Destruction of liver haem by norethindrone. Conversion into green pigments

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Abstract
Factors affecting norethindrone[a contraceptive steroid]-mediated conversion of hepatic heme into green pigments were studied in the rat. Concentrations of heme and green pigments were estimated spectrophotometrically after esterification and separation by silica gel high-pressure liquid chromatography (HPLC). Accumulation of green pigments in the liver was dependent on the dose of steroid and the time after dosing, maximum values being reached after 4-8 h. Phenobarbitone pretreatment of rats resulted in an 8-fold increase in the concentration of green pigments at these times. In microsomal systems in vitro, the formation of green pigments in the presence of NADPH and norethindrone was also dependent on the concentration of steroid and incubation times. Reaction rates became non-linear with time, consistent with the self-catalyzed destruction of the form(s) of cytochrome P-450 responsible for the metabolic activiation of norethindrone. Microsomal mixtures incubated for a short period of time (1 min) with norethindrone gave only 1 green-pigment peak after HPLC. Longer incubation times gave 4 or 5 additional green pigments. Multiple green pigments may arise by metabolic transformation of a single precursor. When liver heme was prelabeled with 14C by using 5-amino[4-14C]levulinic acid, subsequent dosing with norethindrone in vivo gave rise to 3 major 14C green-pigment peaks on HPLC. None of these components had the same retention times as the green pigments produced by microsomal fractions in vitro. When liver heme was prelabeled with 59Fe by using 59FeCl3, norethindrone administration resulted in the detection of 59Fe green pigments if subsequent esterification was carried out under neutral conditions with trimethyloxonium tetrafluoroborate, but not when carried out under acidic conditions with methanol/H2SO4. Green pigments normally contained chelated Fe and metal-free green pigments were not produced by the liver.