Measurement of Xenobiotic Carbonyl Reduction in Human Liver Fractions

Abstract

Carbonyl reducing enzymes are involved in the metabolism of endogenous as well as xenobiotic molecules. Enzymes that catalyze the reversible oxidoreduction of aldehyde and ketone moieties include alcohol dehydrogenases, aldo-keto reductases, quinone reductases, and short-chain dehydrogenases/reductases. These enzymes differ with respect to subcellular location, cofactor dependence, and susceptibility to chemical inhibitors. Thus, it is possible to assess the relative contributions of these enzyme systems in the hepatic metabolism of a particular xenobiotic through simple in vitro experiments with commercially available reagents. The approaches described in this unit assume the availability of analytical procedures for measuring the parent compound and metabolites, such as HPLC with radiochemical, UV, or MS detection. Thus, the purpose of this unit is to outline methods for the study of the enzymatic carbonyl reduction of a drug development candidate or other xenobiotic molecule of interest.

Involvement of microsomal NADPH‐cytochrome P450 reductase in metabolic reduction of drug ketones

“…For the reduction of ketone functions of xenobiotics in the organism, mainly two classes of enzymes are thought to be responsible, namely the AKRs and the SDRs [3][4][5]. Therefore, the effect of published inhibitors of these enzymes [10,11] on the reduction of compound 5 was determined in this former study [9]. Since the AKR inhibitors flufenamic acid and phenolphthalein did not affect the formation of the alcohol 6 from ketone 5 (Table 1), a contribution of AKRs in this metabolic reaction was ruled out.…”

“…In the following inhibition experiments, the activities of the two enzymes were chosen in a way that about 60-70% of substrate 5 was converted to product 6. The concentrations of the inhibitors were selected on the basis of inhibition data published in the literature [10,11]. The results obtained (Table 1) were very surprising.…”

“…In rat liver S9 fractions, compound 5 was even completely metabolized to the alcohol 6. Here, cytosolic SDRs (cSDRs) are held mainly to be responsible for this reaction, because the keto-reduction could be inhibited by certain compounds, which were reported in the literature to be inhibitors of cSDR enzymes [10,11].…”