A comparative study of the chemistry and biology of Heparin

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Content COMPARATIVE STUDY OF THE CHEMISTRY AND BIOLOGY OF HEPARIN by Leon David Freeman A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (Biochemistry and Nutrition) June 1962 UMI Number: DP21587 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing^. UMI DP21587 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106 - 1346 U N IV E R S IT Y O F S O U T H E R N C A L IF O R N IA GRADUATE SCHOOL UNIVERSITY PARK LOS ANGELES 7, CALIFORNIA Vh d q; 0 to* pgss T h is d issertation, w ritte n by 1 .............. JLeoix. D avid . .E x e e m a n .......................... u n d e r the d ire c tio n o f h is .....D isse rta tio n C o m m itte e, a n d a p p ro v e d by a ll its m em bers, has been presented to a n d accepted by the D e a n o f the G ra d u a te S cho o l, in p a r tia l f u lfillm e n t o f re q u irem en ts f o r the degree o f D O C T O R O F P H I L O S O P H Y Dean D a te ., J. \ * & e. , . - .1 .9.6 2 ........ ........................ DISSERTATION COMMITTEE Chairman ACKNOWLEDGMENTS It is with deep appreciation that I acknowledge the patience, encouragement, and assistance of Dr. Walter Marx. ! I am deeply grateful to Dr. Paul Saltman for the in-j Valuable support and assistance throughout the work and the preparation of this dissertation. To the entire Department of Biochemistry and Nutri tion I would like to express my thanks for the help and en couragement I received. I am especially grateful for the encouragement and counsel of Dr. Edwin Hays and the assistance and support of my associates at Riker Laboratories. I owe a debt of gratitude to Nathan Wolfstein, Jr., ::or all the help over the many years this work has taken. To my wonderful wife, whose patience and love made anything and everything possible, I dedicate this disserta tion. TABLE OF CONTENTS Page LIST OF TABLES Vi LIST OF ILLUSTRATIONS V I i LIST OF ABBREVIATIONS Chapter I. HISTORICAL BACKGROUND I ! The Discovery of Heparin and Its Chemistry The Biological Activities of Heparin The Biosynthesis of Heparin Tissue Levels, Storage, and Release of Heparin The Catabolism of Heparin Species Differences Clinical Uses of Heparin The Characterization of Heparin II. STATEMENT OF THE PROBLEMS EXAMINED IN THIS Methods for the Isolation and Determination of Tissue Heparin Methods for the Characterization and Sepa ration of Heparin from Other Substances Chemical Methods for the Detection and Determination of Heparin and Related Substances Methods for the Determination of the Bio logical Activity of Heparin DISSERTATION 23 III. MATERIALS AND METHODS 26 i 1 Chapter IV. V. VI. EXPERIMENTAL RESULTS .................. . Development of the Methods The Comparative Biochemistry of Heparin Heparin Levels, Mast Cell Counts, and Atherosclerosis DISCUSSION............................ . SUMMARY ................................... BIBLIOGRAPHY j (Table s I: II. I III. IV. V. VI. VII. VIII. IX. X. XI. XII. LIST OF TABLES The Digestive Action of Proteolytic Enzymes on Beef Lung Homogenate................. . Yields of Heparin Activity Extracted from Beef Lung Homogenate by Means of Proteo lytic Digestion ............... .. Yield of Heparin Activity Extracted in First Extraction and by Redigestion of the Sedi ment from Beef Lung Homogenate ........... Effects of Variations in Amount of PTC and Period of Digestion on Yield of Heparin Extracted from Beef Lung Homogenate . . . . Heparin Extraction from Beef Lung Homogenate Recovery of Added Heparin .................. Comparison of Results of Heparin Assay Ob tained by Two Individuals Working Inde pendently in Different Laboratories . . . . Heparin Content of Rat and Rabbit Organs . . Heparin Levels in Human Intestinal Tissue . . Distribution of S ^ in the Tissues and Excreta of Rats Following the Administra tion of s35o4 ............................... Analysis of Chromatograms of Rat Tissue Extracts ................................... The Ultracentrifuge Examination of Beef and Hog Heparin........................ .. . The Heparin-Like Substances in Serum or Plasma .................. .................. v fTable XIII. XIV. XV. XVI. XVII. XVIII. XIX. XX. Pagel Clearing and Clotting Response Following j the Administration of Subcutaneous Heparin j to Rats............................ .. 118 Clearing and Clotting Response Following the Administration of Subcutaneous Heparin to Dogs..................................... 12 0 | Clearing and Clotting Data from Human Sub jects Following the Administration of Subcutaneous Heparin...................... 122j 35 Urinary Excretion and Plasma Levels of S j Labeled Heparin after Subcutaneous Injec tion in R a t s ............................... 1241 35 . Excretion of S m the Urine of Dogs Receiv ing Radioactive Heparin by Subcutaneous Administration............................. 1261 Urinary Mucopolysaccharides from Subjects Receiving Heparin.......................... 13 5j Heparin Content and Mast Cell Count of Rat Organs ............. ...... 138 Heparin Content and Mast Cell Count of Rabbit Organs ............................... 139 Figure 1. 2 . 3. 4. 5. 6. 7 . 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. LIST OF ILLUSTRATIONS Page Chromatogram of Beef Heparin on TEAE * • - * * 5S, Chromatogram of Heparin and CSA-A on TEAE • * 59 Chromatogram of Heparin on ECTEOLA . 6G Chromatogram of CSA-A on ECTEOLA 61 Ch romato g r am of CSA-B on ECTEOLA , 62 Chromatogram of Heparitin Sulfate on ECTEOLA . 63 Chromatogram of Heparin and CSA-A on ECTEOLA . 64 Chromatogram of CSA-A on AE-50, pH 4 .3 . . 66 Chromatogram of Heparin on AE-50, pH 4.3 . - ♦ 67 Chromatogram pH 4.3 . . of Heparin and CSA-A on AE-50, 68 Chromatogram Change . . of Heparin on AE-50 with pH 70 Chromatogram Change . . of Heparin on AE-50 with pH 72 Chromatogram of CSA-A on AE-50 . . . 73 Chromatogram of CSA-B on AE-50 . . . 74 Chromatogram of Heparitin Sulfate on AE-50 • • 75 Chromatogram of Heparin and CSA-A on AE-50 • • 76 Chromatogram of N-Desulfated Heparin on AE-50 78 Chromatogram of N-Resulfated Heparin on AE-50 79 Chromatogram of AE-50 . . . . 35 S Labeled Hog Heparin on 80 Figure 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. j 36. 37. 38. 39. vm: Pagej 35 . I Distribution of S m Rat Tissue Following j the Injection of s35o4 ...................... 89j j Chromatogram of Extract of 2-Hour Rat j Intestine ................... 91 Chromatogram of Extract of Rat Intestine at 6 H o u r s ...................................... 92 | Chromatogram of Extract of Rat Intestine at j 24 Hours...................................... 93| - ' I Chromatogram of Rat Liver Extract at 6 Hours . 94j Chromatogram of Rat Liver Extract at 24 Hours 95 Chromatogram of Mixed Rat Tissue M P S ........ 96 35 Chromatogram of S Dog Heparin ........... 102 Chromatogram of Purified and Crude Dog Heparin 103 Chromatogram of Rat Heparin................. 105 Chromatogram of Human Heparin............... 106 Chromatogram of Human Serum #1 M P S ........... 108 Chromatogram of Human Serum #2 M P S .......... 109 Chromatogram of Hog Plasma MPS ........ 112 Chromatogram of Beef Plasma MPS . ........ 113 Chromatogram of MPS from Hyperheparinemic Plasma................................. 115 Chromatogram of MPS from Normal Urine .... 129 Chromatogram of MPS of Subjects Receiving 5,000 Units of Heparin................. 130 Chromatogram of MPS of Group 1 Subjects Receiving 10,000 Units of Heparin ....... 131 Chromatogram of MPS of Group 2 Subjects Receiving 10,000 Units of Heparin ....... 132 Figure Page] 40. Chromatogram of MPS from Subjects who Received 20.000 Units- of Heparin....................... 133 . 41. Chromatogram of MPS from Subjects who Received 30.000 Units of Heparin ..... .. ... 134 42. Heparin Content of Lung, Liver, and Intestine of Several Animals ...................... 149 LIST OF ABBREVIATIONS MPS PAPS LPL 48/80 PTC DEAE TEAE ECTEOLA AE-50 CSA-A CSA-B Mucopolysaccharides 3 1 -pliosphoadenosine 5 ' -phosphosulf ate Lipoprotein lipase A condensation product of p -me tliox i phe ne thy lme thy 1 ami ne wi th formaldehyde Purified trypsin concentrate, a mix ture of trypsin and chymotrypsin Diethylaminoethyl cellulose Triethylaminoethyl cellulose A complex tertiary amino derivative of cellulose Amino ethyl cellulose Chondroitin sulfate-A Chondroitin sulfate-B or -heparin Svedberg flotation units CHAPTER I - HISTORICAL BACKGROUND The Discovery of Heparin and Its Chemistry Heparin was discovered by J. McLean (1) , a second /ear medical student, while working on the problem of puri fying cephalin. His adviser, W. H. Howell, was interested in the clot accelerating properties of cephalin and had as signed McLean the job of purifying it from liver. One of the fractions was found to have anticoagulant activity. We now know that this fraction contained heparin, although by today's standards it was quite impure. It was an important discovery and it was incidental to the work that McLean was doing rather than accidental, as it is often described (2). Because its ability to inhibit clotting was respons ible for its discovery, heparin is still thought of as an anticoagulant, although this may be only a minor aspect of its physiological role. Howell continued to work on the isolation and puri fication of heparin for many years (3,4,5). He was the first to demonstrate the carbohydrate nature of heparin (6). In 1933, Charles and Scott (7) published a procedure for the large scale extraction and purification of heparin. Since 1______________________________________ . that time, adequate quantities of heparin with a high anti- j \ [ coagulant activity have been available to facilitate the I i studies of its chemical and biological nature. I j J. E. Jorpes and associates (8,9) made monumental j contributions to the study of the chemistry of heparin. I I They showed that heparin, as prepared by the Charles and j I Scott procedure, was a polysaccharide containing uronic j acid, hexosamine, sulfate, and acetate. At that time, Jorpes et al. believed that acetate was part of the molecule. They reported a sulfur:nitrogen ratio of 5:2, and postulated that the substance was made up of two components with S:N ratios of 3:1 and 2:1. The more highly sulfated component j was reported to have the biological activity. Jorpes be lieved that heparin was not a discrete compound, but a num ber of compounds of varying degrees of sulfation (10,11). Charles and Scott reported the preparation of a crystalline acid barium salt with a S:N of 5:2 (12). They i ■concluded from the apparent crystallinity that this prepara tion was a homogeneous material. Masamune reported the preparation of heparin free of acetyl groups (13). This was confirmed by Wolfrom and left unexplained the fact that no significant amounts of free I amino groups were found in purified heparin (14). By anal ogy with the chondroitin sulfates, it had been assumed that heparin was N-acetylated on the hexosamine. Wolfrom postu lated that heparin was sulfated on the nitrogen (15). Jorpes and co-workers confirmed this by showing that part ofj j the sulfate was acid labile (16). N-sulfate is much more labile to acid than O-sulfate. Wolfrom elegantly demon strated that the acid hydrolysis constant for part of the sulfate in heparin was the same as that for N-sulfate in some model compounds (17). Other workers have confirmed these findings. The question of the absolute or maximum amount of sulfate in heparin has not been resolved. It appears to be 2.5 sulfate residues per nitrogen atom. Using counter- j current techniques, O'Keefe et al. isolated a preparation with 3 sulfates per nitrogen and with a high anticoagulant activity (18). Another fraction with an S:N of 2:1 was also described which was about one-fourth as active as an anti coagulant. Wolfrom reported that his preparation with an S:N of 5:2 was homogeneous in a similar counter-current sys- | - jtem (19). The complete details of this work have not been published. I The posxtion of the O-sulfates has not been estab lished. Wolfrom postulated a tetrasaccharide repeating ^structure where both amino groups and the 4-positions on the I glucosamine are sulfated (19). The fifth sulfate is on the 2 or 3-position of one of the hexuronic acid residues. If each hexuronic acid residue were sulfated on the 2- or 3- position, a repeating disaccharide would be the basic unit with 3 sulfates. It is possible that some loss of sulfate ■ occurs in the purification of heparin. j f The hexosamine component of heparin appears to be > | glucosamine (14,20,21). After hydrolysis, glucosamine has j been demonstrated in a variety of heparin preparations; how ever, there is the possibility that isomerization may have occurred during hydrolysis. The ultimate solution awaits a j specific enzymatic hydrolysis of heparin. i I There is uncertainty concerning the uronic acid moiety. Wolfrom reported the isolation of saccharic acid Lrom the oxidative hydrolysis of heparin (22). On this basis, he identified the uronic acid as glucuronic acid. Acid hydrolysis, however, destroys the uronic acid. Little other evidence was presented to confirm the identification of glucuronic acid until Cifonelli (23) reported the isola tion of glucuronic acid from the hydrolysis products of heparin and, using reductive techniques, the isolation of glucose. Similar reports from other laboratories have ap peared in the literature. In all cases, the amount of ironic acid isolated was less than 20 per cent of the amount expected. Dische (24) reported that the color intensity of heparin in the carbazole reaction was higher than expected for the calculated uronic acid content using glucuronic acid as a standard. This has been confirmed many times. It has been assumed that this was due to some intrinsic property of the lieparin molecule. It has been reported that when the j i molecule is desulfated, the aberrant color value returns to j normal (25). j j * Brown and associates (26,27) have reported that a similar intensification in color value occurs when the uronic acid content is estimated by the anthrone method. This color enhancement was found to be directly proportional to the anticoagulant potency in a series of heparin prepara tions (28). He has further found that all other natural mucopolysaccharides containing glucuronic acid had the ex pected color value whether sulfated or not. Sulfated hyal uronic acid gave the predicted results for glucuronic acid; no intensification occurred. Using the anthrone reaction, a time and temperature color curve was obtained which was characteristic for each of the available hexuronic acids. These curves were charac teristic for each acid either in free form or as a part of a polysaccharide. Similar or identical curves were found when the polysaccharide was sulfated (29). However, the heparin curve did not correspond to any of the known aldo-hexuronic acids, but appeared to resemble those of ketouronic acids. Degradative studies from Brown's laboratory reveal the presence of what appear to be ketouronic acids (30). Small amounts of glucuronic acid are also present but the quantities were less than that of the other, as yet unidentified, components. One very active preparation j t showed no detectable glucuronic acid (30). i 4 Wolfrom (31) has recently reported that the hydroly-| r i sis of desulfated, acetylated and reduced heparin gave him about 2 per cent glucose and another unknown sugar. Montgomery (32) has reported that a heparin preparation failed to react with periodic acid as would be expected from Wolfrom's structure. If a ketouronic acid were the uronic acid, the reaction with periodic acid would not occur. The proposed structure for heparin by Wolfrom has alternate 1-4 and 1-3 glycosidic bonds (19). This type of structure appears to be present in other acid mucopolysac charides. Reports have appeared which suggest the presence of 1-6 glycosidic bonds (33,34). If present, such a linkage might indicate the presence of branching in the molecule. The homogeneity of heparin is another unresolved question. The primary evidence for the homogeneity of heparin has been the preparation of crystalline salts, in cluding the acid barium salt and the acid benzidine salt. The crystallinity of these preparations has been questioned, hut Wolfrom maintains that the acid barium salt forms true crystals (25). The method of crystallization involves the use of acid conditions and during this procedure the heparin Loses activity. Amorphous preparations have been made which have considerably higher biological activity. Crystallinity, although often a satisfactory criterion for homogeneity of j Tiacromolecules, can often be misleading. I ) The problem of heparin's homogeneity is further com-j plicated by the fact that there are so many methods for its | preparation. In most cases, the commercial processes are unknown to the investigator. Seldom have two reports by iifferent investigators appeared in the literature where the! same heparin sample was used. The reports often fail to in- Llude any significant data on preparation of the heparin used in the study. The macromolecular properties of heparin have re ceived little attention. Its molecular weight has been re ported to be from 10,000 to 20,000 (29). While not a large molecule, it is large enough to have macromolecular charac teristics which might be important for its biological activ ity. It would appear that there is something special about :he spatial arrangement (35) or configuration of heparin which gives it unique activity since no synthetic heparin- oids have been prepared which are as active as heparin as a clot inhibiting agent. The most potent synthetic prepara tions have had as many or more sulfate, groups per repeating unit than heparin. A number of laboratories are actively investigating many aspects of the chemistry of heparin at the present time. r!he nature of the uronic acid component and the type of link ages are the problems receiving the greatest attention. The macromolecular aspects of all the mucopolysac- j * I Jcharides are becoming the subject of study for the molecular biologists. J . The Biological Activities of Heparin (36) j I The ability of heparin to inhibit clotting led to j j its discovery, and this property has been studied the most ! extensively. Although the most important basis for its j clinical usage, its mechanism in coagulation has not been I completely elucidated. Whether endogenous heparin plays a role in the normal balance in blood is unknown. A funda mental barrier to understanding heparin activity is the lack of definitive knowledge of the entire coagulation mechanism. Heparin appears to act upon the clotting system in several ways. One of its primary actions is its ability to inhibit the thrombin generation system and thus to prevent jthe conversion of prothrombin to thrombin (37,38). Further, heparin acts with a serum co-factor to inhibit the action of j thrombin to catalyze the conversion of fibrinogen to fibrin I s (39,40). In addition to the above activities, it has been proposed that it inhibits thromboplastin generation (41,42), platelet agglutination. Factor X activity, and possibly blocks the effects of some of the other recently uncovered fractors (43,44) . I The ability of heparin to effect partial or completej inhibition of blood coagulation occurs in vivo and in vitro. Heparin will inhibit the clottxnq of recalcified plasma and | the clotting of fibrinogen with thrombin when a small amount; of serum is present (45). The serum alone will not inter fere with clotting. A number of different systems have been proposed to quantitatively measure the activity of heparin (45,47,48). The method most generally used is that given in! I the U.S. Pharmacopeia, which involves the inhibition of clott I formation with recalcified sheep plasma under standard