Abstract
The properties of heparin as an inhibitor of blood coagulation have been known for several years(l). However, the physiology and metabolism of heparin has been only slightly clarified. With the preparation of radioactive heparin the possibility for quantitative metabolic studies of this substance can be undertaken. Eiber and Danishefsky(2) reported on the preparation of heparin labeled with S35 by biosynthesis in the dog. With this material they have studied the disposition of heparin after injection by following its disappearance from the circulation(3) and its deposition in various organs(4). More recently Levy and Petracek(5) have prepared heparin-S35 by the chemical reaction of pyri-dine S35O3 or trimethylamine-S35O3 with partially desulfated heparin. Both preparative methods led to biologically active preparations with radioactivity counts of the order of 10 5 dpm/mg. There is still a need for a radioactive heparin labeled with another isotope, e.g., tritium, in positions other than the sulfate moiety which is the most labile bond in the molecule. Recent interest involves the use of graphite-benzalkonium-heparin (GBH) surfaces in artificial organs especially the artificial heart valves(6); an understanding of the mechanism of action of this surface might be facilitated by a stable isotopically labeled heparin molecule.
Tritiation. We used a modification of the Hempel Toepler pump set-up for catalytic tritiation. The apparatus will be described later. Three hundred mg of sodium heparin (157 μ/mg) was suspended in 3 ml of water and 2 ml of diethyleneglycol monoethyl ether. One hundred milligrams of 30% palladium on carbon were then added. The resultant suspension was frozen in a liquid nitrogen bath, evacuated, and the vessel filled with hydrogen. The catalyst was saturated with hydrogen by mixing with a magnetic stirrer for one hour at atmospheric pressure and temperature.
Purification and characterization. The sample was purified by dissolving it in isotonic sodium chloride and precipitating with cetyl-pyridinium chloride (CPC), 3 mg per mg of heparin; the resultant complex was redis-solved in 2 M sodium chloride and the heparin precipitated with absolute alcohol, 2 volumes. The heparin was redissolved in 0.5 M sodium chloride and the entire purification procedure was repeated. The slightly colored product weighed 150 mg, had a specific radioactivity of 8.2 μC/mg and a biological potency in the USP assay of 124 μ/mg.
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