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Absolute Quantitation of NAPQI-ModifiedRat SerumAlbumin by LC–MS/MS: Monitoring Acetaminophen Covalent Binding in Vivo.
- Source :
-
Chemical Research in Toxicology . Sep2014, Vol. 27 Issue 9, p1632-1639. 8p. - Publication Year :
- 2014
-
Abstract
- Acetaminophen is known to cause hepatoxicityvia the formationof a reactive metabolite, N-acetyl p-benzoquinone imine (NAPQI), as a result of covalent binding to liverproteins. Serum albumin (SA) is known to be covalently modified byNAPQI and is present at high concentrations in the bloodstream andis therefore a potential biomarker to assess the levels of proteinmodification by NAPQI. A newly developed method for the absolute quantitationof serum albumin containing NAPQI covalently bound to its active sitecysteine (Cys34) is described. This optimized assay represents thefirst absolute quantitation of a modified protein, with very low stoichiometricabundance, using a protein-level standard combined with isotope dilution.The LC–MS/MS assay is based on a protein standard modifiedwith a custom-designed reagent, yielding a surrogate peptide (followingdigestion) that is a positional isomer to the target peptide modifiedby NAPQI. To illustrate the potential of this approach, the methodwas applied to quantify NAPQI-modified SA in plasma from rats dosedwith acetaminophen. The resulting method is highly sensitive (capableof quantifying down to 0.0006% of total RSA in its NAPQI-modifiedform) and yields excellent precision and accuracy statistics. A time-coursepharmacokinetic study was performed to test the usefulness of thismethod for following acetaminophen-induced covalent binding at fourdosing levels (75–600 mg/kg IP), showing the viability of thisapproach to directly monitor in vivosamples. Thisapproach can reliably quantify NAPQI-modified albumin, allowing directmonitoring of acetaminophen-related covalent binding. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 0893228X
- Volume :
- 27
- Issue :
- 9
- Database :
- Academic Search Index
- Journal :
- Chemical Research in Toxicology
- Publication Type :
- Academic Journal
- Accession number :
- 98249861
- Full Text :
- https://doi.org/10.1021/tx500284g