Simple electrometric method for
determination of blood and tissue
cholinesterase activities in man and animals:
Implications for environmental
biomonitoring
Fouad K. Mohammad
Department of Physiology-Division of
Pharmacology and Toxicology, College of
Veterinary Medicine, University of Mosul,
P. O. BOX 11136, Mosul, Iraq
Sept. 20, 2005
1
Important insecticides in use
Organophosphates •
Carbamates •
Inhibition of acetylcholinesterase (ChE):
Signs of poisoning:
Muscarinic
Nicotinic
CNS
Sept. 20, 2005
2
Cholinesterases
True Cholinesterase (Brain, RBC) •
Pseudo Cholinesterase (Plasma, Liver) •
Sept. 20, 2005
3
Importance of measuring ChE
activity
1. Diagnosis of poisoning with ChE •
inhibitors. •
2. Monitoring exposure to ChE inhibitors. •
3. Monitoring environmental exposure to •
ChE inhibitors. •
Sept. 20, 2005
4
Methods
1. Spectrophotometric: Ellman method •
2. Electrometric: Michel method •
Sept. 20, 2005
5
Modifications of the electrometric
method
1. Increasing the samples volume •
2. Using different buffers •
3. Increasing the reaction temperature •
4. Decreasing the incubation period •
5. Changing substrate concentration •
Sept. 20, 2005
6
Principles of the modified
electrometric method
Cholinesterase + Acetylcholine •
Choline + Acetic acid •
Decrease in pH •
Sept. 20, 2005
7
Electrometric ChE Determination
Mohammad et al., 1997. Vet. Hum. Toxicol.
Vol. 39: 337-339.
0.2 ml blood or tissue homogenate •
+ •
3ml distilled water •
+ •
barbital-phosphate buffer (pH 8.1) •
•
Measure pH (pH1) •
+ 0.1ml acetylcholine iodide (7.1%)
Incubate at 37 ○C for 20 min
•
Measure pH (pH2)
•
•
ChE activity (∆ pH/20 min)= pH1-pH2-∆ pH blank
Sept. 20, 2005
•
8
Applications of the method
Measuring ChE inhibition induced by •
organophosphate and carbamate
insecticides:
Plasma •
Serum •
RBC (mammals) •
Whole blood •
Tissue •
Sept. 20, 2005
9
Correlation with other methods
The described electrometric method •
correlates well with the original Michel’s
method and with the colorimetric
method of Ellman
Sept. 20, 2005
10
Specifications
Simple •
Reproducible •
One-step incubation time •
One type of buffer used •
Can detect carbamate-ChE inhibition •
Reduced reaction mixture handling •
Used in man & animals •
Sept. 20, 2005
11
Normal ChE values in man (Ahmed, 2001;
Ahmed and Mohammad, 2005)
(∆pH/20 min)
M
F
Sept. 20, 2005
PChE
1.05
EChE
1.18
WBChE
1.25
PChE
0.91
EChE
1.19
WBChE
1.23
12
Normal ChE values in mice
(Mohammad et al, 1999)
(∆pH/20 min) •
WBChE 1.10 •
Brain 0.39 •
Liver 0.39 •
Sept. 20, 2005
13
Normal ChE values in rats
(∆pH/20 min) •
PChE 0.29 (Mohammad et al., 2002) •
EChE 0.29 •
Brain ChE 0.19 •
PChE 0.26 (Al-Baggou and Mohammad, 1999) •
EChE 0.30 •
PChE 0.40 (Faris, 2003) •
EChE 0.28 •
Brain ChE 0.26 •
Sept. 20, 2005
14
Chicken Cholinesterase activity
(Δ pH/30 minutes) Abass & Mohammad 2004
Plasma
Brain
Liver
0.56
0.34
0.15
Sept. 20, 2005
15
