Determination of 20-hydroxyeicosatetraenoic acid in microsomal incubates using high-performance liquid chromatography-mass spectrometry (HPLC-MS).
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 microM) kinetics (brain K(m) and V(max): 39.9+/-6.0 microM and 8.7+/-0.6 pM/min per mg; liver K(m) and V(max): 23.5+/-3.2 microM and 775.5+/-39.8 pmol/min per mg; kidney K(m) and V(max): 47.6+/-8.5 microM and 1933+/-151 pM/min per mg). This paper demonstrates HPLC-MS as an efficient method for quantitating 20-HETE enzyme kinetics in microsomes from rat tissues.
Bolcato
Christopher A
CA
Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261, USA.
Frye
Reginald F
RF
Zemaitis
Michael A
MA
Poloyac
Samuel M
SM
D000818:Animals; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism*; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008861:Microsomes / Q000201:enzymology*; D051381:Rats; D017207:Rats, Sprague-Dawley
D006893:Hydroxyeicosatetraenoic Acids; C055987:20-hydroxy-5,8,11,14-eicosatetraenoic acid