con- j ditions. This technique will be described in detail in Chapter III. Some workers prefer systems involving purified* components and feel their advantage is greater specificity and sensitivity (49). Still other workers utilize in vivo systems (50), but these are cumbersome and less reproducible There remains considerable controversy among investigators in the field as to the most satisfactory method (51). I At present, activity is measured in international units or USP units; both are essentially the same. This unit was originally defined as equal to the activity of 1/130 of a milligram of sodium heparin prepared from crystal line barium acid heparin. At the present time the USP ref erence standard is used by most workers in the field. Heparin has been shown to cause the release of Jand/or activation of an enzyme system which was originally jcalled "clearing factor" and which is now known as lipopro- ' jtein lipase (54) . The discovery of this enzyme was a conse-j ’ I guence of Hahn’s (52) observation that injected heparin | i caused clearing of alimentary lipemia. This enzyme has been;] — --------------- — ---------------------------------- ro shown to act upon the very low density lipoproteins, includ ing the chylomicrons which were responsible for the visible lipemia (53). It acts upon the triglyceride moiety of the lipoproteins causing the release of free fatty acids and glycerol and may have other actions as yet unknown (55,56, 57). Lipoprotein triglycerides are the optimum substrate. In contrast, the classical lipase of the pancreas acts best on simple triglycerides (58). LPL is inhibited by cholic I acid, 1M NaCl, eserine, and protamine (56). Recent work has indicated that more than one enzyme may be involved. Lipoprotein lipase appears in the blood only upon the parenteral administration of heparin. Heparin added to blood in vitro will prevent clotting but will not cause the appearance of lipoprotein lipase. However, the plasma from an animal or man who has been heparinized will act upon a suitable substrate in vitro. The measurement of such lipol- ysis has been the basis of the estimation of the lipase levels in serum. The extent of lipolysis can be measured by the reduction in turbidity of the substrate (59), the amount of free fatty acids released (60), or the quantity of glyc erol released (61). The turbidity method is the simplest but less specific because other factors can cause changes in turbidity. Under carefully standardized conditions, the three measurements give parallel values, although the glyc- srol measurement will show a lag until monoglycerides and free glycerol are formed. The glycerol determination is very sensitive and provides the most accurate method of i \ ; [estimating the lipase. i ' Ultracentrifuge studies of the effects of heparin j administration upon the serum lipoproteins have been made j (62). Lipoprotein lipase acts upon the lipoproteins classesj jfrom Sg 12-100,000. These consist of two main groups, the [ 12-400 class and the chylomicrons (S^ 400-100,000). It j does not appear to act upon the S£ 0-12 class {ft lipopro- j fceins) or the high density lipoproteins ) . A serial | study of the effects of the lipase using the ultracentrifuge shows that the higher molecules are converted to those of lower S_£ value (63) . If the reaction is allowed to go to completion in the presence of adequate amounts of acceptor !for the fatty acids, this conversion will continue until all jlipoproteins of values higher than 12 have disappeared. JThe higher fractions appear to have been converted to Sf 0-12 material. The identity of such lipoproteins with jthe endogenous Sf 0-12 fraction has not been established. The same type of changes occur in vivo. These transforma tions are most easily observed in man since most animals normally do not have much, if any, of the greater than Sf 12 I •lipoproteins. Under certain experimental conditions, these •lipoproteins can be produced in animals and usually will re-; 3pond to heparin in the same way. All animals tested to 3ate form lipoprotein lipase even though they have no sub strate (60). Nearly all animals will develop a transient jlipemia after a fat meal which can be reduced more rapidly ’ I ■ jby the administration of heparin. j I j The mechanism by which heparin activates lipoprotein jlipase has not been clarified. It has been suggested that j I ' I it is a prothetic group or co-factor of the active enzyme 1 ! since heparin antagonists will inactivate the enzyme. InorH I ganic salt solutions inactivate the enzyme and most heparin j j complexes can be dissociated with salt. A bacterial hepa- S ; rinase has been shown to inactivate the enzyme from post heparin plasma. However, Robinson (64) has shown that pass ing heparinized plasma containing active lipase through an ion exchange resin will remove heparin, but the enzyme is still active although it is very unstable. The stability can be restored by the addition of heparin. This suggests that heparin is not an essential part of the enzyme but either modifies or stabilizes the protein. A small amount of heparin might remain associated with the enzyme, but evi dence for this has not been presented. ■ Tissue lipoprotein lipases have been demonstrated in the heart and adipose tissue in several species (54,65). Conflicting reports have appeared concerning the presence of lipoprotein lipase in human adipose tissue (66,67). In most cases, the tissue enzyme is detectable without added heparin! and its activity is enhanced with added heparin. The adi- i pose tissue levels are higher m the fed than the fasted j j animals. The release of the tissue enzyme has also been j idemonstrated in the presence of heparin. However, the iden tity of the tissue enzyme with the heparin induced enzyme ini [plasma has not been established. They have many character- j istics in common and appear very similar. i t The site of formation and storage of the serum en- i ! zyme is believed to be in the tissue. Robinson (68) has j [shown that LPL will appear after one passage of heparinized i I ! blood through a number of isolated tissues by perfusion i i techniques. It has also been shown that an in vitro system | for production of the lipase involves a tissue factor, blood, and heparin (63). In vivo, a minimum blood pressure is required for the enzyme to appear in the heparinized ani mal and this can also be demonstrated in perfusion experi- J ments. The most reasonable interpretation of these data is that the enzyme either exists in the blood vessel wall in the active form or as the apoenzyme, and that it will quick- ( j ly enter the circulation under suitable circumstances (64). The ability of heparin induced lipoprotein lipase to hydrolize the triglyceride bearing plasma lipoproteins and thus to reduce or abolish an alimentary lipemia has become s i the basis of another clinical use in man. This will be con-j I sidered in more detail m the next section. Heparin's relation to lipoprotein lipase is the sub ject for active investigation in several laboratories today. Chemically, heparin fits the description of a cation! 1 exchanger since it has sulfate and carboxyl groups on a 'polymeric molecule. Dougherty has proposed that heparin and; other acid mucopolysaccharides can act as biological ion ex-! changers (69). It might, therefore, play an important regu-| latory role in the transport and biological activity of some; cations. Histamine and serotonin, as well as a number of \ other natural organic bases, are physiologically very activej | . 1 cations and have been implicated in many human disorders. 1 It has been demonstrated experimentally that heparin! . j will block the toxic effects of a number of substances whxchj have been characterized as histamine releasers (70). These j i i releasers apparently act by disrupting mast cells and caus- I ing the release of the granules which contain histamine, and in some species, serotonin and perhaps other bases. Such releasers include 48/80, a polymer containing many basic S jgroups, and neomycin. The antibiotic activity of neomycin Administered with heparin has been shown to be present, al- ! though the toxicity was reduced (71). Dougherty suggests that it is part of the function of endogenous heparin to counteract the effect of various j pvents which cause histamine release (72). Where the endog enous material is inadequate, exogenous heparin may serve to facilitate the detoxification of these substances and may pirns be useful in the treatment of allergic disorders and some inflammatory conditions (7 3). Heparin has been shown to possess a number of other ‘ biological activities (74). These include the inhibition of| 1.5 a number of enzymes, such as hyaluronidase (75), trypsin, \ \ ribonuclease (76), and lysozyme (77) . Inhibition of mitosis- (78) and enhancement of fibrinolysis (79) have also been : reported. The Biosynthesis of Heparin I Mast cell tumors have been shown to be capable of synthesizing heparin. This has been demonstrated in vivo in solid and ascites tumors, and in vitro in mast cell tumor cells in culture, in tumor slices, and homogenates (80,81, 82). Radioactive sulfate is incorporated into mast cell granules injected into the intact animals as shown by radio autography. However, radioactivity in the granules does not establish them as the site of synthesis of heparin. Incorporation of labeled sulfate and glucose into heparin has been demonstrated in vivo and in whole cell preparations. Incorporation of labeled sulfate has been shown to occur in whole homogenates and subcellular frac tions. The high speed supernatant (100,000 g for 1 hour) has the highest specific activity (80). Co-factor require- nents for the homogenates include ATP, DPN, glutamine, and Mg. Details of the pathways of biosynthesis are still miss ing. The available evidence suggests that glucose is the precursor of the glucosamine. At what stage sulfation oc curs is not known, but sulfated low molecular weight com pounds other than phosphoadenosine phosphosulfate (PAPS) have been_detected_and-may—well_be—intermediates.. PAPS_has— ibeen shown to be involved in the incorporation of sulfate S I ^ into heparin by mast cell tumor homogenates (80). Further, 1 jmast cell tumors synthesize PAPS (83). j The mechanism of biosynthesis of heparin in normal I animals and tissues has not been studied extensively. The j ! 1 identity of normal tissue heparin with that from mast cell j tumors has not been established. Hence, any extrapolation j from findings in tumor preparations to normal tissue must bej speculative. ; Many laboratories are studying the biosynthesis of heparin and other sulfated mucopolysaccharides and its rela tion to sulfate metabolism. Tissue Levels. Storage, and Release of Heparin The pronounced metachromasia of the mast cell gran- ! ules suggest they are the site of heparin storage in tissue (84,85). However, other acid mucopolysaccharides are also metachromatic. It has been reported that heparin has been isolated from these granules (87). Counter reports have ap peared which indicate that the heparin is not in the gran- ! jules but in the supernatant fraction (8 6 ). In all cases, mixed cell populations have been used since the isolation of pure mast cells is very difficult and the isolation of the acid mucopolysaccharides from pure mast cell preparations from normal tissue has not been accomplished. The mechanism of heparin release from cells and tis-j 'sue is not known at this time. Agents which cause disruption of mast cells cause the release of histamine and are assumed ! to release heparin as well. These include 48/80 and related' bases (8 8 ). Shock in dogs produced by the infusion of largej j amounts of peptone causes the release of heparin into the j circulation. Several cases of hyperheparinemia of unknown I S etiology have been documented in humans (89). j Many reports of the distribution and levels of hepa-j rin in various tissues have appeared (36,90,91). Metachro- j 1 matic activity has been used in some cases to quantitate thej isolated material. In other cases, the weight of the iso lated products have been indicated. The purity of the frac-j tions has not always been measured. In most animals, hepa rin appears to be widely distributed (92,93). The blood of animals has been reported to contain heparin. Essentially no data are available with respect to tissue heparin levels in man. There are many reports that heparin was not present in human serum or plasma (94,95). However, the existence of a circulating heparin-like sub stance in man has been claimed (96,97). Anticoagulant ac tivity, reversible with protamine, has been demonstrated. I jThe isolated material has a component with the same electro phoretic mobility as heparin and which has anticoagulant activity (98,99). The Catabolism of Heparin j Pathways for both exogenous and endogenous heparin i hatabolism is also unknown at present Jagues (100,101) 1 reported the presence of a heparinase in the liver and kid- j j ney of several species. A later report established that thei j japparent destruction of heparin was in reality some type of j binding which reduced the activity (102). Further treatment of the tissue permitted the release of the heparin and es sentially all the heparin activity could be recovered. j A number of workers have shown that only a portion j ! of the injected heparin activity can be recovered in the j urine. An inactive or weakly active substance has been ob tained which has been called uroheparin. Using radioactive 35 S labeled heparin injected i.v., it has been shown m dogs Ithat it leaves the blood stream rapidly and that the bulk of jthe radioactivity is rapidly excreted, a part as free as sulfate ion (103). It is apparent that heparin is catabo- I jjlized prior to excretion in the urine, and that desulfation occurs. Species Differences There is no agreement as to the identity or lack of Lt, of heparin isolated from the tissues of different spe cies. No comparison of heparin from human tissue with other heparins has been reported. Jagues has found differences Ln the unit activity of preparations from different animals | ( 104) . Wolfrom (25) believes that these differences were paused by the different procedures used to prepare the hepa rin samples. He has reported that when heparin is prepared fey one method from different animals, preparations of the j jsame potency are obtained. Other workers have confirmed I ' ' ' this observation. However, no comparative chemical or phys^ ical data have been reported on heparin from different spe- ; i ■ ■ ' . ' ■ ; S ■ i exes. i | Clinical Uses of Heparin (36,106) [ i The most popular clinical use of heparin today is as| i a clot inhibiting agent. It is common procedure to treat immediate post coronary thrombosis or myocardial infarction i with heparin, either intravenously or subcutaneously. It is the practice to maintain the whole blood clotting time at no less than two to three times normal. This can be done by iperiodic subcutaneous injections. A common regimen is to : iinject 10,000-15,000 units every eight hours or 2 0,000 units! |eyery 12 hours (105). Levels which cause complete incoagu- | lability are not used, although this may occur for brief ! periods shortly after an injection. Several courses of treatment are followed with the [acute coronary patient. Heparinization may be used through- I ■ |out the entire acute phase and hospital stay of about three j ' ■ ■ to four weeks. There is evidence to support this approach as the treatment of choice. Another approach is to intro duce a prothrombin depressant at the same time the heparin ‘ administration is started and to stop the heparin injections i ! j I when a therapeutic level of prothrombin depression is ! t ■ • . ■ ■ ■ ■ : ; i ji ' j Reached. Reports have appeared which suggest that the pro- j i i \ ' ! thrombin depressants do not achieve a true thera p e u t i c J I 1 [effect for seven to ten days and the heparin should be used : for that period (105). In addition to the treatment of the acute coronary j patient, heparin is administered for a variety of thrombo embolic diseases. Whenever a thrombotic problem exists, • [heparin seems indicated and is frequently used (107,108). j The second main clinical application of heparin is to lower the serum lipids and to reduce or abolish lipemia from a variety of causes (109). This use is based upon the ability of heparin to cause the release and/or to activate lipoprotein lipase. This therapeutic use of heparin appears! to be growing at the present time. Many dosage schedules have been used. Two common regimens are 5,000-10,000 units daily and 2 0 , 0 0 0 units two to. three times weekly, adminis tered by the subcutaneous route. Some individuals have been treated in this manner for many years. A study has been I published showing that an injection of 2 0 , 0 0 0 units of hepa- I ' ' inn twice a week m post coronary patients significantly de- i [creased the mortality from coronary disease compared with a i Iplacebo group (110). Heparin’s effectiveness has been as sumed to be due to the reduction in serum lipids since a prolongation of the coagulation time would be expected dur- i • • • * |ing less than 2 0 per cent of the time on this regimen. Xanthoma tuberosum patients have a type of essential hyperlipemia involving a massive elevation of serum tri glycerides which has been treated successfully by long term, intermittent heparin therapy (62) . Regression of the most recent lesions in some cases has been demonstrated. New reports, suggesting a relationship between ele- \ vated serum triglycerides (1 1 1 ), atherosclerosis, and coro- j in and use of j H . • : [heparin. It is one of very few agents which can effectively jreduce this class of lipids in the patients. I Other clinical applications of heparin have been re ported. It appears to be of some value in the treatment of ! ' ' ■ ’ ! some types of inflammatory and allergic disturbances (1 1 2 ). The medical literature contains many other references to the clinical usefulness of heparin which may not be due to its antilipemic action or to its clot inhibiting activity. These include senile macular degeneration (113), deafness (114), tuberculosis (115), pyrogenic infections (116), rheu matism (117), nephritis (118), and wound healing (119). In most cases, adequately controlled studies have not been run | I ' ’ ‘ [and definitive work remains to be done to establish the full I ' ' jutility of heparin in these conditions. l i j ! The Characterization of Heparin } Despite a considerable amount of chemical and bio logical work on heparin and its extensive clinical usage, it is not possible to give a satisfactory biochemical defini- i - ' ’ ! tion of heparin. Its complete chemical structure is not 5 known; Its biological activities are not entirely specific j j although quantitatively, the activity of heparin is much nary disease, have increased the interest greater than that of any closely related material. With j these limitations in mind, heparin may be defined in the j following way: j i Heparin is a mucopolysaccharide isolated from j . i mammalian tissue, containing a hexosamine (probably j i i glucosamine), a hexuronic acid moiety, and sulfate. The sulfur to nitrogen ratio is 5:2. Under the condi- j tions of the USP assay procedure, heparin has an ac- j tivity of at least 150 units per mg and preparations ! of 180 units per mg have been seen. j ! Heparin causes the release of lipoprotein lipase; J this action can be quantitated although a standard | assay has not been established. The electrophoretic mobility, R^, and sedimentation constant of heparin can be used to establish its identity. Other biological activities are not sufficiently well understood to permit their use in the characterization of heparin. Other physical measurements which define the Tiacromolecular aspects of the