of 10 mg CSA in 1 ml of saline plus a cholesterol, atherogenic diet. Such aortas are remarkable for their uniquely healthy, glistening appearance. Plate

8. Lateral view of rat myocardial infarction (white patch area), ischemia necrosis produced within twenty-four hours by injection of isoproterenol.

and

Plate 9. Apical view of rat myocardial infarction treated as in Plate 8 with the addition of injections of CSA preceding the isoproterenol. The CSA produced significant reduction in size or area of infarction. Note also the marked increase of the coronary collateral circulation.

CORONARY HEART DISEASE AND THE MUCOPOLYSACCHARIDES (GLYCOSAMINOGLYCANS) LESTER

M. MORRISON,

M.D.

Director and Research Professor Institute for Arteriosclerosis Research

Loma Linda University School of Medicine Emeritus Consultant in Medicine University of California Center for Health Sciences

Los Angeles, California President and Founder, Southern California Hospitals Group and

O.

ARNE

SCHJEIDE,

Ph.D.

Professor of Biological Sciences Northern Illinois University, DeKalb, Illinois Formerly Lecturer in Biophysics

Department of Biophysics and Research Biologist Laboratory of Nuclear Medicine and Radiation Biology University of California School of Medicine Los Angeles, California

With a Foreword by KARL

MEYER,

M.D.,

Ph.D.

Professor of Biochemistry Belfer Graduate School of Science, Yeshiva University Professor Emeritus of Biochemistry

Columbia University, New York, New York

ee — x ay

CHARLES C THOMAS Springfield * Illinois * USA

e PUBLISHER

Published and Distributed Throughout the World by CHARLES

C THOMAS Bannerstone

«+ PUBLISHER House

301-327 East Lawrence Avenue, Springfield, Illinois, U.S.A.

This book is protected by copyright. No part of it may be reproduced in any manner without written permission from the publisher.

© 1974, by CHARLES ISBN

C THOMAS

+ PUBLISHER

0-398-02903-2

Library of Congress Catalog Card Number:

73-7569

With THOMAS BOOKS careful attention is given to all details of manufacturing and design. It is the Publisher's desire to present books that are satisfactory as to their physical qualities and artistic possibilities and appropriate for their particular use. THOMAS BOOKS will be true to those laws of quality that assure a good name and good will.

Printed in the United States of America EE-11

Library of Congress Cataloguing in Publication Data Morrison, Lester M

Coronary heart disease and the mucopolysaccharides (glycosaminoglycans ). 1. Coronary heart disease. 2. Mucopolysaccharides. I. Schjeide, Ole A., joint author. II. Title.

[DNLM: 1. Coronary disease—Therapy. 2. Mucopolysaccharides—Therapeutic

RC685.C6M67 ISBN 0-398-02903-2

use.

616.123

WG300

M879c

73-7569

1973]

Dedicated to our wives Rita and Lynn and to the memory of our late co-investigator (and friend) Dr. Wim A. Loeven (Geron-

tology Division, NIH, the United States), recently fallen in the life and death research struggle against coronary heart disease.

FOREWORD Mucopolysaccharides are glycosaminoglycans, i.e. heteropolysaccharides composed of hexosamines and non-nitrogenous sugars linked by glycosidic bonds; some also contain various substituent groups. The mucopolysaccharides of mammalian tis-

sues may be classified as (1) polycarboxylates (hyaluronic acid, chondroitin), (2) polysulfates (keratan sulfates) and (3) polycarboxy-sulfates (chondroitin-4- and -6-sulfates, previously designated chondroitin sulfate A and C, respectively; dermatan sulfates; and heparitin sulfates). The structure of these various mucopolysaccharides and the nature of their protein linkages is discussed. The functional role of the various mucopolysaccharides in connective tissues is still largely presumptive. They occur in different proportions in different tissues, and the pattern of distribution in the same tissue changes with maturation and aging. The importance of disturbances in the natural distribution of mucopolysaccharides is indicated by the clinical abnormalities characterizing the various mucopolysaccharidoses. I extend to the authors best wishes for the success of their ambitious undertaking with this monograph.

Karl Meyer

ACKNOWLEDGMENTS Grateful acknowledgment is expressed to the following coinvestigators who have helped to make our research program with chondroitin sulfate A possible: Benjamin H. Ershoff, Ph.D.; J.J. Quilligan, Jr., M.D.; K. Murata, M.D., Ph.D.; G.S. Bajwa, D.V.M., Ph.D.; R.B. Alfin-Slater, Ph.D.,; O.J. Dunn, Ph.D.; L. Freeman, Ph.D.; S. Bernick, Ph.D.; P.R. Patek, Ph.D.; W.A. Loeven, Ph.D.; R.C. Robbins, Ph.D.; C. Simp-

son, D.V.M., Ph.D.; H.C. Bergman, Ph.D.; P.G. Rucker, M.S.; H.J. Hernandez, B.S.; R. Holman, M.S.; Janet B. Brown, M.T.; and Elizabeth G. Cogswell, M.T. We also express our deep appreciation to the following for the invaluable collaboration, consultation and/or aid given to the Institute for Arteriosclerosis Research in our mucopolysaccharide research program: A. Whitley Branwood, M.D.; Hans Selye, M.D.; Karl Meyer, M.D.; Serge Renaud, D.V.M., Ph.D.,; S. Srinivasan, Ph.D.; D. Kritchevsy, Ph.D.; G.B. Jerzy Glass, M.D.; S. Bazin, Ph.D.; A. Delaunay, M.D.; M. Samitz, M.D.; L. Robert, Ph.D.; and S. Gero, M.D. Grateful appreciation is also made to the publishers, CHARLES C THOMAS, PUBLISHER, for their full cooperation.