Ruminants Cholinesterase activity (Δ
pH) Mohammad et al., 2005
Species
PChE
EChE
Sheep
30 min
Goats
40 min
Cattle
20 min
0.21
0.63
0.22
0.54
0.10
0.91
Sept. 20, 2005
16
Quail Cholinesterase activity
(Δ pH/30 minutes) Mohammad 2002
Plasma
Brain
Liver
Muscle
1.23
0.39
0.19
0.06
Sept. 20, 2005
17
Rock Dove Cholinesterase activity
(Δ pH/30 minutes) Mohammad 2002
Plasma
Brain
Liver
Muscle
1.28
0.59
0.12
0.08
Sept. 20, 2005
18
Sand Grouse Cholinesterase activity
(Δ pH/30 minutes) Mohammad 2002
Plasma
Brain
Liver
Muscle
1.81
0.37
0.06
0.07
Sept. 20, 2005
19
Starling Cholinesterase activity
(Δ pH/30 minutes) Mohammad 2002
Plasma
Brain
Liver
Muscle
1.10
0.24
0.08
0.08
Sept. 20, 2005
20
Monitoring exposure
In vitro ChE inhibition By: •
1. Organophosphates: •
Dichlorvos •
Diazinon •
2. Carbamates •
Crabaryl •
Sept. 20, 2005
21
Monitoring exposure
In vivo ChE inhibition by: •
1. Organophosphates •
Dichlorvos •
Diazinon •
Malathion •
2. Crabamates •
Carbaryl •
Methomyl •
3. Others •
Diphenhydramine •
Metoclopramide •
Sept. 20, 2005
22
Cholinesterase inhibition in chickens
dosed orally with dichlorvos (7 mg/kg) and
carbaryl (100 mg/kg) Mohammad & Al-Baggou’ 2005
% Cholinesterase inhibition
Carbaryl
Plasma
Brain
Liver
Sept. 20, 2005
28+5
65+3
19+8
Dichlorvos
62+7
29+9
33+9
23
Cholinesterase inhibition in subjects
exposed to insecticides at work
Ahmed & Mohammad 2005
ChE (Δ
pH/20
min)
PChE
Control
1.05+0.024 0.65+0.07
38
EChE
1.18+0.019 0.87+0.09
26
Sept. 20, 2005
Exposed
%
inhibition
24
Conclusion
-The described electrometric method could •
be an added simple method for measuring
blood or tissue cholinesterase activity in man
and animals exposed to organophosphate
and carbamate insecticides.
-The method can also be applied for •
biomonitoring of wild birds exposure to
anticholinesterases in the environment.
Sept. 20, 2005
25
Thank You
College of Veterinary Medicine, University of Mosul
Sept. 20, 2005
26
Normal reference range of
erythrocyte cholinesterase activity of
the cattle, sheep and goat
Paramet Cattle (Δ
pH/20 min)
er
Male Femal
e
35
37
N
0.91 0.89
Mean
0.212 0.118
SD
Minimu 0.32 0.46
m
Maximu 1.27 1.11
m
0.95 0.65
Range
95% CI 0.84, 0.85,
0.98 0.93
Sept. 20, 2005
Sheep (Δ pH/30 Goat (Δ
pH/40 min)
min)
Female Male Femal
Male
e
47
26
35
39
0.54 0.44
0.62
0.63
0.092 0.123 0.169 0.071
0.38 0.29
0.34
0.45
0.68
0.82
0.82
1.04
0.37
0.60,
0.66
0.48
0.58,
0.66
0.66 0.39
0.48, 0.42,
0.60 0.46
27
Normal reference range of plasma cholinesterase activity of the
cattle, sheep and goat
Cattle (Δ pH/20
min)
Sheep (Δ pH/30
min)
Goat (Δ pH/40 min)
Male
Female
Male
Female
Male
Female
N
37
34
35
37
26
47
Mean
0.10
0.17
0.21
0.19
0.22
0.22
SD
0.053
0.029
0.041
0.063
0.084
0.036
Minimum 0.02
0.10
0.12
0.06
0.04
0.16
Maximu
m
0.20
0.23
0.31
0.33
0.38
0.36
Range
0.18
0.13
0.19
0.27
0.34
0.20
95%
CI
0.08,
0.12
0.16,
0.18
0.20,
0.22
0.17,
0.21
0.19,
0.25
0.21,
0.23
Paramet
er
Sept. 20, 2005
28