Heparin was first isolated from beef lung in sufficient purity for clinical use and for chemical study by Scott and Charles [80]. They demonstrated that it is an acidic carbohydrate capable of forming salts with metals (sodium, barium, etc.), and that it was reasonably free of other substances (by crystallizing it as a barium salt [7]). The identification of uronic acid [79], sulfate [48] and glucosa­ mine [50] as the main components demonstrated that heparin is a mucopolysac­ charide, and analyses for these components showed the very high sulfur content of 12% [31]. The quantity of sulfur present, the slowness of reaction with periodate, the very low reducing power, failure to dialyze through cellophane, a mean molecular weight by ultracentrifugation, etc., of about 15,000, led to the conclusion that sulfate was attached to glucosamine and uronic acid by ester linkages (0-sulfate). This data also indicated the compound was a polymer in which alternate hexosamine and hexuronic acid were bound together by the usual glycosidic linkage analogous to other polysaccharides, as postulated for the mucopolysaccharides by P.A. Levene [31, 33]. The demonstration of sulfamino groups [51] distinguished heparin from other mucopolysaccharides, for which, up to then, only acetylhexosamines were known. It was shown that the uronic acid in heparin is not galacturonic acid; instead, it has been believed [21, 88] to be glucuronic acid, the most common uronic acid, in spite of difficulties found in confirming this. Classic isolation technics gave only low yields of glucuronic acid or its breakdown products. Color reactions gave abnormally high values for glucuronic acid as compared with the amount shown by direct determination of carboxyl. It was generally believed that the presence of large amounts of substituent sulfates interfered with the isolation and changed the reactivity of the glucuronic acid [21]. Different investigators suggested iduronic acid was present, but this was only demonstrated in small amounts [8]. The demonstration [24, 32, 38, 45, 89] that trace quantities of heparin and heparinoids produce a metachromatic color change in toluidine blue, changing the dye in dilute solution from blue to red in the presence of concentrations of

It has been generally assumed that the clinical use of heparin is simply an extension of the initial laboratory use to prevent blood coagulation in test tubes and cannulae. Here the effects of mixing heparin with blood appear to be the same in vivo and in vitro. Evidence was obtained early that this was an oversimplification of the situation and the large amount of data that has now accumulated calls for a reevaluation of this basic assumption in a number of aspects which bear on the clinical use of this important drug.

Heparin, hydrogenated dextran sulfate 8000 (Usherdex 8), and dextran sulfate 8000 were administered to rats, and the total drug was separated and determined in endothelium and plasma. A large amount of each drug was recovered from endothelium 2.4 and 6 minutes after intravenous injection. This accounted for the drug missing from plasma. The drugs in water were placed in the stomach by catheter. All three drugs were recovered from the endothelium and identified unchanged by electrophoresis and specific staining. The amounts that were recovered at 2.4 and 6 minutes were equivalent to most of the drug administered. Thus heparin, Usherdex 8, and dextran sulfate 8000 enter the body immediately on oral administration. At longer time intervals after intravenous and oral administration, much of each drug was not demonstrable in endothelium by the method used. Some drug could be detected in endothelium after 4 hours. After oral administration, plasma levels of each drug were rarely more than 0.5% of the dose. Formalin-alcohol was applied to the jugular veins of anesthetized rats to produce a thrombus, (see Blake et al. J Clin Path 1959;12:118-22) and the drugs were immediately introduced into the stomach. Four hours later the injured veins were inspected for thrombi. Incidence of thrombotic plug was 80% in rats that received saline solution, 4% with Usherdex 8, 0% with dextran sulfate 8000, and 0% with heparin. Usherdex 8, dextran sulfate 8000, and heparin demonstrate low, moderate, and high in vitro anticoagulant activity, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Heparin and dextran sulfates 8000 are separated from citrated plasma by absorption on epichlorohydrin triethanolamine cellulose columns followed by elution with 1.1 and 1.4 mol/L NaCl in 0.05 mol/L glycine-HCl buffer. The eluate is desalted with Sephadex G25-40, dried, and dissolved in water. A 1 microliters sample is applied to an agarose gel slide. After electrophoresis, the slide is fixed and stained with toluidine blue. The sulfated polysaccharide band(s) is identified by relative electrophoretic migration. The total amount of drug is estimated by matching its optical density with that of a band on one of a set of slides with graded amounts of heparin or dextran sulfate. The reaction with toluidine blue measures the total polyelectrolyte, not just the small proportion of the drug with anticoagulant activity. Pooled normal plasma showed a trace of chondroitin and no heparin. Recovery of heparin and hydrogenated dextran sulfate that was added to pooled normal plasma was complete (lowest concentration tested was 10 micrograms/ml); however, recovery for unhydrogenated dextran sulfate declined consistently by 9 micrograms/ml for concentrations below 50 micrograms/ml, setting a limit for its recovery. Plasma samples taken from patients for coagulation tests were examined by this procedure, and in so doing, steps were ascertained to improve the procedure for routine use. Results were compared with values for prothrombin time and activated partial thromboplastin times obtained on the same samples by the clinical laboratory. Because the procedure provides an independent parameter for measurement in patients who have received heparin therapy, insight into different patient responses to the drug is therefore possible. With minor modifications, the procedure can be used for heparans, dermatans, and chondroitins, because it allows identification and microscale quantitation on the basis of charge, molecular weight, and carbohydrate structure.

