Acetylcholine and Choline in Neuronal Tissue Measured by HPLC with Electrochemical Detection

Abstract

A simple, rapid method is presented for the determination of acetylcholine (ACh) and choline (Ch) in neuronal tissue using HPLC with electrochemical detection. The method is based on the separation of ACh and Ch by reverse-phase HPLC and mixing the effluent as it emerges from the column with acetylcholinesterase and Ch oxidase, which converts endogenous Ch and Ch produced by the hydrolysis of ACh to betaine and hydrogen peroxide. Production of hydrogen peroxide is continuously monitored electrochemically. The sensitivity of the procedure is 1 pmol for Ch and 2 pmol for ACh. Specificity of the method is based on HPLC, two specific enzymatic reactions, and the detection of hydrogen peroxide.

Immortalized murine striatal neuronal cell lines expressing dopamine receptors and cholinergic properties

“…Background signal was determined by assaying citrate-phosphate buffer instead of cell extract. Acetylcholine (ACh) levels were determined by HPLC-ED with an enzymatic reactor containing acetylcholinesterase and choline oxidase based on the method of Potter et al (1983). The materials used were obtained from Bioanalytical Systems Inc. (West Lafayette, IN).…”

Pharmacokinetics of Succinylcholine in Man.

“…This assay is based on the separation of acetyl Ch and Ch by reverse-phase HPLC (Potter et al 1983). For the electrolytic cell, the dual platinum action electrode and Ag/AgC12 electrode were used as a comparative electrode.…”

Effect of 1,3-Di-n-Butyl-7-(2-oxopropyl)-Xanthine (denbufylline) on metabolism and function of cerebral cholinergic neurons.

“…Recent observations, however, have indicated that the mechanism of endogenous ACh release from brain slices is different from that of preloaded [3H]ACh (23). Since recent advancements in HPLC procedures using electrochemical detection (13,14) have made possible the simultaneous measurements of ACh and choline with reasonable sensitivity, we have used these procedures with a continuous tissue transfer system to minimize enzymatic degradation of released endogenous ACh by AChE. In vitro addition of denbufylline (10-4 M) significantly enhanced 20 mM KC1 (a submaximal dose for ACh release)-evoked endogenous ACh release from striatal slices under these experimental conditions (Fig.…”

“…For the measurements of ACh, ACh was extracted from the tissue samples according to the method of Potter et al (13) and Igarashi et al (14). The samples were then weighed before the homogenization in 2 ml of a 1 N formic acid : acetone (15:85) mixture and then centrifuged at 1,700 X g for 20 min at 4°C.…”

“…The mobile phase consisted of 100 mM phosphate buffer solution (pH 8.0) containing 300 mg/l of sodium 1-decansulfonate and 65 mg/I of tetramethylammonium chloride. The detector potential was maintained at 450 mV against an Ag/AgCI reference electrode (13,19). The endogenous levels of ACh release in the brain was expressed as nmol/g wet weight or percentage of the mean of some control samples taken immediately before the addition of drug or potassium into the incubation medium.…”