molecule/ such as light scat tering, viscosity, and diffusion constant, may ultimately be suitable for use to define this substance. The possibility . exists that heparin as defined above may in fact be a group 3f closely related compounds. CHAPTER II i ! I STATEMENT OF THE PROBLEMS EXAMINED i IN THIS DISSERTATION j i j The main goal of the work to be described is to j jexamine some comparative aspects of the chemistry and biol- j bgy of heparin. Such a study required methods for the de termination of tissue heparin as well as its characteriza tion and separation from related substances. A satisfactory method for the determination of the neparin content of small amounts of tissue was npt available which could determine low levels of heparin in tissue and be •applied to a number of samples at one time. It was neces- jsary, therefore, to undertake the development of a new tech- I . ' ' ' " ’ ' • ■ ■ ■ ■■ mque. The technique for the separation of heparin from re lated substances and for its characterization were limited, rhe paper chromatographic method of Spolter and Marx (120) was an important advance and has been used in these investi gations. However, other methods were needed which could Live more definitive information. For this reason, the i development of a column chromatographic method was under- j taken which would separate heparin from other substances and permit the characterization of different types and sources ! ! ' ■ ■ ■ ■ ' ■ ■ ■ « i [of heparin. ! ! Information about the amount of heparin in particu- j lar tissues and a comparison of tissue levels in different ! animals may be important to understand the biological prop- I ’ ■ . ' . ' ■ ! erties of heparin. Such knowledge can be pertinent in de- I ■ ' ’ ' I fining the role of heparin in ground substance as a biologiq ion exchanger, its relation to lipoprotein lipase in tissue j , f and blood, and its participation in the regulation of blood | coagulation. The kinetics of the incorporation of sulfate into MPS and its subsequent turnover must be known to elucidate the pathways of biosynthesis and catabolism of heparin and other MPS in tissues and ground substance. A study of the incorporation of SJJ as sulfate into the tissues of rats was initiated and its relation to tissue levels examined. Heparin from different animals and different tissues! has been used in the study of its properties, but the ques tion of the identity of these polymers from different sources has not been resolved. Not only do investigators rarely report the source, but it is possible that some in consistencies in the literature arise from the unknown dif ferences which may exist, and might be resolved by the use of well-defined and characterized materials. For these rea sons, a comparison of heparin from several animal sources, including man, was undertaken. Heparin-like substances have been shown to be pres- j ent in plasma. Their circulating levels have been reported j ' ' ‘ i to be inversely proportional to the level of low density [ * lipoproteins of blood (106). The nature of this plasma "heparin" was unknown. With the new and more sensitive ! chemical techniques, it was possible to investigate their properties and to compare both circulating materials with that of tissue and with other species. J The clinical application of heparin has continued to; grow. Little information exists concerning the relationship d £ its metabolism and its biological activity. It is essen tial to understand rates of excretion, mobilization, deposi tion in tissue, and catabolism in relation to its biological activity for proper therapeutic application of this impor tant pharmaceutical agent. By using radioactive tracer and standardized techniques, it should be possible to approach jthese problems more critically. Mast cell counts have been reported to be related to the resistance and susceptibility to atherosclerosis. Be cause heparin is involved in lipid transport and metabolism and may be related to atherosclerosis, a comparison of the heparin content with the mast cell counts of a number of ^issues of different animals was carried out. CHAPTER III | MATERIALS AND METHODS Methods for the Isolation and Determination of Tissue Heparin ! j The heparin content of small amounts of tissue can ! be determined by the method described below. This method was used in the experimental work described in Chapter IV, page 77. Further investigation established that more de pendable results could be obtained with some modifications, Which are described on page 28. Large scale isolation is described on page 29. Analysis of Tissue Samples up to 5 Grams Materials.— 1. Acetone C.P. 2. 5M NH^Cl stock solutions, adjusted to pH 8.5 with concentrated NH^OH C.P. 3. 0.5M NH4 CI pH 8.5. Prepared by dilution of one part of stock solution to 1 0 volumes with distilled water. 4. Purified trypsin concentrate (PTC): 100 mg per ml in 50 per cent glycerol. 26 21{ 5. Frozen sheep plasma as described in U.S. J Pharmacopoeia. j Procedure.— The tissues to be analyzed were removed j from the animal, frozen immediately, and stored frozen. A sample of 1-5 grams (depending on the size of the organ and the expected heparin content) was thoroughly homogenized | with an equal volume of water in a Vertis Homogenizer. The j homogenate was poured into 5 volumes of acetone. After ! standing for several minutes or longer, the suspension was centrifuged and the clear supernatant solution removed and discarded. The sediment was suspended in 5-10 ml of 0.5M NH^Cl buffer and the mixture was heated in a boiling water bath for 15 minutes. The sample was then transferred to a dialysis bag, closed at one end, and fitted with a short piece of glass tubing at the other end which was inserted through the lid of a 1-quart mason jar. The bag was sus pended in the jar containing 0.5 liter of 0.5M NH^Cl buffer. One ml of the PTC solution was added to the bag and the glass tube stoppered. The jar was incubated at 37°C fpr 48 hours. Then a second ml of PTC solution was added and the jar filled with new buffer solution and incubated for an ad ditional 48 hours. The sample was then dialyzed against cold running tap water overnight. The contents of the bag were transferred to a 50 ml centrifuge tube, NaCl was added to a concentration of 1%, and the solution was heated on a boiling water bath for 15 minutes to inactivate the enzyme ^ and coagulate the remaining proteins. The mixture was j I ! cooled to room temperature and centrifuged at 2500 r.p.m. | ! for 5-10 minutes. The supernatant fraction was decanted j carefully into a 2 50 ml centrifuge tube and the sediment j discarded. Five volumes of acetone were added to the solu- j tion and the mixture allowed to stand for 30 minutes. After! centrifugation for 5 minutes at 2500 r.p.m., the supernatant solution was discarded, and the sediment dissolved in a small volume of 1 per cent NaCl and transferred to a 50 ml centrifuge tube. Five volumes of methanol were added and, after standing for 30 minutes or more, the mixture was cen trifuged for 5 minutes at 2 500 r.p.m. The clear supernatant was discarded and the tube was inverted and drained for sev eral minutes. The precipitate was transferred to a tared vial or tube of about 2 0 ml capacity, with a small amount of dis tilled water, not exceeding 5 ml. The solution was then lyophilized and weighed. The samples were dissolved in isotonic saline to give a solution estimated to be 1-5 units per ml. The ac tivity was determined by the procedure to be described on page 42. Modifications of the Above Method The final precipitation with methanol was eliminated since - small losses may occur in this step. In order to in sure the comp1 ete removal of lipid materials which may ' ' 29; . interfere with the anticoagulant assay, the acetone precipi tated tissue was twice suspended in a 1 : 1 mixture of iso- propanol: petroleum ether, (hexane) before proceeding with the procedure as described. High potency pancreatin preparations (which normally contain some intestinal mucosa) have been found to digest the protein more completely than the trypsin concentrate. j , ' ■ i Two hundred mg portions were used instead of the 100 mg of j PTC and each digestion period reduced to 2 4 hours. Pancrea-j tins contain some heparin and this must be determined so that a correction can be applied. If the tissue levels are very low, the accuracy is reduced if the pancreatin correc tion is large in relation to the total heparin. The pan creatin preparation method should not be used for the iso lation of heparin for species characterization. Large Scale Isolation Procedures (50-1000 gms of Tissue) Isolation from tissue.— The tissue was homogenized in a Waring blendor with a minimum, of water and added to 5 volumes of acetone. The acetone was decanted after 3 0 min utes or more and the solids were resuspended in one half the volume of acetone. After a similar period, the tissue was collected by filtration and drained as dry as possible. The tissue was defatted in a mixture of isopropyl alcohol- ipetroleum ether or ethyl ether. The dried tissue was suspended in 0.5M NH^Cl, pH 8.5 suffer and heated to boiling, and then allowed to stand for ’ I 15 minutes.. The mixture was cooled and then homogenized in j a Waring Blendor. One liter of buffer was required for 100 . grams of dried tissue. J The tissue was digested at 37°C with 2 grams of PTC j or 4 grams of pancreatin 5X N.F. per 100 grams of sample. Chloroform and toluene, 1 per cent by volume, were added as preservatives. After 24 hours, a second equal portion of jenzyme preparation was added. After digesting for 24 hours J more, the mixture was heated and maintained at boiling for ! 10-15 minutes. The mixture was then cooled and filtered under vacuum using Celite. The filter cake was suspended in one half the original volume of buffer. The digestion procedure was repeated with one-half the amount of enzyme used above and was terminated as before. After boiling,and cooling, the mixture was filtered without additional filter aid. The cake was washed with small quantities of buffer. The combined filtrates were precipitated with 3-4 volumes of acetone. The mixture was allowed to stand over- bight in the cold. The clear supernatant was decanted and discarded. The oily cake was dissolved in a small amount of jwater, transferred to a dialysis bag, and dialyzed in run ning water overnight. The contents of the bag were transferred to a flask or centrifuge tube, made 1 per cent in NaCl, and precipi tated with 5 volumes of acetone. The mixture was allowed toj |stand in the cold overnight. The clear supernatant was de canted and discarded. The precipitate was dissolved in a . ■minimum of distilled water and was then lyophilized or processed further as described below. r • ' ' ! ; ! Isolation from blood, serum, or plasma.— The plasma j (or serum or blood) was added to 5 volumes of acetone. 'After standing for 30 minutes or more, the acetone super- ; natant was decanted and the precipitate drained as thorough ly as possible. The precipitate was then suspended in one- ] half the original volume of acetone. After standing for 1 j ; ■ ' ! hour or longer, the solids were collected by filtration, I ' ' ■ • ■ j washed with ether, and air dried. The dried solids were suspended in 0„5M NH4 C1 buf fer (100 g per 1 liter) pH 8.5 and heated to boiling for 15 o 1 minutes. After cooling, the mixture was put into a 37 C : bath and 2 grams of PTC were added per 100 grams of solids. Chloroform and toluene (1 per cent by volume) were added. The digestion was allowed to proceed for 24 hours. At this j I jtime, an additional portion of enzyme was added. After j .another 24 hours, the digestion was terminated by bringing | the mixture to 100°C for 15 minutes. The mixture was fil- j j tered using Celite and vacuum. If a substantial amount of j solids were present after boiling, the precipitate was re- jf digested. | ! ' ^ I The clear filtrate was precipitated with 3-4 volumes. i"'~~ .............. ~ ~~ " 32 I ! ! i j ^precipitate was collected by decantation of the clear super natant and dissolved in a small quantity of water. The mix-] ! ■ ■ . . . . . . i jture was transferred to a dialysis bag and dialyzed over- j night against running tap water. The contents of the bag j i ■ ! were made 1 per cent in NaC! and precipitated with 5 volumes of acetone. After standing in the cold overnight, the clear ' i supernatant was discarded and the precipitate dissolved in a i small volume of water and lyophilized. j . - ■ ■ j Other techniques for the isolation of heparin.— The j digested tissue extracts as described above after clarifica-j tion (or other tissue extracts or fluids, such as urine) were treated with cetyl trimethyl ammonium chloride or cetyl pyridinium chloride. Ten parts of the quaternary salt per 1 part of acid mucopolysaccharide were added to the extracts or solutions at a pH 6 .0-8.0. The solid complex was isolated by adding 50-100 ml of methyl isobutyl ketone to the mixture and shaking thor- ' oughly. The mixture was transferred to a separatory funnel and allowed to settle overnight. The clear lower layer was jdrained off and discarded. The interphase layer was then drained off and collected. The clear upper phase was dis carded . The interphase collected above was mixed thoroughly with 2.5M NaCl until the solid fraction was dissolved. The solution was allowed to stand, if turbid. Any residual ke- tone present separated and was removed. The clear solution jwas precipitated with 3-4 volumes of ethanol. The solid was ■ ; ! ‘ collected by centrifugation, dissolved in a small volume of j I ■ “ > Si per cent NaCl, and precipitated with 3-4 volumes of acetonel | ' . ■■■• " ■ ' ■ ' ' * • ■ j [After standing in the cold overnight, the precipitate was ■ I . ■ ■ . . j (collected, dissolved xn a small volume of distilled water, \ j ’ ■ . ' j |and lyophilized. j | When precipitation with quaternary ammonium salts I was used, the ionic strength of the solution of mucopolysac !j ■ . . . . . . . . charides influenced the extent of precipitation. In water jor urine, all the mucopolysaccharides precipitated. In 0.5M i ' ' JNH4 CI, all the sulfated mucopolysaccharides precipitated. The precipitate can be collected by filtration with Celite jor by centrifugation, although the complex will sometimes (tend to float. The flotation procedure was very simple and |the easiest to perform. i( ii i; ij Methods for the Characterization and j Separation of Heparin from Other Substances Paper Chromatography The method used was the procedure of Spolter and Marx (120). Whatman #1 filter paper sheets 7 inches by 22 Inches were used. They-were developed by descending chroma tography . The solvent system was 65 per cent Q.04M ammonium formate, pH 4.3, and 35 per cent isopropanol. In this sys tem, heparin had an Rf of about 0.6-0.65 and chondroitin sulfate-A had an Rf of 1.0. ! 1. The papers were spotted with aliquots of j j ' ► j 10-50 jjl, containing about 30 ug of muco- | I ■ ■ ■ . ■ 5 j polysaccharides. A dryer was used to per- j I ■ mit the application of a small spot. | j 2. The paper was allowed to equilibrate for ' ' • ’ ' ■ ' ' I 3 hours or longer with the solvent system. J ‘ j 3. Solvent was added to the trough and the j chromatogram developed for 16-18 hours at | room temperature (2 5 + 2 °C). j 4. The filter sheet was removed, the front j marked with pencil, immersed in a tray of acetone, and allowed to dry in air. 5. The chromatograms were visualized by stain ing with Alcian blue or Azure A. [Column Chromatocrraphv j j Aminoethvl cellulose columns .— Aminoethyl cellulose- |50 powder is a new cellulose exchanger available from Reeve- Angell and was prepared for use in the columns in the fol- j 1 - lowing manner: J 1. The powder was washed by suspending it in 0.5N HC1 and allowed to stand for several hours. The acid solution was decanted and the cellulose washed several times with distilled water. The suspension settled rapidly after each washing. The exchanger was suspended in several vol umes of methanol or ethanol. After sett ling, the solvent was decanted and the wash ing repeated. The alcohol was decanted and the exchanger suspended several times in distilled water. The exchanger was suspended in 0.5N NaOH made up in 1M NaCl and allowed to stand for several hours. The solution was decanted and the resin washed exhaustively with dis tilled water until the pH was below 8 . The column was prepared by pouring a slurry into the chromatography tube in increments and packed with air pressure without allow ing the column to run dry. After sufficient column height had been built up, the column was compressed further mechanically with a stopper on a glass rod. An additional amount of the exchanger was added if needed. The column was washed with 3M NaCl adjusted to pH 11 with ammonium hydroxide. After the effluent became basic, the washing was con tinued for an additional 1 0 0 mis. The column was washed with 0.012 5M ammonium formate pH 4.3 until the effluent reached a pH of 4.3. The column is now ready for chromatography. j A column of 1.5 x 20 cm can be used to chromatograph! up to 10 mg of sulfated mucopolysaccharides. The sample to j ■ | be chromatographed was dissolved in 0.0125M ammonium formate buffer, pH 4.3. The concentration should be 5 mg per ml or I ■ ' ■ ■ ■ ■ | less. The separation was made as follows: j 1. The mucopolysaccharide solution was placed at the top of the column and forced into the column by connecting the column to the buffer supply (0.012 5 in ammonium fprmate, pH 4.3) and allowing.50 ml or more to run through. 2. The elution was conducted with a continuous gradient. The mix bottle (1000 ml) contained 0.0125M ammonium formate, pH 4.3. The reser voir bottle (1000 ml) cpntained 3M NaCl in 0.1M ammonium formate, pH 9.5. The bottles were connected by a syphon so that the con tents of both bottles were reduced at the same time and at the same rate. This pro vides a linear salt gradient if the bottles are identical. A pH gradient occurs with a rapid change at 500 ml. 3. Using a drop counter, 10 ml fractions were collected in 16 x 150 mm culture tubes. 4. One hundred and forty fractions were col- | lected. Total solids were determined on j every tenth tube to verify the salt gradient. The pH was determined with pH test paper to determine the region of rapid pH change. 5. Mucopolysaccharides were located by the Alcian blue technique described below. When radioactive samples were run, suitable ali quots of each tube were planchetted and the radioactivity determined on a Nuclear.Chicago gas flow D-47 counter. 6 . After localization of the desired fractions, they can be pooled or other methods applied for the estimation of their contents. Direct spectrophotometry was used to detect protein or nucleic acid derivatives. At the termination of the elution, the column was washed with 200 ml of a 3M NaCl solution adjusted to pH 11 with ammonium hydroxide. The column was then washed with |D.0125M ammonium formate pH 4.3 until chloride free. The column was ready for reuse. A column may be used repeatedly If washed properly and not allowed to dry. r ~ ~ “ ' 38' » • i | Chemical Methods for the Detection < and Determination of Heparin j ! and Related Substances ! rir-1-Tiinii , .. . . . . . . , ( I Alcian Blue Stainincr ! • ■ ! This procedure was modified from that of Heremans i et al. and is based upon the reaction of Alcian blue with I YIPS to form an insoluble blue complex. } | Materials.— 1. Alcian blue solution. One per cent Alcian blue in 90 per cent acetic acid. . 2. Glacial acetic acid. 3. Several dilutions of a standard heparin solution in the range 5 to 100 jig per ml distilled water. Store in cold. Procedure.