Acetylcholine, colorimetric measurement

The most generally used method (Warburg manometric method) depends on the evolution of carbon dioxide from bicarbonate solutions when acetic acid is produced by hydrolysis of acetylcholine (3). The conversion of acetylcholine and certain other esters into the corresponding hy-droxamic acids when treated with hydroxylamine has been described by Hestrin (42) and a photometric method, depending on the ferric chloride reaction with acetic acid, has been reported by Abdon and Uvnas (1). A later report by Mitchell and Clark (61) involves the colorimetric measure-... 

An enzymatic assay can also be used for detecting anatoxin-a(s). " This toxin inhibits acetylcholinesterase, which can be measured by a colorimetric reaction, i.e. reaction of the acetyl group, liberated enzymatically from acetylcholine, with dithiobisnitrobenzoic acid. The assay is performed in microtitre plates, and the presence of toxin detected by a reduction in absorbance at 410 nm when read in a plate reader in kinetic mode over a 5 minute period. The assay is not specific for anatoxin-a(s) since it responds to other acetylcholinesterase inhibitors, e.g. organophosphoriis pesticides, and would need to be followed by confirmatory tests for the cyanobacterial toxin. 

Takeuchi et al. published a mechanized assay of serum cholinesterase by specific colorimetric detection of the released acid [40]. The cholinesterase reaction was carried out on a thermostatted rack at 30° C with a reaction mixture of serum (10 pL), 50mM barbitone-HCl assay buffer (pH 8.2 140 pL), and 12.5 mM acetylcholine solution (50 pL). The solutions were prepared by programmed needle actions, and a sample blank was also prepared. The reaction was stopped after 9.7 min by injection of the mixture into a flow injection analysis system to determine the quantity of acetic acid formed. The carrier stream (water, at 0.5mL/min) was merged with a stream (0.5mL/min) of 20 mM 2-nitrophenylhydrazine hydrochloride in 0.2 M HC1 and a stream (0.5 mL/min) of 50 mM 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride in ethanol containing 4% of pyridine. The sample was injected into this mixture (pH 4.5), passed through a reaction coil (10 m x 0.5mm) at 60°C, 1.5M NaOH was added, and, after passing through a second reaction coil (lm x 0.5 mm) at 60°C, the absorbance was measured at 540 nm. 

Colorimetric Methods. Measurement of Unchanged Acetylcholine, This colorimetric technique depends on measuring the unchanged acetylcholine with hydroxylamine to produce acetohydroxamic acid which yields a purple color with excess ferric chloride after acidification. Cook 12) originally applied this method to pesticide residue analysis on paper chromatograms. 

Phenol Red. The first publications of an automated procedure for the measurement of cholinesterase inhibitors are those of Winter 14) and Winter and Ferrari (15). The method employed an Autoanalyzer instrumental system wherein the extracts containing the insecticide were incubated with a standard cholinesterase solution at 37 °G. A continuous sample from the incubation bath is buffered and mixed with acetylcholine iodide. After a second incubation, the acetic acid released by the action of the uninhibited cholinesterase is measured colorimetrically, using phenol red as the indicator. More recently, Fischl et al. 16) reported a method for rapid detection of organic phosphate pesticides in serum. Strips of filter paper were impregnated with a buffered acetylcholine substrate solution containing phenol red as an indicator. When no inhibition is present, the acid released from the action of cholinesterase turns the paper yellow. When cholinesterase has been inhibited, the paper turns pink-to-violet. 

The basis for the standard method for determining blood (ChE) inhibition is the measurement of the enzymatic products derived when either acetylcholine or acetylthiocholine are used as substrates. The rate of formation of acetate is measured by changes in pH, while the formation of thiocholine is determined colorimetrically. A portable device utilizing this method, the Test-Mate OP Kit (EQM Research Incorporated, 2585 Montana Avenue, Cincinnati, OH 45211), provides a rapid, reasonably sensitive assay for ChE inhibition following OP exposure. The kit should be appropriate for emergency contingencies, and only very small blood samples are required. Military forces are viewing such a portable kit as a means to screen... 

The standard methodology for determining blood ChE inhibition is based on the measurement of the enzymatic products derived from either acetylcholine or acetylthiocholine as substrates. The simplest and most convenient method is a colorimetric procedure (Ellman et al., 1961) in... 

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