Simultaneous Absolute Protein Quantification of Carboxylesterases 1 and 2 in Human Liver Tissue Fractions using Liquid Chromatography-Tandem Mass Spectrometry

Drug Metabolism Research Laboratories, Drug Discovery Research, Astellas Pharma Inc., 2-1-6 Kashima, Yodogawa-ku, Osaka 532-8514 Japan.
Drug metabolism and disposition: the biological fate of chemicals (Impact Factor: 3.25). 04/2012; 40(7):1389-96. DOI: 10.1124/dmd.112.045054
Source: PubMed

ABSTRACT The aims of this study were to develop a robust method for simultaneous quantification of carboxylesterases (CESs) 1 and 2 and to quantify those absolute protein levels in human liver tissue fractions. Unique peptide fragments of CES1 and CES2 in tryptically digested human liver microsomes (HLMs) and cytosol (HLC) were simultaneously quantified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) using corresponding stable isotope-labeled peptides as internal standards. Bovine serum albumin was used as a blank matrix for the calibration curve samples. Our procedure showed good digestion efficiency, sensitivity, linearity of calibration curve, and reproducibility. The protein levels of CES1 and CES2 in 16 individual HLMs varied 4.7-fold (171-801 pmol/mg) and 3.5-fold (16.3-57.2 pmol/mg), respectively, that are approximately 10 times higher than the expression levels in HLC. The CES1/CES2 level ratio varied substantially from 3.0 to 25, and the correlation between the protein levels of CES1 and CES2 was negative, indicating significant interindividual variability and independence in their expression levels. CES1 levels significantly correlated with hydrolysis of the CES1 substrates, clopidogrel (5 μM) and oxybutynin (10 μM), whereas CES2 levels correlated strongly with hydrolysis of the CES2 substrate, irinotecan (1 μM), indicating that quantified protein levels are highly reliable. This is the first report to demonstrate the absolute protein levels of CESs quantified by LC-MS/MS.

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    • "Thus, it is very necessary to develop a sensitive and highly selective detection method for the precisely measurement of inter-individual variability in expression and function of hCE2 [22] [23], as well as for high throughput screening of hCE2 inhibitors. Compared with other reported hCE detection methods [22] [24], fluorescence-based assays have received much attention owing to their rapid, selective, sensitive, nondestructive, high temporal and spatial sampling capability features [25] [26] [27] [28]. Up to now, only one selective fluorescent probe for quantitative detection of hCE2 has been reported [1]. "
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    ABSTRACT: Dabigatran etexilate (DABE) is an oral prodrug that is rapidly converted by esterases to dabigatran (DAB), a direct inhibitor of thrombin. To elucidate the esterase-mediated metabolic pathway of DABE, a high-performance liquid chromatography/mass spectrometry based metabolite identification and semi-quantitative estimation approach was developed. To overcome the poor full-scan sensitivity of conventional triple quadrupole mass spectrometry, precursor-product ion pairs were predicted to search for the potential in vitro metabolites. The detected metabolites were confirmed by the product ion scan. A dilution method was introduced to evaluate the matrix effects on tentatively identified metabolites without chemical standards. Quantitative information on detected metabolites was obtained using "metabolite standards" generated from incubation samples that contain a high concentration of metabolite in combination with a correction factor for mass spectrometry response. Two in vitro metabolites of DABE (M1 and M2) were identified, and quantified by the semi-quantitative estimation approach. It is noteworthy that CES1 converts DABE to M1 while CES2 mediates the conversion of DABE to M2. M1 and M2 were further metabolized to DAB by CES2 and CES1, respectively. The approach presented here provides a solution to a bioanalytical need for fast identification and semi-quantitative estimation of CES metabolites in preclinical samples.
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