— For the detection of mucopolysaccharides in the eluate from a chromatographic run, the following pro cedure was used:: Sheets of Whatman #1 were ruled into 6 or 7 rows of 10 one-inch squares. One drop from each tube was applied to a square on the sheet,which was suspended upon suitable sup port. The standard heparin solutions were similarly spotted, The sheet was dried for 3 to 5 minutes in a 90°C air oven. The paper to be stained was immersed in the Alcian blue solution for 5 minutes, removed, and washed with run ning water for 1-2 minutes. The sheet was blotted with I • ' paper towels and immersed in glacial acetic acid for 5______ 1 ij minutes with gentle shaking, then removed and washed in run-; " ’ • I 1 ■ ! ning tap water. This process was repeated twice, or until j . . ■ ■ ■ ■ ■ . ■ | the background was white to a very pale blue. It was then j dried for several minutes in a 90°C air oven. The blue color of the MPS spots appear to be stable indefinitely. | . j The mucopolysaccharide content of each tube can be ! ' - I estimated by this method. The intensity of the spots appear to be proportional to the concentration of MPS up to 100 1 per ml or higher. As little as 5if per ml can be detected, j Metachromatic Staining with Azure A Materials.— -Azure A solution. Two hundred mg of Azure was dissolved in 50 ml of water and then added to 400 ml of acetone. Procedure.— The paper to be stained was immersed in the Azure A solution for 4-5 minutes. The excess solution i ‘ I iwas drained and then allowed to dry in the air. Salt in the [splutions will interfere with the metachromic reaction. The color was not stable and the best interpretation was made immediately after complete drying. The color was not fully developed until all the solvent was evaporated. iCarbazole Color Reaction (from Dische f241) Materials.— 1. 0.5 per cent carbazole in absolute methanol— must be stored in cold. 2. Sulfuric acid C.P. 3. Acid washed 15 x 12 5 mm culture tubes. i •’ S 4. Standard heparin solutions 100 _pg per ml, [ | both in distilled water and in 1M Nad* j j i i i Procedure.— -Pipette 1.0 ml of test solution into a culture tube and slowly add 6 ml of concentrated sulfuric acid. Heat on a boiling water bath for 2 0 minutes. Remove and cool in cold water to room temperature. Add 0.2 ml of carbazole solution. The color was allowed to develop for ! | 1 hour. The optical density was read in a suitable spectro-j photometer or colorimeter at 530 mu. A set of standard solutions were prepared from the standard heparin solution in distilled water. If the un- ! ■ knowns were in NaCl solution, a correction was made by using jstandards containing the same concentration of salt. The heparin content of the unknown solution was calculated by ! jcomparison with the standards. j Sulfuric Acid Reaction for Estimation of Mucopolysaccharides ■ I The procedure as described above for the carbazole | . •reaction was followed to the point of the addition of the carbazole reagent. The cooled solution was transferred to silica cuvettes and the optical density was determined at 320 mp. Other ratios of acid to test solution can be used as 5 well as other heating temperatures and times. Different mucopolysaccharides can be distinguished by variations of [the conditions. The full potentialities of this technique jremain to be developed. Methods for the Determination of the Biological Activity of Heparin The Determination of Anticoagulant Activity ; The method used is a modification of the procedure ^escribed in the USP. Materials.— 1. Sheep plasma. Nineteen volumes of fresh sheep blood were mixed with 1 volume of 8 per cent sodium citrate. The plasma was separated by centrifugation and was frozen in small lots of 2 5-50 ml and held at -10°C. A suitable plasma clots in 5 minutes or less upon the addition of 1 . 0 ml to 0 . 8 ml of isotonic saline and 0 . 2 ml of 1 per cent i j j CaCl9. Upon the addition of 1.0-1.5 units j ^ .-.■■■■■ | of heparin to the above system/ the clotting j should be delayed for approximately 1 hour. 2. 0.89 per cent NaCl in distilled water. 3. 1 . 0 per cent CaCl2 in distilled water. 4. 13 x 100 mm culture tubes. 5. Paraffined corks to fit the above tubes. f Procedure.— A reference standard heparin solution I ' , ^containing 2 USP units per ml was freshly prepared. A ser- j ies of aliquots of this solution were added to the culture ] ■’ j tubes, so that a range of clotting times up to 1 hour would j be obtained. The volume was made up to 0.8 ml with the NaCli !j solution, 1 . 0 ml of plasma was added, and the tubes were j j iplaced in a 37 C water bath. 0.2 ml of CaCl2 solution was j ; added, the tubes were stoppered, inverted gently three itimes, and the time recorded. The time required for each tube to clot was determined. A series of unknown solutions were prepared in the same manner as the standard heparin solution based upon an estimate of the activity present. The clotting times of this series was compared with the standards and the heparin jcontent of the unknown was calculated. Whole Blood Clotting Time i • | This is a version of the Lee-White Method. i i - • • | j Materials; — 1. 12 x 75 mm test tubes. 2. Paraffined corks. 3. 5 ml syringe, rinsed with physiologic saline and the voids filled. Procedure.— Five ml of venous blood was withdrawn j from the subject or animal- The timer was started. One ml : ■ ■ ■ ■ ■ ■ - ' ' ‘ ' ' i | . \ was run down the side of each of three of the test tubes,. ' ' 1 i if Tube 1 was sharply tilted and returned to an upright posi- i i tion every 30 seconds until a clot appeared. Tube 2 was \ I then tilted and returned every 30 seconds until a clot ap- i ■ ’ ' ' ! peared. Then tube 3 was treated in the same way until a j Lot formed. The total elapsed time from the appearance of | j I . . - blood m the syringe until the third tube clots was the whole blood clotting time. Determination of Lipoprotein Lipase I i t . . I ! . , . This is the method of Grossman (59). Materials.— 1. Substrate. Ediol (a 50 per cent coconut oil emulsion, Riker Laboratories) was diluted to j 1 per cent (1:50) with distilled water on the day of use. 2. 12 x 75 mm cuvettes for Coleman Jr. | 3. Coleman Jr. spectrophotometer. Procedure.— A venous blood sample was taken and 9 parts of whole blood were added to 1 part of 4 per cent sodium citrate in a chilled tube (ice bath). Centrifuge at I ' ' 3500 r.p.m. for 5 minutes and draw off the plasma. Use im- i j ■ ■ ' ■ | mediately or freeze. Frozen plasma can be held at -10°C. One ml of thawed plasma was added to a cuvette in a j ! o • [ [ 2 5 C water bath. 0.1 ml of substrate was added. The opti- j t “ ' " ' ’ i pal. density was read at 700 mu with the instrument adjusted i [to zero with distilled water. A stop watch was started at j ' ■ ! fhat time. The reaction was allowed to continue until the i [ - ■ . ■ I [optical density (O.D.) dropped 0.150 and the time recorded, j If the drop in O.D. was less than 0.150 in 10 minutes, the [actual decrease at 10 minutes was recorded. Clearing Units | . . . . . . . . . . . . j (C.U.) or Grossman Units = 1000_______ seconds elapsed for decrease of O.D. of 0.150 br C.U. = 11.1 x O.D. change in 10 minutes. CHAPTER IV | | I EXPERIMENTAL RESULTS Development of the Methods The Method for Tissue Heparin Determination Proteolysis was considered the most suitable way to liberate heparin from tissue. In order to determine the best enzyme for the digestion, three commercial proteinases were tried. These were pepsin (Cudahy 1:15,000), papain (Merclc), and Purified Trypsin Concentrate (Southern Cali fornia Gland Co.). The solution was buffered at the pH optimum of the enzyme and, with papain, Na2 S and KCN were added as activators. Samples of a large lot of beef lung homogenate were used. The procedure used was described in Chapter III with the exception of the pH and buffer. The extent of digestion was estimated by the determination of the total solids which remained after dialysis. The heparin content was determined by its anticoagulant activity as described. The results are shown in Tables I and II. When a combination of enzymes was used, the digestion was carried out in two steps, using the (proper■conditions for each enzyme. PTC, alone, gave the most complete, digestion, as determined by residual solids _________________ ______________45________________________________ TABLE I The Digestive Action of Proteolytic Enzymes on Beef Lung Homogenate Enzyme Papain PTC/papain Pepsin PTC/pepsin PTC ^Samples of 5 grams of beef lung homogenate were treated as described in the text, and the total solids remaining after dialysis were de termined as the percentage of the dry weight of the lung. No. of Total Solids Digested* Experiments Mean % Range 6 337. 22-46 1 50 5 64 48-77 8 63 42-80 13 91 88-94 47 TABLE II i Yields of Heparin Activity Extracted from Beef Lung Homogenate by Means of Proteolytic Digestion Enzyme Papain PTG/papain Pepsin PTG/pepsin PTG No. of Experiments 1 1 2 2 2 Heparin Activity Extracted* Units per Gram** 2 0 27 14, 12 19/21 50/46 * Samples of 5 grams of beef lung were treated as described in text and the anticoagulant activity of the extracts were determined. ** Expressed per gram of wet weight of tissue. .and the highest heparin values. With PTG followed by pep sin, the heparin recoveries were about 40 per cent of that obtained with PTC alone, and the undigested residue after j dialysis was several times greater. It appears that pepsin was binding or inactivating | part of the heparin. In order to determine the fate of the j : I ■ missing heparin, some of the insoluble residues after diges tion and dialysis was redigested with PTC and carried j through the procedure. The results of this experiment are summarized in Table III.. Redigestion of the sediment from PTC alone produced no additional activity, while the residue of the PTC-pepsin digestion contained all the missing activity. The results indicated that the trypsin concentrate was the enzyme of choice. It became necessary to determine the optimal amount of enzyme and the duration of digestion for best results. Using a new pool of beef lung homogenate, the amount of enzyme and the digestion times were varied independently I .The results are summarized m Table IV. The most satisfac tory procedure appeared to be one using 1 . 0 ml of a 1 0 per bent solution of PTC and digesting for 48 hours. Another |l.O ml of enzyme was added, the buffer changed, and the di gestion continued for 48 hours. In order to determine if heparin was degraded or inactivated by the procedure, recovery experiments were 49 TABLE III Yield of Heparin Activity Extracted in First Extraction and by Redigestion of the Sediment from Beef Lung Homogenate Enzyme Experiments PTG/pepsin 4 PTG 5 Total Solids Remaining , Mean - mg . 530 100 Total Heparin Activity* Mean and Range First Ext. Sediment** Total Units Units Units 36 (22-44) 125 (82-155) 88 (56-123) 0 124 125 * Samples of 5 grams of beef lung were treated as described in the text. ** The sediment formed in each case was redigested with PTG and the resulting extract was assayed for heparin. 50 TABLE IV i i Effects of Variations in Amount of PTG and Period of Digestion on Yield of Heparin Extracted from Beef Lung Homogenate Heparin Activity Extracted* Mean No. of Amount of Period of (Individual values) Experiments PTG mg. Digestion Days Units/gm.** 2 2 x 100 4 30 2 x 50 4 26 (24, 25,25,26,28,30) 2 x 100 2 23 (22.24) 2 x 50 2 23 (22.24) * Samples of 5 grams of beef lung were treated as described in the text. ** Expressed per gram of wet weight of tissue. performed. Tissue samples were divided into equal portions j ! j and known amounts of commercial heparin were added to one \ ! ■ ' i S ■ i iportion. The heparin content of the samples were then de- j j termined by the procedure. The results are summarized in j | Table V. The added activity was recovered completely. j Eight 5-gram samples of a beef liver homogenate werej i| assayed separately to determine the reproducibility of the j method. The results were as follows: 12.4, 12.6, 13.0, if ' :! 13.2, 13.6, 13.6, 14.4, and 15.5 units per gram of wet tis sue. The mean was 13.5 + 0.95. J j To further test the dependability and reproducibil ity of the method, four different tissue samples were i jtreated as described by two individuals independently in different laboratories. The results are summarized in ' Table VI. | The final preparations obtained by the proteolytic j extraction described were examined to determine if their pnticoagulant activity was due to heparin. The heparin j ! character of the activity in the extracts was shown in the following ways: | 1- The anticoagulant property of the extracts was completely inhibited by protamine. This ; was true for extracts from rabbit, rat, and beef tissues. 2. The extracts were subjected to paper electro- 1 ! phoresis. A single elongated metachromatic TABLE V Heparin Extraction from Beef Lung Homogenate Recovery of Added Heparin Experiment No. Enzymes PTC/pepsin** PTG Heparin Added Units 0 50 Heparin Activity* Total Added Units Units 160 217 57 0 25 155 180 25 * Samples of 5 grams of beef lung were treated as described in the text. ** The sediment obtained after the pepsin digestion was redigested with PTC and the extracts were combined before assaying for heparin. 53 TABLE VI Comparison of Results of Heparin Assay Obtained by Two Individuals Working Independently in Different Laboratories Sample 1 2 3 4 Tissue Rat intestine Rat lung Rat lung Beef lung Heparin Activity Found* Laboratory A** Laboratory B** Units per Gram Units per Gram 3.0 7.5 8.0 41.1 3.0 7.5 6.5 42.6 * 5 grams of each tissue were treated as described in the text. ** Expressed per gram of wet weight. j spot was obtained exhibiting the same mo- ; i bility as that of commercial beef heparin. j A small weakly staining spot was seen at j i i ' i I ? ' 1 j the origin. Both spots had the typical pink color of heparin, j 3. Aliquots containing about 3-4 units were j ! ■ ■ ' ■ j | spotted on Whatman #1 and subjected to j ! paper chromatography in the system of j ; . j | SpoIter and Marx (120). An elongated spot \ was found with the Rf characteristic of j I’- J - - ; beef heparin. Some metachromasia was ob- served at the origin and in most cases, a j metachromatic area was found at the front j | corresponding to chondroitin sulfate. It is of interest to note that the tissue extracts themselves did not show a metachromatic reaction when (treated with Azure A. Even the addition of purified heparin to the extracts did not produce metachromasia. When some extract was added to a mixture of purified heparin and Azure A, the metachromasia was extinguished, due to the presence of an unidentified component(s). The Column Chromatographic Method j J At the time this work was initiated, a satisfactory j method for the column chromatography of heparin and related | substances had not been reported. Green had indicated the use of an ion cellulose exchanger. ECTEOLA. was promising^ _ _ _ . l ’(122) , Earlier work by the author with the use of IR-45, ah anion exchange resin (12 3), had shown that heparin could be | adsorbed by such materials, but the capacity was low and no I \ 'separation of related substances was achieved. The high j [capacity of the cellulose exchangers for macromolecules made! i i - - ! ■ j them appear attractive, and so they were investigated for ! their possible utility for the chromatography of acid muco- ! polysaccharides. | Three such preparations were commercially available. Diethylaminoethyl cellulose had shown much promise in the i • ! chromatography of proteins. j ECTEOLA, a complex tertiary amine derivative of j cellulose, had been reported to be useful in the chroma- i | I tography of nucleic acids and its derivatives. A third exchanger, triethylaminoethyl cellulose, appeared interest- | I ' ing because of the quaternary ammonium group which would be ! i f ' ' c 'expected to bind sulfated mucopolysaccharides strongly. These three preparations were obtained from Bio Rad ^nd were prepared from Solka-Floc. They were treated as | described in Chapter III. After the alkaline treatment, (they were washed to neutrality with distilled water. These preparations contained a substantial amount of very fine I . . joarticles. During the washing, the slowly sedimenting por- ! Itions were discarded. Almost 50 per cent of the exchanger r ' ' was discarded. After washing, small columns were poured andj J ' . ■ i! packed with a gravity head. Despite the attempt to eliminate n 1 jthe fine particles, the eluant ran very slowly under a grav ity head through these columns. 1 ' * | Heparin (beef, 125 U/mg) and purified cliondroitin j sulfate-A (CSA—A) were used as the test substances. The ? | . I |CSA-A was prepared from a commercial preparation (Wilson | {Labs) and was decolorized and purified by solvent fraction- j ation. Ten mg portions were dissolved in 2-3 ml of dis- ; ; tilled water. They were applied to the columns and 100 ml of distilled water was passed through. No detectable mate- j i i Irial was found in the wash with any of the exchangers. { '! ■ ' ‘ 1 j j The elutions were conducted with a NaCl gradient in {distilled water. In these preliminary runs, Alcian blue {spotting was used to locate the fractions containing the ! ' . ■ test substance. When known quantities of the mucopolysac charide were spotted on the same sheet, an estimate of the quantity of substance in each tube could be made. A summa- I ' ! tion of these estimates gave a total value which corresponded I ' ^ {quite well with the material adsorbed onto the columns, usu ally within 10-15 per cent. j On DEAE, there did not appear to be any separation ' pf CSA—A and heparin. Some separation was observed with I TEAE and ECTEOLA. The latter two exchangers were investi gated further. { | Heparin alone and a heparin-CSA mixture were chro- j matographed on TEAE in an unbuffered system with a shallower! 