Sept. 20, 2005 1

Simple electrometric method for Simple electrometric method for determination of blood and tissue determination of blood and tissue

cholinesterase activities in man and animals: cholinesterase activities in man and animals:

Implications for environmental biomonitoringImplications for environmental biomonitoring Fouad K. MohammadFouad K. Mohammad

Department of Physiology-Division of Department of Physiology-Division of Pharmacology and Toxicology, College of Pharmacology and Toxicology, College of Veterinary Medicine, University of Mosul, Veterinary Medicine, University of Mosul,

P. O. BOX 11136, Mosul, IraqP. O. BOX 11136, Mosul, Iraq

Sept. 20, 2005 2

Important insecticides in useImportant insecticides in use

•OrganophosphatesOrganophosphates•CarbamatesCarbamates

Inhibition of acetylcholinesterase (ChE)Inhibition of acetylcholinesterase (ChE)::

Signs of poisoningSigns of poisoning::

MuscarinicMuscarinic

NicotinicNicotinic

CNSCNS

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CholinesterasesCholinesterases

•True Cholinesterase (Brain, RBC)True Cholinesterase (Brain, RBC)

•Pseudo Cholinesterase (Plasma, Liver)Pseudo Cholinesterase (Plasma, Liver)

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Importance of measuring ChE Importance of measuring ChE activityactivity

•11 . .Diagnosis of poisoning with ChEDiagnosis of poisoning with ChE • inhibitorsinhibitors..

•22 . .Monitoring exposure to ChE inhibitorsMonitoring exposure to ChE inhibitors..

•33 . .Monitoring environmental exposure toMonitoring environmental exposure to • ChE inhibitorsChE inhibitors..

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MethodsMethods

•11 . .Spectrophotometric: Ellman methodSpectrophotometric: Ellman method

•22 . .Electrometric: Michel methodElectrometric: Michel method

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Modifications of the electrometric Modifications of the electrometric methodmethod

•11 . .Increasing the samples volumeIncreasing the samples volume•22 . .Using different buffersUsing different buffers

•33 . .Increasing the reaction temperatureIncreasing the reaction temperature•44 . .Decreasing the incubation periodDecreasing the incubation period•55 . .Changing substrate concentrationChanging substrate concentration

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Principles of the modified Principles of the modified electrometric methodelectrometric method

•Cholinesterase + AcetylcholineCholinesterase + Acetylcholine

•Choline + Acetic acidCholine + Acetic acid

•Decrease in pHDecrease in pH

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Electrometric ChE DeterminationElectrometric ChE DeterminationMohammad et al., 1997. Vet. Hum. Toxicol. Mohammad et al., 1997. Vet. Hum. Toxicol.

Vol. 39: 337-339Vol. 39: 337-339..

•0.20.2 ml blood or tissue homogenateml blood or tissue homogenate•++

•3ml distilled water3ml distilled water• + +

•barbital-phosphate buffer (pH 8.1)barbital-phosphate buffer (pH 8.1)•

•Measure pH (pH1)Measure pH (pH1)

• + +0.1ml acetylcholine iodide (7.1%)0.1ml acetylcholine iodide (7.1%)• Incubate at 37 Incubate at 37 ○○C for 20 minC for 20 min

•Measure pH (pH2)Measure pH (pH2)

•ChE activity (∆ pH/20 min)= pH1-pH2-∆ pH blankChE activity (∆ pH/20 min)= pH1-pH2-∆ pH blank

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Applications of the methodApplications of the method

•Measuring ChE inhibition induced by Measuring ChE inhibition induced by organophosphate and carbamate organophosphate and carbamate insecticidesinsecticides::

• PlasmaPlasma• SerumSerum

• RBC (mammals)RBC (mammals)• Whole bloodWhole blood

• TissueTissue

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Correlation with other methodsCorrelation with other methods

•The described electrometric method The described electrometric method correlates well with the original correlates well with the original Michel’sMichel’s method and with the colorimetric method and with the colorimetric method of method of EllmanEllman

