Hepatic microsomal metabolism of indole to indoxyl, a precursor of indoxyl sulfate

Abstract

The aim of our study was to determine which microsomal cytochrome P450 isozyme(s) were responsible for the microsomal oxidation of indole to indoxyl, an important intermediate in the information of the uremic toxin indoxyl sulfate. Indole was incubated together with an NADPH-generating system and rat liver microsomes. Formation of indigo, an auto-oxidation product of indoxyl, was used to determine the indole-3-hydroxylation activity. Apparent Km and Vmax values of 0.85 mM and 1152 pmol min(-1) mg(-1) were calculated for the formation of indoxyl from indole using rat liver microsomes. The effects of various potential inducers and inhibitors on the metabolism of indole to indoxyl by rat liver microsomes were studied to elucidate the enzymes responsible for metabolism. Studies with general and isozyme-specific P450 inhibitors demostrated that P450 enzymes and not FMO are responsible for the formation of indoxyl. In the induction studies, rate of indoxyl formation in the microsomes from untreated vs induced rats correlated nearly exactly with the CYP2E1 activity (4-nitrophenol 2-hydroxylation). These results suggests that CYP2E1 is the major isoform for the microsomal oxidation of indole to indoxyl.

Fig. 1

Metabolic pathways showing role of indole 3-hydroxylation in vivo (A) and in vitro (B).

Fig. 2

Initial velocity for the oxidation of indole to indoxyl by the uninduced rat liver microsomes prepared in our laboratory. Symbols represent observed values while the line represents non-linear fitting of the observed data to the Michaelis-Menten equation.

Fig. 3

Effect of potential cytochrome P450 inhibitors on the metabolic formation of indoxyl using rat liver microsomes. Bars represent the mean ± SD of two determinations, and 100% activity represents activity in the absence of inhibitor (325 pmol min⁻¹ mg⁻¹). SKF 525-A, 2-diethyl-aminoethyl-2,2-diphenylvalerate; DDTC, diethyldithiocarbamic acid.

Fig. 4

Effect of potential cytochrome P450 inducers on the metabolic formation of indoxyl using rat liver microsomes (A) and human liver microsomes (B) and comparison with CYP2E1 activity. The left axis represents indoxyl formation, and the right axis represents CYP2E1 activity (4-nitrophenol 2-hydroxylation). Bars represent the mean ± SD of two-four determinations. Rat liver microsomes were prepared in our lab from untreated animals or obtained from In Vitro Technologies from untreated, Arochlor 1244-treated, or isoniazid-treated animals. Insert shows the correlation of CYP2E1 activity with 3-hydroxylation of indole in all samples.