Finally, to Mrs. Monica R. Stevens, B.A., executive secretary of the Institute for Arteriosclerosis Research, our lasting gratitude for many years of dedication, as technologist and editor of all published and unpublished investigations at the Institute for Arteriosclerosis Research and with patients in the clinical studies with chondroitin sulfate A since 1946.

CONTENTS Foreword=—Karl Meyer frou. cso reassert era cre rare

ACKOWIER 2MENES spe crests

ical ais 18 Mee oe aeenen e ceeneet

Chapter [sap Il.

INTRODUCTION Sone ee CHEMISTRY

OF THE

ee

MUCOPOLYSACCHARIDES

(GLYCOSAMINOGLYCANS ) AND THE CHONDROMUCOPROTEINS (PROTEOGLYCANS) ...-ccccccceceeeeeeseeeesees III.

Occurrence or Actip MUCOPOLYSACCHARIDES

(GLYCOSAMINOCLYCANS IV.

GENERAL

BIOLOGICAL

0) 20 cant ROLES

see

ee

OF CHONDROMUCO-

PROTEINS AND ACID MUCOPOLYSACCHARIDES V.

Errects or Appinc Actin MUCOPOLYSACCHARIDES TOMMSSUEMAND

VI.

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

ORGAN

CULTURES

Ne

ABSORPTION, DISTRIBUTION, METABOLISM

ee

AND

EXCRETION OF ACID MUCOPOLYSACCHARIDES

ADMINISTERED VII.

TO ANIMALS

AND PATIENTS

ANTITHROMBOGENIC AND ANTICOAGULANT Errects oF ActiD MUCOPOLYSACCHARIDES

VIII. IX.

Toxicrry Stupres on Actin MUCOPOLYSACCHARIDES ANTIATHEROGENIC

EFFECTS OF CHONDROITIN-4-

SULFATE, CHONDROITIN-6-SULFATE POLYSULFATE

X.

IN E.XPERIMENTAL

PREVENTION AND TREATMENT Heart DIsEASE IN HUMAN

XI.

CHONDROITIN ANIMALS

OF ISCHEMIC

PATIENTS

THEORETICAL CONSIDERATIONS REGARDING PosstinBLE MECHANISMS OF ACTION OF CHONDROITIN

SULFATES IN CELLS AND TISSUES

CORONARY HEART DISEASE AND THE MUCOPOLYSACCHARIDES (GLYCOSAMINOGLYCANS)

CHAPTER

I

INTRODUCTION Fo THOUSANDS of years extracts from human and animal tissues have been utilized for medicinal purposes. It is likely that such techniques originated even prior to recorded history.

The Ebers Papyrus of the Rosetta Stone (approximately 1550 B.C.) provides explicit instructions for preparation of extracts from various organs including liver, testes, etc. Usually, these are prescribed as cures for afflictions of corresponding organs.’ An example of the validity of at least some of these extremely ancient medicinal prescriptions is one described for night blind-

ness (xeropthalmia). Instructions for the cure of this disorder call for preparation and oral administration of an extract from ox liver. As the reader is aware, contemporary studies have revealed that vitamin A, which is present in abundance in mammalian liver, is a vitamin specifically required for optimal night vision. Interestingly, an extract containing this vitamin was suc-

cessfully employed by Sara bat Kaftibah, a physician-ophthalmologist, in treatment of the three hundred blind crusader knights imprisoned for ransom in the dungeons of Acre by Saladin following the battle of the Horns of Hattim in 1167.?% Specific exploitation of the acid mucopolysaccharides, which are extracted from connective tissues, is of much more recent origin. Chondroitic acid was first isolated and identified by Fischer and Boedeker in 1861. The 1861 publication refers to a 1G.M. Ebers, The Papyrus Ebers, trans. by B. Ebbell Milford, 1937), p. 26.

(London,

Humphrey

*§.J. Gotein, A Mediterranean Society Jewish Community of the Arabian World as Found in Gineza Papers (Oxford, Union Theological Seminary Press, 1967), Vols. 1,2,3.

51,.M. Morrison, Sarah of Toledo. To be published. ‘G. Fischer and C. Boedecker, Kiinstliche bildung von zucker aus knorpel (chondrogen), und iiber die umsetzung des genossenen knorpels in menschlichen kérper. Liebigs Ann Chem, 117:111, 1861.

3

Coronary Heart Disease and the Mucopolysaccharides

4

report by Boedeker dealing with chondroitic acid in 1854. How-

ever, the reference is incomplete. During the same year (1861), Rudolph Virchow* proposed that arteriosclerosis might be due to defects in—or deficiencies of—constituents comprising the ground substance of the connective tissue of the arterial wall— molecules which are now known to include a large proportion of acid mucopolysaccharides. However, it was not until 1909

that Elie Metchnikoff, in his Nobel Laureate address, crystalized the concept of the cells of connective tissue as comprising a system which can respond in a positive manner to infection and inflammation. He spoke specifically of the leukocyte as constituting a natural connective tissue defense against such factors. Subsequently, he applied certain of his stated principles to facets of development of—and resistance to—arteriosclerosis*® and the process of aging. Fischer and Boedeker’s extraction and isolation of chondroitin sulfate in 1861,'° the same year in which Virchow brought forth his concept of arteriosclerosis in his text on pathology, inspired other biochemists to further elucidate chemical and _ biologic

ramifications of this newly discovered substance. Krukenberg (1884),"* Morner (1889, 1895)1*'* and Schmiedeberg (1891) were explorers of the nineteenth century in the area of the chondroitin sulfates. Krukenberg should be especially recognized 5R. Virchow, Gesammelte Meidinger, 1858), p. 492.

