Measurement of Carboxylesterase (CES) Activities

Masakiyo Hosokawa1, Tetsuo Satoh2

1 Chiba University, Chiba, 2 Biomedical Research Institute, Chiba
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.7
DOI:  10.1002/0471140856.tx0407s10
Online Posting Date:  February, 2002
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Abstract

Measurement of Carboxylesterase (CES) Activities (Masakiyo Hosokawa, Chiba University, Chiba, Japan and Tetsuo Satoh, Biomedical Research Institute, Chiba, Japan). Mammalian carboxyesterase (CES) efficiently catalyzes the hydrolysis of a variety of ester‐ and amide‐containing chemicals. CES is involved in detoxification or metabolic activation of various drugs, environmental toxicants, and carcinogens. This unit contains three protocols for measuring CES activities.

     
 
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Table of Contents

  • Basic Protocol 1: Photometric Assay for CES Using p‐Nitrophenyl Acetate
  • Alternate Protocol 1: Photometric Assay for CES Using Malathion and Other Ethyl Esters
  • Alternate Protocol 2: Photometric Assay for CES Using Butanilicaine
  • Alternate Protocol 3: Photometric Assay for CES Using ACYL‐CoA Ester
  • Basic Protocol 2: Fluorometric Assay for CES Using Phenacetine, Acetanilide
  • Basic Protocol 3: HPLC Methods to Measure CES Activity
  • Support Protocol 1: Preparation of Microsomes from Tissue
  • Support Protocol 2: Preparation of Microsomes from Cultured Cells
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Photometric Assay for CES Using p‐Nitrophenyl Acetate

  Materials
  • Microsomes (see protocol 7Support Protocol 1 or protocol 82)
  • GET solution (see recipe), ice cold
  • Bio‐Rad DC protein assay kit II
  • 1 mM p‐nitrophenyl acetate solution (see recipe for substrate solutions)
  • 1 M Tris⋅Cl buffer, pH 8.0 ( appendix 2A)
  • 5‐ml Potter‐Elvehjem homogenizer with Teflon pestle
  • 5‐ml test tubes
  • 30°C water bath
  • 1.0‐ml semimicro cuvettes, 1‐cm path length
  • UV/VIS spectrophotometer with a computer and temperature‐controlled Peltier cuvette holder

Alternate Protocol 1: Photometric Assay for CES Using Malathion and Other Ethyl Esters

  • p‐iodonitrophenyltetrazolium violet (INT) solution (see recipe)
  • 72 mM NAD (see recipe)
  • 1000 U/ml alcohol dehydrogenase
  • 4 U/ml NAD diaphorase
  • 60 mM malathion (see recipe for substrate solutions)
  • 37°C water bath
  • 2.0‐ml cuvette, 1‐cm path length

Alternate Protocol 2: Photometric Assay for CES Using Butanilicaine

  • CES assay solution (see recipe)
  • 10 mM butanilicaine solution (see recipe for substrate solutions), prewarm to 30°C

Alternate Protocol 3: Photometric Assay for CES Using ACYL‐CoA Ester

  • 120 mM HEPES buffer, pH 7.4 (see recipe)
  • 5 mM EDTA, pH 7.4 ( appendix 2A)
  • 3 mM DTNB solution (see recipe)
  • 400 µM long‐chain acyl‐CoA solution (see recipe for substrate solutions)

Basic Protocol 2: Fluorometric Assay for CES Using Phenacetine, Acetanilide

  Materials
  • Microsomes (see protocol 7Support Protocol 1 or protocol 82)
  • GET solution (see recipe), ice cold
  • Bio‐Rad DC protein assay kit II
  • 250 mM Tris⋅Cl, pH 8.6 ( appendix 2A)
  • Substrate solution, e.g., 10 mM phenacetin solution or 20 mM acetanilide solution (see recipe), 30°C
  • 1.2 M TCA solution (see recipe)
  • 1 N NaOH
  • 5‐ml Potter‐Elvehjem homogenizer with Teflon pestle
  • 37°C water bath with shaker
  • Fluorometric quartz cuvette, 1‐cm path length
  • Spectrofluorometer with a computer

Basic Protocol 3: HPLC Methods to Measure CES Activity

  Materials
  • Microsomes (see protocol 7Support Protocol 1 or protocol 82)
  • GET solution (see recipe), ice cold
  • Bio‐Rad DC protein assay kit II
  • 120 mM HEPES buffer, pH 7.4 (see recipe)
  • 200 µM irinotecan hydrochloride (CPT‐11; see recipe for substrate solutions), 37°C
  • 0.05 N HCl containing 200 nM CPT as an internal standard (see recipe for substrate solutions)
  • 5‐ml Potter‐Elvehjem homogenizer and Teflon pestle
  • 37°C water bath with a shaker
  • HPLC system consisting of model L‐6000 pump (Hitachi), model L7480 FL detector (Hitachi), model AS‐2000 auto‐sampler (Hitachi), model D‐2500 integrator (Hitachi), and a 4.6 × 150–mm YMC‐Pack Ph A‐402 column (YMC Co.) or equivalent.

Support Protocol 1: Preparation of Microsomes from Tissue

  Materials
  • Organ tissue, freshly recovered or frozen
  • SET solution (see recipe)
  • GET solution (see recipe)
  • Potter‐Elvehjem homogenizer with a motorized Teflon pestle
  • 50‐ml centrifuge tubes
  • 30‐ml ultracentrifuge tubes
  • Ultracentrifuge
NOTE: Perform all steps at 4°C.

