Mass Spectrometric Characterization of an Acid-Labile Adduct Formed with 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and Albumin in Humans

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a carcinogenic heterocyclic aromatic amine formed during the high temperature cooking of meats. The cytochrome P450-mediated N-hydroxylation of the exocyclic amine group of PhIP produces 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (HONH-PhIP), an electrophilic metabolite which forms adducts with DNA and proteins. Previous studies conducted by our laboratory showed that the reaction of N-oxidized-PhIP metabolites with human albumin in vitro occurs primarily at the Cys34 residue, to produce an acid-labile linked sulfinamide adduct. Based on these findings, we developed a sensitive ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) method to measure acid-labile albumin-PhIP adducts in humansvolunteers administered a dietary-relevant dose of 14C-labelled-PhIP (Dingley et al., Cancer Epidemiol. Biomarkers Prev. 8:507–512). Mild acid-treatment of albumin (0.1N HCl, 37 °C for 1 h) or proteolytic digestion with pronase (50 mM ammonium bicarbonate buffer pH 8.5 at 37 °C for 18 h) released similar amounts of covalently bound PhIP, which was characterized by multistage scanning (MS3) and quantified by Orbitrap mass spectrometry. The amount of 14C-PhIP recovered by acid-treatment of albumin 24 hour following dosing accounted for 7.2 to 21.3% of the 14C-PhIP bound to albumin based on accelerator mass spectrometry measurements. 2-Amino-1-methyl-6-(5-hydroxy)-phenylimidazo[4,5-b]pyridine (5-HO-PhIP), a hydrolysis product of the Cys34 S−N linked sulfenamide adduct of PhIP, was not detected in either acid-treated or protease treated samples. These findings suggest that a portion of the PhIP bound to albumin in vivo probably occurs as an acid labile-sulfinamide adduct formed at the Cys34 residue.