*E. Metchnikoff,

abhandlungen

zur

wissenschaftlichen.

Sur Vetat actuel de la’ question

(Frankfort,

de Vimmunite

dans

les

maladies infectieuses. Bulletin de L’Institut Pasteur, VII (13) :545, 1909. Thid., VII(14) :593, 1909.

°E. Metchnikoff, Lecons sur Pathologie Comparee de UInflammation Faites a l'Institut Pasteur en Avril et Mai 1891, Masson, Ed. (Paris, 1892), Vol. 1.

°E. Metchnikoff,

Life of Elie Metchnikoff,

(London,

Constable,

1921),

p.

152.

Fischer and Boedecker. “C.F.W. Krukenberg, Die chemischen

bestandteile des knorpe is zeitschr. F

Biol, 20:307, 1884.

¥C.T. Morner,

Chemische

studien iiber den trachealknorpel.

Skand Arch

F

Physiol, 1:210, 1889. “C.T. Mérner, Einge beobachtungen iiber die verbreitung der chondroitin‘schwefelsaure. Z Physiol Chem, 20:357, 1895.

“O. Schmiedeberg, Uber die chemische zusammensetzung des knorpels. Arch Exp Pathol Pharmacol, 28:355, 1891.

Introduction

5

for his isolation of chondroitin-4-sulfate (chondroitin sulfate A) from mammalian cartilage by extraction with dilute sodium hydroxide. He termed the isolate “chondroitic acid.” However, as Bearn has pointed out,’* Schmiedeberg deduced that Kruken-

berg’s isolate (chondroitin-4-sulfate) is linked to protein and is present in such a complex in mammalian cartilage. In the present century, among pioneers who have blazed a lasting trail in elucidating new aspects of the properties of the mucopolysaccharides, the names of Karl Meyer'® in the United

States, Brante’’ in Sweden and Albert Dorfman’ in the United States are especially distinguished. Meyer and his colleagues,’ opened up new biological and biochemical territories by first — extracting chondroitin sulfate from cartilage with neutral solutions of calcium chloride, then easily cleaving the chondroitin

sulfates from their proteins (associated by O-glycosidic linkages) by the use of a mild alkali. Since their initial breakthrough, Meyer and his collaborators have continued to build an extraordinary foundation of vital knowledge on basic aspects of the glycosaminoglycans. Brante’s classic description, entitled Gargoylism—A Muco-

polysaccharidosis” signaled a new era of recognition of the role of mucopolysaccharides in the heritable diseases of the Inborn Error of Garrod. His isolation of chondroitin sulfate B from the livers of patients with Hurler’s syndrome opened up an entirely new clinical field. When Dorfman and his co-workers?! discovered an increased excretion of mucopolysaccharides in the urines of patients with this disease, they supported the concept that the mucopolysaccharidosis was inherited according to Mendelian law. ~ 354 'G. Bearn, Symposium

on

mucopolysaccharides.

Foreword,

Am

J Med,

47(5):661, 1969. *K. Meyer, The chemistry and biology of mucopolysaccharides and glycoproteins. Cold Spring Harbor Symposia Quant Biol, 6:91, 1938. “G, Brante, Gargoylism. A mucopolysaccharidosis. Scand J Clin Lab Invest, 4:43, 1952.

#84. Dorfman

and A.E. Lorincz:

Occurrence

of urinary acid mucopolysac-

charides in the Hurler syndrome. Proc Nat Acad Sci, 43:443, 1957.

*K. Meyer, ibid. 16 and Meyer. Brante.

=Dorfman and Lorincz.

Coronary Heart Disease and the Mucopolysaccharides

6

In modern times, the concept of the ground substance and against disease processes

as

tissue

in connective

its constituents

a defense

comprising

has been further developed

by such

investigators as Delaunay and Bazin,22 the late T. Gillman,” Morrison*! and many others. As a result of innumerable investigations, the biologic and therapeutic properties of some acid

mucopolysaccharides (e.g. hyaluronic acid and heparin) are now well known and are in wide clinical use. However, the biologic and therapeutic properties of the chondroitin-4- and -6-sulfates

(chondroitin sulfate A and chondroitin sulfate C) have only very recently been explored. Chondroitin-4-sulfate (or chondroitin sulfate A—CSA) was reported on by Crandall*’ in 1936 with respect to its efficacy in treatment of migraine headache. This relatively

crude (according to current biochemical specifications) product was administered to patients in oral form. However, the medical profession, as a whole, failed to adopt the method of treatment. During the past ten years Dr. John Prudden, Professor of Surgery at the Columbia University Presbyterian Medical Center has found chondroitin-4-sulfate to be a potent agent in wound healing of experimental animals and human patients.*° A number of reports have originated from university clinics in West Germany which describe therapeutic values of intraarticular injec-

tions of chondroitin-4-sulfate in arthritis (osteoarthritis in particular ).?*°S In Italy, CSA has been marketed for treatment of *A. Delaunay and S. Bazin, Mucopolysaccharides,

collagen and nonfibrillar

proteins in inflammation. Int Rev Connect Tissue Res, 2:301, 1964.

“T. Gillman,

Connective

Tissue

Symposium

(Oxford,

Blackwell,

1957),

/p.

120.

“L.M. and B.H.

Morrison, J.J. Quilligan, Jr., K. Murata, O.A. Schjeide, L. Freeman, Ershoff, Treatment of atherosclerosis with acid mucopolysaccharides.

