Determination of Cholinesterase in Blood and Tissue

Barry W. Wilson1, John D. Henderson1

1 University of California, Davis, California
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 12.13
DOI:  10.1002/0471140856.tx1213s34
Online Posting Date:  November, 2007
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The widespread use of organophosphate and organocarbamate pesticides and the dangers of related chemical warfare agents dramatize the importance of rapid, accurate, and sensitive assays for blood and tissue cholinesterases (ChEs), important targets for neurotoxic chemicals. Two ChE enzymes used as biomarkers of exposure are the specific acetylcholinesterases (AChE, EC and the nonspecific plasma cholinesterases (BChE, EC This unit contains two protocols for measuring ChE activity: (1) a colorimetric kinetic method and (2) a radiometric endpoint assay and selective inhibitors that are used to distinguish between the two classes of enzymes. Curr. Protoc. Toxicol. 34:12.13.1‐12.13.16. © 2007 by John Wiley & Sons, Inc.

Keywords: cholinesterases; colorimetric assay; radiometric assay; delta pH assay

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Table of Contents

  • Introduction
  • Basic Protocol 1: Colorimetric Determination of Cholinesterase
  • Basic Protocol 2: Radiometric Determination of Cholinesterase
  • Support Protocol 1: Preparing Tissue for Cholinesterase Assays
  • Support Protocol 2: Preparing Free Sulfhydryl Standard Curves
  • Support Protocol 3: Prepare a Substrate Concentration Curve
  • Support Protocol 4: Using Specific Inhibitors of ChEs
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Colorimetric Determination of Cholinesterase

  • DTNB color reagent (see recipe)
  • ChE assay buffer (see recipe)
  • ATCh substrate (see recipe)
  • Automated microtiter plate reader (with plate shaker and temperature control, capable of kinetic measurements and electronic data storage)
  • Heating block
  • Temperature probe (small enough to fit into a well of a 96‐well microtiter plate)
  • Repeater pipettors and disposable syringe‐type tips (Eppendorf or equivalent)
  • 96‐well flat‐bottomed microtiter plates

Basic Protocol 2: Radiometric Determination of Cholinesterase

  • ChE enzyme sample
  • 100 U/ml eel AChE (see recipe)
  • ChE assay buffer (see recipe)
  • 10 µCi/ml (10 mM) 3H‐acetylcholine (ACh) substrate (see recipe)
  • Stopping mixture (see recipe)
  • Fluor (see recipe)
  • 5‐ml disposable scintillation vials with caps (e.g., Packard Pony vials cat. no. 6000292)
  • Vial racks
  • Tabletop shaker
  • Scintillation counter

Support Protocol 1: Preparing Tissue for Cholinesterase Assays

  • 1 mM glutathione (see recipe)
  • ChE assay buffer (see recipe)
  • 10.3 mM DTNB color reagent (see recipe)
  • ATCh substrate (see recipe)
  • 96‐well microtiter plates
  • Microtiter plate reader

Support Protocol 2: Preparing Free Sulfhydryl Standard Curves

  • ATCh or 1 mCi/ml 3H‐Ach substrate (see reciperecipes)
  • ChE assay buffer (see recipe)
  • Unlabeled ACh iodide stock (Sigma‐Aldrich cat. no. A7000)
  • 50 mM sodium phosphate buffer, pH 7 (see recipe)

Support Protocol 3: Prepare a Substrate Concentration Curve

  • Inhibitor: 1,5‐bis(4‐allyldimethyl‐ammoniumphenyl)pentan‐3‐one dibromide (BW284c51) or tetraisopropyl‐pyrophosphoramide (iso‐OMPA) (see reciperecipes)
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Literature Cited