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SpecificationsSpecifications

•SimpleSimple•ReproducibleReproducible

•One-step incubation timeOne-step incubation time•One type of buffer usedOne type of buffer used

•Can detect carbamate-ChE inhibitionCan detect carbamate-ChE inhibition•Reduced reaction mixture handlingReduced reaction mixture handling

•Used in man & animalsUsed in man & animals

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Normal ChE values in man (Ahmed, 2001; Normal ChE values in man (Ahmed, 2001; Ahmed and Mohammad, 2005)Ahmed and Mohammad, 2005)

(∆pH/20 min)(∆pH/20 min) MMPChEPChE1.051.05

EChEEChE1.181.18

WBChEWBChE1.251.25

FFPChEPChE0.910.91

EChEEChE1.191.19

WBChEWBChE1.231.23

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Normal ChE values in mice Normal ChE values in mice (Mohammad et al, 1999)(Mohammad et al, 1999)

•∆)∆)pH/20 minpH/20 min((

•WBChE 1.10WBChE 1.10•Brain 0.39Brain 0.39•Liver 0.39Liver 0.39

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Normal ChE values in ratsNormal ChE values in rats

•∆)∆)pH/20 minpH/20 min((•PChE 0.29 PChE 0.29 (Mohammad et al., 2002)(Mohammad et al., 2002)

•EChE 0.29EChE 0.29•Brain ChE 0.19Brain ChE 0.19

•PChE 0.26 PChE 0.26 (Al-Baggou and Mohammad, 1999)(Al-Baggou and Mohammad, 1999)•EChE 0.30EChE 0.30

•PChE 0.40 PChE 0.40 (Faris, 2003)(Faris, 2003)•EChE 0.28EChE 0.28

•Brain ChE 0.26Brain ChE 0.26

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Chicken Chicken Cholinesterase activity Cholinesterase activity (Δ pH/30 minutes) (Δ pH/30 minutes) Abass &Abass & Mohammad 2004Mohammad 2004

PlasmaPlasma BrainBrainLiverLiver

0.560.560.340.340.150.15

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Ruminants Ruminants Cholinesterase activity Cholinesterase activity (Δ pH) (Δ pH) Mohammad et al., 2005Mohammad et al., 2005

SpeciesSpeciesPChEPChEEChEEChE

SheepSheep

3030 minmin

0.210.210.630.63

GoatsGoats

4040 minmin

0.220.220.540.54

CattleCattle

2020 minmin

0.100.100.910.91

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QuailQuail Cholinesterase activity Cholinesterase activity (Δ pH/30 minutes) (Δ pH/30 minutes) Mohammad 2002Mohammad 2002

PlasmaPlasma BrainBrainLiverLiverMuscleMuscle

1.231.23 0.390.39 0.190.19 0.060.06

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Rock Dove Rock Dove Cholinesterase activity Cholinesterase activity (Δ pH/30 minutes) (Δ pH/30 minutes) Mohammad 2002Mohammad 2002

PlasmaPlasma BrainBrainLiverLiverMuscleMuscle

1.281.280.590.590.120.120.080.08

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Sand GrouseSand Grouse Cholinesterase activity Cholinesterase activity (Δ pH/30 minutes) (Δ pH/30 minutes) Mohammad 2002Mohammad 2002

PlasmaPlasma BrainBrainLiverLiverMuscleMuscle

1.811.810.370.370.060.060.070.07

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StarlingStarling Cholinesterase activity Cholinesterase activity (Δ pH/30 minutes) (Δ pH/30 minutes) Mohammad 2002Mohammad 2002

PlasmaPlasma BrainBrainLiverLiverMuscleMuscle

1.101.100.240.240.080.080.080.08

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Monitoring exposureMonitoring exposure

•In vitro ChE inhibition ByIn vitro ChE inhibition By::•11 . .OrganophosphatesOrganophosphates : :

