tx1213

7/28/2019 tx1213

http://slidepdf.com/reader/full/tx1213 1/16

UNIT 12.13Determination of Cholinesterase in Bloodand Tissue

Barry W. Wilson1 and John D. Henderson1

1University of California, Davis, California

ABSTRACT

The widespread use of organophosphate and organocarbamate pesticides and the dan-gers 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 3.1.1.7) and the nonspecific plasma cholinesterases

(BChE, EC 3.1.1.8). 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. C 2007 by John Wiley & Sons, Inc.

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

INTRODUCTION

Acetylcholine (ACh) is an important neurotransmitter. Its action at synapses is regulated

by its hydrolysis by acetylcholinesterase (AChE, EC 3.1.1.7). AChE and related enzymes,

known as nonspecific or pseudocholinesterases (BChE, EC 3.1.1.8), are found in nerve,

muscle, blood, and other tissues. Natural organocarbamates, such as physostigmine

found in the calabar bean, synthetic organophosphate (OP) and organocarbamate (OC)

pesticides, and chemical warfare agents act by inhibiting AChE. AChE and BChE activity

levels are recognized biomarkers of exposure to these agents and their determinations

are important tools in neurotoxicology.

Basic Protocol 1 describes colorimetric determination of ChE and Basic Protocol 2

describes radiometric determination of ChE. Basic Protocol 1 uses 96-well microtiter plates, the synthetic substrate acetylthiocholine (ATCh), and is designed for efficient

throughput of samples. Basic Protocol 2 uses the natural substrate ACh. It is useful

for samples with low ChE activity, but it takes longer to produce results and generates

radioactive waste. Support Protocol 1 describes tissue preparation and Support Protocol

2 provides an instrument-specific calculation factor for generating a free sulfhydryl

standard curve resulting from the colorimetric method. Support Protocol 3 optimizes

the concentration when measuring samples from a new species and produces a substrate

concentration curve. Support Protocol 4 lists useful inhibitors of ChEs.

BASIC

PROTOCOL 1

COLORIMETRIC DETERMINATION OF CHOLINESTERASE

The rate of hydrolysis of acetylthiocholine (ATCh) catalyzed by cholinesterases (ChEs) isdetermined colorimetrically with an automated microtiter plate reader and a colorimetric

reagent that detects sulfhydryl groups (–SH). ATCh is incubated with the tissue sam-

ple and the color reagent, 5,5-dithiobis-2-nitrobenzoic acid (DTNB). The thiocholine

produced by the hydrolysis of the substrate reacts with DTNB to yield a yellow colored

anion, 5-thio-2-nitrobenzoate (TNB). The formation of TNB is followed kinetically using

the microtiter plate reader. There are two different blanks. An enzyme blank (no substrate

present) detects free sulfhydryls that may be present in the sample. The SH-compounds

Current Protocols in Toxicology 12.13.1-12.13.16, November 2007

Published online November 2007 in Wiley Interscience (www.interscience.wiley.com).

DOI: 10.1002/0471140856.tx1213s34

Copyright C 2007 John Wiley & Sons, Inc.

Biochemical andMolecularNeurotoxicology

12.13.1

Supplement 34

7/28/2019 tx1213


Determination of Cholinesterase inBlood and Tissue

12.13.2

Supplement 34 Current Protocols in Toxicology

react directly with DTNB to give TNB. A substrate blank (no sample present) shows the

amount of color formation due to non-enzymatic hydrolysis of ATCh.

Materials

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

1. Turn on power to the microtiter plate reader. Allow the instrument to warm up

according to manufacturer’s recommendations.

2. Set the temperature control on the microtiter plate reader to maintain the plate at

25◦C.

This will need to be determined for each particular instrument. It is usually a temperature>25◦C (30◦C for some instruments).

3. Set the wavelength on the microtiter plate reader to 412 nm.

For instruments using narrow-band interference filters, the commonly supplied 405-nm

filter is satisfactory.

4. Set the time interval between absorbance readings to 2 min and the number of

readings to six.

This provides a determination with a low background. Samples with high activity may

need a shorter time interval (although dilution of the sample is preferable).

5. Turn on the heating block. Set the temperature to warm a microtiter plate and its

contents to 25◦C.

Determine this temperature for the specific equipment (the authors use 34◦

to 35◦

C).

6. Turn on the temperature probe and calibrate according to the manufacturer’s recom-

mendations.

7. Diagram the specific sample layout in the 96-well microtiter plate on a grid template.

The microtiter plate contains 96 wells arranged in eight horizontal rows (lettered Athrough H) and twelve vertical columns (numbered 1 through 12). In the authors’ assays,

the samples are usually run in triplicate in consecutive wells within a row (e.g., B7 to B9,C4 to C6, etc.). Fill in a template as shown in Figure 12.13.1 to show which samples or

blanks are in which wells.

8. Add solutions to the wells, referring to Table 12.13.1 for appropriate volumes.

a. Add DTNB color reagent and ChE assay buffer.

b. Add the samples.

c. Add the ATCh substrate using a repeater pipettor (with syringe-type tip) to start

the assay.

The repeater pipettor with syringe-type tip provides better mixing of the well contentsthan a plate-shaker.

9. Place the microtiter plate into the plate reader without delay.

10. Check the temperature with the probe and record.

7/28/2019 tx1213



12.13.3

Current Protocols in Toxicology Supplement 34

Figure 12.13.1 A 96-well microtiter plate assay form template. Microtiter plate well designations

(e.g., sample IDs, controls, blanks) should be labeled on the form and referred to while performing

the colorimetric assay.

