Aldehyde Reduction by Cytochrome P450

Abstract

This protocol describes the procedure for measuring the relative rates of metabolism of the α,β-unsaturated aldehydes, 9-anthracene aldehyde (9-AA) and 4-hydroxy-trans-2-nonenal (4-HNE); specifically the aldehyde reduction reactions of cytochrome P450s (CYPs). These assays can be performed using either liver microsomal or other tissue fractions, spherosome preparations of recombinant CYPs, or recombinant CYPs from other sources. The method used here to study the reduction of a model α,β-unsaturated aldehyde, 9-AA, by CYPs was adapted from the assay used to investigate 9-anthracene oxidation as reported by Marini et al. (Marini et al., 2003). For experiments measuring reduction of the endogenous aldehyde, 4-HNE, the substrate was incubated with CYP in the presence of oxygen and NADPH and the metabolites were separated by High Pressure Liquid Chromatograpy (HPLC), using an adaptation of the method of Srivastava et al. (Srivastava et al., 2010). For study of 9-AA and 4-HNE reduction, the first step involves incubation of the substrate with the CYP in appropriate media, followed by quantification of metabolites through either spectrofluorimetry or analysis by HPLC coupled with a radiometric assay, respectively. Metabolite identification can be achieved by HPLC GC-mass spectrometric analysis. Inhibitors of cytochrome P450 function can be utilized to show the role of the hemoprotein or other enzymes in these reduction reactions. The reduction reactions for CYP’s were not inhibited by either anaerobiosis or inclusion of CO in the gaseous phase of the reaction mixture. These character of these reactions are similar to those reported for some cytochrome P450-catalyzed azo reduction reactions.

Cytochromes P450 Catalyze the Reduction of α,β-Unsaturated Aldehydes

“…The differences in fluorescence emission wavelengths and the differential extraction with ethyl acetate allows for measurement of both the carboxylic acid and alcohol products in a single assay procedure as described by Amunom et al . 23 .…”

“…The emission wavelengths for both 9-A-MeOH and 9-A-MeOH were strikingly different, thus allowing measurement of these products in organic extracts from either acidified or alkalinized incubation reactions using appropriate standard curves for the two fluorescent products, respectively. 23 For example, 9-A-MeOH was measured in the ethyl acetate phase after raising the pH of the microsomal reaction mixture with a base. The fluorescence intensity of 9-anthracene methanol was found to be highest in this organic phase after a single extraction step, demonstrating the high extraction efficiency of 9-A-MeOH into the ethyl acetate organic phase (data not shown).…”

“…The fluorescence excitation and emission spectra of the metabolites was nearly identical to authentic 9-ACA and 9-A-MeOH. 15,23 The 9-AA metabolism assay was used as an initial indication of aldehyde oxidation or reduction by P450s.…”

CYP4F13 is the Major Enzyme for Conversion of alpha-Eleostearic Acid into <i>cis</i>-9, <i>trans</i>-11-Conjugated Linoleic Acid in Mouse Hepatic Microsomes

“…Unlike the well-established oxidation reaction, the reduction reaction catalyzed by CYP has not been characterized in detail. The stoichiometry is written as: R X NADPH H RH XH NADP 55 . This reaction is mostly seen when the substrates contain quinones, azo-, halogenated, nitro-, N-hydroxy-, and hydroperoxide functional groups.…”

“…This reaction is mostly seen when the substrates contain quinones, azo-, halogenated, nitro-, N-hydroxy-, and hydroperoxide functional groups. Amunom et al 55,56 have reported that α,β-unsaturated aldehydes 9-anthracene aldehyde and 4-hydroxy-trans-2-nonenal are reduced/hydrogenated to their corresponding carboxylic acid by several human and murine CYP enzymes. They also identified that this CYP-dependent reduction occurred in the presence and absence of molecular oxygen.…”