Harold B. Eiber

Investigations on the chemistry of heparin. I. Desulfation and acetylation

Abstract Desulfation of heparin with 0.06 M anhydrous methanolic HCl resulted in the formation of a polysaccharide containing equimolar amounts of sulfur and nitrogen, wherein the amino groups are free. When this was N-acetylated and again submitted to methanolysis, an additional sulfate per tetrasaccharide was cleaved yielding a product containing nitrogen, sulfur, acetyl, and methoxy in the ratio of 2:1:2:2. The rate of hydrolysis in 1 N H2SO4 of the glycosidic linkages of this product is appreciably higher than that of heparin. This desulfated, N-acetylated derivative would, therefore, be more suitable for graded hydrolysis studies than heparin itself, since the conditions required for hydrolysis of the latter also bring about excessive destruction of the uronic acid moiety.

Studies on the metabolism of heparin

Abstract Heparin-S 35 was injected into dogs, and the properties of the radioactive material excreted in the urine were studied. It was found that after administration of small doses, all the label is excreted as inorganic sulfate. On the other hand, after injection of larger amounts the urine contains an appreciable portion of the S 35 associated with heparin and other mucopolysaccharide material. It is concluded that as a result of metabolic degradation the sulfate linkages of heparin are cleaved. However, the capability of the animal to perform this hydrolysis is limited so that when excessive amounts are administered, a significant portion is excreted unchanged or only partially desulfated.

Studies Made With Radioactive Heparin in Humans

The present study was conducted in order to gain more insight into the disposition and metabolism of heparin after intravenous injection in humans. The major pharmacologic effects of heparin, i.e., anticoagulant and clearing action, are well established, and although almost nothing is known concerning its metabolism, advantage may be taken of its clinical properties. Nonetheless, an understanding of the metabolism and, ultimately, the exact mechanism of the action of heparin may show the way for additional applications or possible limitations of this substance. Since heparin is a comparatively complex molecule which can interact with various body constituents, the problem of its chemical determination is extremely difficult and in many cases unreliable. This problem is further complicated by the fact that a number of substances

Heparin in Blood.

Summary The presence of heparin in normal blood is demonstrated by the “carrier” technic, using radioactive sulfate as precursor. It appears to be found in blood in a combined form so that preliminary decomposition of the complex is required before it can be isolated.

Physiological disposition of heparin.

Summary After intravenous administration of heparin-S35 to dogs, it is rapidly cleared from the bloodstream. The highest radioactivity is found in liver and lung. However, no measurable radioactivity is detected in any organ after 48 hours.

Investigations on the chemistry of heparin. II. presence of a uronidic linkage with carbon-6 of glucosamine

Abstract The comparative resistance of the glycosidic linkages of heparin to acid hydrolysis is probably the major source of difficulty in the determination of its structure by graded hydrolysis experiments. Thus the conditions required to effect appreciable reaction also cause extensive degradation of the uronic acid. A previous paper from this laboratory described the conversion of heparin to a desulfated N-acetylated derivative containing nitrogen, sulfur and acetyl in a molar ratio of 1:0.5:1 (Danishefsky et al., 1960). It was also shown that the acid hydrolysis of this product proceeds at a significantly higher rate than heparin itself. A subsequent study was, therefore, carried out on the desulfated acetamido heparin. This report describes the isolation of a disaccharide from the hydrolyzate and presents evidence as to some of its structural characteristics.

Studies with clearing factor V. State of tissue lipases after injection of heparin.

Summary Injection of heparin into rats induced an increase in lipase activity of the plasma and a decrease in lipase activity of the tissues. The recovery of lipase levels in the tissues was observed after 2 to 3 hours. Before and after heparin injection the ratio of lipase activity inhibited by protamine sulfate and NaCl to lipase activity resistant to these inhibitors was not constant. After heparin injection increased activity of β-monoglyceridase in the tissues was observed. The tissues (of the No-Heparin-Injected rats) treated with epinephrme exhibited significant increases in lipase activity. However, no marked increase in lipase activity was observed in the tissues (of Heparin-Injected rats) treated with epi-nephrine.

Studies with clearing factor. 3. Application of column and thin layer chromatography for separation of lipid hydrolysis products by clearing factor.

