Stereoselective reversible ketone formation from 10-hydroxylated nortriptyline metabolites in human liver

Abstract

1. E- and Z-10-hydroxynortriptyline are major metabolites of amitriptyline and nortriptyline in man. Upon incubation with human liver microsomes or cytosol, these metabolites were oxidized to the corresponding ketones, E- and Z-10-oxonortriptyline. (+)-E- and (+)-Z-10-hydroxynortriptyline were distinctly preferred over the (-)-isomers as substrates. NADP⁺ supported the oxidation in cytosol, whereas in microsomes NAD⁺ was the best cofactor.

2. Incubation of E- and Z-10-oxonortriptyline with NADPH and cytosol resulted in the nearly exclusive formation of (+)-E- and (+)-Z-10-hydroxynortriptyline. Kinetic analysis revealed high-affinity reduction (K_m 1–2 μM) of the two ketones and an additional low-affinity component with the E-isomer. 10-oxonortriptyline reduction was also catalysed by rabbit, but not by rat or guinea pig liver cytosol.

3. With [4-³H]NADPH as cosubstrate, tritium was incorporated into E- and Z-10-hydroxynortriptyline preferentially from the pro-4R position. Redox cycling of (+)-E- and (+)-Z-10-hydroxynortriptyline in cytosol in the presence of NAD₋ and NADPH was indicated by ³H incorporation from [pro-4R-³H]NADPH.

4. Recombinant human carbonyl reductase catalysed low-affinity reduction of E-10-oxonortriptyline with preferential transfer of the pro-4S-³-H of labelled NADPH.

5. Ketone reduction in cytosol was strongly inhibited by 9,10-phenanthrenequinone and dehydrolithocholic acid and moderately by other 3-oxo steroids and some anti-inflammatory drugs.

6. The high-affinity reduction of E- andZ-10-oxonortriptyline and the oxidation of the alcohols in cytosol are probably mediated by a member of the aldo-keto reductase family of enzymes.