Abstract
An assay using radioactive substrates is described that permits rapid determinations of glycerophospholipid-hydrolyzing activity in postheparin plasma or its fractions. Optimal conditions are described for hydrolysis of phosphatidylcholine and phosphatidylethanolamine. A minimum of only 2 mul of normal postheparin plasma is used and no extraction of the reaction products is required before their separation by thin-layer chromatography. We found that after an optimal heparin dose of 60 units/kg body weight the rate of hydrolysis for diacyl glycerophosphocholine and diacyl glycerophosphoethanolamine is 1.16 mumoles/ml per hr and 22.4 mumoles/ml per hr, respectively.
Highlights
Minimum of only 2 p1 of normal postheparin plasma is used and no extraction of the reaction products is required before their separation by thin-layer chromatography
The unique features are that only 2-20 p1 of postheparin plasma are used and no extraction of the reactionproducts is required before their separationby thin-layer chromatography (TLC).Thisreport describes a procedurefor synthesis of the radioactive substrates, data on the assay, and biological reproducibility and optimal heparin dosage
X- 100, deoxycholate, and cetyltrimethylammoniumbromide were obtained from Sigma Chemical Company, St
Summary
Summary Aanssay using radioactive substrates is described that permits rapid determinations of glycerophospholipid-hydrolyzing activity inpostheparin plasma or its fractions. Optimal conditionsare described for hydrolysis of phosphatidylcholine and phosphatidylethanolaminAe. minimum of only 2 p1 of normal postheparin plasma is used and no extraction of the reaction products is required before their separation by thin-layer chromatography. Thisapproachpermits simple andrapid serial sampling of glycerophospholipid hydrolyzing activity in postheparin plasma or its fractions, andeither 1,2-diacyl-sn-glycero-3-phosphoethanolamin(eGPE) or 1,2-diacyl-sn-glycero-3-phosphocholin(eGPC) can be used as substrates. The unique features are that only 2-20 p1 of postheparin plasma are used and no extraction of the reactionproducts is required before their separationby thin-layer chromatography (TLC).Thisreport describes a procedurefor synthesis of the radioactive substrates, data on the assay, and biological reproducibility and optimal heparin dosage.
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