Simple electrometric method for determination of blood and tissue cholinesterase activities in man a

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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

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Important insecticides in use

• Organophosphates
• Carbamates
• Inhibition of acetylcholinesterase (ChE)
• Signs of poisoning
• Muscarinic
• Nicotinic
• CNS

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Cholinesterases

• True Cholinesterase (Brain, RBC)
• Pseudo Cholinesterase (Plasma, Liver)

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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.

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Methods

• 1. Spectrophotometric Ellman method
• 2. Electrometric Michel method

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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

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Principles of the modified electrometric method

• Cholinesterase Acetylcholine
• Choline Acetic acid
• Decrease in pH

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Electrometric ChE DeterminationMohammad 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

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Applications of the method

• Measuring ChE inhibition induced by
  organophosphate and carbamate insecticides
• Plasma
• Serum
• RBC (mammals)
• Whole blood
• Tissue

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Correlation with other methods

• The described electrometric method correlates
  well with the original Michels method and with
  the colorimetric method of Ellman

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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

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Normal ChE values in man (Ahmed, 2001 Ahmed and
Mohammad, 2005)(?pH/20 min)
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Normal ChE values in mice (Mohammad et al, 1999)

• (?pH/20 min)
• WBChE 1.10
• Brain 0.39
• Liver 0.39

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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

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Chicken Cholinesterase activity (? pH/30
minutes) Abass Mohammad 2004
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Ruminants Cholinesterase activity (? pH) Mohammad
et al., 2005
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Quail Cholinesterase activity (? pH/30 minutes)
Mohammad 2002
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Rock Dove Cholinesterase activity (? pH/30
minutes) Mohammad 2002
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Sand Grouse Cholinesterase activity (? pH/30
minutes) Mohammad 2002
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Starling Cholinesterase activity (? pH/30
minutes) Mohammad 2002
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Monitoring exposure

• In vitro ChE inhibition By
• 1. Organophosphates
• Dichlorvos
• Diazinon
• 2. Carbamates
• Crabaryl

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Monitoring exposure

• In vivo ChE inhibition by
• 1. Organophosphates
• Dichlorvos
• Diazinon
• Malathion
• 2. Crabamates
• Carbaryl
• Methomyl
• 3. Others
• Diphenhydramine
• Metoclopramide

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Cholinesterase inhibition in chickens
dosed orally with dichlorvos (7
mg/kg) and carbaryl (100 mg/kg)
Mohammad Al-Baggou 2005
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Cholinesterase inhibition in subjects exposed to
insecticides at work Ahmed Mohammad 2005
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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.

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Thank You
College of Veterinary Medicine, University of
Mosul
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Normal reference range of plasma cholinesterase
activity of the cattle, sheep and goat