Exp Med Surg, 25:61, 1967.

*T,.A.

Crandall,

G.M.

Roberts,

and

L.D.

Snorf,

The

use

of chondroitin

in

idiopathic headache (including Migraine). Am J Dig Dis Nutr, 1936, p. 289. “].F. Prudden, P. Nigel, P. Hanson, L. Friedrich, and L. Balassa, The discovery of a potent pure chemical wound-healing accelerator. Am J Surg, 119:560, 1970.

“K. Viernstein, The

effect of Eleparon

when

administered

intraarticularly.

. Med Klin, 59(8):3, 1964. *“H. Greiling, and H.W. Stuhlsatz, Biochemical studies on the mode of action

of a polysaccharide sulphate on degenerative articular disease.

25(3/4):3, 1966.

Z Rheumaforsch,

Introduction

a

peptic ulcer. However, the pharmaceutical manufacturer distributing this oral form of CSA has recently discontinued its sale in favor of other products.” Reports have appeared in the Japanese literature with respect to the therapeutic values of chondroitin sulfates and polysulfates in a large group of diseases or ailments, including hepatic, renal, vascular, ophthalmologic, otologic and nervous system conditions, and even psychosomatic

disturbances such as enuresis! Regrettably, statistically controlled clinical trials with Japanese chondroitin sulfates have not yet been carried out on human subjects, although such studies are currently underway, especially with chondroitin polysulfate, de-

rived from shark cartilage.*° One of the most important considerations in the use of any medicine is the absence of toxic symptoms and undesirable side effects. That this is the case for the chondroitin-4- and -6-sulfates is attested to by the fact that in many millions of doses of chondroitin-4-sulfate and chondroitin-6-sulfate administered to patients and the general public over a ten-year period in both oral and injectable forms, no reports of serious toxic or dangerous side effects have been reported in the scientific literature. How-

ever, some allergic reactions appear to have been observed (albeit very rarely), specifically in association with injection of CSA or CSC as a result of impurities in certain of the preparations used. The latter constitutes an additional argument for the use of natural food products as oral therapeutic agents where possible. CSA and CSC are both essentially extracts from bone, cartilage or connective tissue.

The senior author has employed acid mucopolysaccharidecontaining extracts of calf and hog aorta in 1938. In 1945 he reported the efficacy of mucosa in treatment of peptic ulcer.** As able to describe beneficial effects of aortic

clinical practice since extracts from gastric early as 1955 he was extracts in the treat-

*— Pollavini, Medical Director of Instituto de Angeli, Milan, Italy. Personal ’

communication.

“Biochemistry

and

Medicine

of Mucopolysaccharides,

Oshima, Eds. (Tokyo, Maruzen, 1962).

F. Egami,

and

Y.

=], M. Morrison, Treatment of experimental peptic ulcer with a hog stomach

extract. Am J Dig Dis, 12:328, 1945.

8

Coronary Heart Disease

and the Mucopolysaccharides

ment of cardiovascular disease.*? These results are described in further detail in subsequent chapters. Beginning in 1918, under the name of “Telatuten,’ crude arterial extracts were used in Europe for treatment of vascular disorders.** Relatively uncharacterized, crude extracts of muco-

polysaccharides are even now employed in oral form in Europe for the treatment of human hyperlipemias, hypercholesterolemias and vascular disorders.** No scientific corroboration of positive claims made for these products have been published by American scientists or clinical investigators.

In the area of disorders of mucopolysaccharide metabolism, recent extensive studies of the mucopolysaccharidoses have opened up a Pandora’s box which includes gargoylism or Hurler’s syndrome, Hunter’s, Marquio’s, Schiei’s, Marateux-Lamy syn-

dromes and related deforming, blinding, mentally arresting, cardiovascular disease syndromes, i.e. a parade of devastating clinical afflictions. Although infancy and childhood bear the brunt of these mucopolysaccharide disorders, adults are also potential victims, particularly when the recessive forms of the above syndromes are present. Although such disorders as Hurler’s syndrome represent a

pure dramatic example of disordered mucopolysaccharide metabolism, other more subtle connective tissue disorders appear to be indigenous to the general population. Among these disorders of universal occurrence are those which take place as a

function of physiological age, e.g. those of the connective tissue system of the arterial wall.

Indeed,

the disease

processes

of

athero- and arteriosclerosis and the process of aging are intimately intermingled and to some

extent each probably contributes

to

the other. The adage that “you are as old as your arteries” is “L.M. Morrison, Data submitted in Patent Application U.S.A., 1955. *Telatuten (1918). Preparation from the blood vessels of young slaughtered animals using the physiologically active components of the cell walls (intima, media, adventitia)

for arteriosclerosis.

Ampoules

1 cc., tablets 0.25 g. Manu-

facturer: Luitpold-Werk, Munich 25, Germany. Information from Gehe’s Codex (German drug encyclopedia). _

“P. Bianchini, Crinos ae

Patent).

a

heparinoid

Industria

Farmacobiologica,

anti-cholesterolemic

factor.

Como,

Italy. Producer

(Description

from

of

Letter

Introduction

9

a homily founded in the concept of the role of the arterial wall in the vital processes of aging and disease due to performance decrements of the heart, the brain and other vital organs such as the kidneys.

