Fouad K. Mohammad

1. 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

2. Important insecticides in use • Organophosphates • Carbamates Inhibition of acetylcholinesterase (ChE): Signs of poisoning: Muscarinic Nicotinic CNS

3. Cholinesterases • True Cholinesterase (Brain, RBC) • Pseudo Cholinesterase (Plasma, Liver)

4. 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.

5. Methods • 1. Spectrophotometric: Ellman method • 2. Electrometric: Michel method

6. 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

7. Principles of the modified electrometric method • Cholinesterase + Acetylcholine • Choline + Acetic acid • Decrease in pH

8. 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

9. Applications of the method • Measuring ChE inhibition induced by organophosphate and carbamate insecticides: • Plasma • Serum • RBC (mammals) • Whole blood • Tissue

10. Correlation with other methods • The described electrometric method correlates well with the original Michel’s method and with the colorimetric method of Ellman

11. 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

12. Normal ChE values in man (Ahmed, 2001; Ahmed and Mohammad, 2005)(∆pH/20 min)

13. Normal ChE values in mice (Mohammad et al, 1999) • (∆pH/20 min) • WBChE 1.10 • Brain 0.39 • Liver 0.39

14. 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

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

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

17. Quail Cholinesterase activity (Δ pH/30 minutes) Mohammad 2002

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

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

20. Starling Cholinesterase activity (Δ pH/30 minutes) Mohammad 2002

21. Monitoring exposure • In vitro ChE inhibition By: • 1. Organophosphates: • Dichlorvos • Diazinon • 2. Carbamates • Crabaryl

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

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

24. Cholinesterase inhibition in subjects exposed to insecticides at workAhmed & Mohammad 2005

25. 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.

26. Thank You College of Veterinary Medicine, University of Mosul

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