Table 12.13.1 Colorimetric Assay Volumesa

ReagentChE sample

(µ l)

Tissue blank

(µ l)

Substrate

blank (µ l)

Final

concentration

ChE assay buffer 250 280 280 —

DTNB color reagent 10 10 10 0.32 mM

Sample 30 30 — —

ATCh substrate 30 — 30 1.0 mM

aFinal volume = 320µl.

11. Start measurements.

12. When the plate is finished being read, check the temperature with the probe again

and record.

13. Calculate activity by performing the following:

a. Take the mean of the slopes for each triplicate set of samples. This gives the mean

A /min (the change in absorbance per minute).

b. Convert the A /min to µ mol/min/ml:

Equation 12.13.1

7/28/2019 tx1213



12.13.4


where the factor is an instrument-specific calculation factor that must be deter-

mined (see Support Protocol 2).

c. If total ChE and AChE assays were run on the same sample, the nonspecific ChE(BChE) is calculated by subtracting the AChE value from the total ChE value(see Support Protocol 4).

d. Divide the value by the protein concentration of the sample (mg/ml) to yieldenzyme activity as µ mol/min/mg protein.

BASIC

PROTOCOL 2

RADIOMETRIC DETERMINATION OF CHOLINESTERASE

Tritiated ACh is incubated with the sample for a predetermined time in this end-point

assay. The reaction is stopped by adding a solution that is basic, high in salt, and contains

an excess of an analog of acetic acid (one of the reaction end-products). A toluene-based

fluor is mixed with the sample and there is a separation of the aqueous and organic

phases. The unhydrolyzed ACh preferentially enters the aqueous phase, quenching its

radioactivity. The hydrolyzed labeled acetate remains in the fluor solvent phase, where it

can be measured with a scintillation counter. Commercially available electric eel AChE

is added in excess to control vials to hydrolyze all the ACh to determine the total counts

per million (cpm) in the vials.

Materials

ChE enzyme sample

100 U/ml eel AChE (see recipe)


10 µ Ci/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

1. Set out three 5-ml vials in a rack for each sample, eel AChE control, and blanks (no

sample).

2. Add to each vial: ChE enzyme sample and ChE assay buffer to a volume of 80 µ l as

listed in Table 12.13.2.

A useful way to determine if solutions were added is to tilt the rack containing the vials,checking the meniscus visually in each vial to be certain no reagent addition was missed.

3. Initiate the assay by adding 20 µ l of 10 µ Ci/ml 3H-ACh substrate to each vial. Begin

timing when the first vial is started.

4. Run the assay for 15 to 40 min at room temperature in a shaker. Note temperature.

5. Stop the reaction by adding 100 µ l stopping mixture to each vial in the same order

in which the assay was begun.

6. Add 4 ml of fluor to each vial.

7. Cap each vial. Shake vigorously. Allow phases to separate for 30 min.

8. Count samples in a scintillation counter set for the 3H energy spectrum.

7/28/2019 tx1213



12.13.5


Table 12.13.2 Radiometric Assay Volumesa

Reagent Eel AChE (µ l) Blank (µ l) ChE sample (µ l)

Assay buffer 70 80 60

Eel AChE 10 — —

Sample — — 20

3H-ACh substrate 20 20 20

aFinal volume = 100µl; final substrate concentration is 2.0 mM.

9. Calculate the activity (µ mol ACh hydrolyzed/min/ml):

Equation 12.13.2

The amount of micromoles of substrate added is 0.2.

The sample cpm should not be >50% of the completely hydrolyzed eel cpm to keep theassay reaction within its linear range.

It is prudent to determine the dpm and use it in the calculations (see Critical Parametersand Troubleshooting).

SUPPORT

PROTOCOL 1

PREPARING TISSUE FOR CHOLINESTERASE ASSAYS

Tissues should be kept cold during transport, processing, and handling. While ChEs are

stable enzymes, some ChE inhibitors are readily reversible and warmer temperatures can

speed up this process. Such a reversal could result in underestimation or non-detection

of ChE inhibition in a sample. When assaying samples from a given species for the first

time, the appropriate dilution should be determined using a subset of the samples.

ChE can be determined in whole blood, or separated plasma and red blood cells (RBCs).

Serum is not recommended because of the warmer temperature used in processing.

Blood should be collected in the presence of an anticoagulant, preferably heparin or

EDTA. Blood is separated by centrifuging 10 min at 1000 × g, 4◦C. The plasma fraction

supernatant may be clear, white to yellowish in color, and may look milky if lipid levels

are high (such as in laying hens). A reddish color indicates some degree of RBC lysis

has occurred (suggesting that gentler handling of samples is required). One consequence

of RBC lysis is contamination of ChEs between fractions. Plasma itself may be used

directly in ChE determinations or diluted if activity is too high (see Critical Parameters

and Troubleshooting). The cell pack is the RBC fraction. The RBCs should be washed by

resuspending the cell pack with ChE buffer, or a commercial normal saline, at a ratio of

1:1 buffer/RBCs (up to 5:1). The higher ratio of buffer to RBCs provides a more effective

wash. The suspension is recentrifuged 10 min at 1000 × g, 4◦C and the supernatant is

discarded. Whole blood and RBCs are diluted with solubilization buffer (see recipe) to

lyse the RBCs, and to reduce the hemoglobin concentration when performing the colori-

metric assay (see Critical Parameters and Troubleshooting). A typical dilution of human

blood is 1:50 for the colorimetric assay. Some species, such as birds, do not have ChE

in their RBCs, therefore, only the plasma fraction is necessary for ChE determinations.

