Heparin and sulfated mucopolysaccharides--a micro system for quantitative differentiation and determination.

Heparin (Hep), hyaluronic acid, chondroitins (sulfate) A, B, and C, and heparins (sulfate) A, B, C, and D were subjected to microelectrophoresis in barbital-agarose gel, fixed with cetylpyridinium chloride and stained with toluidine blue. The optical densities of the resulting bands were compared with optical densities obtained upon reaction with azure A in aqueous solution and with the carbazole reagent. A linear relation was obtained between optical density and concentration of purified sulfated mucopolysaccharide (SMP). Less than 1 microgram of Hep and 2 microgram of other SMPs are required for measurement by electrophoresis, while about 30 microgram of each is required with the carbazole reagent. The optical density of a mixture of SMPs was equal to the sum of the densities for the individual SMPs upon microelectrophoresis. It was demonstrated that the individual SMPs in mixtures were distinguishabed by reaction with specific enzymes and by changes in migration in agarose with barbital, phthalate, ethylenediamine, or propanediamine buffers, permitting ready demonstration and quantitation of various SMP species. Examples are shown of the application of the procedure to measure the total SMPs and individual SMPs in tissue extracts. The method is sensitive, reproducible, flexible, and measures quantities 1/30th of those measured colorimetrically, yet is relatively unaffected by protein, carbohydrate, or inorganic electrolytes.

Susceptibility to experimental atherosclerosis: relation to mast cells and heparin.

Fifteen chicken tissues were examined for mast cells. Numerous mast cells were found in peritoneum and spleen. The sulfated mucopolysaccharides extracted from these two tissues, corresponding in amount to that in mast cells, were found to be dermatan sulfate, chondroitin sulfate and heparitin sulfate but no evidence of heparin. We have shown a similar situation occurs in the rabbit which is also highly susceptible to the production of atherosclerosis by diet. These observations provide further evidence of a role for heparin and mast cells in limiting atherogenesis.

Effects of acidity, cations and alcoholic fractionation on absorption of heparin from gastrointestinal tract.

Heparin was introduced into the stomach or duodenum of mice separately in doses of ca. 250 mg/kg. A slight anticoagulant effect in the systemic circulation was detected in whole blood clotting times and factor X inhibition. In contrast to most drugs, more heparin was absorbed from the stomach than from the intestine. Suppressing ionization of heparin by simultaneous administration of acid resulted in improved absorption of heparin from the small intestine. Heparin was separated with ethanol into five molecular weight fraction: I, 17 999; II, 13 i99; III, 10800, IV, 8 700; and V, 6 700. Each was introduced into the duodenum of mice with citric acid. The maximum hypocoagulability was produced with fraction IV. When administered in distilled water instead of in citric acid, this heparin fraction did not produce an anticoagulant effect. These studies demonstrated that improvement of heparin absorption from the gastrointestinal tract can be obtained by the combination of suppressing ionization and selecting molecular size.