Identification of glutathione conjugates of the dimethyl ester of bilirubin in the bile of gunn rats

The Gunn rat, which is deficient in the UDP-glucuronosyltransferase for bilirubin, promptly excreted polar conjugates of the dimethyl ester of bilirubin in bile after intravenous infusion of this ester. The conjugates proved to be monoglutathione thioether adducts of the vinyl groups of the parent tetrapyrrole. High performance liquid chromatographic analysis of the conjugates as their dipyrrolic azosulfanilates demonstrated that only one of the dipyrroles of each tetrapyrrole was conjugated. The nonconjugated dipyrrole eluted as either the methyl endo- or exovinyl azodipyrrole. The amino acid composition of the pigments was consistent with that of a monoglutathione conjugate. NMR spectroscopy of the two major pigments demonstrated the loss of the proton signals of the C-18 vinyl group, indicating it to be the site of conjugation. Cation fast atomic bombardment tandem mass spectrometry demonstrated a molecular ion, [M + H]+, of m/z 937, which fragmented with a loss of 307 atomic mass units, consistent with glutathione. A molecular ion of m/z 807 was observed for the conjugate treated with gamma-glutamyltranspeptidase, consistent with the loss of glutamate. The mass spectrometry data indicated that the conjugates also contained a functional group whose mass was equivalent to hydroxyl, suggesting initial formation of an epoxide, which then reacts with glutathione. Pretreatment of the rat with 2,3,7,8-tetrachlorodibenzo-p-dioxin to induce cytochrome P-450 resulted in a 6-fold increase of the biliary excretion of the glutathione conjugates. Such induction also resulted in the excretion of a glutathione conjugate of bilirubin itself.