Among the three layers of the arterial wall (intima, media and adventitia), the ground substance structures and contributes to the metabolic life of this vital organ, thus influencing life and death processes of all tissues of the body. Accordingly, the wall of the artery can no longer be considered to be an inert tube or conduit for the coursing of blood throughout the body. Within the walls of pulsating arterial viaducts, complex and delicate life processes are in operation, creating vital metabolic interplay that can determine the course of aging and disease. In arterial

connective

tissue the roles of collagen, elastin, mucopolysac-

charides, enzymes,

organic and inorganic substances

such as

calcium have only begun to be elucidated. The understanding of such systems presents a fascinating challenge because of their potential impact on aging and disease. The genetic programming

or blueprint of the life and death cycle of arterial tissues may be mediated by such agents, including especially the mucopolysaccharides. In the chapters which follow, the authors explore the possi-

bilities that these (natural signaling?) factors can be judiciously employed by the physician to intervene in the so-called normal course of degeneration so that it may be possible for formerly hopeless patients to be returned to the mainstream of society.

They (the authors and their colleagues) are of the opinion that the data are sufficiently encouraging so that strong consideration should be given to initiation of a program of prevention of

arteriosclerosis

(and/or possibly premature aging) by therapy

with those acid mucopolysaccharides whose characterization and action form the main thrust of this monograph—chondroitin-4-

sulfate (CSA), chondroitin-6-sulfate (CSC) and chondroitin polysulfate. The old proverb, “an ounce of prevention is worth a pound of cure,” represents a sparing of human suffering in a ratio

of 1:16! This is a goal to be earnestly sought and ardently worked for and it is in this spirit that the following pages are presented.

CHAPTER

I

CHEMISTRY

OF THE

MUCOPOLYSACCHARIDES

(GLYCOSAMINOGLYCANS) AND THE CHONDROMUCOPROTEINS (PROTEOGLYCANS) A.

LTHOUGH

Structures

THE

of the Mucopolysaccharides

MUCOPOLYSACCHARIDES,

including

the chon-

droitin sulfates, are mainly present (both as secretions from chondrocytes, fibroblasts, intestinal epithelium, vascular connective tissue and endothelium, etc., and as intracellular constituents

of such cells) in combination with protein, these products of hydrolysis will be considered first by themselves, since their structures have been relatively well established. Further, it appears that the polysaccharide moieties of the proteoglycans may exist physiologically as separate entities in minor proportion and

that they may be biologically active (in some of the systems discussed in this monograph) from the protein moiety.

after they have been split away

Concomitant with verification of structure of the mucopoly-

saccharides, new nomenclature has been forthcoming for them, chiefly by virtue of the efforts of Jeanloz.’ Accordingly, the nonspecialist in the biochemistry of connective tissue may find the tabulation of old and new synonyms, as presented in Table I useful at this point. *R.W. Jeanloz, The nomenclature of mucopolysaccharides. Arthritis Rheum, 3: 233, 1960.

10

Chemistry of the Mucopolysaccharides & the Chondromucoproteins 11 TABLE

I. OLD

AND

NEW

NOMENCLATURE

FOR

MUCOPOLYSACCHARIDES

OLD

NEW

ME EURV GROOT

oR

Mbmneoitinecmiratee Asan (OLS SIRT Tas TEEENS) Eley BernremaCHEOIEIEL SULTALE” Gree.

go. esc escy i ccvsn vss swindle ccaresvevins Glycosaminoglycan .. ee

eee oo Chondroitin-4-sulfate a Dermatan sulfate ce cece ake aoe cccvecccerccnekeee. Wevcsineccns Chondroitin-6-sulfate COUN TSONSESE LES ee Chondroitin polysulfate OES ES pe ee ane ee ee ar Heparan sulfate

Oe

ea

[oad ARTS

Faas Wie eee an

a

eh

ee

akc

a

RO

icdaves tog sknece esd Geamns Heparin

a

ES en

Keratan sulfate

Hyaluronic acid

RM TRIAMRINEMCIEIEORORE, 65655555005 chaisonseccvnsdnsouseesisceveounneceas Proteoglycan

I.

Chondroitin-4-sulfate

(Chondroitin

Sulfate A)

In general, polysaccharides may be regarded as condensation polymers of monosaccharides, i.e. formation of glycosidic linkages are accomplished by elimination of water between carbons bear-

ing hydroxyl groups. As is the case for all acid mucopolysaccharides (the polysaccharides of vertebrate connective tissues), chondroitin-4-sulfate has been found, by molecular dissections with specific hyaluronidases such as those obtained from Proteus

vulgaris (including chondroitinase-ABC as prepared by Seikagaku Kogyo Company, Tokyo, Japan), flavobacterium, streptococcus,

mammalian testes, etc.,>° to be comprised for the most part of an unbranched chain of repeating units of disaccharides. The molecular weights of such chains appear to vary greatly, even within the same tissue, ranging from approximately 5,000 to °§. Suzuki, Introduction

to the chemistry and biochemistry

of mucopolysac-

charides. In Biochemistry and Medicine of Mucopolysaccharides, F. Egami and Y. Oshima, Eds. (Tokyo, Maruzen, 1962), pp. 1-11.

°K. Meyer, Biochemistry and biology of mucopolysaccharides. Am J Med, 47: 664, 1969. *P. Hoffman,

A. Linken,

and K. Meyer, Transglycosylation

during the mixed

digestion of hyaluronic acid and chondroitin sulfate by testicular hyaluronidase. J. Biol Chem, 219:653, 1956. ®P, Hoffman, A. Linker, K. Meyer, P. Sampson, and E.D. Korn, The degrada-

tion of hyaluronidase, the chondroitin sulfates and heparin by bacterial enzymes ‘flavo-bacterium). Biochim Biophys Acta, 25:658, 1957. °K, Murata, and Y. Oshima, Chondroitin sulfates in atherosclerotic aorta. Atherosclerosis, 14:121, 1972.

human

12

Coronary Heart Disease and the Mucopolysaccharides

coo7

CH2OH

Ficure 1. Repeating unit of chondroitin-4-sulfate.

