Basal concentrations of free and esterified monohydroxylated fatty acids in human blood platelets

Monohydroxylated fatty acids (HO-FA), namely 12-hydroxyeicosatetraenoic and 12-hydroxyheptadecatrienoic acids, are enzymatically formed in response to platelet activation. Different techniques, including gas chromatography (GC) and liquid chromatography-mass spectrometry (LC-MS), have been described to measure HO-FA in activated cells, but they are not well-adapted to resting cells. Measurements of free and esterified HO-FA at basal concentration require the prevention of platelet activation. For this purpose, such an activation was minimized by adding various inhibitors to the anticoagulant. Platelet recovery was greater in the protected group than in controls (473 x 10(9) +/- 4.0 x 10(9) platelets/L vs 410 x 10(9) +/- 4.53 x 10(9) platelets/L, respectively) (mean +/- SEM, n = 9, P < 0.05). Lipids were extracted and immediately hydrogenated to avoid fatty acid autoxidation occurring during the workup. Unesterified and esterified HO-FA were analyzed by GC-MS, and the former were lower in the protected group (1.52 +/- 0.84 pmol/10(9) platelets) than in the unprotected one (12.63 +/- 10.52 pmol/10(9) platelets) (mean +/- SEM, n = 9, P < 0.05). Interestingly, only traces of HO-FA were detected in both the triglyceride and sterol ester fractions, and they were also weakly esterified in phospholipids.