Absolute Quantitation of NAPQI-Modified Rat Serum Albumin by LC–MS/MS: Monitoring Acetaminophen Covalent Binding in Vivo

Abstract

Acetaminophen is known to cause hepatoxicity via the formation of a reactive metabolite, N-acetyl p-benzoquinone imine (NAPQI), as a result of covalent binding to liver proteins. Serum albumin (SA) is known to be covalently modified by NAPQI and is present at high concentrations in the bloodstream and is therefore a potential biomarker to assess the levels of protein modification by NAPQI. A newly developed method for the absolute quantitation of serum albumin containing NAPQI covalently bound to its active site cysteine (Cys34) is described. This optimized assay represents the first absolute quantitation of a modified protein, with very low stoichiometric abundance, using a protein-level standard combined with isotope dilution. The LC-MS/MS assay is based on a protein standard modified with a custom-designed reagent, yielding a surrogate peptide (following digestion) that is a positional isomer to the target peptide modified by NAPQI. To illustrate the potential of this approach, the method was applied to quantify NAPQI-modified SA in plasma from rats dosed with acetaminophen. The resulting method is highly sensitive (capable of quantifying down to 0.0006% of total RSA in its NAPQI-modified form) and yields excellent precision and accuracy statistics. A time-course pharmacokinetic study was performed to test the usefulness of this method for following acetaminophen-induced covalent binding at four dosing levels (75-600 mg/kg IP), showing the viability of this approach to directly monitor in vivo samples. This approach can reliably quantify NAPQI-modified albumin, allowing direct monitoring of acetaminophen-related covalent binding.

Absolute quantitation of acetaminophen‐modified human serum albumin in acute liver failure patients by liquid chromatography/tandem mass spectrometry

“…Ultra‐pure water was prepared from a Millipore Synergy UV system (Billerica, MA, USA). Iodo‐APAP ( N ‐(4‐hydroxyphenyl)‐2‐iodoacetamide) and d 4 ‐iodo‐APAP ( N ‐(4‐hydroxy[2,3,5,6‐d 4 ]phenyl)‐2‐iodoacetamide) were synthesized in‐house for chemical labeling of HSA and standard peptide to produce surrogate standard and internal standard (IS, see Figure B).…”

“…(N-(4-hydroxy[2,3,5,6-d 4 ]phenyl)-2-iodoacetamide) were synthesized in-house 30 for chemical labeling of HSA and standard peptide to produce surrogate standard and internal standard (IS, see Figure 1B).…”

“…Our group has previously introduced a liquid chromatography/multiple reaction monitoring (LC/MRM) assay using a rat model of acetaminophen covalent binding in vivo . The rat assay was used as a starting point for the human model, using the same principle of protein‐level standard and isotope dilution.…”

“…29 Our group has previously introduced a liquid chromatography/multiple reaction monitoring (LC/MRM) assay using a rat model of acetaminophen covalent binding in vivo. 30 The rat assay was used as a starting point for the human model, using the same principle of protein-level standard and isotope dilution. However, based on differences in protein sequence between rat and human albumins, the peptide formed following digestion yields a different target analyte to monitor for the human assay.…”

Biomonitoring Human Albumin Adducts: The Past, the Present, and the Future

“…An alternative approach employed pepsin to digest NAPQI-Alb adducts in the mouse and also showed that the Cys34 was a major site of adduct formation. 117 …”