5 NaCl gradient. The results of these runs are shown in { Figs. 1 and 2, Both the Alcian blue method and the carba- | zole color were used to estimate the material in the frac- | i ^ itions. The correspondence was reasonably good. The results! Iwere somewhat difficult to interpret. The elution pattern | {for the mixture showed two peaks while heparin alone had ! ■ ■ ■ s ’ only one peak. However, heparin was eluted over the same \ range of salt concentration as the mixture. On this basis, \ ! r jit did not appear that a good separation had occurred. In > jview of the greater promise with ECTEOLA, no further work j was undertaken with TEAE. I f . With ECTEOLA, a separation of heparin and CSA ap- j peared to have occurred. There was also some indication of resolution of heparin itself into more than one component. New supplies of ECTEOLA with a better flow rate and higher I capacity were obtained from the Brown Co. They were pre- j pared from Solka-Floc, with a capacity of 0.9 m e<g/g. Col umns of 1.5 x 20 cm were used for the bulk of the work re ported. Runs were made on 2 mg portions of heparin, CSA-A, jcSA-B (B-heparin), and heparitin sulfate. The results are Jshown in Figs. 3, 4, 5, and 6 . Figure 7 shows a run made on | p mixture of CSA-A and heparxn. All materials showed some ; i ■ evidence for heterogenexty. A mxxture containxng all the pbove mucopolysaccharxdes would give a chromatogram whxch inight be difficult to interpret. > While this work was underway, a new exchanger becamej f : available. This was aminoethyl cellulose (AE-50), made by | L _____ _ _________________ ____________ _— _ — 1 Fig. 1 Beef Heparin - TEAE-No buffer NaCl 30 Alcian Blue 20 0.5M Carbazole 10 0 Fraction No. Ui 00 50 Fig. 2 CSA-A and Heparin TEAE-No buffer 40 -- 30 20 - - e >e 10 65 75 80 Fraction No. 85 -r- 1.5M 1.0M --0.5M o $ a Fig. 3 2 mg. Heparin-ECTEOLA-No buffer 30 — 1.0M --0.5M 10 __ 100 Fraction No c r > o Fig. 4 2 mg. CSA-A-ECTEOLA-No buffer 40 -- — 1.0M 0.5'M Fraction No Fig. 5 3 mg. CSA-B (p-heparin)-ECTEOLA-No buffer --1.0M 20 - - --0.5M 100 Fraction No. ^/ml. Fig. 6 2 mg. Heparitin Sulfate-ECTEOLA-No buffer 1 .0M 30 4- 20 + 4 - 0.5M 1 0 4- 100 Fraction No. Fig. 7 2 mg. Heparin and 2 mg. CSA-A-ECTEOLA-N© buffer 30 -- 20 - - 10 - - Heparin CSA-A Fraction No. -- 1.0M o 1 3 0.5M -ife . ’ Mhatman. Preliminary examination of this material estab- i i I lished a substantially higher capacity and greater reten- j * ! . i jtiveness for the acid mucopolysaccharides. The results of j the first runs are shown in Figs. 8 , 9, and 10. The AE-50 appeared to have greater potential than ECTEOLA and the de- Lision was made to work with the former exchanger for the j work to be described. More work with ECTEOLA might very well have permitted its use, but practical considerations j ! required selection of the most suitable exchanger. A variety of elution systems were considered. The system most commonly used consists of a reservoir which reeds into a mixing chamber, and the eluant flows from the mixing chamber onto the column. The volume in the mixing j^hamber does not change, and this leads to a gradient with a decreasing rate of change as the limit concentration is ap- . proached. In most types of chromatography, such a gradient | yay cause the accentuation of tailing. This system, there fore, was not used. In order to try to reduce tailing, another system was explored which consisted of three 500 ml bottles of identical dimensions. Two of the bottles, which must be flat-bottomed, were mixed with magnetic stirrers. The third bottle contained the limit concentration of eluant The bottles were connected by syphons so that the volumes in all three containers were always the same. This gave a gra dient where the rate of change increased during the elution. This system was used in a number of runs (Figs. 1-10), Fig. 8 CSA-A-AE-50, pH 4.5 40 -- 30 e 20 - - 10 -- 80 90 Fraction No. 100 110 120 01 _oa_ Fig. 9 Heparin-AE-50, pH 4.5 a -r 3M 40 30 -- 2M 20 10 100 110 120 Fraction No c r > <i Fig. 10 CSArA and Heparin-AE-50 pH 4.5 -r- 3 M 30 2M 20 10 Heparin CSA-A 40 50 60 70 80 90 100 110 Fraction No. 0 0 jhowever, it was not completely dependable and did not appear to offer a substantial advantage over the next system to be \ j described. I I I ( A linear gradient was obtained by the use of two | - • ' ' - i ( 1 0 0 0 ml bottles of identical dimensions, where one func- j • f \ tioned as the reservoir and the other was the mixing cham- j ber. This system proved to be the most dependable and was the simplest to reproduce, so it was used for the bulk of the work to be described. The gradient is shown in each of the figures. Earlier work with ECTEOLA had established that; the retention of heparin was greater at pH 4.5 than at neu trality or the pH of distilled water. A pH of 4.5 was used in the first work with AE-50; however, the salt concentra tion required to elute heparin at this pH was 2.0-2.5M in NaCl. This led to some practical difficulties. A very steep gradient was needed and the limit concentration had to be 4M or greater (saturated NaCl). In the system described, this required the collection of 150 or more 10 ml fraction to be certain all the heparin was eluted. The effect of introducing a pH change during the course of the run was investigated to see if heparin could be eluted at a lower salt concentration. The first system investigated involved the use of 3M NaCl at pH 9.5 in the reservoir. A chromatogram of hepa-j rin using this shallower gradient with the pH change is 1 ji shown in Fig. 11. The salt and pH gradients are shown. { Fig. 11 Heparin-AE-50, pH change pH curve 1M 20 - - 6 0.5M 10 --5 100 Fraction No. _ Cu jFigure 12 shows a repeat of this run using the same heparin j preparation and another identical column. The same pH gra- I dient was present here and in all subsequent runs. In this i case, the heparin was quantitated by means of the reaction with sulfuric acid as described. The data from the Alcian j blue method were also plotted and the correspondence between) i |the two methods of estimation serves to confirm the useful- | ness of the Alcian blue technique. ■ " ' i Under these conditions, heparin is eluted between j I : 0.8 and 1.2M NaCl in the region of basic pH. This permits a! ’ ' ' I total run to be completed in 80-100 10 ml fractions. The remaining work to be described was performed in this manner. This system appeared to be adequate for the purposes of the . . . studies planned. j Using the same system, chromatograms were made of J CSA-A, CSA-B, heparitin sulfate, and a mixture of CSA-A and j heparin. The results are shown in Figs. 13, 14, 15, and 16.j r ' " ' ' CSA-A and CSA-B appear to give very similar elution patterns. The samples used are not homogeneous in this system. The I ' ' I heparitin sulfate chromatogram has some similarity to that i i I j j s f heparin, but they are not identical. The major componentj |in heparitin sulfate corresponds with a minor component in j all the commercial heparin samples examined. A sample of commercial hog heparin which had been ' ' ’ ' '' ! | partially N-desulfated and whose anticoagulant activity had j j been reduced from .140 units per mg to 35 was chromatographed! Fig. 12 Heparin-AE-50, pH change — = — Alcian Blue H2SO4 color at 320 n^i -- 1. 0M 5M 1 0 0 Fraction No. Fig. 13 CSA-A AE-5Q, pH change 40 30 1. 0M r “ 4 6 20 0.5M 1 0 100 Fraction No. U) Fig. 14 CSA-B ((3-heparin) AE-50, -pH change 40 - 30 -- -- 1. 0M 20 - - -- 0.5M 10 - - 100 Fraction No. Fig. 15 Heparitin Sulfate-AE-50, pH change -r- 1.5H 40 -- 1. 0M T — t -- 0.5M 10 - - 100 110 Fraction No Fig. 16 CSA-A and Heparin-AE-50, pH change 30 __ -- 1. 0M -- 0.5M Heparin 60 Fraction No <i Ch ! oc : ISome of the same material was resulfated with and the ! original activity was restored. This was also chromato- * graphed. These results are shown in Figs. 17 and 18. The i N-desulfation (about 30 per cent) did not have a very great ! : ] effect on the elution pattern, although there is an indica- j tion that a larger proportion of this material was eluted at . . . . . . .. , ' j the lower salt concentrations. This would be the effect ex-! ' i pected. Figure 19 shows the chromatogram of a sample of hog | 35 ^ heparin labeled biosynthetically with S . The radioactiv- j ity of the fractions was determined by direct planchetting j of 0.5 ml of each tube and counting in the Nuclear Chicago D-47 gas flow counter. This was done for both the N-resul- ! sfated and biosynthetically labeled heparin samples and j j ■ ■ . 1 ’ I! plotted along with the Alcian blue data. The correspondence! of the radioactivity data with the Alcian blue further con- | . ’ ' ■ • i firms the usefulness of the latter method. The two types of labeled heparin appear chromatographically very similar. ■ • ■ • - I ji The Comparative Biochemistry of Heparin Tissue Heparin Levels I A comparison of rats and rabbits.— The heparin con- j jtent of several organs of rats and rabbits was determined byj jthe method described above. Twenty adult male rats of the i pniversity of Southern California strain and 12 rabbits from a commercial breeder were used as obtained. The animals ! ■ ■■■ ' • ' ■ ' yere anesthetized with nembutal, and the liver, spleen. Fig. 17 12 Mg. N-desulfated Heparin-AE-50, pH change -rl.5M 30 -- 1.0M -- a 20 - - --0.5M 10 - - 100 110 Fraction No. Fig. 18 8 mg. N-resulfated Heparin-AE-50^ pH change -- 1.5M 40-- Alcian Blue --S 30-- l.QM 20- - -- 0.5M 10- - 100 110 Fraction No. Fig. 19 Biosynthetic S Labeled Hog Heparin-AE-50 Alcian Blue 40 ^ 30 — 1.0 20 -- --0.5 80 110 •intestine, kidneys, lungs, and thymus were excised and I I . . . . . . , |veighed. A small slice was removed from each organ for his-: ! i feological examination and samples weighing up to 5 grams ! " * ■ were frozen immediately. Duplicate samples could be ob- i jtained from most of the rabbit tissues. In the case of the | rat tissues, it was necessary to pool the spleens and thymusi ! - ' - ' p | ' ■ ■ I I glands of several animals to perform the tissue analysis for! I I ‘ ' • ' i | ^ . ' i i heparin. Single analyses were made on the larger organs.* S | ■ ■ - • : ' ' ■ ' ’ i Data comparing the tissue heparin levels are shown ! in Table VII. In all cases, the rat organs contained more activity than those of the rabbit. The liver had the lowest! i k f tissue heparin level in both species. The kidney and thymus; were the richest in the rats, while the kidney was the high est in the rabbit, and the thymus was one of the lowest. It is of interest to note that the beef tissues examined had a ! much higher heparin content than did either of these animals*. Heparin levels in a human tissue.— Many difficulties! were encountered in obtaining normal human tissues. As a j ® I consequence, the number and types of tissues were much more * ! . j Limited than had been planned. Through the courtesy of j Dr. Edwin Boyle/ Jr., a series of samples of human intes- J I ;ines were obtained. None of the subjects had any pathology; • j in this tissue. A small portion of the duodenum was ground | and immersed in acetone as soon as possible after the post- i _______________ i i This work was performed by R. Posthuma, L. Gordon, j and P. Rucker.______ j 82 TABLE VII j Heparin Content of Rat and Rabbit Organs Heparin Content** No. of No. of No. of Units per Gram Organ Rats Measurement s* Rabbits Rats Rabbits Liver 19 19 12 1.73± .12 1.111.14 Spleen 20 8 10 4.84± .35 3.501.49 Intestine 18 18 12 5.55± .58 2.801.35 Lung 20 9 12 6.32±1.04 5.011.92 Kidney 18 15 12 12.84+1.69 5.221.81 Thymus 19 2 10 13.3 1.341.23 * The smaller organs required pooling of tissue; when the number of measurements is less than number of rats, some pooling is indicated. ** Mean heparin content ± the standard error. mortem. The acetone was drained and the samples were shipped to Los Angeles for analysis. The tissues, still wet with acetone, were homogen ized in more acetone, filtered, and air dried. Samples 1-15: '• ' ' ■ ■ ' ‘ ‘ : ' ’ ■ i were then treated as described in Chapter III, page 29. The' i solids were determined after digestion by drying a small j aliquot. j j Samples 16-26 were homogenized with acetone, drained, defatted with hexane, and air dried- The dry weights were recorded. They were then extracted by the same method. The digestion appeared to be more complete in this group. The heparin content was estimated by the anticoagu lant assay* The remainder of the samples were pooled for isolation of the heparin. The results of the heparin analy- ! ' jses are shown in Table VIII. ! The results are listed in two groups in order of in-| jCreasing chronological age. Samples 1-15 had not been ade- j guately digested during the proteolysis. This may have been ! ■ ' ■ ■ (due to the failure to defat these tissues with hexane after [acetone drying. Acetone alone apparently did not extract jail the lipids, and the samples were infiltrated with con siderable fat. In addition, the estimate of total solids was not dependable. This led to lower values and greater variability in this group as compared to the second group of samples, 16-26. 84 TABLE VIII Heparin Levels in Human Intestinal Tissue Heparin Activity No. and Subject Pathology No. Age Units per gram Dry Weight 1. M.S. 59-42 18 mo. 35 2. J.R. 59-49 3 mo. 33 3. C.B. 59-51 22 yrs. 8.8 4. W.J. 59-41 35 Lost 5. J.E. 59-63 38 26.5 6. J.K. 59-40 44 16 7. C.B. 59-55 53 14.5 8. H.B. 59-52 58 24 9. W.H. 59-45 58 14.5 10. A.W. 59-53 59 12 11. J.W. 59-43 60 16 12. D.E. 59-39 61 24 13. E.E. 59-60 67 20 14. T.G. 58-44 76 60 15. W.H. 59-50 78 10 16. Girl S. 59-54 1 day 190 17. A.W. 59-61 3 mo. 44 18. B.C. 59-58 8 mo. 35 19. E.M. 59-65 6 yrs. 38 20. E.H. 59-67 10 36 21. T.K. 59-67 24 28 22. C.B. 59-62 40 60 23. J.J. 59-68 46 60 24. H.Y. 59-65 55 42 25. T.B. 59-64 66 45 26. W.L.W. 59-59 67 42 I The data are inadequate as the basis for any conclu- ! ' sions. The results computed for wet weight would give a ■ ■ ■ i range of 3-8 units per gram. The most interesting observa- ! I tion is the high value for the tissue of the new born baby„ j (22 units per gram, wet weight). Since this is a single de-j I termination, it would have to be repeated before any conclu sions can be drawn. j The Incorporation of into the Tissues of Rats I 35 A series of rats were injected with S C>4 and sacri ficed at various time intervals to determine the optimum {time for maximum incorporation into heparin, i f t | Each rat received 1.14 x 10 c.p.m. by a single sub- - - 35 cutaneous injection. The was used carrier-free. The rats were kept in pairs in metabolic cages and the urine and feces were collected. Pairs were sacrificed at 2, 6 , 24, knd 48 hours following the injection. The blood was col lected by cardiac puncture and citrated. The liver, lungs, intestine, kidneys, injection site, and skin were removed. Fhe radioactivity in the urine was determined by direct planchetting. The feces, an aliquot of blood, and portions of the carcasses were wet digested and counted. All count- I ing was done at infinite thinness on the Nuclear Chicago D-47 gas flow counter with a thin window. The internal or gans, injection site, and skin were acetone dried, defatted | with isopropanol:hexane (H:IPA), and digested as in the tis-j s u e—hep a rin mebho d-.— —S-imu Ib-ane ou s-«diaiy s is —was—no • f e - ' T i s e d-s-~— J ’ The radioactivity was determined in the total digests. They were then dialyzed and the activity was again determined. ? {The acetone used for drying and the H:IPA from the defatting Istep were also counted in order to permit accounting for all the radioactivity injected. The data are;, shown in Table IX. The radioactivity recovered varied from 23.7 to 63 per cent. The counts in the carcass were rechecked by total digestion \ with KOH, but the values were no higher. It would appear \ that the low recoveries were primarily due to losses in the { urine collection. j After two hours, the internal organs and skin con tained the largest part of the total radioactivity. At 6 1 hours and later, the largest fraction of total counts was pound in the urine. Very little radioactivity was excreted j 1 * Jin the feces. From 6 to 48 hours, the activity in the blood, remained quite constant. The nature of the substantial ‘ amount of radioactivity in the carcass was not determined. j jit is likely that much of. this was incorporated into the connective tissue mucopolysaccharides - j The nature of the counts in the acetone was not de termined. It is likely that much of this is due to inor- jganic radiosulfate and small molecular weight sulfates; how- 1 ever, some lipid-like material may have been extracted. The presence of a substantial amount of counts in the defatting 'solvent (H:IPA) in the short time interval was unexpected. I - 35 This apparently represents incorporation of S . into i 87 TABLE IX OC Distribution of S in the Tissues and Excreta of Rats Following the Administration of Urine c.p.m. x 10 Feces c.p.m. x .10 Blood c.p.m. x 10- * Internal organs** c.p.m. x 10' Carcass c.p.m. x 107 Total c.p.m. x 10 8 7o recovered of administered dose 2.28 x 108 2 hours .86 (6.5)* 1.6 13.3 7.7 1.43 1.43 63 Time after Injection 6 hours 24 hours 2.2 (17)* lost .38 1.75 1.5 .54 23.7 9.2 (19)* 10 .30 .30 1.0 1.05 46 48 hours 10.8 (20)* 83 .39 .20 .39 1.14 50 * The values in parenthesis are the estimated urine values, if all the missing activity were in the urine. ** Includes the lungs, liver, kidney, intestine and the injection site and skin. Ssulfolipids, as they are not very soluble in acetone. The I ' : s pcidney and skin lipids were labeled the most rapidly. In- corporation of into the lung lipids was slower while thej liver lipids did not appear to have any label. I A number of interesting facts appear from an exami- j : ' "' ' ! nation of the data on the internal organs shown in Fig. 2 0. j The various internal organs examined show different patterns of incorporation with time. The intestine accumulated the ! I largest amount of the total activity at 2 hours. However, only 2 0 per cent of the activity in the dried, defatted tis sue was non-dialyzable. The proportion of non-dialyzable radioactivity increased with time and at 48 hours; nearly 60 per cent of the total activity and 80 per cent of the ac tivity in the tissue extract was in the non-dialyzable frac tion. The pattern in the liver was similar, but the frac tion of total activity incorporated into the non-dialyzable Ssxtract was higher at 2 hours. In the lungs, all the activity in the tissue extract |vas non-dialyzable. This fraction seemed to have a peak at I 6 hours and did not change between 24 and 48 hours while , i' liver and intestine radioactivity decreased at each succeed ing interval. The kidneys showed a very different pattern. ! ■ ■ ■ ■' The total amount of radioactivity accumulated was substantial j i ' " ■ ' • £>n a weight basis and did not decrease with time to the ex- jtent observed in the other organs. Both the relative and absolute counts in the non-dialyzable fraction increased .p.m. X10 per gram of tissue Fig. 20 Distribution of in Rat Tissue Following Subcutaneous Injection in c.p.m. XIO5 per gram □ c.p.m. in acetone + H: IPA c.p.m. dialysate “ 891 non-dialyzable c.p.m. 13 12.4 2 6 24 48 Liver 2 6 24 48 Intest. 2 6 24 48 Lung 2 6 24 48 Kidney 2 6 24 48 Skin i • i With time. The values at 24 hours are somewhat anomalous. There are two possible explanations for the different behav ior of the kidney. Since the excretion of sulfate is via i 3 5 * the kidney, it will continue to get exposed to the S for a longer period than the other organs. It was also the tis-l ■ • ' I ■ - I Jsue which had the highest heparin content of the organs j ' I • examined in rats. \ \ The skin actively accumulated sulfate. However, \ \ bnly about 1 0 per cent of the total activity was in the non-- pialyzable fraction at 48 hours due to the large amount of I 35 acetone extractable and HrIPA soluble S Several of the digested, dialyzed samples were ex- i amined by column chromatography. The results of these anal-jj yses are shown in Figs. 21-25. A sample of pooled tissue j Lxtracts retained from the determination of tissue heparin j i ' I levels was also chromatographed and the results of this run ; t ■ ‘ . . . . . I I ' is shown m Fig. 26. The total radioactivity, the amount off I ■ I MPS, and the specific activity are shown in Table X. I j Part of the radioactivity was not retained by the jcolumn and appeared in the washings. This material con- ! - jtained no MPS, but did have uv absorbing (280 mu) substances! 'the radioactivity in this fraction was substantial in the 2 lour intestine sample, but small in the 6 - and 24-hour sam ples . In the intestine samples, a split peak with maxima at fraction 12-13 and 17 with a minima at 15 appears at all j - Rat Intestine Extract Alcian Blue 2 0 - £ 0 1. 0M 10 -<40 ■■ 0.5M --0.25M --0.125M Fraction No Rat Intestine Extract - - S Alcian Blue 20 --80 1. 