Determination of Acetylcholine

Acetylcholinesterase hydrolyzes ACh to choline and acetate. One mole of H+ is formed per mole of ACh by dissociation of acetate  

The increase of the concentration of H+ can be indicated by means of glass electrodes. Durand et al. (1978) entrapped AChE around the active tip of a glass electrode in a gelatin layer of 50 pm thickness and, after drying, crosslinked the layer with glutaraldehyde. In 0.01 mol/1 phos- 

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Tor [7] developed a new method for the preparation of thin, uniform, self-mounted enzyme membrane, directly coating the surface of glass pH electrodes. The enzyme was dissolved in a solution containing synthetic prepolymers. The electrode was dipped in the solution, dried, and drained carefully. The backbone polymer was then cross-linked under controlled conditions to generate a thin enzyme membrane. The method was demonstrated and characterized by the determination of acetylcholine by an acetylcholine esterase electrode, urea by a urease electrode, and penicillin G by a penicillinase electrode. Linear response in a wide range of substrate concentrations and high storage and operational stability were recorded for all the enzymes tested. 

The same group reported in 1986 a sensitive and selective HPLC method employing CL detection utilizing immobilized enzymes for simultaneous determination of acetylcholine and choline [187], Both compounds were separated on a reversed-phase column, passed through an immobilized enzyme column (acetylcholine esterase and choline oxidase), and converted to hydrogen peroxide, which was subsequently detected by the PO-CL reaction. In this period, other advances in this area were carried out such as the combination of solid-state PO CL detection and postcolumn chemical reaction systems in LC [188] or the development of a new low-dispersion system for narrow-bore LC [189],... 

On the other hand, several oxidases are known to generate hydrogen peroxide, acting as an oxidant in the CL system, from corresponding substrates. IMERs in which the oxidases are immobilized on adequate supporting materials such as glass beads have been developed. IMERs are often used for flow injection with CL detection of uric acid and glucose, and are also applicable to the CL determination of acetylcholine, choline, polyamines, enzyme substrates, etc., after online HPLC separation. 

Kehr J, Yoshitake T, Wang FH, Wynick D, Holmberg K, et al. 2001. Microdialysis in freely moving mice Determination of acetylcholine, serotonin and noradrenaline release in galanin transgenic mice. J Neuro Sci Method 109 71-80. 

Fig. 9.7. Determination of acetylcholine by differential pulse polarography with hanging electrolyte drop electrode. Acetylcholine concentrations 0-0, 1-0.5 ppm, 2-1 ppm, 3-2 ppm, 4-5 ppm.

Ellman, G L, Courtney, K, Andres, V., and Featherstone, R (1961) A new and rapid colorimetric determination of acetylcholine esterase activity. Biochem Pharmacol 7, 88-95... 

Rather more specialized are the chemical methods for the determination of acetylcholine, but a short account has been given of their historical development in a brief correspondence in 1968 between Sir Henry Dale, D. J. Jenden, and B. Holmstedt, who wrote it up for publication.192... 

Suzuki, M., Nitsch, C., Wunn, W., Schmude, B., and Hang, P. (1980). Selected ion monitoring determination of acetylcholine during methoxypyridoxine seizures. Biomed. Mass Spectrom. 7, 537-539. 

The recent developments in the determination of acetylcholine and choline, the advantages and the disadvantages of a variety of analytical methods used in the analysis, were reviewed and discussed by Maruyama et al. [7]. Hanin published an overview for the methods used for the analysis and measurement of acetylcholine [8], Shimada et al. presented a review, including the applications of some reactors in the analysis of choline and acetylcholine. The immobilized enzyme reactors were used for detection systems in high performance liquid chromatography [9],... 

Kakutani et al. described an ion-transfer voltammetry and potentiometry method for the determination of acetylcholine with the interface between polymer-nitrobenzene gel and water [13]. The PVC-nitrobenzene gel electrode was prepared as described by Osakai et al. [14]. The transfer of acetylcholine ions across the interface between the gel electrode and water was studied by cyclic voltammetry, potential-step chronoamperometry, and potentiometry. The interface between the two immiscible electrolyte solutions acted as the ion-selective electrode surface for the determination of acetylcholine ions. 

Baum reported a potentiometric determination of acetylcholine activity using an organic-cation-selective electrode [15], The performance of a liquid membrane electrode selective for acetylcholine (Corning No. 476.200) was investigated. Measurements of the potential difference at various concentration of acetylcholine were made against a calomel reference-electrode. 

An immobilized enzyme flow injection conductimetric system was used by Godinho et al. for the determination of acetylcholine [17]. Aliquots... 

A quantitative method was reported by Maslova for the determination of acetylcholine in biological tissues by polarographic analysis utilizing a rotating platinum electrode [18]. The principle of the method is based upon a polarographic analysis of the iron ions which remain after the formation of a specific Fe-acetylhydroxamic acid complex. Using this method, it was shown that 1 mL of peripheral blood of a healthy adult man contained 6.6 ng of acetylcholine. 