The plasma is typically assayed undiluted or diluted 1:10. Fresh blood treated with an

anti-clotting agent may be stored for several days in a refrigerator before processing

unless readily rehydrolyzable anti-ChE chemical exposures are suspected. Processed

samples may be stored in a low-temperature (−80◦C) freezer for extended periods.

7/28/2019 tx1213



12.13.6


Muscle samples are prepared by removing fascia and fatty tissue. Brain samples are

prepared by removing outer membranes. Since different regions of the brain have different

AChE activities, care must be taken to prepare consistent samples. Homogenize the

entire, or one half of the brain (cut longitudinally) to measure whole brain activity.

Wet weight of samples is determined by blotting them prior to weighing. Samples are

homogenized in solubilization buffer, typically at ratios of 1:5 to 1:50 of tissue to buffer.

Homogenization may be carried out in glass homogenizers with pestles (such as conical

ground glass mortar/ground glass pestle or Potter-Elvehjem grinder with PTFE pestle),

or by motorized tissue grinders. Homogenization should be carried out on ice. Ten to

twenty strokes is sufficient to homogenize soft tissues such as the brain. Firmer tissuessuch as muscle may require up to 50 strokes to complete the homogenization. If not

processed fresh, tissue samples should be stored in a low-temperature (−80◦C) freezer.

Processed samples should be stored in a low-temperature (−80◦C) freezer for extended

periods. Typical dilutions of sample used in ChE determinations are in mammal tissue,

1:500 for the brain and 1:50 for muscle; in bird tissue, 1:500 for the brain and 1:50 for

muscle; and in fish tissue, 1:500 for the brain and 1:500 for muscle.

SUPPORT

PROTOCOL 2

PREPARING FREE SULFHYDRYL STANDARD CURVES

Known amounts of free sulfhydryl (−SH) in the form of reduced glutathione are added

to the colorimetric assay system in the absence of enzyme to determine the change in

absorbance per unit of –SH for the conditions of a specific laboratory (Padilla et al., 1999).

Materials

1 mM glutathione (see recipe)


10.3 mM DTNB color reagent (see recipe)

ATCh substrate (see recipe)

96-well microtiter plates

Microtiter plate reader

1. Add reagents to wells of a 96-well microtiter plate as shown in Table 12.13.3. Run

each concentration of −SH in triplicate.

2. Incubate 5 to 15 min at room temperature.

3. Measure the absorbance of the microtiter plate wells at 412 nm (the commonly

supplied 405-nm narrow-band interference filter is satisfactory).

Table 12.13.3 Glutathione Standard Curve Volumes

–SH (nmol)1 mM glutathione

volume (µ l)

ChE assay buffer

volume (µ l)

DTNB volume

(µ l)

ATCh substrate

volume (µ l)

0 0 280 10 30

2 2 278 10 30

5 5 275 10 30

10 10 270 10 30

15 15 265 10 30

20 20 260 10 30

30 30 250 10 30

50 50 230 10 30

7/28/2019 tx1213



12.13.7


Figure 12.13.2 Glutathione standard curve for colorimetric ChE assay. The slope of the regres-

sion line ( absorbance/ −SH nmol) is used in calculating ChE activity. This factor is instrument-

specific and must be determined in each laboratory.

4. Plot the absorbance versus the concentration of −SH.

The slope of the regression is the change in absorbance per nanomole of free sulfhydryl.

5. Use this value in calculating ChE activity (Equ. 12.13.1 and Fig. 12.13.2).

SUPPORT

PROTOCOL 3

PREPARE A SUBSTRATE CONCENTRATION CURVE

The optimal substrate concentration should be verified when measuring ChE activity

in a new species or tissue. It has been found that a concentration of 1 to 2 mM to be

optimal in the mammal and bird species tested, while 2 to 3 mM was optimal in some fish

species. Since AChE exhibits a characteristic substrate inhibition with excess substrate,

it is important to be certain that the substrate concentration is not too high.

Additional Materials ( also see Basic Protocols 1 and 2 )

ATCh or 1 mCi/ml 3H-Ach substrate (see recipes)


Unlabeled ACh iodide stock (Sigma-Aldrich cat. no. A7000)

50 mM sodium phosphate buffer, pH 7 (see recipe)

1. Prepare substrate stock solutions at the concentrations shown in Table 12.13.4.

a. For colorimetric assay: start with the highest ATCh stock concentration listed

(which is ten times the normal stock concentration) and make appropriate dilutions

with ChE assay buffer.

b. For radiometric assay: start with the highest unlabeled ACh stock concentration

listed (which is ten times the normal stock concentration) and make appropriate

dilutions with 50 mM sodium phosphate buffer, pH 7. Add 1 part of 1 mCi/ml3H-ACh stock solution to 100 parts of each of the unlabeled ACh substrate stock

dilutions.

2. Run the assay as usual (see Basic Protocols 1 and 2) with a single sample as the

enzyme source and each substrate concentration in triplicate.

3. Plot the ChE activity versus the log substrate concentration. Determine the optimal

substrate concentration giving the highest ChE activity.