0M 10 --40 r\ -- 0.5M Fraction No Fig. 23 24 Hour Rat Intestine Extract an Blue 1.0M r-l 10-- 40 5 -- Fraction No Fig. 24 6 Hour Rat Liver Extract 100 — S Alcian Blue 20-- 80 IH 10 - 40 --0.5M I 0 10 20 30 40 Fraction No Fig. 25 24 Hour Rat Liver Extract Alcian Blue 15-- 60 ; V S3 10-- 40 --0.5M 5 -- 10 Fraction No. vo Fig. 26 Mixed Rat Tissue MPS-AE-50, pH change 50 ^ 40 .. --1.0M - -0.5M 50 Fraction No 97 TABLE X Analysis of Chromatograms of Rat Tissue Extracts Tissue Radioactivity Radioactivity MPS* and Applied Recovered Recovered c.p.m. per** Time c.p.m. x 103 c.p.m. x 103 mg. mg. MPS Intestine 2 hours 6 hours 24 hours 33.0 18.0 12.5 32.1 16.6 13.0 1.81 .87 3.27 11.1 16.9 4.13 Liver 6 hours 24 hours 18.0 6.6 18.7 6.75 2.62 2.40 7.0 2.5 Mixed tissue extract 5.51 * Calculated from the alcian blue staining method. ** Only the radioactivity associated with the MPS was used for this calculation. ; (three time intervals. A trace of Alcian blue staining mate-| rial was found in this region in the 2 -hour sample, but was ! absent at 6 and 24 hours. The radioactivity in this peak | I ' ' ' ! was high at 2 hours and much lower at 6 and 24 hours. It is: ! ' ! possible this material was some sort of intermediate which i ' ‘ ‘ ; - ' ■ was non-dialyzable. In the liver, a very small amount of | material appeared in this region. : ■ - ! An Alcian blue staining and radioactive peak ap- j peared in all chromatograms at fractions 25-26. No known i mucopolysaccharide examined was eluted in this region. In the intestinal samples, a substantial part of the total 'counts were found in this peak at 2 and 6 hours, and to a lesser extent at 24 hours. Very little radioactivity was found in this peak in the liver samples. The specific ac tivity of the MPS in this peak was not higher than the CSA- • ■ ... ■ heparin peaks except at 24 hours in the intestine. It does hot appear to be a precursor of heparin. The results indicate that the heparin with the high- 2St specific radioactivity would be obtained at 6 hours in jthe intestine. The specific activity of the 2-hour sample is higher than that of the 24-hour sample. The 2-hour sam ple does not appear to contain very much heparin. This may he an experimental error. In the liver, the 6 -hour sample has a higher specific activity than the 24-hour sample. The distribution of radioactivity and mucopolysaccharides was _ 99' I different in intestine and liver. The liver data are more i difficult to interpret. i f The mixed rat tissue extract pattern is similar to aj partially purified heparin sample with a substantial amount j of CSA present. A very small peak also appears at fraction I 2 3 which corresponds to the peak seen in the individual tis-j ! sue runs. It is proportionally much smaller. j ! The general correspondence of the Alcian blue and radioactivity curves is good. This method should be useful for further studies of sulfate metabolism and MPS biosynthe sis . A Comparison of Heparin and Heparin-Like Materials in Plasma Derived from Several Species Heparin from several species.— Beef and hog heparin are commercially available and such preparations were exam ined by the column chromatographic methods described earlier. Typical elution patterns are shown in Pig. 12 for beef hepa rin and Figs. 18 and 19 for hog heparin. There was no ap- I parent chromatographic difference between hog and beef hepa rin. A sample of beef heparin and two samples of hog hepa rin were examined in the analytical ultracentrifuge by Dr. Earl Frieden. The results are shown in Table XI. Under jphe conditions, there do not appear to be any differences in the samples. The molecular weight calculated from these I 3ata is about 10,000-12,000. This value is in agreement with the literature. The samples appeared to be homogeneous 100 TABLE XI The Ultraeentrifuge Examination of Beef and Hog Heparin* S20 -13 Source Sample Designation (in 10 ) Beef SN 60058 125 units per mg. 1.73 Hog Lot #31543 154 units per mg. 1.65 Hog Lot #31353 147 units per mg. 1.71 o Conditions: 20 G., ionic strength (NaCl) = 1.0 M pH 6.2, PO4 Buffer = 0.01 M Heparin Concentration = 1.00% Comments No evidence of heterogenity No evidence of heterogenity No evidence of heterogenity *Data by courtesy of Dr. E. Frieden, Florida State University in the ultraeentrifuge, although they did not in the column | I chromatographic systems. This would be the case if the var-| ious components were very close in molecular weight. j Two samples of dog heparin were prepared from the j lungs, liver, and intestine. One was biosynthetically la- J 3 5 i beled with S and had an anticoagulant activity of 160 USP j units per mg? the other was not labeled and had 135 units I per mg. These samples were examined by the column chromato-j ) graphic technique. A third sample of crude dog heparin was J obtained from Hynson, Wescott and Dunning with a unit activ ity of 12.5 units per mg. Twenty, mg of the crude heparin preparation containing 2 50 units were chromatographed. A total of 2.66 mg of MPS was found by the Alcian blue stain ing method, with 2 . 1 2 mg eluted in the same fractions as the purified dog heparin samples. The elution patterns of these I ' dog heparin samples are shown m Figs. 27 and 28. The dog heparin samples gave different results from e of beef and hog in that the elution began at a higher I NaCl concentration and the main peaks were all eluted at higher concentrations of salt than any other type of heparin 2xamined. The sulfur content of the purified dog heparin samples was found to be the same as the beef and hog heparin and so the difference in the chromatographic pattern cannot I oe explained by a higher degree of sulfation. Samples of rat and human heparin were prepared in small quantities by acetone fractionation from the tissue fhos Fig. 27 Dog Heparin 160 u/mg 5.0 mg. 1.5M -S Alcian Blue 60 g • r l 1.0M 0 0.5M roo 110 H o to Fig. 28 Dog Heparin 135 u/mg. ---- Crude Dog Heparin HMD 12.5 mg. — ---- I.5M 1.0M 0.5M 110 Fraction No [extracts obtained in the work described earlier. These ; i s preparations were purified to 100 units per mg. Further j I fractionation would have resulted in additional losses and \ ' ' \ the amounts available were limited. Both samples were ex- j amined in the column chromatographic system,, and the resultsj are shown in Figs. 29 and 30. The elution patterns were | \ very similar to thosq of beef and hqg heparin except for {presence of larger amounts of MPS in the early part of the j I, ■ \n.- „ . „ v , ■ I elution. This was to be expected, due to the low unit ac tivity of the samples and the probable contamination with heparitin sulfate-like substances. All these preparations were subjected to paper chromatography in the system described in Chapter III. There was no difference except that the spot was more elon gated in the case of the beef, rat, and human heparin sam ples. The purified dog heparin and the hog heparin had the npst compact spots which corresponded to the lowest R f (.63) of the range in which they all fell (.60-.70). A trace of material running with the front was observed in the rat, human, and crude dog.heparin samples. The data suggest that only dog heparin is different from the others by the criteria applied. The quantities of material from rat, human, and dog were inadequate for an ex amination of the biological activity of these materials in ^ivo. Prior experience had shown that there was no detect- Fig. 29 2 Mg. Rat Tissue Heparin (100 u/mg.)-AE-50; pH change 1.5M 30 -- 1.0M 20 - - 0.5M 100 110 Fraction No. o j un. Fig. 30 2 Mg. Human Heparin (Intestine 100 u/mg.)-AE-50, pH change I.5M -- 1.0M 30 -- -- 0.5M 100 110 Fraction No jable difference in the biological activity of tbe hog and i beef preparations. j ! i | Heparin-like substances in the serum or plasma of | man and animals.— The MPS were isolated from two larqe sam- ! f ■ ! pies of outdated human plasma which were obtained from j Hyland Laboratories through the courtesy of Dr. Roy Fisk. I Four liters were obtained and they were processed in two portions of 2 liters each as described in Chapter III, ex- i i jcept the residues after the initial proteolytic digestions | were redigested two times. 1.3 grams of lyophilized solids j jwere obtained from one sample (No. 1) and 1.5 grams from the |bther (No. 2), which represented about 1 per cent by weight Lf the original plasma solids. The solids were tested for |anticoagulant activity and found to be less than 0.5 unit per mg. ( | Each of the samples were subjected to column chroma tography. The samples were dissolved in about 50 ml of di- Lute buffer (0.0125M ammonium formate pH 4.3), and washed into the column. The solution was yellow-brown in color, irhe washings showed a considerable amount of the same color. I ' " ■ " " ' ‘ In the course of the elution, a peak appeared centering at fraction 15 which was yellow and had an ultraviolet absorp tion maxima at 280 mu. No Alcian blue positive substances were found in the wash or in the early yellow peak. The two elution patterns are shown in Figs. 31 and 32. Using Alcian blue staining, the MPS in each sample was j Fig. 31 MPS from 2 liters Human Plasma #1 40 -- 30 -- --1.0M u 20 -- •-0.5M Fraction No. Fig. 32 MPS from 2 liters Human Plasma #2 30 -- 20 • - --0.5M 10 -- Fraction No o I I [estimated to be 11.0 mg in No. 1 and 6 . 8 mg xn No. 2. In ; [both cases, about 5.5 mg appeared after fraction 60 which isj ' * ! the region where heparin and heparitin sulfate would be j eluted. i In run 1, fractions 38-49, 50-58, 59-69, 70-74, and j j I 75-83 and in run 2, 47-57, 58-68, and 69-80 were pooled, j evaporated to a small volume (5-1Q ml), dialyzed, and lyo- j j philized. They were then dissolved in a small amount of ! j | distilled water, subjected to paper chromatography, and tested for anticoagulant activity. The paper chromatograms failed to show the presence of a metachromatic component with a characteristic of I (heparin. A component was found in the fractions eluted first which had a similar to. that of heparin, but not identical and stained blue. In the fractions which were. |eluted last and resembled heparin or heparitin sulfate, a pomponent was seen xn the paper chromatograms whxch had a j similar mobility to that of heparitin sulfate and was meta- chromatic. It did not run with the front as does CSA-A. There were signs of a small amount of material in the early fractions which had a mobility close to CSA-B (B-heparin). The amount of anticoagulant activity was small and | ; ' • jno defxnxte fxgures were obtaxned. It was much less than jvould have been expected from the amount of plasma used if 110 unit per cent had been present. j In a similar manner, 1.5 liters of beef serum and i 1 . 2 liters of bog serum was processed. 1 . 1 grams of solids ‘ ‘ ' « were obtained as the final product from beef and 1 . 0 gram I for tbe bog serum. These samples were subjected to column j ii J : . 1 chromatography and tbe elutions patterns are shown in | I I I ! jFigs. 33 and 34. These plasma extracts behaved very simi- j ! larly to those of the human extracts, including the manner j in which the chromagenic substances were eluted. \ i The beef serum showed the presence of 4.5 mg of MPS | i with 1.5 mg appearing after fraction 60. The hog serum sam-J pie had 3.3 mg of MPS with 0.8 mg appearing after fraction j i |60. Fractions 41-59 and 60-79 of the beef and 41-50, 52-58, jand 60-70 of hog were pooled, concentrated, dialyzed, and | lyophilized. The beef samples were lost due to faulty dial ysis bags. The hog samples were paper chromatographed and Itested for anticoagulant activity. The first pooled sample from the hog showed a long streak almost the entire length of the paper, none of which jwas metachromatic, with the exception of a slightly purplish region with a mobility of CSA-B. The middle portion showed p . slightly metachromatic spot at the front and resembled pSA-A. The last portion showed a metachromatic region very I " similar to the one in human plasma with a Rf of 0.85. A sample of plasma from a patient with suspected hyperheparinemia had been obtained from Dr. G. Lanchantin several years ago and had been held frozen since that time. Fig. 33 MPS from 1.2 liters of Hog Plasma 40 -- --1.0M 30 -- 20 - ■ --0.5M 10 - - Fraction No. Fig. 34 MPS from 1.5 liters of Beef Plasma 40-- 30.. --1.0M a 20-- --0.5M 50 Fraction No. jThe MPS were isolated by the techniques described for the i i 1 other plasma samples. Approximately 150 ml of plasma was j available. The product isolated which weighed 0.150 grams j ' ■ ’ I was column chromatographed. The elution pattern is shown in; Fig. 35. The total MPS was found to be 0.92 mg with 0.60 mg after fraction 65. Fractions 52-65 and 66-78 were pooled and prepared as described above. They were then examined in the paper chromatographic system. The first portion showed two spots, one with an just ahead of heparin but not metachromatic, and another at the front which was metachro- matic. The second portion showed a metachromatic streak with a mobility about that of heparitin sulfate. No meta chromatic spot with a mobility of heparin was found. The paper chromatographic data, on the MPS from all these samples are summarized in Table XII. The data from a chromatographic analysis of a human intestinal extract ^treated in the same manner are included in the table. I The Biological Activity and Metabolic Fate of Exogenous Heparin in Several Species The biological response to the administration of subcutaneous heparin was determined in rats, dogs, and human subjects. The activities measured were the prolongation of the whole blood clotting time by the Lee-White method and Lipoprotein lipase by the method of Grossman. Urines were collected in the same or similar experiments and examined for the excretion of heparin and its degradation products. Fig. 35 MPS from Plasma of Hyperheparinemic Patient 150 ml. ACD Plasma -- 1.0M 30 -- 20 - - 0.5M 10 - - Fraction No. H H tn 1U> TABLE XII The Heparin-like Substances in Serum or Plasma Source and Amount Human Plasma #1 2 liters Total MPS Mg. 11.0 Pooled Fractions Ho. 38-49 50-58 59-69 70-74 75-83 MPS in Pooled Samples Mg. 3.0 2.5 3.5 1.5 0.5 Rf* .67(b) .67(b) ♦80(p) .80(p) .85(pk) Human 6.8 47-57 1.3 No spot Plasma #2 58-68 4.0 .67(b); .85(p) 2 liters 69-80 1.5 -85(p) Hyper heparin emic Plasma 150 ml. .92 52-65 66-78 0.32 0.60 .7(b); 1.0(p) .85(p); .90(p) Hog Plasma 1.2 liters 3.3 41-50 52-58 60-70 2.0 0.5 0.8 •4(p); .5(b).80(b) 1.0(p) .85(pk) Beef Serum 1.5 liters 4.5 41-59 60-79 3.0 1.5 lost lost Human Intestine 11.2 Extract 45-55 56-66 67-74 75-82 83-91 1.0 4.7 2.3 2.5 0.7 1.0(p) 1.0(p); .80(p) •80(p); .65(p) .65(pk) .65(pk) *Metachromasia is indicated by p for purple and pk for pink; b indi cates blue spot, not metachromatic. The Rf indicated is the center of the region of the spot. 135 ;S labeled heparin was used in the urinary studies in rats * ( j Urinary data from dogs are included through the courtesy of jDr. L. Levy. j The biological activity of heparin in three j ■ i i species.— s j 1. Rats: The male rats, 150-200 grams, were ob- j tained from Carworth Farms. Heparin sodium, USPj, i of hog origin and having a potency of 140 units j per mg was used. Solutions of 500-1000 units per ml were prepared just before use. The hepa rin was injected into the subcutaneous tissue in the back. The doses ranged from 2 00 to 1000 units per kg. The animals were sacrificed at intervals from 0 to 7 hours. The blood was j taken by cardiac puncture and the Lee-White clotting times measured immediately. Similarly | . treated groups of animals were used for the mea surement of clearing. The results are shown in Table XIII. The data indicate that the response of rats was of short duration and, even at the high dose of 1 0 0 0 units per kg, most of the ac tivity in blood was gone at 7 hours. | • ■ ii 2. Dogs: The animals were obtained from the Los ! ■ i l ; Angeles City Pound and conditioned for at least j two weeks in the animal quarters of Riker Labo- j i! ratories. The same heparin preparations were J TABLE XIII Dose per Kg. No.* . 200 9 250 9 500 9 1000 20 Dose per Kg. No.* 200 9 250 6 500 13 ; 1000 30 *Number of animals. Clearing and Clotting Response Following the Administration of Subcutaneous Heparin to Rats Lee-White Clotting Times (in minutes) Hours after Injection 0 .5 1 2 3 7.1 10.8 13.4 10.8 8.9 14.8 28.0 >60 >60 34.9 Clearing Response in Grossman Units 0 .5 1 2 3 .20 1.20 .75 .45 .25 1.34 3.34 4.30 4.61 4.05 2.10 7.5 11.3 .06 .40 24.5 .85 7.1 7.0 17.8 .05 .22 .45 118 119 used that were applied to the rats. Solutions of 5000-10,000 units per ml were prepaxed and injected into the subcutaneous tissue in the back of the animal at dose levels of 2 50-1000 units per kg. Blood samples were taken by veni puncture at various time intervals. A portion was tested by the LeeVWhite method and another portion was converted to plasma and the clearing determined. The results are shown in Table XIV. The dogs responded to an equivalent dose to a greater extent than the rats, particularly with respect to the clotting effect, and the duration of activity was longer. 3, Human subjects: Volunteers were obtained from the work force at Riker Laboratories. The sub jects were all male, from ages 30 to 45, and clinically healthy. Their serum lipids ranged from normal to slightly elevated levels. The experiments were run in groups of 5 or 6 sub jects. The heparin used was Riker Lipo-Hepin Lot No. 02454, and doses from 2,500 to 30,000 units per individual were injected subcutaneous- ly in the ileac crest region. Blood samples were taken by venipuncture at 0, 1, 2, 3, 6 , and/or 8 hours. In the 20,000 unit dose experi ment, a 24-hour sample was also taken. The TABLE XIV Clearing and Clotting Response Following the Administration of Subcutaneous Heparin to Dogs Lee-White Clotting Times (in minutes) Dose per Kg. No.* 0 1 Hours after Injection 2 3 4 5 6 7 9 16 24 250 8 8.8 23.8 33.3 17.8 12.0 500 8 10.3 49.7 >60 >60 22.9 10.3 1000 20 10.5 39.5 >60 >60 30 10.3 Dose per Kg. No.* 0 Clearing Response in Grossman Units 1 2 3 4 5 6 7 9 16 24 250 8 .05 1.00 .71 .32 .20 .04 .04 500 8 .02 2.81 2.05 1.13 .39 0.0 0.0 1000 20 ,05 2.30 3.64 3.22 2.03 1.52 .24 .07 *Number of dogs at each level. 120 30,000 unit experiment was run in two parts witTi 6 subjects in each case. The heparin was admin- 3 J istered at 4 P.M. and 16- and 20-hour samples j were taken. In all other cases, the heparin wad ' ' ' . ' ‘ ” i. given at 8 A.M. to fasting subjects. The data j from these experiments are summarized in | Table XV. Man appeared to be more sensitive than either rats or dogs both in clotting and clearing response to equivalent doses of hepa rin. The duration of the effect was greater. J Since absolute doses per subject were used rather than a dose per unit weight, it is not possible to make a precise comparison, but as suming an average weight of about 75 kg, the highest dose in man was 400 units per kg and the lowest was about 35 units per kg. It is of in terest to note that the two lowest doses used in the human subjects failed to produce any effect on clotting as measured by the Lee-White method,] but clearing could be demonstrated. Urinary excretion and metabolic fate of injected leparin.— 35 1. Rats: Two types of S labeled heparin were used in these experiments. One was biosyntheti- cally labeled heparin prepared by the subcutane-j 3 5 ^ ous injection of young hogs with ■S_ro4- and______ I TABLE XV Clearing and Clotting Data from Human Subjects Following the Administration of Subcutaneous Heparin Dose No. Sub j. 0 Lee-White Clotting Times 1 2 3 (minutes) 6 8 16 20 24 2,500 5 12.2 11 y6 12.3 12.1 12.1 5,000 5 13.9 13.7 13.0 12.4 12.7 11.8 10,000 11 11.4 17.1 17.7 17.1 16.3 16.1 20,000 5 10.9 18.7 28.5 20.9 10.6 30,000 12 12.5 33.9 17.6 Dose No. Subj. 0 Clearing : 1 2 in Grossman 3 Units 6 8 16 20 24 2,500 5 0.0 .13 ± .14 .11 ± .09 .11 ± .06 ± .10 .08 5,000 5 0.05 ± .08 .38 ± .05 . 34 ± .06 .25 ± .03 ± .14 .12 .18 ± .11 10,000 11 0.05 + .11 .78 ± . 17 .73 ± .16 .65 ± .18 ± .49 .14 .37 ± .10 20,000 5 0.10 ± .06 1.26 ± .37 .95 ± .25 .63 ± .18 .21 ± .12 30,000 12 0.03 ± .06 .62 ± .23 .28 ± .25 122 isolating the heparin from the tissues (lung, j liver, and intestine).^" The other was prepared ! O ; by chemically N-sulfating heparin which had s been partially N-desulfated. The biosynthetic I i O I heparin (B-H) was only 10 cpm/mg while the j 4 ‘ chemically labeled material (CL-H) was 10 cpm/mg. One thousand units per kg of heparin | • 3 ! was administered subcutaneously to the rats. In! 35 addition to the two types of hepapn, S was j also administered for comparison. Animals were j sacrificed at 1, 4, 7, and 24 hours and the blood withdrawn to permit the plasma radioactiv ity to be determined. The rats were placed in metabolic cages for urine collection. The urine was counted by direct planchetting. A portion was also dialyzed and then counted. These data are shown in Table XVI and indicate that only a small portion of the heparin excreted is desul- fated while the main part of the radioactivity is still part of a macromolecule. No anticoagu lant activity was found in the urine. All the evidence suggests the modification of the hepa rin before excretion in the urine. The urinary data at 24 hours with both types of labeled ^Supplied by P. Rucker and L.. Levy (Riker Labs.). 