Mass Spectrometric Characterization of an Acid-Labile Adduct Formed with...

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, an electrophilic metabolite that 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 primarily occurs at the Cys³⁴ residue, to produce an acid-labile linked sulfinamide adduct. On the basis of these findings, we developed a sensitive ultraperformance liquid chromatography-mass spectrometry method to measure acid-labile albumin-PhIP adducts in human volunteers administered a dietary-relevant dose of ¹⁴C-labeled PhIP [Dingley, K. H., et al. (1999) Cancer Epidemiol., Biomarkers Prev. 8, 507-512]. Mild acid treatment of albumin (0.1 N 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 and quantified by Orbitrap mass spectrometry. The amount of [¹⁴C]PhIP recovered by acid treatment of albumin 24 h following dosing accounted for 7.2-21.3% 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, 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.