Some mass-spectral and n.m.r. analytical studies of a glutathione conjugate of aflatoxin B1

Abstract

A system for the formation of an aflatoxin B1-reduced glutathione conjugate in vitro was developed, capable of yielding 80% conversion of aflatoxin B1 into the conjugate. A reverse-phase high-pressure-liquid-chromatography system was also devised that facilitated improved resolution of the compound, and by manipulation of the pH was also capable of an extensive purification of the compound from other aflatoxin B1 metabolites in a single step. Material produced by these techniques, after further purification, was used in 1H-NMR and mass-spectroscopic studies. Results were obtained that supported the proposed linkage of the aflatoxin B1 to reduced glutathione in a 1:1 molar ratio via a thioether linkage. Amino acid analyses were also consistent with this structure. The absence of a Schiff-base linkage of aflatoxin B1 8,9-dihydrodiol to glutamate was further demonstrated by the presence of a .gamma.-glutamyltransferase-catalyzed-transferable glutamate moiety. These data were consistent with the structure 8,9-dihydro-8-(S-glutathionyl)-9-hydroxy-aflatoxin B1. [Aflatoxin B1 is a hepatocarcinogen.].