Received for publication March 17, 2009.
Revised June 16, 2009.
Accepted for publication June 17, 2009.

Contrasting Influence of NADPH and a NADPH-regenerating System on the Metabolism of Carbonyl-Containing Compounds in Hepatic Microsomes

Abstract

Carbonyl containing xenobiotics may be susceptible to NADPH-dependent cytochrome P450 (CYP) and carbonyl-reduction reactions. In vitro hepatic microsome assays are routinely supplied NADPH either by direct addition of NADPH or via an NADPH-regenerating system (NRS). In contrast to oxidative CYP transformations, which occur on the periphery of a microsome vesicle, intraluminal carbonyl reduction depends on transport of cofactors across the endoplasmic reticulum (ER) membrane into the lumen. Glucose-6-phosphate, a natural cofactor and component of the NRS matrix, is readily transported across the ER membrane and facilitates intraluminal NADPH production, whereas direct addition of NADPH has limited access to the lumen. In this study, we compared the effects of direct addition of NADPH and use of an NRS on the CYP-mediated transformation of propiconazole and 11-HSD1 carbonyl reduction of cortisone and the xenobiotic triadimefon in hepatic microsomes. Our results demonstrate the use of NADPH rather than NRS can underestimate the kinetic rates of intraluminal carbonyl reduction, whereas CYP-mediated transformations were unaffected. Therefore, in vitro depletion rates measured for a carbonyl-containing xenobiotic susceptible to both intraluminal carbonyl reduction and CYP-processes may not be properly assessed with direct addition of NADPH. Additionally, we used in silico predictions to: (1) show 11-HSD1 carbonyl reduction was energetically more favorable than oxidative CYP transformation and (2) calculate chemical binding score and the distance between the carbonyl group and the hydride to be transferred by NADPH to identify other 11-HSD1 substrates for which reaction kinetics may be underestimated by direct addition of NADPH.

Key words: cytochrome P450 function, endoplasmic reticulum, enzyme kinetics, in vitro-in vivo prediction, in vitro-in vivo scaling, liver microsomes, microsomes, transporters