High pressure liquid chromatography and mass spectrometry characterization of the nephrotoxic biotransformation products of cisplatin

Previous studies have shown that cisplatin requires metabolic activation to become nephrotoxic. The activation is proposed to be via the metabolism of a glutathione-platinum conjugate to a cysteinyl-glycine-platinum conjugate, which is further processed to a cysteine conjugate. Preincubating cisplatin with glutathione (GSH), cysteinylglycine, or N-acetylcysteine (NAC) results in a transient increase in the toxicity of cisplatin toward renal proximal tubular cells. In this study, the preincubation solutions were analyzed by high pressure liquid chromatography (HPLC), atomic absorption spectrometry, and mass spectrometry (MS) to characterize the formation and structure of the platinum conjugates. HPLC analysis of the cisplatin-GSH, cisplatin-cysteinyl-glycine, and cisplatin-NAC preincubation solutions revealed two new platinum-containing peaks in each of the solutions. MS-MS analysis of the peaks revealed a diplatinum- and a monoplatinum conjugate in each of the solutions. Analysis of the composition and toxicity of the solutions with time showed that the transient increase in toxicity correlated with the formation of the monoplatinum conjugate whereas prolonged preincubation decreased toxicity and correlated with the formation of the diplatinum conjugate. The monoplatinum-monoglutathione conjugate is a substrate for gamma-glutamyl transpeptidase, an enzyme that is essential for the nephrotoxicity of cisplatin. The monoplatinum-mono-NAC conjugate can be deacetylated to a cysteine conjugate, which is a substrate for pyroxidol phosphate (PLP)-dependent cysteine S-conjugate beta-lyase. This PLP-dependent enzyme is proposed to catalyze the final step in the metabolic activation of cisplatin. Identification of the structure and toxicity of these conjugates further elucidates the metabolism of cisplatin to a nephrotoxin.