Determination of CYP4A11-catalyzed lauric acid 12-hydroxylation by high-performance liquid chromatography with radiometric detection.
Lauric acid serves as an endogenous substrate for the cytochrome P450 enzyme CYP4A11. A reverse-phase, high-performance liquid chromatography method is described for the quantification of 12-hydroxylauric acid formed enzymatically by incubation of 14C-labeled lauric acid with cDNA-expressed CYP4A11 or human liver microsomes. Analytical separation is achieved using a C18 column and a gradient of 30% acetonitrile and 2 mM perchloric acid to 100% methanol, using a detection scintillation counter. This method is applicable to enzymatic studies for determination of lauric acid 12-hydroxylation activity.
Crespi
Charles L
CL
Discovery Labware, BD Biosciences, Woburn, MA, USA.
Chang
Thomas K H
TK
Waxman
David J
DJ
D002384:Catalysis; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006801:Humans; D006900:Hydroxylation; D007850:Lauric Acids / Q000378:metabolism*; D011874:Radiometry / Q000379:methods*
D007850:Lauric Acids; C030358:lauric acid; D003577:Cytochrome P-450 Enzyme System; C465478:CYP4A11 protein, human; D042926:Cytochrome P-450 CYP4A