Determination of in vivo adducts of disulfiram with mitochondrial aldehydedehydrogenase

Extensive use for disulfiram (DSF) has been found in the aversion therapy t reatment of recovering alcoholics. Although it is known to irreversibly inh ibit hepatic aldehyde dehydrogenase (ALDH), the specific mechanism of in vi vo inhibition of the enzyme by the drug has not been determined yet. We hav e demonstrated in this report a novel, but simple and rapid method for stru cturally characterizing in vivo derived protein-drug adducts by linking on- line sample processing to HPLC-electrospray ionization mass spectrometry (H PLC-MS) and HPLC-tandem mass spectrometry (HPLC-MS/MS). Employing this appr oach, rats were administered DSF, and their liver mitochondria were isolate d and solubilized, Both native and in vivo DSF-treated mitochondrial ALDH ( mALDH) were purified in one step with an affinity cartridge. The in vivo DS F-treated mALDH showed 77% inhibition in enzyme activity as compared with t hat of the control. Subsequently, thr control and DSF-inhibited mALDH were both subjected to HPLC-MS analyses. We were able to detect two adducts on D SF-inhibited mALDH, as indicated by the mass increases of similar to 71 and similar to 100 Da. To unequivocally determine the site and structure of th ese adducts, on-line pepsin digestion-HPLC-MS and HPLC-MS/MS were performed . We observed two new peptides at MH+ = 973.7 and MH+ = 1001.8 in the pepsi n digestion of DSF-inhibited enzyme, These two peptides were subsequently s ubjected to HPLC-MS/MS for sequence determination. Both peptides possessed the sequence FNQGQC(301)C(302)C(303), derived from the enzyme active site r egion, and were modified at Cys(302) by N-ethylcarbamoyl (+71 Da) and N-die thylcarbamoyl (+99 Da) adducts. These findings indicated that N-dealkylatio n may be an important step in DSF metabolism, and that the inhibition of AL DH occurred by carbamoylation caused by one of the DSF metabolites, most li kely S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO). Finally, there was no evidence of the presence of an intramolecule disulfide bridge modif ication on the peptide FNQGQCCC. (C) 2001 Elsevier Science Inc. All rights reserved.