The optimal substrate concentration is represented by the plateau of the curve shown

in Figure 12.13.3 (i.e., 1 to 2 mM ATCh for human blood AChE determined with thecolorimetric assay). Note the drop of AChE activity when the substrate concentration is

>2 mM, demonstrating the characteristic substrate inhibition of AChE.

7/28/2019 tx1213



12.13.8


Table 12.13.4 Substrate Test Concentrations

Stock concentration

of substrate (mM)

Colorimetric assay: Final

concentration of substrate

(mM)

Radiometric assay: Final

concentration of substrate

(mM)

1.07 0.1 0.2

2.14 0.2 0.4

3.21 0.3 0.6

5.35 0.5 110.7 1 2

21.4 2 4

32.1 3 6

53.5 5 10

107 10 20

Figure 12.13.3 Acetylthiocholine (ATCh) substrate curve for colorimetric assay. Human blood

AChE was measured over a range of ATCh substrate concentrations. The optimal concentra-

tion is 1 to 2 mM. There is a characteristic substrate inhibition of AChE activity at higher ATCh

concentrations. Values are mean ± SD, n = 3. Adapted from Wilson et al. (1995).

SUPPORT

PROTOCOL 4

USING SPECIFIC INHIBITORS OF ChEs

Specific inhibitors can be used to distinguish between AChE and BChE in a given species.

1,5-Bis(4-allyldimethyl-ammoniumphenyl)pentan-3-one dibromide (BW284c51) in-

hibits AChE and tetraisopropyl-pyrophosphoramide (iso-OMPA) inhibits BChE. Quini-

dine is an inhibitor of mammalian BChE. The appropriate inhibitor concentrations should

be determined by running dose/response curves.

7/28/2019 tx1213



12.13.9


Additional Materials ( also see Basic Protocols 1 and 2 )

Inhibitor: 1,5-bis(4-allyldimethyl-ammoniumphenyl)pentan-3-one dibromide(BW284c51) or tetraisopropyl-pyrophosphoramide (iso-OMPA) (see recipes)

1. Prepare inhibitor stock solutions at the concentrations shown in Table 12.13.5.

2. Replace part of buffer volume with inhibitor stock: 10 µ l for radiometric assay or

30 µ l for colorimetric assay. Add all reagents except substrate. Incubate mixture 15

min at room temperature.

3. Add substrate at end of 15 min to start enzyme reaction and continue to assay asusual (see Basic Protocol 1 or 2).

Table 12.13.5 ChE Inhibitor Stock Concentrations

Final inhibitor

concentration (M)

Colorimetric assay

stock concentration

(M)

Radiometric assay

stock concentration

(M)

1.0 × 10 –3 9.7 × 10 –3 8.0 × 10 –3

3.0 × 10 –4 2.9 × 10 –3 2.4 × 10 –3

1.0 × 10 –4 9.7 × 10 –4 8.0 × 10 –4

3.0 × 10 –5 2.9 × 10 –4 2.4 × 10 –4

1.0 × 10 –5 9.7 × 10 –5 8.0 × 10 –5

3.0 × 10 –6 2.9 × 10 –5 2.4 × 10 –5

1.0 × 10 –6 9.7 × 10 –6 8.0 × 10 –6

3.0 × 10 –7 2.9 × 10 –6 2.4 × 10 –6

1.0 × 10 –7 9.7 × 10 –7 8.0 × 10 –7

3.0 × 10 –8 2.9 × 10 –7 2.4 × 10 –7

1.0 × 10 –8 9.7 × 10 –8 8.0 × 10 –8

Figure 12.13.4 Dose-response curve for a specific ChE inhibitor. Iso-OMPA inhibits human

plasma BChE, but not RBC AChE,at 10–4 M. This inhibitor concentration can be used to distinguish

between the two enzymes in a human whole blood sample. Values are mean ± SD, n = 3.

7/28/2019 tx1213



12.13.10


4. Plot activity versus inhibitor concentration.

The appropriate inhibitor concentration is the plateau of the curve where activity is lowest (Fig. 12.13.4).

REAGENTS AND SOLUTIONS

Use Milli-Q-purified water or equivalent in all recipes and protocol steps. For common stock

solutions, see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX .

3 H-Acetylcholine (ACh) stock solution (1 mCi/ml)

Add 1 ml of 50 mM sodium phosphate buffer, pH 7.0 (see recipe) to 1 mCi 3H-

ACh iodide (Perkin-Elmer cat. no. NET113001MC). Store up to 1 year at −20◦C

desiccated.

3 H-Acetylcholine (ACh) substrate (10 mM ACh; 10 µCi/ml)

68.3 mg unlabeled ACh iodide (Sigma-Aldrich cat. no. A7000)

25.0 ml 50 mM sodium phosphate buffer, pH 7.0 (see recipe)

250 µ l 1 mCi/ml 3H-ACh stock solution (see recipe)

Store up to 1 year at −20◦C

ATCh substrate (10.7 mM acetylthiocholine iodide)

Dissolve 30.8 mg ATCh per 10.0 ml final volume in 0.1 M sodium phosphate buffer,pH 8.0 (see recipe). Store 1 month at −20◦C.

A fresh solution is recommended. It may precipitate if kept on ice during assay.

