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

Endothelial cells in vivo and in vitro take up heparin following administration. In in vitro systems, cellular and pericellular extracts of monolayers exposed to heparin, demonstrate internalization as well as cell surface attachment. To further investigate the characteristics of this exogenous heparin partitioning, we have exposed two types of endothelial cells to media containing cold and 125I labelled bovine lung, porcine mucosal or CY222 heparin. Heparin uptake, in pericellular and intracellular fractions of porcine cells, increased with concentration at 24 hrs exposure for all heparins. Only bovine heparin was preferentialy accumulated intracellularly. Cultured murine LE-II endothelial cells showed a greater accumulation in the intracellular fraction than porcine cells when exposed to porcine heparin. When bovine or CY222 heparin was administered for varying times, total heparin uptake increased with time. Heparin in the pericellular fraction was greater than intracellular at exposure durations from 5 mins to 6 hrs. At 16 hrs intracellular heparin markedly increased and far exceeded pericellular. The possibility of release of previously internalized heparin was studied by washing cultures previously exposed to heparin with heparin-free media. Heparin could be recovered up to 96 hours post exposure. A concurrent decrease in pericellular and cellular heparin was observed. These results show a differing distribution of heparin across endothelial cell membranes that is heparin source, concentration and time dependent. A previously unsuspected gradient-time mechanism is suggested. The degree of internalization differs for different endothelial cell sources. The protracted retention of heparin intracellularly by endothelial cells and subsequent release may be of therapeutic and physiological consequence.

Heparins from bovine or porcine sources were fed in media for 48 hrs to cultured porcine aortic and human umbilical vein endothelial cells. Heparin was found in pericellular and cellular fractions after extraction by chemical methods and 125I radiolabelled heparins were recovered when radiolabelled heparin was included in the feed. Even after washing and media changes heparin was detected in media and cell fractions up to 6 days post feeding. Metachromatic vacuoles within cells were demonstrated histologically up to 7 days post feeding after staining with toluidine blue. This is the first report of protracted internalization of exogenous heparin by cultured endothelial cells with concurrent prolonged release of the heparin to the media. This clearly demonstrates that the endothelium plays an important role in the distribution and metabolism of heparin.

A micro-method for the identification of most acidic mucopolysaccharides by agarose gel electrophoresis with three different buffer systems is described. In barbital buffer the mucopolysaccharides are fractionated from each other as a function of their net charge, whereas in a diamine buffer the fractionation is probably achieved according to the degree to which they are bound to the diamine. A combination of barbital and diaminopropane buffers in two-dimensional electrophoresis for the identification of mucopolysaccharides is also described.

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.