Osakai et al. used a microcomputer-controlled system for the ion transfer voltammetry procedure [20]. The system used is based on a NEC PC-9801 microcomputer, which was designed by using a polarizable oil-water interface as an ion-selective electrode surface. The system was applied to the determination of acetylcholine ion by cyclic, differential pulse, and normal pulse voltammetry at the PVC-nitrobenzene gel electrode. The amperometric measurement was carried out with voltage pulses of short durations and constant amplitude. 

Hale et al. reported the use of an enzyme-modified carbon paste for the determination of acetylcholine [21], The sensor was constructed from a carbon paste electrode containing acetylcholineesterase and choline oxidase, and the electron transfer mediator tetrathiafulvalene. The electrode was used for the cyclic voltammetric determination of acetylcholine in 0.1 M phosphate buffer at +200 mV versus saturated calomel electrode. Tetrathiafulvalene efficiently re-oxidized the reduced flavin adenine dinucleotide centers of choline oxidase. The calibration graph was linear up to 400 pM acetylcholine, and the detection limit was 0.5 pM. 

Yao et al. reported a flow injection analytical system for the simultaneous determination of acetylcholine and choline that made use of immobilized enzyme reactors and enzyme electrodes [25]. Acetylcholineesterase-choline oxidase and choline oxidase were separately immobilized by reaction with glutaraldehyde onto alkylamino-bonded silica, and incorporated in parallel as the enzyme reactors in a flow injection system. The sample containing acetylcholine and choline in 0.1 M phosphate buffer (pH 8.3) carrier solution was injected into the system. The flow was split to pass through the two reactors, recombined, and mixed with 0.3 mM K4Fe(CN)6 reagent solution before reaching a peroxidase immobilized electrode. Because each channel had a different residence time, two peaks were obtained for choline and total acetylcholine and choline. Response was linear for 5 pM-0.5 mM choline, and for 5 pM 1 mM acetylcholine plus choline. The detection limits were 0.4 pM for choline and 2 pM for acetylcholine. 

Mynka et al. reported a method for the infrared spectrophotometric identification and the determination of acetylcholine in the presence of other esters in pharmaceuticals [3]. Acetylcholine was determined at the maximum absorbance of 1755 cm-1. Beer s law was obeyed in the concentration range 1-10 pg/mL, and the relative standard deviation was less than 2.7%. Results obtained were reported to be reliable and reproducible. 

Eksborg and Persson reported a photometric method for the determination of acetylcholine in rat brain after selective isolation by ion-pair extraction and microcolumn separation [28]. The same authors also reported a photometric method for the determination of acetylcholine and choline in brain and urine samples after selective isolation by ion-pair column chromatography [29]. 

Sakai et al. reported a novel flow injection method for the selective spectrophotometric determination of acetylcholine using thermochro-mism of ion associates [38]. Samples (0.14mL) containing acetylcholine were injected into a flow injection system with a buffered (pH 11) carrier stream and a reagent stream (10 mM tetrabromo-phenolphthalein ethyl ester in dichloroethane) at 0.8 mL/min. The temperature of the flow cell was 45°C which reduced interference and improved recovery, and the detection was at 610 nm. 

Sakai et al. reported the use of batchwise and flow-injection methods for the thermo-spectrophotometric determination of acetylcholine and choline with tetrabromophenolphthalein ethyl ester [39]. An aqueous sample... 

O Neill and Sakamoto reported an enzymatic fluorimetric method for the determination of acetylcholine in biological extracts [41]. Nanomolar amounts of acetylcholine were determined in perchloric acid extracts of biological materials (brain tissues) by use of a system containing acetylcholineesterase, acetyl CoA synthetase, maleate dehydrogenase, and citrate synthase. The production of NADH2 was stoichiometrically related to the amount of acetylcholine in the system, and was followed fluorimetrically. Interfering fluorescent substances in the brain extracts were removed with acid-washed Florisil. 

A fluorimetric assay method for the determination of acetylcholine with picomole sensitivity was reported by MacDonald [44]. The method is based on the hydrolysis of acetylcholine to choline and acetate, catalyzed in the presence of acetylcholineesterase, oxidation of choline to betaine, and H202 in the presence of choline oxidase, and oxidation of 4-hydroxyphenylacetic acid by H202 to a fluorescent product, catalyzed by peroxidase. The interference in the analysis of brain homogenates was discussed. 

Ternaux and Chamoin described an enhanced chemiluminescence assay method for the determination of acetylcholine [48]. Reaction medium was prepared by mixing 250iu/mL of choline oxidase (100 pL), 2mg/mL of horse-radish peroxidase (50 pL) and 10-120 pM luminol in 100 pL of 0.1 M Tris buffer (pH 8.6), or 100 pL of 10-100 pM 7-dimethylamino-naphthalene-l,2-dicarbonic acid hydrazide, for 10min in 5mL of 0.1 M sodium phosphate buffer (pH 8.6). Aqueous 0.325-80 pmol of acetylcho-lineesterase (50 pL) purified on a Sephadex G50 coarse column was mixed with 450 pL of reaction mixture, and the chemiluminescence was measured at 21° C. 