For over 100 years heparin has attracted interest because of its anticoagulant powers. Commercial heparin has now been shown to be a mixture of over 100 different closely related sulfated polysaccharides of which only 10% activate antithrombin-III. Fifty years ago the original research teams in Toronto and Stockholm in demonstrating the clinical uses of heparin observed that antithrombotic activity did not correspond to levels of anticoagulation. It has been shown that: (a) Heparin accumulates rapidly and specifically in the endothelium against a concentration gradient of hundreds- to thousands-fold. (b) Experimental thrombosis, however produced, is accompanied by a marked decrease in the electronegative charge of the vessel wall and the charge is restored in all cases by heparin. (c) The normal electronegative charge is due to glycosaminoglycans. Heparin possesses the strongest electronegative charge of these substances and is present in the vessel wall as a component of a larger heparitin (sulfate) proteoglycan molecule. (d) Maintenance of the normal electronegative charge depends on adequate supply of oxygen (adequate blood flow). (e) Commercial heparin releases enzymes from the endothelium, lipoprotein lipase and histaminase (D.A.O.). Lipoprotein lipase changes the composition of plasma lipids and lipoproteins and histaminase provides a check for fat absorption. The release of these enzymes decrease and prevent atherosclerotic changes. (f) After administration of commercial heparin, heparin isolated from the plasma has higher antithrombin activity than that injected. The heparin taken up by the endothelium is returned with greater activity. The anticoagulant effect of administered heparin does not produce hemorrhage since this requires simultaneous occurrence of defects in the vascular factor of hemostasis (the result of stress or pituitary-adrenal imbalance) or platelet defect. Thus, clinical effectiveness of heparin is an expression of its close relationship to the vessel wall.

Heparin and heparinoids constitute the one drug group shown to arrest and reverse atherosclerotic changes in rabbits on a high cholesterol/fat diet. Little use has been made of this finding because heparin has been administered by routes which produce anticoagulation and thus bleeding. Inhalation once a fortnight results in a high concentration of heparin in endothelium with low plasma concentrations. No toxicity has been demonstrated with long term heparin but this is not true for heparinoids.

