UNIT 4.37 Aldehyde Reduction by Cytochrome P450
Published Online: 1 MAY 2011
Copyright © 2011 by John Wiley & Sons, Inc.
Lab Protocol Title
Current Protocols in Toxicology
How to Cite
Amunom, I., Srivastava, S. and Prough, R. A. 2011. Aldehyde Reduction by Cytochrome P450. Current Protocols in Toxicology. 48:4.37:4.37.1–4.37.15.
- Published Online: 1 MAY 2011
- Published Print: MAY 2011
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). More specifically, these assays measure the aldehyde reduction reactions of cytochrome P450s (CYPs). They can be performed using liver microsomal or other tissue fractions, spherosome preparations of recombinant CYPs, or recombinant CYPs from other sources. The method for reduction of 9-AA (a model α,β-unsaturated aldehyde) by CYPs was adapted from an assay for 9-anthracene oxidation published by Marini et al. (2003). For 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 HPLC, using an adaptation of the method by Srivastava et al. (2010). For both 9-AA and 4-HNE, the first step involves incubation of the substrate with the CYP in an appropriate medium. This is followed by quantification of metabolites through by spectrofluorometry (9-AA) or HPLC coupled with a radiometric assay (4-HNE). Metabolite identification can be achieved by HPLC GC/MS analysis. Inhibitors of cytochrome P450 can be utilized to show the role of the hemoprotein or other enzymes in these reduction reactions. The reduction of CYPs is not inhibited by either anaerobiosis or inclusion of CO in the gaseous phase of the reaction mixture. These characteristics are similar to those reported for some cytochrome P450−catalyzed azo reduction reactions. Curr. Protoc. Toxicol. 48:4.37.1-4.37.15. © 2011 by John Wiley & Sons, Inc.
- Cytochrome P450;
- reduction reactions;
- α,β-unsaturated aldehydes;
- inhibitors 4-hydroxy-2-nonenal;
- 9-anthracene aldehyde