BW284c51

For 10 –2 M stock: Add 28.3 mg 1,5-bis(4-allyldimethyl-ammoniumphenyl)pentan-3-one dibromide (BW284c51, Sigma-Aldrich cat. no. A9013) in 5.0 ml finalvolume of water

10 –3 M solution: Dilute 10 –2 M BW284c51 stock 1:10 with 0.1 M sodiumphosphatebuffer, pH 8 (see recipe)

Store up to 1 month at 4◦C

CAUTION: BW284c51 is a selective AChE inhibitor and very toxic.

ChE assay buffer

0.1 M sodium phosphate, pH 8.0 (see recipe)

Store up to 6 months at room temperature

Cloudiness indicates fungal or bacterial growth, if this occurs, prepare fresh solution.

DTNB color reagent (10.3 mM)

Dissolve 20.5 mg 5,5-dithiobis-2-nitrobenzoic acid (DTNB) per 5.0 ml final vol-

ume in 0.1 M sodium phosphate buffer, pH 7.0 (see recipe). Store up to 1 month at

−20◦C.

A fresh solution is recommended. It may precipitate if kept on ice during assay.

Eel AChE (100 U/ml)

1000 U eel AChE (Sigma-Aldrich cat. no. C2888)

10.0 ml H2O

Store up to 1 year at –20◦C

7/28/2019 tx1213



12.13.11


Fluor

3.0 liters toluene (scintillation-grade)

15.0 g 2,5-diphenyloxazole (PPO)

0.6 g 1,4-bis[5-phenyl-2-oxazoyl]benzene; 2,2-p-phenylene-bis[5-phenyloxazole](POPOP)

330 ml isoamyl alcohol

Store up to 2 years at room temperature

Glutathione stock (1 mM)

3.1 mg glutathione (reduced form; Sigma-Aldrich cat. no. G4251) brought to a

10.0-ml volume with 0.1 M sodium phosphate, pH 8.0 (see recipe). Prepare fresh.

Iso-OMPA

For 10 –2 M tetraisopropylpyrophosoramide (iso-OMPA; Sigma-Aldrich cat. no.

T1505) stock: Add 17.1 mg iso-OMPA in 5.0 ml final volume of water.

For a 10 –3 M iso-OMPA solution: Dilute 10 –2 M stock 1:10 with 0.1 M sodium

phosphate buffer, pH 8 (see recipe). Store 1 month at 4◦C.

CAUTION: Iso-OMPA is a selective BChE inhibitor and extremely hazardous.

Sodium phosphate buffer, 0.1 M (pH 7.0 or 8.0)

Prepare 0.1 M monobasic sodium phosphate (12.0 g/liter in water) and 0.1 M

dibasic sodium phosphate (14.2 g/liter in water). Mix solutions at a ratio of 55 parts

monobasic to 945 parts dibasic for pH 8.0 or 39 parts monobasic to 61 parts dibasic

for pH 7.0. Check pH with a pH meter and adjust with appropriate phosphate stock

solution. Dilute 1:1 with water for 50 mM phosphate buffer. Store for 6 months at

room temperature.

Cloudiness indicates fungal or bacterial growth.

Solubilization buffer

0.5 ml Triton X-100 (0.5% final concentration; Sigma-Aldrich cat. no. T9284)

99.5 ml ChE assay buffer (see recipe)

Store up to 6 months at 4◦C

Stopping mixture

9.45 g monochloroacetic acid (1 M)

2.0 g NaOH (0.5 M)

11.6 g NaCl (2 M)

Bring to a final volume of 100.0 ml with H2O

Store up to 1 year at room temperature

COMMENTARY

Background Information

Acetylcholine (ACh) is an important neu-rotransmitter to humans and animals. A part

of transmission regulation is the destruc-

tion of ACh by special esterases (acetyl-

cholinesterase, AChE, EC 3.1.1.7; nonspe-

cific cholinesterase or pseudocholinesterase,

BChEs, EC 3.1.1.8) that hydrolyze it into

choline and acetic acid (Fig. 12.13.5). In addi-

tion to its presence in the nervous and muscle

systems, AChE and BChE are found in the

blood and other tissues. AChE is found in the

red blood cell (RBC) membranes of mammals.AChE and BChEs are found in the plasma of

some mammals and other vertebrates (only

BChE is present in human plasma; Wilson,

1999, 2001).

ChE and other esterase activities in the

nervous system, their cellular regulation, and

physiological functions were first studied early

in the 20th century. The development of ChE

inhibitors for use as chemical warfare agents

7/28/2019 tx1213



12.13.12


Figure 12.13.5 Cholinesterases catalyze the hydrolysis of acetylcholine.

just before World War II and the synthesis of

organophosphate and organocarbamate ChE

inhibitors as pesticides after the war created

a need for rapid and clinically suitable as-

says to screen for ChE inhibiting pesticides

and to monitor human exposures to these

agents. The first ChE assays were research

tools; they relied on slow but precise meth-

ods such as macro-Warburg manometry and

micro-Cartesian divers (Wilson, 1999, 2001).

Three techniques in use today are the delta

pH method of Michel (1949), the radiomet-

ric method of Johnson and Russell (1975),

and the thiocholine method of Ellman et al.

(1961). The delta pH method currently is used

by CHPPM (U.S. Army Center for Health

Promotion and Preventive Medicine), the U.S.

