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

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 mu M) 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-H-3]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 H-3 incorporation from [pro-4R-H-3]NADPH. 4. Recombinant human carbonyl reductase catalysed low-affinity reduction of E-10-oxonortriptyline with preferential transfer of the pro-4S-H-3 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 antiinflammatory drugs. 6. The high-affinity reduction of E- and Z-10-oxonortriptyline and the oxidation of the alcohols in cytosol are probably mediated by a member of the aldo-keto reductase family of enzymes.