Heparin was developed as a drug for clinical use 50 years ago by research groups in Toronto and Stockholm, headed respectively by Professors Charles H. Best and Erik Jorpes. 1 Jorpes E Its chemistry, physiology and application in medicine. 1st ed. Oxford University Press, London1939 Google Scholar Since then, the drug has been used for millions of patients and has been the subject of intensive clinical and laboratory study. There has been no question of the clinical effectiveness of this remarkable drug, but the results of studies on how the drug acts have often disappointed investigators. Undoubtedly, this is related to investigators' assumptions which were found to be invalid by the original research teams. These teams recognized the chemical complexity of heparin and realized that the property of preventing blood from clotting in a test-tube was insufficient to explain clinical effectiveness. Recent studies using new techniques have resolved the enigmas and now provide the clinician with enough basic knowledge to use the drug satisfactorily. I had the privilege of working with the late Gordon Murray in Toronto on the initial testing of the drug's effects in thrombosis; with the late A. F. Charles on the chemistry of the drug; and I discussed these problems with the late Professor Jorpes. I have recently published an extensive review 2 Jaques LB Heparins—anionic polyelectrolyte drugs. Pharmacol Rev. 1980; 31: 99-166 Google Scholar of the total literature, together with a new overall view of what heparin is and does. The present article presents an organized synopsis of the knowledge of heparin that has been accumulated over the past 50 years. For detailed presentation of the observations and literature citations, the reader is directed to the first eight references. 1 Jorpes E Its chemistry, physiology and application in medicine. 1st ed. Oxford University Press, London1939 Google Scholar , 2 Jaques LB Heparins—anionic polyelectrolyte drugs. Pharmacol Rev. 1980; 31: 99-166 Google Scholar , 3 Jaques LB The pharmacology of heparin and heparinoids. Prog Med Chem. 1967; 6: 139-198 Crossref Scopus (43) Google Scholar , 4 Jaques LB The premises involved in the clinical use of heparin. Semin Thromb Hemost. 1978; 4 (275): 275 Crossref PubMed Scopus (7) Google Scholar , 5 Jaques LB McDuffie NM Heparin: the chemical and anticoagulant nature of heparin. Semin Thromb Hemost. 1978; 4: 277-297 Google Scholar , 6 Jaques LB Mahadoo J Pharmacodynamics and clinical effectiveness of heparin. Semin Thromb Hemost. 1978; 4: 298-325 PubMed Google Scholar , 7 Jaques LB Endogenous heparin. Semin Thromb Hemost. 1978; 4: 326-349 PubMed Google Scholar , 8 Jaques LB Addendum: the discovery of heparin. Semin Thromb Hemost. 1978; 4: 350-353 Crossref PubMed Scopus (18) Google Scholar

A single large dose of heparin (2000 units/kg) was administered to dogs by intratracheal instillation. Whole blood clotting times and plasma heparin concentrations were measured at intervals. At each interval the calculated dose of protamine required to neutralize the circulation heparin was given intravenously and the measurement of plasma heparin concentration repeated. The authors found that the whole blood clotting time was prolonged for 24 to 48 hours and there was a detectable concentration of heparin in the plasma for 96 hours. On each occasion the protamine eliminated the circulation heparin, but more heparin continued to enter the circulation. It is hypothesized that after rapid absorption from the lung, heparin is stored temporarily in a cellular pool throughout the body and then released into the circulation. At any given time the anticoagulant effect can be reversed by intravenously administered protamine sulfate if this should become necessary, but repeated administration would be required. Intrapulmonary heparin may have useful clinical applications but further clinical and laboratory investigations are required.

Heparin releases diamine oxidase (DAO, histaminase) from binding sites in the intestinal vasculature. Histamine is involved in a number of pathological lesions. In this study we have examined the effect of intrapulmonary administration of heparin on plasma DAO activity in mice. For comparative purposes the same parameter was measured following the administration of an intravenous heparin regimen. The time course and dose-response were examined with the two heparin regimens. The doses of heparin were based on appropriate clinical equivalents. Both heparin regimens showed a dose dependent response (correlation coefficientr=0.9). The dose of intrapulmonary heparin (10 mg/kg) was 12 times greater than the dose injected intravenously but the DAO response lasted 48 times longer than that obtained from the intravenous heparin regimen.

