ENZYMATIC FLUOROMETRIC DETERMINATION OF ACETYLCHOLINE IN BIOLOGICAL EXTRACTS

Abstract— Acetylcholine was determined fluorometrically by the following enzymic reactions: (1) ACh = Acetate + Choline (2) Acetate + ATP + CoASH = Acetyl-SCoA + AMP + PPi (3) Malate + NAD+= Oxalacetate + NADH (4) Oxalacetate + Acetyl-SCoA = CoASH + Citrate The fluorescence produced by NADH was stoichiometric with the ACh present and the citrate formed. The complete system contained acetylcholinesterase (EC 3.1.1.7), acetyl-CoA synthetase (EC 6.2.1.1), malate dehydrogenase (EC 1.1.1.37), and citrate condensing enzyme (EC 4.1.3.7). The acetyl-CoA synthetase was rate limiting in the system. Authentic samples of ACh (10−8 to 10−9mol) were measured with ±5 per cent reproducibility; this corresponds to the content of an 80 mg (fresh weight) sample of brain. Tissue levels of ACh in this concentration range, within normal biological variation, were determined with ±15 per cent reproducibility. The method can also be employed to measure acetate and acetyl-SCoA with the same degrees of sensitivity and reproducibility. The method can be used to measure ‘total’ and ‘bound’ ACh and thus to estimate ‘free’ ACh, by varying the extraction procedure. Values obtained in the manner described agree with those previously reported in the literature. Troublesome fluorescence in brain extracts was effectively removed with acid-washed Florisil.