Electrochemical detection of acetylcholine and choline: application to the quantitative nonradiochemical evaluation of choline transport.

The development of analytical methods for determining the cholinergic biomarkers acetylcholine (ACh) and choline (Ch) is important for assessing their role in neurological and cognitive functions. In this review, electrochemical (EC) strategies to detect ACh and Ch are summarized and compared to other analysis methods. Recent research focusing on the development of a versatile nonradiochemical in vitro assay to evaluate Ch transport is also described. The assay coupled to analysis by capillary electrophoresis (CE) with indirect EC detection at an enzyme-modified microelectrode affords exceptional selectivity and sensitivity. Femtomole or lower mass detection limits for ACh (1 fmol) and Ch (100 amol) have been readily achieved, opening up a new range of possible experiments for investigating transport or turnover of Ch and ACh in neurobiological systems. The value of this method is illustrated through the evaluation of the pharmacological efficacy and mode of inhibition of a new class of quaternary ammonium alkyl-substituted catechol-based inhibitors of high-affinity choline transport (CHT). This microanalytical approach is particularly useful when knowledge of endogenous concentrations of Ch or ACh is desired or when the amount of available compounds or the sample size is limited. A brief description of the principles of CE is also provided.

Classification of the mode of inhibition of high-affinity choline uptake using capillary electrophoresis with electrochemical detection at an enzyme-modified microelectrode.

A nonradiochemical in vitro assay using capillary electrophoresis with electrochemical detection at an enzyme-modified microelectrode has been developed to evaluate the inhibition of high-affinity choline transport in synaptosomes. Quantitative analysis of high-affinity choline transporter rates as a function of inhibitor and substrate concentrations allowed determination of the mode of inhibition for the quaternary ammonium-catechol-based inhibitors 3-[(trimethylammonio)methyl]catechol, N,N-dimethylepinephrine, and 6-hydroxy-N,N-dimethylepinephrine. The results are compared to the well-characterized inhibitor of choline transport, hemicholinium-3.

Evaluation of the inhibition of choline uptake in synaptosomes by capillary electrophoresis with electrochemical detection.

A direct method for evaluating choline uptake by the high-affinity choline transport system in synaptosomes was developed using capillary electrophoresis (CE) with electrochemical (EC) detection. On-column EC detection of choline and the internal standard, butyrylcholine, was accomplished with a 25 microm platinum electrode modified with the enzymes, choline oxidase and acetylcholinesterase. Choline uptake was evaluated as a function of choline concentration and a KM value of 1.7 microM was determined. The method was also used to evaluate a new class of redox affinity inhibitors of choline transport. In particular, the effectiveness of 3-[(trimethylammonio)methyl]catechol (TMC) as an inhibitor of choline uptake was examined independently and relative to the inhibition of the well-known inhibitor of choline transport, hemicholinium-3. The IC50 and KI for TMC were determined to be 30 microM and 14 microM, respectively. The combination of the selectivity and sensitivity afforded by CEEC provides a relatively straightforward approach for monitoring choline transport in synaptosomes.

Internal standard method for the measurement of choline and acetylcholine by capillary electrophoresis with electrochemical detection.

An internal standard method has been developed for the determination of the neurotransmitter acetylcholine and/or its metabolic precursor choline. This approach couples the high separation efficiency of capillary electrophoresis with the sensitivity and selectivity of electrochemical detection at an enzyme-modified electrode. Indirect electrochemical detection is accomplished at a 25 microm platinum electrode modified by cross-linking the enzymes choline oxidase and acetylcholinesterase with glutaraldehyde. Although in this simple form of electrode fabrication there is a gradual loss of response from the electrochemical detector with time, accurate quantitation is achieved by the addition of butyrylcholine, which is also a substrate for acetylcholinesterase, as an internal standard. A linear response is achieved between 0 and 125 microM with a limit of detection of 2 microM (25 fmol). The utility of this method was demonstrated by monitoring the kinetics of choline uptake in synaptosomal preparations.