Israel and Lesbats reported a chemiluminescence method for the determination of acetylcholine, and continuous detection of its release from torpedo electric organ synapses and synaptosomes [55], Birman described a new chemiluminescence assay method for the determination of acetylcholineesterase activity with the natural substrate [56], The method involved monitoring the increase in light emission produced by accumulation of choline, or measuring the amount of choline generated. 

Karlen et al. analyzed acetylcholine and choline by an ion pair extraction and gas phase method [134]. The gas chromatographic estimation of the isolated acetylcholine and choline was carried out after demethylation either with benzenethiolate or by controlled pyrolysis. Acetylcholine was quantitated by flame ionization detection at the nmol level. Mass fragment analysis was employed for the determination of acetylcholine in pmol amounts. 

Maruyama et al. reported the use of a simple pyrolysis gas chromatographic method for the determination of choline and acetylcholine in brain tissue [136]. Schmidt and Speth reported a simultaneous analysis of choline and acetylcholine levels in rat brain tissue by a pyrolysis gas chromatographic method [137], Kosh et al. reported an improved gas chromatographic method for the analysis of acetylcholine and choline in rat brain tissue [138], Mikes et al. used a syringe micro-pyrolyzer for the gas chromatographic determination of acetylcholine, choline and other quaternary ammonium salts [139],... 

Duan et al. reported the use of a rapid and simple method for the determination of acetylcholine and choline in mouse brain by high performance liquid chromatography, making use of an enzyme-loaded post column and an electrochemical detector [144]. Perchloric acid extracts of small brain tissue were injected onto the HPLC system with no prior clean-up procedure. Detection limits for both compounds were 1 pmol, and this method was successfully applied to the measurement of acetylcholine in discrete brain areas of the mouse. 

Tao et al. described an HPLC method for the determination of acetylcholine in a pharmaceutical preparation [145]. Utilizing reverse phase ion pairing, acetylcholine was determined in lyophilized ophthalmic preparations. Analysis of degraded commercial samples showed the utility of the method in quantitation, being stability indicating, and useful in separating acetylcholine from choline. 

Other high performance liquid chromatography methods reported for the determination of acetylcholine are summarized (together with their conditions) in Table 3 [4, 117, 147-193],... 

Polak and Molenaar described a method for the determination of acetylcholine from brain tissue by pyrolysis-gas chromatography-mass spectrometry [200]. The deuterium-labeled acetyl-choline is pyrolytically demethylated with sodium benzenethiolate, followed by quantitative GC-MS analysis. In this method, care must be taken so that the samples do not contain appreciable amounts of choline since exchange of deuterium-labeled groups between acetylcholine and choline during pyrolysis may yield erroneous results. The same authors have also reported a method for the determination of acetylcholine by slow pyrolysis combined with mass fragment analysis on a packed capillary column [201]. 

Watanabe et al. used a pyrolysis gas chromatography-mass spectro-metric method for the determination of acetylcholine in human blood... 

Bluth et al. described a simplified radio-enzymatic assay method for the determination of acetylcholine and choline in discrete structures of rat brain [211],... 

Spector et al. developed a specific radioimmunoassay method for the determination of acetylcholine [216]. The described method is suitable for determining 2-137 pmol of acetylcholine the radioligand used is... 

Ladinsky and Consolo used an enzymatic radioassay method for the determination of acetylcholine and choline [218], The method was based on the electrophoretic separation of acetylcholine and choline, hydrolysis of acetylcholine to form choline, and acetylation of the choline with labeled AcCoA and choline acetyltransferase. The labeled acetylcholine formed was isolated and quantitated. The method was sensitive and specific, and permitted the routine handling of a large number of samples in a single experiment. The standard curves were linear up to at least 42.5 ng (0.4 nmol) choline and 45 ng (0.3 nmol) acetylcholine. The lower limit of sensitivity was 2ng, and the recovery of acetylcholine was 95% when carried through the entire procedure. 

Keski-Rahkonen, R et al. Quantitative determination of acetylcholine in microdialysis samples using liquid chromatography/atmospheric pressure spray ionization mass spectrometry. Rapid Commun. Mass Spectrom. 2007, 21, 2933-2943. 

Nanoporous ZnO film has recently been deposited on Au electrode by rf sputtering under high pressure and used it for cholesterol detection. A well preferred c-axis oriented ZnO film with porous surface morphology exhibits linearity as 25-400 mg/dl, response time of 15 s and stability of 75 days [35], Khan et al. have been fabricated a nanocomposite film of ZnO nanoparticles containing chitosan (CH) for cholesterol estimation. This biosensor exhibits linearity from 5-300 mgdL 1 with detection limit of 5mg dF1 and the value of Km as 8.63 mgdL 1 [36]. Sol-gel derived ZnO film has been deposited on Pt electrode for development of an amperometric biosensor to determination of acetylcholine (ACh) and choline (Ch). The resulting biosensor shows linear response from 1.0 x 10"6 to 1.5 x 10 3 M to ACh with detection limit of 6.0 x 10"7 M and a linear response upto 1.6 x 10 3 M to Ch with detection... 

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