100,000 daltons.’ As can be seen in Figure 1, the repeating disaccharide unit of chondroitin-4-sulfate consists of a hexuronic acid residue in B 1 —> 3 linkage with D-galactosamine. The galactosamine moiety is N-acetylated and, as indicated by the nomenclature, the sulfate ester group is on the 4 position of the galactosamine group. A B 1 —> 4 linkage combines the galactosamine residue with the glucuronic acid residue of the next disaccharide unit. The f designation in these cases refers to the

position of the hydroxyl group of the number 1 carbon atom of the hexose which is rendered asymetric by the formation of a

ring involving oxygen

and the number

5 carbon

atom.

(By

convention, when the OH group on the asymetric carbon points upward in two-dimensional projection, the monosaccharide is

termed Beta.) In those

cases

in which

the disaccharide

chain

has

been

digested with hyaluronidase, it has been found that the region of linkage to protein is resistant to the enzyme. It is mainly from "G, Gutinov, E. Yee, K. Granath, and T. Horton, Determinations under Institute

for Arteriosclerosis aegis performed at the University of California, Los Angeles, Pharmacia,

Uppsala, Sweden and Pharmacia, Piscataway, New Jersey.

*K. Meyer, E. Davidson,

A. Linker, and P. Hoffman,

The

acid mucopoly-

saccharides of connective tissue. Biochim Biophys Acta, 21:506, 1956.

Chemistry of the Mucopolysaccharides & the Chondromucoproteins 13 the studies of Meyer and co-workers® in the United States, Muir® in England and Roden and Swedish colleagues,’ that information has been forthcoming with respect to the exact nature of the linkage region among them.

They discovered that, following proteolysis of proteoglycans containing chondroitin-4-sulfate, a short peptide remains attached

to the main polysaccharide chain. If this procedure is preceded

by extensive degradation of the polysaccharide moiety with hyaluronidases, only the linkage region (plus a few extra amino acids ) remains. Unequivocal establishment of the nature of the linkage region was achieved by observing the action of special

enzymes on it, followed by fractionation methods, such as high voltage electrophoresis, column chromatography and comparison with standards of known structure. As can be seen in Figure 2, the terminal amino acid of the peptide moiety is serine, which

serves as a site of linkage to xylose, the first sugar of the potential reducing end of the polysaccharide chain. This type of bond (an O-glycosidic linkage to a hydroxyamino acid) is labile to alkali so that treatment of proteoglycans, containing chondroitin4-sulfate, with base yields a moiety comprised entirely of carboCH20H
"3 linkage to glucosamine. At this writing, no details are known with respect to the exact nature of the reducing end of the chain. Although possessing a high negative charge by virtue of its

hexuronic residues, the absence of additional negative charges due to lack of sulfation leads to the prediction that it may have

lesser although somewhat similar biologic properties (as described in the hyaluronidase article of Maroko et al.’*) to the chondroitin sulfates. Hyaluronic acid appears to facilitate gliding of surfaces in articular spaces and in tendon sheaths. In the vitreous humor and joints it also cushions and maintains turgor, being able to sequester approximately one hundred times its own weight of water.”° 5.

Keratan

Sulfate

(Kerato-sulfate)

Keratan sulfate is the only mucopolysaccharide to exhibit considerable variation in terms of chemical composition, molecular weight and associations with specific proteins.**** Indeed, the exact details of its composition are not available at this writing. A major part of the chain appears to be made up of a repeat-

ing disaccharide (see Fig. 6) differing from the disaccharides of the chondroitin sulfates and hyaluronic acid in that (a) galactose is present in place of uronic acid, (b) the glycosidic linkages are B 1 —> 3 and 8 1 —> 4 and (c) the sulfate groups vary in their positions and numbers.**° Further, it has been demonstrated by methylation experi*P.R. Maroko, P. Libbey, C.M. Bloor, B.E. Sobel, and E. Braunwald, Reduc-

tion by hyaluronidase of myocardial necrosis following coronary artery occlusion. Circulation, 46:430, 1972.

“Meyer. “Suzuki. “Meyer. *H. Muir, The structure and metabolism of mucopolysaccharides (glycosaminoglycans) and the problem of the mucopolysaccharidoses. Am J Med, 47:673, 1969. *“Meyer. *Muir, The structure and metabolism of mucopolysaccharides glycans) and the problem of the mucopolysaccharidoses.

(glycosamino-

Chemistry of the Mucopolysaccharides & the C hondromucoproteins 19 ments***” that keratan sulfate is a molecule with branches. An excess of galactose over hexosamine is observed and has led to the suggestion that galactose may be present in duplicate at points of branching. As in the case of the other mucopolysaccharides, xylose is present but small amounts of such monosaccharides as sialic acid, fucose and mannose have also been detected. Keratan CHoOH

Ho COSO3”

6

OH

NHCOCH3

Ficure 6. The probable major repeating unit of keratan sulfate.

sulfate obtained from corneal tissue (keratosulfate I) is thought to link to protein by virtue of a glucosamine-asparaginine or glucosamine-glutamine bonding.** (As Meyer” has pointed out, the N-glycosidic bond between N-acetylglucosamine and asparaginine is present in many glycoproteins.) On the other hand, in keratan sulfate obtained from cartilage (keratosulfate II), the bond is probably between a terminal sugar, galactosamine and either or both, threonine and serine.*” The N-acetylgalactosamine, which serves as the linking sugar in this instance, is also the linking monosaccharide of maxillary mucin and blood group substances.*! Further relating both types of keratan sulfates to *VP. Bhavandan,

desulfation

J Biol Chem,

VP.

and

K.

and acid hydrolysis 243:1052,

Bhavandan,

Meyer,

studies

Studies

on

keratosulfates:

in old human

methylation,

cartilage keratosulfate.

