Quantitative Aspects of Lipoprotein Lipase Release by Heparin in Mice 1

Abstract

Although heparin has been known to release lipoprotein lipase (diacylglycerol acylhydrolase, EC 3.1.1.34) since 1943 (1), the quantitative aspects related to dosage have never been fully determined. Furthermore, only the data of Robinson et al. (2) are available concerning the half-life of enzyme action, once released into the blood. Olsson et al. (3) and Estes et al. (4) published data on the volume of distribution of heparin and kinetic data on heparin's anticoagulant action in man. More recently, McAvoy (5) determined the biologic half-life of heparin. In this paper, we present quantitative dose-response data on the release of lipoprotein lipase by heparin in mice, as well as information on time-duration action of released lipoprotein lipase in serum.

Materials and Methods. Seventy male Swiss-Webster mice (weighing 20–30 g) were used in this investigation. Heparin sodium was injected intravenously in the tail vein in a volume of 0.1 ml. Axillary blood was obtained from etherized mice, allowed to clot, and then spun down in a refrigerated centrifuge at 2500 rpm for 30 min at 5°. (In previous experiments we found that etherization did not affect lipoprotein lipase release.) The clear serum was either tested immediately or frozen at −10° and used on subsequent days. The enzyme was found to be quite stable and showed no discernible diminution in activity on storage at −10° for 20 days. It was always tested either immediately or within a few days after collection.

Lipoprotein lipase assay. To determine the level of lipoprotein lipase, 0.6 ml of serum was added to a flask in which 5.4 ml of [¹⁴C]triolein substrate (see below) was placed; it was incubated in a shaking incubator for 20 min at 27° (according to a modification of the method of Schotz (6, 7)). At intervals of 1.5, 10, and 20 min, 0.5-ml samples were removed from the flask and added to isopropanol-sulfuric acid (40:1) solution to stop the reaction. Then 5 ml of hexane and 1 ml of water were added to each aliquot and the tubes were shaken for 1 min in a mechanical shaker. The upper layer containing the hexane phase was separated and shaken with 2 ml of 0.1 N KOH for 10 min. The lower phase contains the potassium oleate generated by enzymic action on the triolein substrate. The 1-ml aliquots of the lower phase were added to 10 ml Aquasol and counted in an Intertechnique liquid scintillation spectrometer, Model SL-30.