Determination of 20-hydroxyeicosatetraenoic acid in microsomal incubates using high-performance liquid chromatography-mass spectrometry (HPLC-MS)

摘要

20-HETE is a potent, vasoconstrictive arachidonic acid metabolite with a limited number of published methods for quantitative assessment of microsomal formation rate. The purpose of this study was to evaluate the utility of HPLC-MS (negative ESI) for quantitation of rat microsomal 20-HETE enzyme kinetics. Calibration curves were linear over 0.75-16 ng on-column (r~2 > 0.996). The intra- and inter-assay precision and accuracy were <15%. Microsomal 20-HETE revealed saturable (100 μM) kinetics (brain K_m and V_(max): 39.9 ± 6.0 μM and 8.7 ± 0.6 pmol/min per mg; liver K_m and V_(max): 23.5 ± 3.2 μM and 775.5 ± 39.8 pmol/min per mg; kidney K_m and V_(max): 47.6 ± 8.5 μM and 1933 ± 151 pmol/min per mg). This paper demonstrates HPLC-MS as an efficient method for quantitating 20-HETE enzyme kinetics in microsomes from rat tissues.