Many researchers have employed column and thin layer chromatography for the separation of lipids. Qualitative thin layer chro-matographic separation of cholesterol esters, triglycerides, fatty acids, cholesterol, di- and monoglycerides, and phospholipids on silica gel plates of normal plasma and of patients with lipid disorders has been reported(1). Also, separation of 1,3- and 1,2-diglycerides and α- and β-monoglycerides by thin layer chromatography, after pancreatic lipase digestion of fats, has been demonstrated(2). Silicic acid column chromatography was used for a similar purpose by Borgstrom(3), Fillerup and Mead(4), and Hirsch and Ahr-ens(5). In this report, the digestion products of the action of Clearing Factor on coconut oil were studied. The presence of α-, β-mono-glycerides and 1,2- and 1,3-diglycerides intermediates were indicated by various thin layer and column chromatographic procedures. The free fatty acids were the major end product of hydrolysis. Among several separation methods of complex lipid mixtures, column chromatography in combination with thin layer chromatography produced the best information and is recommended. Materials and methods. Post-heparin Clearing Factor was prepared as reported previously (10). Tripalmitin, triolein, and cholesterol were purchased from Mann Biochemical Laboratories. Monoolein, monopal-mitin, 1,2- and 1,3-dipalmitin were supplied by Dr. J. Hirsch. Conditions of hydrolysis of Ediol with Clearing Factor was the same as previously reported(9,10). Five ml aliquots were taken at zero time, 1 hr, 3 hr, and overnight from the incubation mixtures. Each aliquot was extracted with 50 ml of Bloor solution (3 parts ethanol:1 part diethyl ether). Thin layer plates were (20 × 20 cm glass) coated with a 225-250 layer of silica gel G and air dried(6). The plates were then activated for one hour at 160° and placed in a desiccator.

Studies with clearing factor IV. Fatty acid exchange reaction catalyzed by clearing factor.

It was previously reported (1) that postheparin Clearing Factor catalyzes the exchange of fatty acids with triglycerides, di-glycerides and monoglycerides. This exchange as measured by incorporation of C14 oleic acid, was postulated to take place only on the 1 and 3 position of the glyceride. It was thus suggested that the position specificity of Clearing Factor for the glyceride ester bonds was such that the exchange took place only in the 1, 3 positions. In these experiments no attempt was made to demonstrate that actual distribution of C14 fatty acids among the isomers of diglycerides and monoglycerides. These conclusions were rejected later by Korn(7). He used adipose tissue Clearing Factor and demonstrated random hydrolysis of fatty acids from various natural triglyc-erides and chylomicrons. These results were possibly affected by the presence of β-mono-glyceridase(8) and lipases other than the lipo-protein lipase in adipose tissue. Similar exchange reactions with C14 fatty acids and dibutyrine, catalyzed by adipose tissue lipoprotein lipase, were demonstrated (5). In this report, it was claimed that 2 to 8 carbon fatty acids were more readily incorporated in the 3-position of 1,2-dibutyr-ine and ester bonds were formed. On the other hand, with long chain fatty acids exchange reaction occurred only at C-1. The exchange reaction and hydrolysis had an optimum at pH 7 with an apparent increase of activity with increasing pH. The present report describes the exchange reaction of plasma Clearing Factor with C14 stearic and oleic acids and glyceride fractions of coconut oil and its hydrolysis products, i.e., 1,2- and 1,3-diglycerides, a- and β-monoglycerides. Methods and materials. The postheparin plasma Clearing Factor was prepared as previously reported (9). It had an activity of 16 μ E free fatty adds/ml postheparin plas-ma/hr. Stearic acid 1-C14, with a specific activity 21.4 mC/mM, and oleic acid-1-C14, 7.7 mC/mM were purchased from Volk isotopes.

Free Electrophoresis of Lipoproteins

Summary Analyses by free electrophoresis have been made on the low density lipopro-teins obtained by centrifugation of serum at a solution density of 1.063. At pH 8.6 the lipo-proteins give a clear electrophoretic pattern showing concentration dependence, but no change in mobility with increase in concentration. Addition of this material to normal serum or lipoprotein-poor serum results primarily in an increase in the β-globulin peak. On the other hand, when the lipoproteins are removed from the serum a significant decrease in this peak is observed. This method is suggested for possible metabolic studies with these lipoproteins.