Department of Defense’s monitoring facility,

to keep track of ∼15,000 servicemen and em-

ployees annually. The radiometric and colori-

metric methods presented here are drawn from

the authors’ laboratory.The U.S. Department of Defense chose the

delta pH method of Michel to measure red

blood cell AChEs before the thiocholine-based

Ellman method was developed. The Michel

assay is relatively inexpensive and capable of

standardization using multiple pH meters. It

is useful for a laboratory that does not have

access to automated colorimetric or to radio-

metric instruments. The radiometric method

uses radiolabeled acetylcholine. It is the most

sensitive of the assays but also requires facil-

ities that handle radiolabeled chemicals and

their disposal. Being end-point assays, the pHand radiometric methods are less suitable for

kinetic studies than the Ellman assay. The Ell-

man assay is the most widely used and has

spawned many variants.

It is important to consider the properties of

the ChE inhibitors, which may be present in

exposed tissue samples, whether they are read-

ily or very slowly reversible (Wilson, 1999,

2001). See Nostrandt et al. (1993) for consid-

erations when working with readily reversible

carbamates.

Critical Parameters andTroubleshooting

It is important to establish the enzyme as-

say conditions. A few minutes of testing an as-

say can save untold hours of grief. The assay

conditions for enzymes printed in peer- andnon-reviewed papers may not apply to the lab-

oratory specific systems. Determining the rate

of an enzyme is not quite the same as cleaning

up a residue and running gas chromatogra-

phy with appropriate standards. Enzymes are

catalysts and they are affected by a number

of conditions that are not entirely predictable.

The following short discussion contains sug-

gestions on settingup enzymeassays forChEs.

Use the desired experimental tissues to be

measured to determine the optimum substrate

concentration and range of sample amounts.

The purpose of an enzyme assay is to deter-mine the activity of an enzyme in a sample.

This means that the conditions must be such

that if the amount of sample is doubled, the

activity doubles and if the amount of sample is

halved, then the activity is also halved. If this

does not occur, then the conditions of the assay

must be adjusted so that the sample itself is the

rate-limiting factor. It is important to note that

some widely used commercial ChE kits do not

use optimal reagent conditions (Wilson et al.,

1997). It is critical to establish that the levels

of substrate and other reagents (such as DTNB

in the Ellman method) are not rate limiting inthe assay. Use the values presented here as a

starting point.

Basic Protocol 11. Any compound in the sample that ab-

sorbs at 410 nm can interfere with the as-

say (e.g., hemoglobin in blood samples). A

small amount of such a compound will have

a negligible effect since its absorbance will

7/28/2019 tx1213



12.13.13


not increase over time (and, therefore, will not

add to the A /min). A large amount of inter-

fering compound will have a relatively large

absorbance that could mask the smaller in-

creased absorbance due to ATCh hydrolysis.

Whole blood or RBC samples should be di-

luted so that the initial absorbance reading in

the assay is <1.0.

2. Free sulfhydryls present in the sample

can react directly with DTNB to form the col-

ored TNB. Check this by using control tissue-

only blanks.

3. Adjusting sample activity.

The r 2 value of the slope is routinely≥0.99.

A value lower than this is seen when activity is

too low (near the noise level of the sensitivity

of the assay)or activity is too high (absorbance

near the upper limit of detection of the instru-

ment or substrate is becoming rate limiting).

If the activity is too low, the value of the

slope will be highly variable and prone to er-

ror. Change the timer in the plate reader proto-

col to give longer intervals between repeatedreadings. This will increase the sensitivity of

the assay system.

If the activity is too high, the measured

absorbance can become too high and the ob-

served slope can become nonlinear. Dilute the

sample to give an appropriate level of ac-

tivity (i.e., final absorbance should not ex-

ceed 3.0 and its slope should be linear; r 2 >

0.99). Higher activities can be measured with

a shorter reading interval, but this is not prefer-

able. Blank values are more variable and can

appear higher when measured with a shorter

interval (e.g., 1 min rather than 2 min).4. The quality of the sample is another fac-

tor that could produce a low r 2 value. Par-

ticulates in the sample may scatter light and

interfere with the absorbance measurements.

These can be removed by centrifugation, but

ensure that the ChE fraction of the sample is

not removed. Ensure that samples are mixed

well and homogenous in the microtiter plate

well, especially viscous samples such as sol-

ubilized RBCs. These can also scatter light if

not mixed well.

Basic Protocol 21. The assay is useful when interferences

to the Ellman assay exist (such as high

hemoglobin or sample particulates that remain

in the aqueous phase) or high sensitivity is re-

quired. Although this assay is simple enough

and can be used on a routine basis in labo-

ratories possessing a scintillation counter, the

problems associated withthe useof radioactiv-

ity, including the cost of removing hazardous

waste, promotes its use only for special situa-

tions.

2. When running the samples in triplicate,

up to 30 sets can be assayed at one time. Given

the large number of vials being prepared, it

is easy to lose track during a pipetting step.

Tilting the rack, while holding the vials, makes

it possible to readily detect the absence of as

low as 10 µ l of solution by comparing the

meniscus from one vial to another.

3. It is crucial that the toluene-based fluor

is used. Common commercial fluors have

been designed to avoid using solvents such

as toluene, and they contain surfactants to

achieve homogenous mixtures. This would

ruin the radiometric assay, which relies on the

phase separation between the aqueous buffer

(containing the intact labeled substrate) and

the solvent fluor (containing the labeled reac-

tion product, acetic acid).

4. Check the level of counts in the blanks.

The cpm in the blank should be ≤5% of the

total cpm measured in the eel AChE vials.A higher percentage in the blank indicates a

breakdown of the substrate during storage. If

this is observed, the assays should be repeated

using a new substrate preparation.

