Destruction of liver haem by norethindrone. Conversion into green pigments

1. Factors affecting the norethindrone-mediated conversion of hepatic haem into green pigments have been studied in the rat. Concentrations of haem and green pigments were estimated spectrophotometrically after esterification and separation by silica gel high-pressure liquid chromatography (h.p.l.c.). 2. Accumulation of green pigments in the liver was dependent on the dose of steroid and the time after dosing, maximum values being reached after 4–8h. Phenobarbitone pretreatment of rats resulted in an 8-fold increase in the concentration of green pigments at these times. 3. 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 very rapidly became non-linear with time, consistent with the self-catalysed destruction of the form(s) of cytochrome P-450 responsible for the metabolic activation of norethindrone. Microsomal mixtures incubated for a short period of time (1min) with norethindrone gave only one green-pigment peak after h.p.l.c. Longer incubation times gave four or five additional green pigments. Results suggested that multiple green pigments may arise by metabolic transformation of a single precursor. 4. When liver haem was prelabelled with ¹⁴C by using 5-amino[4-¹⁴C]laevulinic acid, subsequent dosing with norethindrone in vivo gave rise to three major ¹⁴C-labelled-green-pigment peaks on h.p.l.c. None of these components had the same retention times as the green pigments produced by microsomal fractions in vitro. 5. When liver haem was prelabelled with ⁵⁹Fe by using ⁵⁹FeCl₃, norethindrone administration resulted in the detection of ⁵⁹Fe-labelled 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. These results suggested that green pigments normally contain chelated iron and that metal-free green pigments are not produced by the liver.