2 i Supplied by F. Petrachek (Riker Labs.). i 124 Substance B-H* CL-H*** #13655 CL-H #13763 S350, TABLE XVI Urinary Excretion and plasma Levels of S33 Labeled Heparin After Subcutaneous Injection in Rats Time after Injection 1 4 7 24 24 48 % of Administered Radioactivity in Urine 2.1 5.4 6.5 15.0 14.0 30.0 1 4 7 24 24 48 4.2 5.2 10.6 10.0 25.0 31.8 % of Radioactivity in Urine After Dialysis 0.0 72 88 63 100 lost 86 88 100 78 % of Dose in Plasma*** 5.8 9.6 0 0 1 0 0 0 0 * Biosynthetically labeled heparin; 2.6 x 10^ GPM administered. ** Chemically labeled heparin by N-resulfation #13655 3.0 x 105CPM administered #13763 3.3 x 105CPM administered 1000 units per kg. administered in all cases *** Radioactivity isolated by the procedure for serum heparin of . Engelberg et. al. . _ _ * - 12 51 lieparin indicate that even though all evidence j of systemic biological activity was gone, most of the radioactivity was still in the body. The data on radioactive sulfate suggest that if the administered heparin was extensively desulfated, more inorganic sulfate would have been found in the urine and a larger proportion of the in jected radioactivity would have been excreted. 2. Dogs: A number of dogs were given a subcutane ous injection of 2 0 0 units per kg of chemically labeled heparin. Some were given 1 dose and others were given 2 doses one day apart. The urines were collected and counted and the data are shown in Table XVII. A number of the ani mals were sacrified 24 hours after 1 dose and some after shorter intervals. Various tissues were samples and counted. The results suggest that following an injection, the heparin became widely distributed in the body fluids throughout the body. With time, a substantial portion ac cumulated in the liver. The urinary excretion was rapid at first and became very slow after the first several hours. The distribution of the radioactivity which remained was not deter mined. Dr. Levy, who performed the dog experi ments and was kind enough to make these data 126 TABLE XVII 35 Excretion of S in the Urine of Dogs Receiving Radioactive Heparin by Subcutaneous Administration* % OF COUNTS IN URINE AFTER A SINGLE INJECTION OF 200 UNITS OF S3504 LABELED N-RESULFATED HEPARIN Time after Injection 24 hours 7 days** 12.0 37.7 52 24.4 % OF COUNTS IN URINE FOLLOWING 2 DAILY INJECTIONS OF 200 UNITS Time after Second Injection 24 hours** 7 days** 31.6; 28.3; 46.7; 56.8; 75.3; 47.6; 50.0; 41,5; 14,4; 54.1; 76.7; 67.7 48.0; 31.0; 28.3; 30.0; 37.4; 30.4; 21.3 * Data by courtesy of Dr. L. Levy, Research Division, Riker Labora tories, Inc. ** Accumulated radioactivity 12 7j I available, feels that the extent of urinary ex- ] t cretion is dose dependent. j I Human subjects: In the experiment with human volunteers, urine collections were made. The urine was collected in two portions--from 0 to 8 hours and 8 to 24 hours. Toluene was used as a preservative. . In the case of the 30,000 unit dose experiment, a 24-hour collection was made. Twenty-four-hour urines were collected from two groups of subjects who had not received heparin. Many of these individuals had participated in the heparin experiments at another time. The urines from each group and time period were pooled and the MPS were isolated by complexing with a quaternary amine salt and floating with methyl isobutyl ketone, as described in Chapter III. The isolated complex was dissolved in 3M NaCl and the MPS precipitated with 3 volumes of methanol. The precipitate obtained was dissolved in a small volume of water, clarified by centri fugation and lyophilized. The weight of the crude MPS and the anti coagulant activity were determined. A qualita tive biuret test was run and found to be posi tive in all cases. The isolated solids were chromatographed and the of the metachromatic spots was determined. Normal urine showed one j spot at the front (Rf of 1.0) and was apparently CSA-A or C. A similar pattern was found with j the 2500 and 5000 unit experiments. At all the : I higher levels, a spot with a characteristic j of heparin (0.65) was seen along with the CSA-A j spot. A portion of the isola.ted MPS was chro- ! ! matographed by the column method. These chro- j matograms are shown in Figs. 36-41. The total Alcian blue positive material was estimated and the actual MPS were calculated for each prepara tion. These data are shown in Table XVIII. . . I The chromatographic analyses indicate that ! ] normal urine contains MPS which resemble CSA-A. f ’ ' ' . ‘ ! In the experiments where the subjects received j 10,000 units or more, the MPS appear to contain i ■ ' ! a heparin-like component in addition to the CSA.j Although very little heparin activity was recov- ered, the MPS found in the urine of these sub- j jects resembled heparin both in its behavior on J paper chromatography and in column chromatogra phy. The anticoagulant activity recovered in the 10,000, 2 0,000, and 30,000 unit experiments were highly comparable in terms of percentage of dose. However, a higher percentage of MPS was found in the 10,000 unit experiment. The I Fig. 36 Normal Urine MPS No. 1 1.0M --0.5M 10 -- 20 30 40 50 60 Fraction No. H fo J£>. Fig. 37 Urinary MPS from Subjects Receiving 5,000 Units Heparin - 0-8 hrs — 8~24 hrs - -1.OM 30 -- o 20 -- --0.5M 10 - - 30 40 50 60 70 80 90 Fraction No. U) o Fig. 38 Urinary MPS from Subjects Receiving 10,000 units Heparin - Group 1 0--8 hrs, 8-24 hrs. 40 -- r~ 30 --1.0M --0.5M 10 - Fraction No. H ( jO H Fig. 39 Urinary MPS from Subjects Receiving 10,000 units Heparin - Group 2 0- 8 hrs. - 8-24 hrs. 40 -t- --1.0M 20 -- --0.5M 10 - - Fraction No. H W Fig. 40 Urinary MPS from Subjects Receiving 20,000 Units 0- 8 hrs. 8-24 hrs. -- 1.0M 30-- 20-- -- 0.5M 10- - Fraction No. C O U) ~ n Fig. 41 Urinary MPS from Subjects Receiving 30,000 Units Group 2 - — - Group 1 Part 1 Group 1 Part 2 40 ^ -- 1.0M t \ -- u --0.5M 10 Fraction No. TABLE XVIII Urinary Mucopolysaccharides from Subjects Receiving Heparin Acid Mucopolysaccharides Isolated Hep. Wt. of No. of Dose Time of Crude Est. % Recovery Subjects Units Collection Isolate MPS* Wt. Units Rf 6 - 0-24 144 mg. 17.7 mg. « • 1.0 5 0-24 146 mg. 13.0 1.0 4 2,500 0-8 70 mg. •• 0 0 1.0 3 2,500 8-24 100 mg. “ - 0 1.0 6 5,000 0-8 75 mg. 6.7 mg. 0 0 1.0 8-24 100 mg. 9 mg. 0 0 1.0 6 10,000 0-8 900 mg.** 97 mg. 0 o 4.2 .65; 1.0 8-24 890 mg. 68 mg. 33 0 .65; 1.0 5 10,000 0-8 250 mg. 100 mg. 2.8 .65; 1.0 8-24 360 mg. 40 mg. 34 0 .65; 1.0 5 20,000 0-8 104 mg.*** 22 mg. 2.7 0.1 .65; 1.0 8-24 175 mg. 49 mg. 5.3 1.0 .65; 1.0 6 30,000 0-24 547 mg. 144 mg. 11 4.4 .65; 1.0 6 30,000 0-24 576 mg. 173 mg. 13 3.6 .65; 1.0 * Estimated from the column chromatograms. ** Not filtered, contains insoluble material. *** Collection or processing incomplete. The Rj of CSA-A was 1.0 and heparin was 0.65. 135 I \ I absolute weight of the MPS found was similar in i the 10,000 and 30,000 unit experiments. The j 0 - to 8 -hour collection in the 2 0 , 0 0 0 unit ex- j J periment was either incomplete or the processing! was inadequate. It now is apparent that a j longer period of urine collection would have J been desirable. It appears that most of the ad-1 ' I } ministered heparin was modified to some extent, i ■ ! The animal data indicate that a large portion of I 1 the administered heparin is not excreted in 24 hours, and the same may be true in people. Heparin Levels. Mast Cell Counts, and Atherosclerosis In the experiments described in Chapter III, the heparin levels in rat and rabbit tissues were determined, and mast cell counts were made on the same organs. The rat "is known to be resistant to atherosclerosis and the rabbit Jis a susceptible animal. The mast cell counts were performed by Dr. I». Marx. The slices of tissue were fixed in a mixture of formalin and sthyl alcohol, embedded in paraffin and cut into 5u sections. These were then stained in 1 per cent aqueous toluidine blue I for 10-15 minutes. The slides were then dehydrated with \ Lsopropanol. Eight to ten days later the metachromasia was j well developed. The mast cells of the rat stained a very dark purplish-black. Those of the rabbits were a pink color. The mast cell counts were made by counting 20 fields corre- ■ 0 *sponding to a total area of 3.5 mm When there appeared to be considerable irregularity, the counts were repeated. | . j Pink lumps in the rabbit kidney were counted as mast cells, j I although they may have been cell fragments. i I In some organs, a diffuse, metachromasia could be de-j ‘ : • • ‘ I - tected which was not associated with a specific cell. The ..... . j results of the heparin analyses and the mast cell counts of ! • ’ ' ' ‘ ' ' ■ ' ■ ■ ''1 the rat tissues are summarized in Table XIX. Those for the j rabbit tissues are shown in Table XX. The data show that the rat tissues examined were higher in heparin content than any of the comparable rabbit I ' ' ' ' jtissues, although the differences were not great. This was not true of the mast cell counts. In neither animal was there a consistent relationship between the mast cell counts and the heparin content of these tissues. In the rats, the highest heparin content was in the kidney and thymus. How- S ' I ever, the kidney had the lowest mast cell count while the ! ■ . . . . . . thymus had the highest. Similar disparities were found in | . . . . - j:he comparison of the mast cell count and heparin content of rabbit tissues. | The heparin levels of several beef and hog tissues Tjiave been determined. In beef the lungs are the highest in heparin content--30-50 units per gram, with the intestine j next at 25-30 units per gram, and the liver considerably i i.ess at 10-15 units per gram. The mast cell counts are very 138 TABLE XIX Heparin Content and Mast Cell Count of Rat Organs Mean Heparin Mean Mast Cell Number of Content Count M a C! T i n 4 4-0 n a t * t~r n o n *3 ^ in m ^ 1 Organ Rats* Measuremen t s Units per g per 3..5 y mm Liver 19; 20 19 1.73 .12 10.8 + 1.6 Spleen 20; 19 8** 4.84 + .35 3.6 + 1.4 Intestine 20; 20 18 5.55 + .58 12.5 ± 4.2 Lung 20; 19 9** 6.32 + 1.04 17.2 ± 1.7 Kidney 18; 19 15** 12.84 ± 1.69 3.5 ± .9 Thymus 19; 16 2** 13.3 66.2 ± 9.5 * First figure, heparin assay; second figure, mast cell count. ** The smaller organs required pooling of tissues. The standard error was computed for the number of measurements shown. TT9 TABLE XX Heparin Content and Hast Cell Count of Rabbit Organs Mean Heparin Content Mean Mast Cell Count Organ No. of Rabbits* Units per g. per 3.5 Liver 12 11 1.11 + .14** 28.7 ± 4.4** Thymus 10 12 1.34 + .23 21.8 ± 3.8 Intestine 12 12 2.80 + .35 42.3 ± 3.0 Spleen 11 11 3.50 + .49 25.2 ± 5.4 Lung 12 12 5.01 ± .92 30.9 ± 5.6 Kidney 12 12 5.22 + .81 27.5 ± 4.5 * First figure, heparin assay; second figure, mast cell count. ** ± standard error 2 ' ' high in the intestine (approximately 150-300/3.5 cm ), while £ , jj {those in the lungs are about 60-120. The liver is quite low • ' ' ' ’ ' { in mast cells. Thus, there appears to be a disparity be- j ■ . ‘ } tween counts and heparin content. The heparin levels and | mast cell counts are substantially higher than the corre- j sponding levels for the rats and rabbits. { In hogs, the intestine is very similar to that of \ ■ - I the lung-— about 2 5-35 units per gram. The liver is very ! ■ ■ ■ ■ | similar to that of beef. No mast cell counts were made on ' ' ' i hog tissues. No real data exist as to the susceptibility of cattle and hogs to atherosclerosis. The dog, which is a resistant animal, has the highest heparin content in the liver of the organs examined. The lungs and intestine were relatively high, but lower than the liver. The levels are much higher than those of rats and J rabbits, but not quite as high as those of beef and hogs. The intestine is very rich in mast cells with the lung next and the liver the lowest. The lack of correlation of mast cell counts with the heparin levels of the tissues is again apparent. The available data on the heparin levels in tissues of man was presented earlier. Human intestine contains from 3-8 units per gram of heparin. Some very preliminary data jon human lungs and liver indicated levels of about 10-15 junits per gram in lungs and 5-10 units per gram in liver. j The same sample of human liver showed very few mast cells. { } The autopsy data for the subjects did not seem to i j indicate any relationship between age or pathology and hepa-i . ' ! rin levels. If the total organ weight and the fraction re- j t ceived had been known, the results might have been more meaningful. CHAPTER V | .! DISCUSSION j The study of tissue heparin levels, as well as othej . I aspects of biochemistry of heparin, was limited by the lack J of suitable methods for the isolation from and estimation of heparin in small amounts of tissue. The available tech niques for the characterization of heparin and for the sepa ration from other tissue constituents were not sufficiently [definitive for comparative studies. For these reasons, the jdevelopment of new methods was undertaken. j The isolation of heparin from tissue requires de struction of the protein binding. The techniques previously employed used autolysis or alkaline extraction or both to effect this separation. However, autolysis does not com pletely free the heparin, and alkaline conditions may de grade or modify the heparin. There were reports of a hepa- rinase (1 0 0 ,1 0 1 ) in several tissues, which were erroneous (1 0 2 ); however, tissue enzymes might be expected to destroy or degrade heparin during autolysis. In the. work described, the use of proteolytic enzymes was found to readily free heparin from protein. Acetone drying and thorough defatting were used to prepare the tissue for proteolysis, and heating 14-3 _________ I . |was intended to inactivate the enzymes as well as to dena- I ■ ture the proteins to facilitate the digestion. PTC, which contains both trypsin and chymotrypsin, was shown to be the most effective purified proteolytic en- ! < ! i izyme preparation examined. Potent commercial pancreatm was!; more effective, but contains some heparin. Simultaneous ! ■' ’ ■ 1 iialysis with continuous removal of the hydrolysed products ! : . : l permits more complete digestion. Extensive dialysis is 1 j • • • ' ‘ ii I • ^ necessary to remove split products which may have some ef- \ feet on the clotting system. This system also maintains a | constant pH due to the large amount of buffer present. j The concentration of the dilute solutions obtained after dialysis was best accomplished by acetone precipita tion. However, there were reports of lipid anticoagulants in tissue which were acetone insoluble, and a thorough de- | f ’ | fatting of the acetone dried tissue was introduced to effect jtheir removal. This method has been used for the determina-j i ‘ I tion of tissue heparin levels and also m the study of its | • J i | ! biosynthesis in mast cell tumors and other tissues. j I The available techniques for the separation and j i characterization of heparin and other MPS were limited to a 1 I ' ' '' I paper chromatographic procedure ( 1 2 0 ) and fractional precip-j I ' I itation which are neither sensitive or convenient on a micro? or macro scale (124). For this reason, the development of aj column chromatographic method was undertaken. Green had re ported the use of ECTEOLA with NaCl elution for the chroma- I . I tography of heparin from mast cell tumors (125). Ringertz I f has also reported the use of this exchanger for the chroma- ; tography of MPS using a NaCl:HCl elution system (126). The ; bellulose exchangers had proven very useful in the chroma tography of proteins and polynucleotides, and so the anion exchange cellulose derivatives were investigated. \ In the early trials, DEAE did not appear to be use- ; jful, although a recent paper reports the use of this ex- jghanger in the chromatography of heparin (134) . TEAE showed! some promise, but ECTEOLA seemed to be more satisfactory. j Green had reported that there was no effect of pH and that j 2M NaCI was required to elute heparin with this exchanger j (122). Neither of these observations could be confirmed. ! - ■ ■ - ■ ■ ■■■ The exchanger was more retentive for heparin at an acid pH (4.3) than at neutrality and even at this pH, heparin was j jeluted at about 1M NaCI. The system of Ringertz was not explored because heparin is not stable, even in the cold, at the pH used in his system. The most satisfactory exchanger found was aminoethyl I cellulose, AE-50, a new preparation from Whatman. The meth- pd developed in this work provides a basis for the charac- if fcerization of MPS in mixtures, although as described, it I I - ■ . ■ . . i | does not permit complete separations. However, it should bej possible to extend this technique to permit such resolution.} j h more complete examination, of other pH conditions and other - " ~ - M S J ! salts for elution may be useful for modifications of this ; method. J A simple procedure was.needed to locate the MPS in j j the eluate fractions in the development of the column method;!. [The carbazole and other colorimetric methods were cumbersome and were also less sensitive in the presence of the NaCI. j Spotting on paper and using the metachromatic reaction with ] ;j Azure A did not work when the salt concentration was above j about 0.2 5M. Alcian blue staining, which had been used in histological work (130) and had been reported to be useful j for the detection of MPS in paper electrophoresis, was ex- j plored and found to be very satisfactory. Using suitable |standards, it was possible to use the technique to locate and to make a reasonable estimate of the MPS concentration in the eluate. The correspondence of the Alcian blue data with the carbazole method, the radioactivity measurments, i ' ' I and the sulfuric acid procedure was quite good. The reaction of MPS with sulfuric acid to produce a Ichromogen which absorbs at 320 mu was examined in a very preliminary way in this work. It appears very likely that a Jfurther exploration of this approach could be fruitful as a possible way to distinguish between different MPS in a simi lar manner to the way Brown has used the anthrone method. [It xs a sxmpler method than the carbazole method for the I ' " ' ' estimation of MPS in the fractxons. j The anticoagulant method for the estimation of hepa rin activity was used because at the time there were no I l ! [Chemical methods for distinguishing heparin from other MPS. | | a h MPS are met a chromatic and exhibit the same color reac- 1 i - i I ' ‘ tions (carbazole, anthrone, etc.). Heparin is very potent I ii j |as a clot inhibiting substance and, while heparitin sulfate ! i | has been reported to have weak anticoagulant activity (131),1 ; • ' ! i . Ireports to the contrary have also appeared (12 7) . CSA-B has; I I ‘ • ! 