1968.

and K. Meyer, Studies on keratosulfates:

methylation

and

partial acid hydrolysis of bovine corneal keratosulfate. J Biol Chem, 243:4352, 1968. *N. Seno, K. Meyer, B. Anderson, and P. Hoffman, Variations in keratosulfates. J Biol Chem,

240:1005,

1965.

™Meyer. *°Seno, Meyer, Anderson, and Hoffman. Meyer.

20

Coronary Heart Disease and the Mucopolysaccharides

glycoproteins is that both contain mannose in about the same ratio as do the glycoproteins.” As already maintained, keratan sulfate II and chondroitin-6-sulfate are attached to the same protein backbone. 6.

Heparin,

Heparan,

Heparinoids

Heparin (Fig. 7) appears to differ from heparan (heparitin) sulfate only in that no N-acetyl groups are present. The latter are mostly substituted by sulfate groups. This similarity implies that a close synthetic relationship exists between the two substances. Unlike heparan sulfate, heparin is readily extracted from tissues

HeCOSO0z 6 5 0 4

| OH 3

Osos

2 HNSOs

Ficure 7. Repeating unit of heparin.

and may be present in small amounts in plasma.** Both heparin and heparan sulfate contain a residual peptide rich in serine and their end groups possess the sequence of neutral sugars that have already been described for the chondroitin sulfates and dermatan sulfates. The essential similarity of heparin and heparan sulfate is further indicated by the presence of some iduronic acid in both substances. The hybrid nature of heparan sulfate is also evidenced by a variable content of N-acetylglucosamine and “[bid.

“Muir, The structure and metabolism of mucopolysaccharides glycans) and the problem of the mucopolysaccharidoses.

(glycosamino-

“A. Serafini-Fracossini, J.J. Durwand, and L. Floreani, Heparin protein complex of ox liver capsule, Isolation and chemical characterization, Biochem J, 112;167, 1969.

Chemistry of the Mucopolysaccharides & the Chondromucoproteins 21 N-sulfated glucosamine. Meyer* and co-workers envision the two moieties as occurring in bunches giving rise to a branched carbohydrate in which the inner core is N-sulfated and the outer branches are N-acetylated. As will be demonstrated later in this monograph, the mechanisms of inhibition of blood clotting or thrombosis in which they are involved are quite different in the cases of heparin and heparan sulfate on the one hand, and chondroitin-4-sulfate on the other. Chondroitin-4-sulfate inhibits clot or thrombus formation more effectively when it is administered in vivo. Certain European

pharmaceutical manufacturers

do market

oral heparinoid products for the treatment of hyperlipemia, cor-

onary heart disease, arteriosclerosis and blood platelet aggregation, citing numerous references in Italian, German and French scientific literature in support of the manufacturers’ claims for

therapeutic efficacy of their heparinoids derived from gastrointestinal mucosa of swine, sheep or cattle. Most investigators in Britain, Scandinavia or other countries, such as the United States, have failed to find therapeutic value for such oral heparinoids. At the Institute for Arteriosclerosis Research no oral heparinoid samples from commercial sources abroad have shown experimental therapeutic efficacy.

B.

Structure

of Proteoglycans

(Chondromucoproteins)

According to the original proposals of Mathews and Lozaityte®® and Partridge et al.,*” the proteoglycans consist of a protein core to which are attached a number of polysaccharide chains.

Essentially, the model has not been refuted by subsequent investigations. However, questions remain with respect to the num-

bers of polysaccharide chains, possible variations in their lengths and their distributions on the protein moiety and an exception ¢

Meyer. *M.B.

Mathews,

and

Ҥ.M.

Partridge,

H.F.

I. Lozaityte,

Sodium

chondroitin

sulfate-protein

com-

plexes of cartilage. 1. Molecular weight and shape. Arch Biochem, 74:158, 1958. Davis,

and G.S. Adair,

The

chemistry

of connective

tissue and the constitution of the chondroitin sulfate protein complex in cartilage. Biochem J, 79:15, 1961.

22

Coronary Heart Disease and the Mucopolysaccharides

to the single protein core concept in the case of association keratan sulfate II and chondroitin-6-sulfate.*®

of

Extraction of proteoglycans from cartilage can be achieved by homogenization of the tissue in distilled water or weak salt solution followed by centrifugation so as to remove collagen fibers. The supernatant of such a preparation is quite viscous and is found—by light scattering properties, ultracentrifugation,

etc.—to contain a complex approaching 5,000,000 daltons in molecular weight.*® About 29 to 30 percent protein is present. Electron microscope studies by Dr. Sylvia Fitton-Jackson, University of London, on the supernatant material has revealed the presence of globular particles of about 60 A in diameter.*’ Occasionally such particles are present in rings of five or six and at other times

they are present in straight chains. Since the particles are untouched by hyaluronidases but are eliminated by exposure to the protease, papain, they appear most certainly to be protein in

nature and could, indeed, represent different types of proteins. Partridge and co-workers*'

carried out column fractionation

of cartilage supernatant (using DEAE at a pH of 7.0 and an increasing gradient of KCl). They found that extraneous globulins were present in a complex which included true chondroitin sulfate proteins. If the globulin proteins are subtracted from the total protein of the complexes, the individual complexes appear to contain about 7 to 8 percent of true core peptide, i.e. protein to which polysaccharide chains are attached. The nonprotein material is all carbohydrate. Although the complexes obtained from cartilage contain a mixture of mucopolysaccharides, digestion of

the protein(s) with papain has indicated the presence of about eight to nine side chains of mucopolysaccharides. These average Meyer. *S.M. Partridge, The chondroitin sulfate-protein complex from bovine cartilage. In The Chemical Physiology of Mucopolysaccharides, G. Quintarelli, Ed. (Boston, Little, Brown,

1968), pp. 51-62.