Support Protocol 2: Preparation of Microsomes from Cultured Cells

  Materials
  • Cultured cells, such as V79, COS7, or HepG2 cells, expressing CES after transfection with a mammalian expression vector with CES cDNA
  • PBS ( appendix 2A)
  • SET solution (see recipe)
  • GET solution (see recipe)
  • 5‐ml round‐bottom tubes
  • Sonicator with a narrow probe
  • Microscope
  • Potter‐Elvehjem homogenizer with a motorized Teflon pestle
  • 2‐ml centrifuge tubes
  • 5‐ml ultracentrifuge tubes and ultracentrifuge
NOTE: Perform all procedures at 4°C.
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Figures

Videos

Literature Cited

Literature Cited
   Berge, R.K. 1979. Purification and characterization of a long‐chain acyl‐CoA hydrolase from rat liver microsomes. Biochim. Biophys. Acta. 574:321‐333.
   Heymann, E. and Mentlein, R. 1981. Carboxylesterases‐amidases. Methods Enzymol. 77:333‐344.
   Heymann, E., Mentlein, R., and Rix, H. 1981. Hydrolysis of aromatic amides as assay for carboxylesterases‐amidases. Methods Enzymol. 77:405‐409.
   Hosokawa, M. and Satoh, T. 1993. Differences in the induction of carboxylesterase isozymes in rat liver microsomes by perfluorinated fatty acids. Xenobiotica. 23:1125‐1133.
   Hosokawa, M., Maki, T., and Satoh, T. 1987. Multiplicity and regulation of hepatic microsomal carboxylesterases in rats. Mol.Pharmacol. 31:579‐584.
   Hosokawa, M., Maki, T., and Satoh, T. 1988. Differences in the induction of carboxylesterase isozymes in rat liver microsomes by xenobiotics. Biochem. Pharmacol. 37:2708‐2711.
   Hosokawa, M., Maki, T., and Satoh, T. 1990. Characterization of molecular species of liver microsomal carboxylesterases of several animal species and humans. Arch. Biochem. Biophys. 277:219‐227.
   Hosokawa, M., Hirata, K., Nakata, F., Suga, T., and Satoh, T. 1994. Species differences in the induction of hepatic microsomal carboxylesterases caused by dietary exposure to di(2‐ethylhexyl)phthalate, a peroxisome proliferator. Drug Metab. Dispos. 22:889‐894.
   Kurita, A. and Kaneda, N. 1999. High‐performance liquid chromatographic method for the simultaneous determination of the camptothecin derivative irinotecan hydrochloride, CPT‐11, and its metabolites SN‐38 and SN‐38 glucuronide in rat plasma with a fully automated on‐line solid‐phase extraction system, PROSPEKT. J. Chromatogr. B. Biomed. Sci. Appl. 724:335‐344.
   Mentlein, R., Heiland, S., and Heymann, E. 1980. Simultaneous purification and comparative characterization of six serine hydrolases from rat liver microsomes. Arch. Biochem. Biophys. 200:547‐559.
   Mentlein, R., Lembke, B., Vik, H., and Berge, R.K. 1986. Different induction of microsomal carboxylesterases, palmitoyl‐CoA hydrolase and acyl‐L‐carnitine hydrolase in rat liver after treatment with clofibrate. Biochem. Pharmacol. 35:2727‐2730.
   Mori, M., Hosokawa, M., Ogasawara, Y., Tsukada, E., and Chiba, K. 1999. cDNA cloning, characterization and stable expression of novel human brain carboxylesterase. F.E.B.S. Lett. 458:17‐22.
   Satoh, T. and Hosokawa, M. 1998. The mammalian carboxylesterases: From molecules to functions. Annu. Rev. Pharmacol. Toxicol. 38:257‐288.
   Satoh, T., Hosokawa, M., Atsumi, R., Suzuki, W., Hakusui, H., and Nagai, E. 1994. Metabolic activation of CPT‐11, 7‐ethyl‐10‐[4‐(1‐piperidino)‐1‐piperidino]carbonyloxycamptothecin, a novel antitumor agent, by carboxylesterase. Biol. Pharm. Bull. 17:662‐664.
   Talcott, R.E. 1979. Hepatic and extrahepatic malathion carboxylesterases. Assay and localization in the rat. Toxicol. Appl. Pharmacol. 47:145‐150.
   Yamada, T., Hosokawa, M., Satoh, T., Moroo, I., Takahashi, M., Akatsu, H., Yamamoto, T. 1994. Immunohistochemistry with an antibody to human liver carboxylesterase in human brain tissues. Brain Res. 658:163‐167.
Key References
   Berge, 1979. See above.
  A modification of this method is described in .
   Heymann and Mentlein, 1981. See above.
  A modification of this method is described in .
   Heymann et al., 1981. See above.
  A modification of this method is described in and .
   Hosokawa et al., 1987. See above.
  A modification of this method is described in .
   Mori et al., 1999. See above.
  A modification of this method is described in .
   Satoh et al., 1994. See above.
  A modification of this method is described in .
   Talcott, 1979. See above.
  A modification of this method is described in .
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