   Arrieta, D., Ramirez, A., DePeters, E., Bosworth, D., and Wilson, B.W. 2003. Bovine red blood cell ghost cholinesterase as a monitoring standard. Bull. Environ. Contam. Toxicol. 71:447‐452.
   Burruel, V.R., Raabe, O.G., Overstreet, J.W., Wilson, B.W., and Wiley, L.M. 2000. Paternal effects from methamidophos administration in mice. Toxicol. Appl. Pharmacol. 165:148‐157.
   Ellman, G.L., Courtney, K.D., Andres, V. Jr., and Feather‐Stone, R.M. 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7:88‐95.
   Hamm, J.T., Wilson, B.W., and Hinton, D.E. 1998. Organophosphate‐induced acetylcholinesterase inhibition and embryonic retinal cell necrosis in vivo in the teleost (Oryzias latipes). Neurotoxicology 19:853‐869.
   Henderson, J.D., Higgins, R.J., Dacre, J.C., and Wilson, B.W. 1992. Neurotoxicity of acute and repeated treatments of tabun, paraoxon, diisopropyl fluorophosphate and isofenphos to the hen. Toxicology 72:117‐129.
   Johnson, C.D. and Russell, R.L. 1975. Rapid simple radiometric assay for cholinesterase, suitable for multiple determinations. Anal. Biochem. 64:229‐238.
   Johnson, C.S., Schwarzbach, S.E., Henderson, J.D., Wilson, B.W., and Tjeerdema, R.S. 2005. Influence of water temperature on acetylcholinesterase activity in the Pacific tree frog (Hyla regilla). Environ. Toxicol. Chem. 24:2074‐2077.
   Michel, H.O. 1949. Electrometric method for the determination of red blood cell and plasma cholinesterase activity. J. Lab. Clin. Med. 34:1564‐1568.
   Nostrandt, A.C., Duncan, J.A., and Padilla, S. 1993. A modified spectrophotometric method appropriate for measuring cholinesterase activity in tissue from carbaryl‐treated animals. Fundam. Appl. Toxicol. 21:196‐203.
   Padilla, S., Lassiter, T.L., and Hunter, D. 1999. Biochemical measurement of cholinesterase activity. In Methods in Molecular Medicine, Vol. 22: Neurodegeneration Methods and Protocols. (J. Harry and H.A. Tilson, eds.) pp. 237‐245. Humana Press Inc., Totowa, N.J.
   Stein, R.W., Yamamoto, J.T., Fry, D.M., and Wilson, B.W. 1998. Comparative hematology and plasma biochemistry of red‐tailed hawks and American kestrels wintering in California. J. Raptor Res. 32:163‐169.
   Wilson, B.W. 1999. Cholinesterases. In Clinical Chemistry of Laboratory Animals. (F. Quimby and W. Loeb, eds.) pp. 430‐440. Taylor and Francis Inc., Philadelphia.
   Wilson, B.W. 2001. Cholinesterases. In Handbook of Pesticide Toxicology; Volume 2. Agents. (W.J. Hayes Jr. and E.R. Laws Jr. eds.) pp. 967‐985. Academic Press Inc., San Diego.
   Wilson, B.W. and Nieberg, P.S. 1983. Recovery of acetylcholinesterase forms in quail muscle cultures after intoxication with diisopropylfluorophosphate. Biochem. Pharmacol. 32:911‐918.
   Wilson, B.W., Sanborn, J.R., O'Malley, M.A., Henderson, J.D., and Billitti, J.R. 1997. Monitoring the pesticide‐exposed worker. Occup. Med. 12:347‐363.
   Wilson, B.W., Henderson, J.D., Chow, E., Schreider, J., Goldman, M., Culbertson, R., and Dacre, J.C. 1988. Toxicity of an acute dose of agent VX and other organophosphorus esters in the chicken. J. Toxicol. Environ. Health 23:103‐113.
   Wilson, B.W., Padilla, S., Sanborn, J.R., Henderson, J.D., and Billitti, J.E. 1995. Clinical blood cholinesterase measurements for monitoring pesticide exposures. In Enzymes of the Cholinesterase Family. (D.M. Quinn, A.S. Balasubramanian, B.P. Doctor, and P. Taylor, eds.) pp. 329‐336. Plenum Press, New York.
Key References
   Ellman et al., 1961. See above.
  Original method paper for the colorimetric assay.
   Johnson and Russell, 1975. See above.
  Original method paper for the radiometric assay.
   Wilson, 2001. See above.
  Review of cholinesterase structure and function, distribution, determinations, inhibitions, and reactivations, and risk assessment.
Internet Resources
  Website for reaching analytical techniques used by the U.S. Department of Defense including the delta pH ChE method.
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