5. Some compounds (e.g., colored com-

pounds) may enter the toluene phase of the

scintillation mixture and cause quenching and

interfere with an accurate assessment of the

cpm. In this case, the dpm should be deter-

mined to account for quenching. This can be

done by using an external standard present

in most scintillation counters (consult specific

instrument’s manual). This will increase thecounting time per vial. The dpm are used in

place of the cpm in the calculations. It is pru-

dent to alwaysdetermine the cpmand usethem

in calculating the ChE activities.

Expressing activityBlood activities are generally expressed in

terms of volume per milliliter of whole blood

or plasma, or per milliliter RBC based on

hematocrit determinations. Activities of tissue

are usually expressed in terms of weight per

gram of wet weight or per milligram protein.

Species and developmental differencesDifferences in AChE/BChE content be-

tween species, tissues, and ages are all too

often ignored, leading to gross errors in exper-

imental analysis. For example, many determi-

nations of rodent blood (an important matter

in toxicology studies) assume that the AChE is

in the RBC and the serum contains only BChE

activity. Instead, almost half the serum activity

7/28/2019 tx1213



12.13.14


Table 12.13.6 ChE Values of Various Species Determined According to These Protocols

Species Tissue n Total ChEa AChEa BChEa Assayb Reference

Mammals

Human

( Homo sapiens)

Whole blood 10 5.32 ± 0.65 C Unpublished

RBC 10 12.9 ± 2.2

Plasma 10 1.61 ± 0.29

Rhesus monkey

( Macaca mulatta)

Whole blood 4 2.64 ± 0.17 R Unpublished

Plasma 4 0.065 ± 0.023

Dog

(Canis familiarus)

Whole blood 4 0.87 ± 0.084 R Unpublished

Plasma 4 0.34 ± 0.028

Dairy cow

( Bos taurus)

Whole Blood 20 1.98 ± 0.37 C Arrieta

(2003)

Swiss mouse

( Mus musculus)

Plasma 5 2.07 ± 0.08 C Burruel et al.,

(2000)

BirdsRed-tailed hawk

( Buteo jamaicensis)

Plasma 44 0.66 ± 0.03c 0.14 ± 0.01c 0.52 ± 0.02c C Stein et al.

(1998)

American kestrel

( Falco sparverius)

Plasma 86 1.9 ± 0.05c 1.6 ± 0.05c 0.26 ± 0.01c C

Pigeon

(Columba livia)

Plasma 23 1.65 ± 0.26 C Unpublished

Brain 23 282 ± 18.5

White Leghorn

chicken (Gallus

domesticus)

Plasma 24 0.22 ± 0.01 C Henderson

et al. (1992)

Muscle 3 1.26 ± 0.27 Wilson et al.(1988)

Brain 3 296 ± 12

Japanese quail

(Coturnix coturnix

japonica)

Muscle

culture

64 51.7 ± 7.8c R Wilson and

Nieberg

(1983)

Fish

Mudsucker

(Gillichthys

mirabilis)

Brain 122 217 ± 31 C Unpublished

Medaka (larva)

(Oryzias latipes)

Retina 3 × 10

pooled

126 ± 2.3c C Hamm et al.

(1998)

continued

7/28/2019 tx1213



12.13.15


Table 12.13.6 ChE Values of Various Species Determined According to These Protocols, continued

Species Tissue n Total ChEa AChEa BChEa Assayb Reference

Amphibians

Pacific tree frog

( Hyla regilla)

Whole body,

bred 8◦C

30 28.2 ± 10.2 C Johnson et al.

(2005)

Whole body,

bred 19◦C

30 42.3 ± 17.3

aUnits areµmol/min/ml for blood fractions, and nmol/min/mg protein for other tissues. Values are means ± SD.bC, colorimetric assay; R, radiometric assay.cValues = SEM.

in the rat is AChE (Wilson, 1999, 2001). Sera

from embryos of species such as the bovine

may differ from that of the adult, in this case,

the bovine fetal blood is high in AChE and the

adult serum has little of either AChE or BChE

(Arrieta et al., 2003).

VariabilityAll samples are routinely run in triplicate

and the mean activity rate is used for further calculations. If the standard deviation is >10%

of the mean, the sample is routinely re-run.

Samples with very low activity (i.e., highly

inhibited) will be near the noise level of the

assay. Activities near zero are likely to be more

variable and will usually not meet the 10%

threshold criterion.

StandardsA triplicate set of bovine RBC ghost stan-

dards prepared by the laboratory (Arrieta et al.,

2003) is routinely run with each plate in the

colorimetric assay. Its use or a preparation of electric eel AChE or other commercial stan-

dards is recommended.

ReagentsIf the assay does not appear to be working

and the reagents are suspect, discard reagents

and prepare fresh reagents. Do not waste time

checking reagents.

Anticipated Results

Basic Protocol 1The basic unit of data determined from

the colorimetric assay is the calculated

slope of the kinetic reaction, with units of

change in absorbance per minute. The sub-

strate blank provides the background rate of

non-enzymatic substrate hydrolysis, with a

typical value of 0.001. The minimum slope

for a sample well should be at least three times

the background, or 0.003. The maximum lin-

ear slope for a sample well is ∼0.25. This

yields a change of absorbance of 2.5 over the

10-min assay time, near the limit of accurate

absorbance measurements. Typical slopes are

in the range of 0.01 to 0.15. It is important to

check the correlation coefficient of the slope as

an indication of a successful assay (see Critical

Parameters and Troubleshooting).