• DichlorvosDichlorvos• DiazinonDiazinon•22 . .CarbamatesCarbamates

• CrabarylCrabaryl

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Monitoring exposureMonitoring exposure

•In vivo ChE inhibition byIn vivo ChE inhibition by::•11 . .OrganophosphatesOrganophosphates

• DichlorvosDichlorvos• DiazinonDiazinon

• MalathionMalathion•22 . .CrabamatesCrabamates• CarbarylCarbaryl

• MethomylMethomyl•33 . .OthersOthers

• DiphenhydramineDiphenhydramine• MetoclopramideMetoclopramide

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Cholinesterase inhibition in chickens dosed orally with dichlorvos (7 mg/kg) and carbaryl (100 mg/kg) Mohammad & Al-Baggou’ 2005

% Cholinesterase inhibition Carbaryl Dichlorvos

Plasma 62+7 28+5Brain 29+9 65+3Liver 33+9 19+8

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Cholinesterase inhibition in subjects Cholinesterase inhibition in subjects exposed to insecticides at workexposed to insecticides at work

Ahmed & Mohammad 2005Ahmed & Mohammad 2005

ChE ChE (Δ (Δ pH/20 min)pH/20 min)

ControlControlExposedExposed % %inhibitioninhibition

PChEPChE1.051.05++0.0240.0240.650.65++0.070.073838

EChEEChE1.181.18++0.0190.0190.870.87++0.090.092626

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ConclusionConclusion•--The described electrometric method could The described electrometric method could

be an added simple method for measuring be an added simple method for measuring blood or tissue cholinesterase activity in man blood or tissue cholinesterase activity in man and animals exposed to organophosphate and animals exposed to organophosphate and carbamate insecticidesand carbamate insecticides..

•--The method can also be applied for The method can also be applied for biomonitoring of wild birds exposure to biomonitoring of wild birds exposure to anticholinesterases in the environmentanticholinesterases in the environment..

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Thank YouThank You

College of Veterinary Medicine, University of MosulCollege of Veterinary Medicine, University of Mosul

Sept. 20, 2005 27

Normal reference range of erythrocyte cholinesterase activity of the cattle, sheep and goat

Cattle (Δ pH/ 20 min)

Sheep (Δ pH/ 30 min)

Goat (Δ pH/ 40 min)

Parameter

Male Female

Male Female Male Female

N 37 35 39 35 26 47 Mean 0.91 0.89 0.63 0.62 0.54 0.44 SD 0.212 0.118 0.092 0.123 0.169 0.071 Minimum

0.32 0.46 0.45 0.34 0.38 0.29

Maximum

1.27 1.11 0.82 0.82 1.04 0.68

Range 0.95 0.65 0.37 0.48 0.66 0.39 95% CI 0.84,

0.98 0.85, 0.93

0.60, 0.66

0.58, 0.66

0.48, 0.60

0.42, 0.46

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Normal reference range of plasma cholinesterase activity of the cattle, sheep and goat

ParametParameterer

Cattle (Cattle (ΔΔ pH/20 pH/20 min)min)

Sheep (Sheep (ΔΔ pH/30 pH/30 min)min)

Goat (Goat (ΔΔ pH/40 min) pH/40 min)

MaleMale FemaleFemaleMaleMaleFemaleFemaleMaleMaleFemaleFemale

NN373734343535373726264747

MeanMean0.100.100.170.170.210.210.190.190.220.220.220.22SDSD0.0530.0530.0290.0290.0410.0410.0630.0630.0840.0840.0360.036

MinimumMinimum0.020.020.100.100.120.120.060.060.040.040.160.16

MaximuMaximumm

0.200.200.230.230.310.310.330.330.380.380.360.36

RangeRange0.180.180.130.130.190.190.270.270.340.340.200.20

95%95% CICI

0.080.08 , ,0.120.12

0.160.16 , ,0.180.18

0.200.20 , ,0.220.22

0.170.17 , ,0.210.21

0.190.19 , ,0.250.25

0.210.21 , ,0.230.23