1 also been reported to be a weak anticoagulant, but in the recalcified sheep plasma system used, it has. very little ac-j tivity, only 2-3 per cent of heparin. It is more active in i ! a purified system using dilute thrombin and fibrinogen, or in diluted plasma (128). Despite the small effect of re- j jlated substances, the anticoagulant method used was the most definitive method for estimating heparin in crude mixtures such as the tissue extracts. ! In the course of the development of the column meth- I pd, many chromatograms were made of various samples of hepa- ! f rin and other MPS. Under these conditions, CSA-A and CSA-B I appeared to be homogeneous with indications of small amounts 'of impurities. Heparin and heparitin sulfate, in all cases, showed patterns which suggested heterogeneity. The repro- jlucibility of these patterns indicated that they were not (artifacts of the system. Jorpes1 (10,11) contention that ! | heparin is a mixture of closely related substances is sup- j I ; I ported by these observations. The differences may reflect ; 'small differences in the degree of sulfation, molecular f ''' ' - ' weight, or other differences in macromolecular configura- ! i tions, such as branching and cross-linking (35). Further | investigation of this question is planned. If the chromato-l graphic system can be scaled up in size, it should be possi ble to prepare sufficient quantities of the various compo- . • • • . . _ • j nents to permit more extensive chemical and biological char acterization . I Our method for the isolation and estimation of the ' heparin content of tissue was applied to several organs of rats and rabbits. In all cases, the amount in rat tissue was higher than the corresponding tissue of the rabbit (93).j There were some differences in the distribution within each | 1 i (animal. The liver had the lowest level in both species and i ,the kidney was among the highest. The thymus was the rich- f est in the rat, and one of the lowest in the rabbit. The lvalues obtained for the heparin content of these animals are higher than other reports in the literature (90,92). This may be due to differences in strain of animals, but it is more likely that it is due to the more complete extraction by this method. The very complete digestion of the tissue, ' junder conditions which are very mild and do not degrade the heparin, should lead to higher values than other techniques I which depend on extraction by alkaline solutions. Using alkaline extraction alone, it is difficult to isolate hepa- ■ ' - i rin from beef lung, and autolysis has been shown to increase} ....... - — — _ .... ~ - ’ - 158’ ithe extraction yield by 100 per cent. A second extraction | i i jof the tissue residue with some proteolysis gives additional^ jheparin and activity. In contrast, the heparin is quite j easy to extract from beef intestinal tissue. ! J Further evidence for the effectiveness of our methodj } i !of isolation has been demonstrated by its application to J tissues which have been used commercially for the production! bf heparin. The levels obtained by this procedure were I - ■ ! f ' ' 110-2 5 per cent higher than those found by commercial proc- I ' i ■ esses, which m turn were higher than many of those re- 5 ported in the literature for some beef tissues (90,91), j A summary of the data for the heparin levels in the j liver, intestine, and lungs of several animals and man are jShown in Fig. 42. The values for human tissues are based on (limited data for liver and lungs. No conclusions can be 1 ■ drawn from these data. The total heparin content of the body of different animals is not known, but would be useful information and will be undertaken. 35 The examination of the incorporation of injected S into the tissues of intact rats indicates that the synthesis | bf heparin and other MPS occurs in many organs. Alterna tively, it is possible that there is very rapid transport of MPS from its site of synthesis to storage tissues and that sach organ has its own pattern of incorporation. The data j shown in Fig. 2 0 indicate that all the organs examined con centrated radiosulfate as compared to the whole animal. | 30 " 25 -- 20 -- 15 -- 10 *- 5 -• ‘ 40- Fig. 42 Heparin Content in Units per g. in the Lung, Intestine and Liver of Several Animals Lung H Rabbit □ ■ p; Intestine Liver 16ET I j phis was most striking at the early time intervals. The in- 35 jtestine had the highest concentration per gram of S at 2 hours with the kidney next, followed by the skin, liver, andj - ■ I f lung, in that order. The kidney, which was one of the organs with the highest heparin content in the rat, appeared to be incorpor- 35 atmg S into macromolecular form after the non-dialyzable radioactivity of the other tissues had begun to decrease. B This may relate to the function of kidney as excretory organl and some of this radioactivity may have arisen from release O C ■ by other tissues. However, exogenous S 3 labeled heparin j does not accumulate in the kidney. A possible key role for the kidney in heparin metab olism was suggested by some preliminary observations made by jsome associates of the author'*' in the course of preparing biosynthetically labeled heparin. An attempt to keep the 3 S injected S from being excreted rapidly was made by tying 35 off the kidneys. S was found in the heparin, but the spe cific radioactivity was not as great as in normal animals. A related MPS, which was extracted along with the heparin and had very little anticoagulant activity, was, however, much more radioactive than the heparin. The amount of data was limited and can only be considered suggestive until fur-i ther studies are made. I _ _ _ _ _ _ _ _ _ _ _ ! ^P. Rucker and L. Levy (Riker Laboratories) . 151 i ) The lung was also somewhat unique in that all the ! I O C . 1 S which was not extracted by the acetone or hexane:iso propanol was non-dialyzable and hence was macromolecular even at 2 hours. This was not true of any of the other tis sues examined. 'DC The incorporation of into the lipid fraction was rapid in many organs. The kidney and skin had the most ra- ' dioactivity. The liver did not apparently incorporate (into its lipids. The results with kidney and liver parallel some preliminary observations by Spolter and Rice (132) in -3 c the in vitro incorporation of S~'-J into homogenates of these tissues. The nature of the lipid moieties which were la beled were not determined. However, in view of the reports O C by Benson (133) on the extremely rapid incorporation of S into some newly discovered sulfolipids of plants, this rep resents an interesting area for future exploration. The chromatographic data suggest a precursor rela tionship to heparin may exist for the components which were eluted at 0.1M to 0.15M NaCI (Figs. 21-23). The nature of jthis material was not determined, but they must have been high molecular weight substances as they were not dialyzable rhey appeared to consist of two components, slightly sepa rated by the column chromatography. An ultraviolet absorb ing peak (260 mu) occurred with a maxima corresponding to the second component. This suggests a nucleotide moiety may be involved. No other relationships were suggested by the : I ' ■ data. ; One of the purposes of the experiment on the incor- I i i O C ? iporation of S-5- * into the tissues of rats was to find the op-; jtimum time for obtaining heparin with the highest specific j [activity. The chromatographic data indicate that in the intestine, the 6-hour sample had the highest radioactivity I - per mg. Similarly, the liver was higher at 6 hours than at ; I 2 4 . It is not possible to draw any conclusions with regard | I ■ I I ' I jto the other organs since, even though the non-dialyzable j radioactivity is highest at 2 hours, except for the kidney, j |a substantial portion of this radioactivity is not heparin, j It is possible that in the kidney, the heparin might have j been the most active at the last time interval. This ques- 1 tion will be explored further. [ The examination of heparin derived from several spe cies showed that by the criteria used, only the dog heparin was different than other types examined. The difference ap peared only in the column chromatographic method, but was lot discernable in the paper chromatographic examination. The basis for this difference and the reason for the higher ! molarity of NaCI required to elute dog heparin is not known. The sulfate content was not different from that of beef and hog. If the molecular weight was greater than beef or hog, I its mobility in the paper system might be expected to be af fected. However, molecular weight determination, viscosity,j and other macromolecular aspects of dog heparin might pro- \ vide some clues as to the nature of the difference- The jj chemical composition of dog heparin should be compared with j \ those of other species. f Circulating heparin-like substances have been re- | ported by several groups. The material isolated had anti- jcoagulant activity which was reversible with protamine; it was metachromatic and had a mobility the same as heparin and other MPS in paper electrophoresis. Engelberg (99) has re ported that 10-2 0 unit per cent of heparin activity is nor- nally present in human plasma. This would give 100-2 00 junits per liter, or at 100 units per mg, 1-2 mg per liter, pur data indicate the presence of 3-6 mg per liter of MPS. I jThis appears to consist of two main components, one of about 2.75 mg per liter which resembles heparitin sulfate, and the -remainder which is unlike any MPS examined. In the test I system used, a very small amount of anticoagulant activity was found. A sample of plasma from a patient diagnosed as lyperheparinemic contained a greater quantity of the same type of materials. These data appear to be in conflict with reports in the literature (97,98). There are two possible explanations Cor this discrepancy. One is that the MPS found are anti coagulant in the test systems used to determine the activity Ln plasma, but not in the USP method. Heparitin sulfate has been reported to have some anticoagulant activity and CSA-B has been shown to have a considerable activity in test sys- I ( t i terns of diluted plasma or where the thrombin concentration j ■ t is low. The test system used by Engelberg (99) is a dilutedj } plasma system. * It is also possible that the activity was destroyed I i or lost in the course of the handling of the samples. Our j experience with crude extracts of human tissue do not sup- | port this explanation as they showed both anticoagulant ac- I tivity and a heparin component when examined in the same way| as the plasma extracts. The question of the nature of the circulating MPS in human plasma requires further study to permit resolution of the conflicting data now available. A better understanding will be possible with the isolation of larger amounts of these substances, their separation into the various compo- lents, and characterization by chemical and biological means, The serum of beef and hog also contained MPS. Hog serum appeared to contain CSA-A, CSA-B, and heparitin sul fate, as well as some unidentified materials, but no heparin- like component. The biological responsiveness of rats, dogs, and man so equivalent doses of injected heparin was similar with the main difference being the duration of activity. In rats, she activity disappeared quickly. Dogs showed a more pro longed response, while man had even a longer period of ac- jsivity. If the area under the activity curve is compared, 155) the yield from the rat is the least and man is the greatest.) J 1 rhis was particularly true of the lipoprotein lipase activ ity. The area under the curve showed a response propor tional to dose in each of the three species. j The use of the subcutaneous fat tissue, which is poorly vascularized, as the injection site in man may cause slower release of the injected heparin. However, the injec tion site in dogs and rats was the loose connective tissue, and was comparable. The earliest samples taken in dogs and nan were at 1 hour, at which time the maximum response had been achieved, while the rat data do not suggest any more rapid appearance of activity. There is no satisfactory ex planation of the difference in species. When heparin is injected intravenously, the response is very rapid and the duration is less than by the subcu taneous route. As little as 500 units i.v. in man had been reported to permit the demonstration of both clearing and anticoagulant activity (106). Similar results have been shown in animals. A comparison of the excretion of heparin following Its administration to rats, dogs, and man indicated a gen eral similarity in the manner in which these three species handle exogenous heparin. Very little of the heparin activ ity was found in the urine, less than 5 per cent at any dose 35 used in man and none m rats or dogs. When S labeled heparin was administered to rats or dogs, only a small jamount of inorganic S^O. was found, indicating that little | desulfation had occurred. Eiber and Danishefsky (103) re- | 3 5 i ported that considerable desulfation did occur when S j I heparin was administered i.v. to dogs. However, their use j of dog heparin was an important difference in experimental | technique. As noted above, dog heparin appears chemically distinct from other species and may be handled by the dog differently than hog heparin, which was used in our work. Rats excreted about 15 per cent of the radioactivity administered in 24 hours, and the data for dogs suggest a similar figure at a dose one-fifth that given to the rats. 35 After the early rapid excretion of S , the rate of excre tion was slow and in dogs, only a total of 50-60 per cent of the dose was excreted in 7 days. The nature of the retained activity was not determined. In man, the administration of 5000 units per subject ' produced no MPS in the urine above the normal amount. At higher doses, material was found which resembled heparin in the paper and column chromatographic systems. If urinary spillage were occurring, a higher proportion of weight would have been expected at higher doses. The opposite was true and the fraction of anticoagulant activity was the same. |This meant that the substances excreted in the 30,000 units per subject experiment had a higher unit activity than that found when 10,000 units were used. These observations might 157 be explained if the kidney had a restricted capacity to ex- ! 1 crete these materials. | i Limited observations in dogs and rats indicate that the radioactivity of injected heparin is rapidly distributee all over the body. After 24 hours, the largest amount of the retained activity is in the liver. In man it was not possible to distinguish between retention in the tissues and catabolic destruction to the point where recognizable MPS dc 35 not exist. Since only S was measured m the animal ex periments, it is possible that sulfate transfer to some other slowly turning over component had occurred. It is also possible that the exogenous heparin became part of the total body pool and, after the first rapid excretion, the amounts found in the urine reflected the half-life of hepa rin. If the amount of heparin entering the tissue pool were greater at higher circulating levels, this might explain the human data. If extensive desulfation had occurred in the animal experiments, a greater amount of free sulfate and total ra dioactivity would have been found in the urine. The possible relationship of heparin to atheroscle rosis had been suggested by the finding of less mast cells in the atherosclerotic aorta as compared to normal (129) . Reports have also appeared which indicate that mast cell counts of rat tissues were higher than those of rabbits, the I former being a resistant and the latter a susceptible animalj. jThe assumption lias been made that the mast cell is the pri- i 1 i r ary and possibly the only site of storage of heparin (85). S i I The data reported do not support a relationship be- ■ tween mast cell counts and the heparin content. It appears j that the concept of the mast cell as the primary site of | storage of heparin is not valid. The presence of diffuse J | metachromasia in some of the tissues examined and the low j i r ast cell counts xn some txssues with relatively high hepa- ! rin content suggest that other cells and/or non-cellular compartments, such as the ground substance, contain heparin. The mast cell counts in rats and rabbits we obtained do not agree with other reports in the literature (134). The reasons for this are unknown at present, although they may be related to differences in technique and strains of animals. The variability was great within a given organ and even in individual sections, and this may contribute to the (Conflicting reports. At present, one cannot correlate the heparin content . bf tissue with resxstance or susceptxbilxty to atherscle- {rosis. The differences between rats and rabbits were small, 1 jwhxle the dxfferences between these animals and hog and beef jwere great. The determination of the total heparin of the body might provide useful information, but this will be dif ficult to achieve. The small series of human intestinal samples assayed! did not offer any basis for suggesting a relationship of the 159 l I heparin content with, age or pathology. Studies are under way to examine a series of human tissue samples of known pathology and various ages and compare them with tissue from normals. i I ! j i CHAPTER VI ! SUMMARY One of the first requirements of this study was the development of methods for the determination of tissue hepa-j rin and for the characterization and separation of heparin J I . from related substances. A method for tissue heparin was developed, based upon total proteolysis with simultaneous dialysis. The anticoagulant activity of heparin was the basis for its es timation. The lack of definitive techniques for the character ization of heparin in mixtures of mucopolysaccharides led to the exploration of column chromatography. The anion cellu lose exchangers were examined for this purpose and amino- ethylcellulose was found to be the most satisfactory. The method as reported will permit the characterization of hepa rin and other mucopolysaccharides, but does not effect a complete separation in mixtures. In conjunction with other procedures, such as paper chromatography, important informa tion can be gained with this method. An examination of the heparin levels in several or gans showed that those of the rat were higher than the IfiO ________________________________________________________________________________ — _ comparable one of the rabbit. There were both similarities \ ! i and differences in the distribution of heparin in the tis- j I sues examined. The results of the analysis of a series of j I i human intestinal samples indicate that this tissue has a j heparin content of 3-8 units per gram. j O C S as sulfate is rapidly incorporated into macro- molecular forms in the tissues of rats. The pattern of in corporation was different in the various tissues examined j I and no relationship between heparin levels and incorporationj was found. An incidental finding was the rapid labeling of the lipids of some of the tissues, but not that of liver. A comparison of heparin derived from human, beef, hog, rat, and dog was made and, by the criteria of column I chromatography, only that from the dog appeared to be unique. Heparitin sulfate-like substances were found in the serum or plasma of man, beef, and hog, but no heparin-like components were detected. Plasma from a hyperheparinemic patient was found to have more mucopolysaccharides than nor- Lal plasma, but there was no qualitative difference. The biological response to exogenous heparin in man, dog, and rat was compared, and man had the greatest duration of response. Dog was second, and rat, third. The metabo- I jlism and excretion of exogenous heparin was similar in the jthree species. A substantial proportion of the administered i heparin remains in the body for some time. It was not de termined if this was the original heparin or some modified 162 form. 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Creator Freeman, Leon David (author)

Core Title A comparative study of the chemistry and biology of Heparin

Contributor Digitized by ProQuest (provenance)

Degree Doctor of Philosophy

Degree Program Biochemistry and Nutrition

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Advisor Saltman, Paul (committee chair), [illegible] (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-605730

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