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.
"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  , as well as for high throughput screening of hCE2 inhibitors. Compared with other reported hCE detection methods  , fluorescence-based assays have received much attention owing to their rapid, selective, sensitive, nondestructive, high temporal and spatial sampling capability features    . Up to now, only one selective fluorescent probe for quantitative detection of hCE2 has been reported . "
[Show abstract][Hide abstract] ABSTRACT: A new ratiometric fluorescent probe derived from 4-hydroxy-N-butyl-1,8-naphthalimide (HNN) has been developed for selective detection of human carboxylesterase 2 (hCE2). The probe is designed by introducing benzoyl moiety to HNN, based on the intramolecular charge transfer (ICT) mechanism. The probe displays satisfying stability under physiological pH conditions with very low background fluorescence signal, but it can be rapidly hydrolyzed by hCE2 and release of HNN which leads to a remarkable red shift in emission spectra (148 nm). The newly designed probe exhibits excellent selectivity towards hCE2 over other human hydrolases, while the interference from various biologically relevant chemicals can be negligible. Its potential biological applications including inhibitor screening using human tissue preparations as enzyme sources, as well as fluorescence imaging of endogenous hCE2 in human living cells, have also been demonstrated.
Sensors and Actuators B Chemical 12/2014; 205:151–157. DOI:10.1016/j.snb.2014.08.066 · 4.10 Impact Factor
"However, human CES1 is also expressed in the lung, heart, and kidney, though to a lesser extent (Satoh et al., 2002). This dominant CES1 expression relative to CES2 in the liver was also confirmed by proteomic data, with an average protein expression level of 402 and 30 pmol/mg protein reported in human liver microsomes for CES1 and CES2, respectively (Sato et al., 2012). In addition to the small intestine, human CES2 mRNA data have also been reported for the kidney and to a minor extent for the colon and heart (Satoh et al., 2002; Quinney et al., 2005). "
[Show abstract][Hide abstract] ABSTRACT: Hydrolysis plays an important role in metabolic activation of prodrugs. In the current study, species and in vitro system differences in hepatic and extrahepatic hydrolysis were investigated for 11 prodrugs. Ten prodrugs in the dataset are predominantly hydrolyzed by carboxylesterases (CES), whereas olmesartan medoxomil is also metabolized by carboxymethylenebutenolidase (CMBL) and paraoxonase. Metabolic stabilities were assessed in cryopreserved hepatocytes, liver S9 (LS9), intestinal S9 (IS9), kidney S9 (KS9) and plasma from human, monkey, dog and rat. Of all the preclinical species investigated, monkey hydrolysis CLint,hepatocytes were the most comparable to human hepatocyte data. Perindopril and candesartan cilexetil showed the lowest and highest CLint,hepatocytes, respectively regardless of the species investigated. Scaled hydrolysis CLint,LS9 were generally higher than CLint,hepatocytes in all species investigated, with the exception of dog. In the case of human and dog intestinal S9, hydrolysis CLint could not be obtained for CES1 substrates, whereas hydrolysis for CES2 and CMBL substrates was detected in IS9 and KS9 from all species. Pronounced species differences were observed in plasma; hydrolysis of CES substrates was only evident in rat. Predictability of human CLint,h was assessed for 8 CES1 substrates using hepatocytes and LS9; extrahepatic hydrolysis was not considered due to high stability of these prodrugs in intestinal and kidney S9. On average, predicted oral CLint,h from hepatocyte data represented 20% of the observed value; the under-prediction was pronounced for high clearance prodrugs, consistent with predictability of P450/conjugation clearance from this system. Prediction bias was less apparent with LS9, in particular for high clearance prodrugs, highlighting the application of this in vitro system for investigation of prodrugs.
Drug metabolism and disposition: the biological fate of chemicals 07/2014; 42(9). DOI:10.1124/dmd.114.057372 · 3.25 Impact Factor
[Show abstract][Hide abstract] 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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