“S. Fitton-Jackson, '

In Structure and Function of Connective

and Skeletal

Tissue, S, Fitton-Jackson, R.D. Harkness, $.M. Partridge, and G.M. Tristam, Eds. (London, Butterworth, 1965).

“Partridge.

Chemistry of the Mucopolysaccharides & the Chondromucoproteins 23 560 A units in length and exhibit molecular weights of about 28,000.*? Other estimates** indicate that there may be up to sixty chondroitin sulfate chains on a single peptide with the molecular weights of these ranging to 50,000. Even in the same, as well as different, tissues, some variations in lengths and compositions of individual types of polysaccharides are found. Meyer** has remarked that such a degree of heterogeneity, so unlike the invariant compositions

of specific types of proteins, is explained

by the circumstance that, unlike proteins, direct translations from a code does not take place. Thus—depending on local conditions —variations in chain lengths, etc., are likely to occur. Digestion of chondroitin sulfate proteoglycans with hyaluronidases, which attack only the polysaccharide chains, frees the specific core peptides of the proteoglycans. The core peptides exhibit molecular weights averaging about 16,000. Proteoglycans of the chondroitin sulfates in cartilage have been found to contain some keratan sulfate. Seno and co-workers*® have demonstrated

(with isolated peptido-polysaccharide)

that the proteoglycans

migrate as a single component. However, following alkaline cleavage, two polysaccharide bands are seen. One of those is keratan sulfate II whereas the other is chondroitin-6-sulfate. Correlation appears to exist among the sizes of proteoglycans and the amount of keratan sulfate—the larger the complex the more keratan sulfate is present.***" Also a higher ratio of protein to polysaccharide is present in these cases. Both of the poly“[bid. “M.B. Mathews, The macromolecular organization of connective tissue. In The Chemical Physiology of Mucopolysaccharides, G. Quintarelli, Ed, (Boston, Little, Brown, 1968), pp. 189-197.

“Meyer. “Seno, Meyer, Anderson, and Hoffman.

“C.P. Triganoo, and H. Muir, Studies on protein-polysaccharide from pig laryngeal cartilage. Heterogeneity, fractionation and characterization. Biochem J, 113:885, 1969.

“K.B. Brandt, and H. Muir, Differences in composition and size of proteinpolysaccharides extracted from pig articular cartilage. FEBS Letters, 4:16, 1969.

24

Coronary Heart Disease and the Mucopolysaccharides

saccharides appear to be present on the same protein backbones in the case of the proteoglycan containing chondroitin-6-sulfate, whereas the core peptide bearing chondroitin-4-sulfate side chains apparently has no sites where keratan sulfates are attached. Meyer*® has postulated further that keratan sulfate II and peptides or protein are linked to keratan sulfate II by a second bond stable to alkali so that keratan sulfate II, in effect, cross links two sites of the same protein molecule or two separate protein chains. Further differences in the various proteoglycans have been found. Several fractions exhibit different amino acid composition sequences.*® Several N-terminal amino acids are present and proteoglycans can be distinguished on the basis of antigen-anti-

body reactions.5° As previously indicated, hyaluronic acid may not exist in close association with any particular protein moiety.

C.

Physical and Chemical Properties of Proteoglycans and Chondroitin Sulfates

In tissue, most of the proteoglycans appear to play structural roles. They are present as large complexes and Partridge™ enyisions at least certain of them as forming cross linkages with

proteins (collagen) of cartilage, etc., thus maintaining the elasticgel nature of the intracellular matrix in various connective tissues.

Coulombic (electrostatic) interactions have been observed among the chondroitin sulfates and collagen under in vitro conditions.™ Despite their roles in maintenance of structure, most of the proteoglycans are readily extractable with water or weak salt

solutions, indicating that any cross bridges with collagen are in the nature of electrostatic bonding. The dermatan “Meyer.

sulfate proteoglycans

at

“Triganoo and Muir. *°Serafini-Francossini, Durwand, and Floreani. *'Triganoo and Muir.

“Partridge. “Ibid.

are much

more

difficult

Chemistry of the Mucopolysaccharides & the Chondromucoproteins 25 to extract. Toole and Lowther have employed hydrogen bondbreaking reagents in order to obtain these proteins. A higher protein-to-polysaccharide ratio appears to be a property of the dermatan sulfate proteoglycans and they exhibit lower overall molecular weights than do the chondroitin sulfate proteoglycans. The dermatan sulfate and chondroitin sulfate proteoglycans precipitate soluble tropocollagen even in the range of four to zero degrees centigrade. In aqueous solution, the proteoglycans tend to assume spherical configurations with the relatively hydrophobic protein moiety

situated centrally and the hydrophyllic (negatively charged) carbohydrate chains extended outward (see Fig. 8). The sedimentation constants of the proteoglycans are on the order of 20 to 30 S

ao

-

a

-_

oo

-

2

“Y,

/

prtiiitll

-

=

Ba oe

rd

Pa

7

nf

A:

-

~=

~


~ rs

>

xz!