Intrapulmonary heparin calls for the administration of a single large dose of heparin. This results in a prolonged but low grade heparinemia lasting for many days. These features are quite different the response obtained from any of the currently used heparin regimens. A toxicity study was therefore conducted on mice exposed to heparin aerosol(7ppm) for 20 min, one a week for ten weeks. No deleterious effects were observed on physical and macroscopic examinations of the internal organs as well as on histological examination of tissues from most organs of the body.

Several hypotheses have been advanced for disorders of lipid metabolism such as atherosclerosis. However, these have not led to any significant means of control or cure. A reappraisal of the situation in the light of recent observations has led us to believe that lipid absorption and redistribution is under the control of a histamine-glycosaminoglycan-histaminase system which regulates vascular permeability. Failure of this control mechanism may lead to pathological conditions.

The lipoprotein lipase (LPL) activity obtained from the intrapulmonary administration of 2-10 mg of heparin in mice was compared with the same parameter measured for intravenously administered heparin. The doses administered were based on appropriate clinical equivalents. A relatively large dose of intrapulmonary heparin produced a peak LPL activity which was a third of the maximum response obtained from a small dose of i.v. heparin. This was followed by a moderate LPL activity (twice the control level) which persisted for the next 4 days while the response obtained from i.v. administered heparin lasted only 2 h. Both the intrapulmonary and the i.v. administration of heparin produced dose-dependent increases in plasma LPL activity (correlation coefficient r = 0.9). This study indicates that intrapulmonary heparin causes a prolonged antilipemic effect.

Annals of the New York Academy of SciencesVolume 370, Issue 1 p. 650-655 VASCULAR DISTRIBUTION OF INTRATRACHEALLY ADMINISTERED HEPARIN* J. Mahadoo, J. Mahadoo Department of Surgery, College of Medicine, University of Saskatchewan Saskatoon, Saskatchewan, Canada S7N 0X0Search for more papers by this authorL. M. Hiebert, L. M. Hiebert Department of Surgery, College of Medicine, University of Saskatchewan Saskatoon, Saskatchewan, Canada S7N 0X0Search for more papers by this authorC. J. Wright, C. J. Wright Department of Surgery, College of Medicine, University of Saskatchewan Saskatoon, Saskatchewan, Canada S7N 0X0Search for more papers by this authorL. B. Jaques, L. B. Jaques Department of Surgery, College of Medicine, University of Saskatchewan Saskatoon, Saskatchewan, Canada S7N 0X0 Department of Oral Biology, College of Dentistry, University of Saskatchewan Saskatoon, Saskatchewan, Canada S7N 0X0Search for more papers by this author J. Mahadoo, J. Mahadoo Department of Surgery, College of Medicine, University of Saskatchewan Saskatoon, Saskatchewan, Canada S7N 0X0Search for more papers by this authorL. M. Hiebert, L. M. Hiebert Department of Surgery, College of Medicine, University of Saskatchewan Saskatoon, Saskatchewan, Canada S7N 0X0Search for more papers by this authorC. J. Wright, C. J. Wright Department of Surgery, College of Medicine, University of Saskatchewan Saskatoon, Saskatchewan, Canada S7N 0X0Search for more papers by this authorL. B. Jaques, L. B. Jaques Department of Surgery, College of Medicine, University of Saskatchewan Saskatoon, Saskatchewan, Canada S7N 0X0 Department of Oral Biology, College of Dentistry, University of Saskatchewan Saskatoon, Saskatchewan, Canada S7N 0X0Search for more papers by this author First published: June 1981 https://doi.org/10.1111/j.1749-6632.1981.tb29771.xCitations: 12 † This work was supported by funds from the Saskatchewan Heart Foundation. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume370, Issue1Contributions to HemostasisJune 1981Pages 650-655 RelatedInformation