Preparation and Characterization of Tritiated Heparin

Abstract

The properties of heparin as an inhibitor of blood coagulation have been known for several years ( 1 ) . 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-Sa5 by the chemical reaction of pyridine S3503 or trimethylamine-S3503 with partially desulfated heparin. Both preparative methods led to biologically active preparations with radioactivity counts of the order of lo5 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 p/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. Five curies of tritium gas were pumped into the 10 ml reaction vessel after freezing and evacuation. The reaction mixture was then adjusted to roo,m temperature and atmospheric pressure with hydrogen. The reaction for the catalytic exchange tritiation was 48 hours with constant mixing. After 48 hours the catalyst was separated from the reaction mixture by filtration. Labile tritium was removed by distilling five 2501 ml portions of water from the product. The crude product weighed 275 mg and assayed 137 pCJmg by scintillation counting.Purification and characterization. The sample was purified by dissolving it in isotonic sodium chloride and precipitating with cetylpyridinium chloride (CPC), 3 mg per mg of heparin; the resultant complex was redissolved in 2 M sodium chlo'ride and the heparin precipitated with absolute alcohol, 2 volumes. The heparin was redissolved in 0.5 R/I sodium chloride and the entire purification procedure was repeated. The slightly colored product weighed 1.50 mg, had a specific radioactivity of 8.2 ,pC/mg and a biological potency in the USP assay of 124 p/mg.The radioactive powder diluted with cold startin...