Identification of arachidonate P-450 metabolites in human platelet phospholipids

• Published in: Hypertension (Dallas, Tex. 1979) Tex. 1979), 1995-04, Vol.25 (4), p.854-859
• Main Authors: YING ZHU, SCHIEBER, E. B, MCGIFF, J. C, BALAZY, M
• Format: Article
• Language: English
• Subjects: 8,11,14-Eicosatrienoic Acid - analogs & derivatives; 8,11,14-Eicosatrienoic Acid - metabolism; Arachidonic Acid - metabolism; Biological and medical sciences; Blood Platelets - metabolism; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme System - metabolism; Fundamental and applied biological sciences. Psychology; Gas Chromatography-Mass Spectrometry; Humans; Hydroxyeicosatetraenoic Acids - metabolism; Phospholipids - metabolism; Vertebrates: blood, hematopoietic organs, reticuloendothelial system
• ISSN: 0194-911X; 1524-4563
• DOI: 10.1161/01.HYP.25.4.854

Phospholipase A2 (Naja mocambique) catalyzed release of epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) from phospholipids of isolated human platelets. The amount of EETs released by phospholipase A2 measured by gas chromatography/mass spectrometry (GC/MS) was 4.3 +/- 0.9 pmol/10(6) platelets. No EETs were detected when phospholipase A2 was omitted from the incubations. The relative abundance of EET isomers (14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET) from human platelets was 5.4:4.5:3.7:1, respectively, as established by a new method based on particle-beam liquid chromatography/mass spectrometry (LC/MS). Fractionation of platelet phospholipids by normal-phase high-performance liquid chromatography followed by hydrolysis and GC/MS analyses indicated that the amount of EETs was highest in fractions containing phosphatidylinositol and phosphatidylserine (142 and 61 pmol/nmol of phosphorus, respectively) while low in phosphatidylcholine and phosphatidylethanolamine (19 and 11 pmol/nmol of phosphorus, respectively). The majority of EETs associated with phosphatidylcholine was found in fractions containing 1-O-alkylphosphatidylcholine. Human platelet phospholipids also released 20-HETE on phospholipase treatment (9.7 +/- 1.6 fmol/10(5) cells) and at least three other HETEs, one of which was tentatively identified as 16-HETE. Activation of human platelets by thrombin or platelet-activating factor released 5 to 7 fmol EET/10(6) cells. Receptor-mediated hydrolysis of phospholipids containing EETs and 20-HETE may play a role in stimulus-response coupling in platelets.