The use of high pressure liquid chromatography (HPLC) for the separation of radiolabeled arachidonic acid and its metabolites produced by thrombin-treated human platelets. II. Establishment of optimal assay conditions.
We have utilized HPLC to develop optimal conditions for assaying the transformation of arachidonic acid in thrombin-treated human platelets. In the presence of increasing amounts of albumin, the total amount of radioactivity released from thrombin-treated platelets pre-labeled with 3H-arachidonic acid is first enhanced and then inhibited. Maximal release, reflecting primarily enhanced amounts of free labeled arachidonic acid, occurs at a final albumin concentration of 0.5 mg/ml. Calcium promoted the release of all radiolabeled metabolites, but it specifically enhanced HETE formation and release. Magnesium was without effect. Cyclo-oxygenase derived products constituted the bulk of released label at short time intervals, but after ten minutes exposure to thrombin in the presence of albumin (0.5 mg/ml) and 3 mM calcium, radioactivity in the released products was equally distributed among cyclo-oxygenase derived products (TXB2 + PGD2 + HHT), HETE and free arachidonic acid.
D000418:Albumins / Q000494:pharmacology; D001095:Arachidonic Acids / Q000302:isolation & purification* / Q000378:metabolism; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D012636:Secretory Rate / Q000187:drug effects; D013917:Thrombin / Q000494:pharmacology*
D000418:Albumins; D001095:Arachidonic Acids; D011451:Prostaglandin-Endoperoxide Synthases; D013917:Thrombin; D008274:Magnesium; D002118:Calcium