Basic Protocol 2The cpm determined in the radiometric

assay range from ∼3000 in the blanks to

∼100,000 in the eel AChE total hydrolysis

controls. The cpm in the sample should range

from three times background to 50% of the

total (eel) cpm, i.e., 10,000 to 50,000 cpm.

Examples of ChE activity levels in human,

other mammalian, avian, fish, and amphibian

tissues determined using these assay protocols

are shown in Table 12.13.6.

Time Considerations

Sample preparationPreparing samples can take up to 1.5 hr

depending upon the type of sample (blood or

solid tissue), type of preparation (centrifuga-

tion or homogenization), and dilution. Label

sample tubes prior to beginning sample prepa-

ration.

Basic Protocol 1Preparing fresh substrate and colorimetric

reagents takes∼30 min.Pipettingreagents and

samples into a 96-well microtiter plate takes

∼20 min. The time to acquire the kinetic read-

ings depends on the time interval and number

of readings (the typical six readings at 2-min

intervals takes 10 min, the first reading is time

zero).

7/28/2019 tx1213



12.13.16


Basic Protocol 2Preparation of radiometric reagents is time

consuming and they should be prepared ahead

of time. The radioactive substrate is typically

storedfrozen and thawed for the assay. Adding

reagents and samples takes ∼15 min per rack

of vials. Assay time takes 15 to 40 min, de-

pending on activity levels. Adding stopping

reagent and fluor takes ∼10 min per rack of

vials. Phase separation takes 30 min. Scintil-

lation counting can take 5 to 10 min per vial

and is typically done overnight.

Literature CitedArrieta, D., Ramirez, A., DePeters, E., Bosworth,

D., and Wilson, B.W. 2003. Bovine red bloodcell ghost cholinesterase as a monitoring stan-dard. Bull. Environ. Contam. Toxicol. 71:447-452.

Burruel, V.R., Raabe, O.G., Overstreet, J.W.,Wilson, B.W., and Wiley, L.M. 2000. Paternaleffects from methamidophos administration inmice. Toxicol. Appl. Pharmacol. 165:148-157.

Ellman, G.L., Courtney, K.D., Andres, V. Jr., andFeather-Stone, R.M. 1961. A new and rapid col-orimetric determination of acetylcholinesteraseactivity. Biochem. Pharmacol. 7:88-95.

Hamm, J.T., Wilson, B.W., and Hinton, D.E. 1998.Organophosphate-induced acetylcholinesteraseinhibition and embryonic retinal cell necrosis invivo in the teleost (Oryzias latipes). Neurotoxi-cology 19:853-869.

Henderson, J.D., Higgins, R.J., Dacre, J.C., andWilson, B.W. 1992. Neurotoxicity of acute andrepeated treatments of tabun, paraoxon, diiso-propyl fluorophosphate and isofenphos to thehen. Toxicology 72:117-129.

Johnson, C.D. and Russell, R.L. 1975. Rapid sim-ple radiometric assay for cholinesterase, suit-able for multipledeterminations. 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 acetyl-cholinesterase activity in the Pacific tree frog( Hyla regilla). Environ. Toxicol. Chem. 24:2074-2077.

Michel, H.O. 1949. Electrometric method for thedetermination of red blood cell and plasmacholinesterase activity. J. Lab. Clin. Med.34:1564-1568.

Nostrandt, A.C., Duncan,J.A.,and Padilla, S. 1993.A modified spectrophotometric method appro-priate for measuring cholinesterase activity intissue from carbaryl-treated animals. Fundam. Appl. Toxicol. 21:196-203.

Padilla, S., Lassiter, T.L., and Hunter, D. 1999.Biochemical measurement of cholinesterase ac-tivity. 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., andWilson, B.W. 1998. Comparative hematologyand plasma biochemistry of red-tailed hawksand American kestrels wintering in California. J. Raptor Res. 32:163-169.

Wilson, B.W. 1999. Cholinesterases. In ClinicalChemistry of Laboratory Animals. (F. Quimbyand W. Loeb, eds.) pp. 430-440. Taylor andFrancis Inc., Philadelphia.

Wilson, B.W. 2001. Cholinesterases. In Handbookof 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. Recoveryof acetylcholinesterase forms in quail mus-cle cultures after intoxication with diisopropy-lfluorophosphate. Biochem. Pharmacol. 32:911-918.

Wilson, B.W., Sanborn, J.R., O’Malley, M.A.,

Henderson, J.D., and Billitti, J.R. 1997. Mon-itoring the pesticide-exposed worker. Occup. Med. 12:347-363.

Wilson, B.W., Henderson, J.D., Chow, E.,Schreider, J., Goldman, M., Culbertson, R., andDacre, J.C. 1988. Toxicity of an acute dose of agent VX and other organophosphorus esters inthe 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 bloodcholinesterase measurements for monitoringpesticide exposures. In Enzymes of theCholinesterase Family. (D.M. Quinn, A.S.Balasubramanian, B.P. Doctor, and P. Taylor,eds.) pp. 329-336. Plenum Press, New York.

Key ReferencesEllman 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 reac-tivations, and risk assessment.

Internet Resources

http://chppm.com/ Website for reaching analytical techniques used bythe U.S. Department of Defense including the delta pH ChE method.

Documents

tx1213