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

Yi Wang, Peter W. Villalta, Lijuan Peng, Karen Dingley, Michael A. Malfatti, Ken W Turteltaub, Robert J. Turesky

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)705-714
Number of pages10
JournalChemical Research in Toxicology
Volume30
Issue number2
DOIs
StatePublished - Feb 20 2017
Externally publishedYes

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Albumins
Acids
Mass spectrometry
Mass Spectrometry
Metabolites
Amines
2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine
Hydroxylation
Pronase
Meats
Liquid chromatography
Cooking
Biomarkers
DNA Adducts
Cytochrome P-450 Enzyme System
Particle accelerators
Liquid Chromatography
Hydrolysis
Meat
Buffers

ASJC Scopus subject areas

  • Toxicology

Cite this

Mass Spectrometric Characterization of an Acid-Labile Adduct Formed with 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and Albumin in Humans. / Wang, Yi; Villalta, Peter W.; Peng, Lijuan; Dingley, Karen; Malfatti, Michael A.; Turteltaub, Ken W; Turesky, Robert J.

In: Chemical Research in Toxicology, Vol. 30, No. 2, 20.02.2017, p. 705-714.

Research output: Contribution to journalArticle

Wang, Yi ; Villalta, Peter W. ; Peng, Lijuan ; Dingley, Karen ; Malfatti, Michael A. ; Turteltaub, Ken W ; Turesky, Robert J. / Mass Spectrometric Characterization of an Acid-Labile Adduct Formed with 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and Albumin in Humans. In: Chemical Research in Toxicology. 2017 ; Vol. 30, No. 2. pp. 705-714.
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abstract = "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.",
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AU - Villalta, Peter W.

AU - Peng, Lijuan

AU - Dingley, Karen

AU - Malfatti, Michael A.

AU - Turteltaub, Ken W

AU - Turesky, Robert J.

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N2 - 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.

AB - 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.

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