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

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Abstract

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 μm 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 μM with a limit of detection of 2 μM (25 fmol). The utility of this method was demonstrated by monitoring the kinetics of choline uptake in synaptosomal preparations.

Introduction

Impaired cholinergic neurotransmission has been implicated in neurodegenerative diseases such as Alzheimer’s disease and in the neuromuscular disease, myasthenia gravis. Acetylcholine (ACh) and its metabolic precursor, choline (Ch), are important neurotransmitter components in a properly functioning cholinergic system. Therefore, monitoring the levels of ACh and Ch is important in characterizing these cholinergic abnormalities.

Detection of ACh and Ch is a challenging analytical problem because they are neither UV-active, fluorescent, nor electroactive. For this reason, bioassays, radiochemical methods, or derivatization methods have often been employed even with their inherently complicated procedures and difficulties in quantitation [1]. Detection is further complicated by the complexity of the biological fluids in which ACh and Ch are typically found. Therefore, highly selective analytical techniques that also exhibit low detection limits are necessary for investigative purposes.

Electrochemical detection methods have received significant attention for the sensitive determination of ACh and Ch. Indirect electrochemical sensing is accomplished by taking advantage of the selective reactions of ACh and Ch with the corresponding enzymes, acetylcholinesterase (AChE) and choline oxidase (ChO) to generate hydrogen peroxide, which is detected by electrochemical means. Based on these concepts, numerous approaches have been reported that describe either stand-alone micro [2], [3], [4], [5], [6] or macro [7], [8], [9], [10], [11], [12], [13], [14] biosensors for ACh and Ch. Further selectivity for complex samples is gained when the enzyme reactions are coupled to a separation technique with electrochemical detection. Potter et al. [15] used a post-column reaction coil to mix the enzymes with ACh and Ch in the effluent, which had been separated by high-performance liquid chromatography (HPLC), followed by detection of hydrogen peroxide at a bare gold electrode. Subsequently, post-column reactors containing immobilized enzymes have reduced enzyme consumption and thus, have been widely used with HPLC coupled to a variety of detector electrode types and configurations [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27]. In cases where only ACh is to be determined, a pre-column enzyme reactor has been added to remove Ch [28], [29], [30]. As an alternative to the use of a separate enzyme reactor and electrochemical detector, AChE and ChO have also been directly immobilized on the detector electrode surface [31], [32], [33].

One of the challenges of using the enzyme reactions in combination with electrochemical detection is the loss of electrode sensitivity with time [25], [26], [31]. Loss of enzyme, reduction in enzyme activity, or electrode fouling have been discussed as contributing factors leading to a reduction in sensitivity in various ACh and Ch detection systems. In our efforts to use an AChE/ChO-modified microelectrode as a detector for capillary electrophoresis, we have observed a similar loss of sensitivity with time. To counteract the decaying response to ACh and Ch, we have employed butyrylcholine (BuCh) as an internal standard [26] and describe the results herein.

Section snippets

Materials

Acetylcholinesterase (EC 3.1.1.7, Type III from electric eel), choline oxidase (EC 1.1.3.17, Alcaligenes species), acetylcholine chloride (>99%), choline chloride (>98%), and butyrylcholine chloride (>98%) were purchased from Sigma (St. Louis, MO), stored in a desiccator at −10 °C, and weighed in a drybox when used. Choline was vacuum-dried overnight before use. N-Tris[hydroxymethyl]methyl-2-aminoethane-sulfonic acid (TES) (>99%), bovine albumin (>98%), Bradford reagent, and glutaraldehyde

Results and discussion

To utilize electrochemistry for detection of ACh and Ch, the enzymes AChE and ChO were used as biocatalysts to selectively recognize the substrates and generate hydrogen peroxide, which is easily detected electrochemically at +0.600 V vs. Ag/AgCl. The basis for this indirect approach to detect ACh and Ch is described in Fig. 1 and has been previously demonstrated to be both selective and sensitive [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19]

Conclusions

Capillary electrophoresis with electrochemical detection at an enzyme-modified electrode has been used for the separation and determination of the neurotransmitter ACh and its metabolic precursor, Ch. The electrochemical detector was prepared by chemically crosslinking ChO and AChE on a 25-μm platinum wire with glutaraldehyde. Under the hydrodynamic conditions of CE, the adsorbed enzyme layer is gradually lost and results in a decrease in electrode response with time. To counteract this

Acknowledgements

This work was funded by the National Institutes of Health through Grant R15 NS35305. Additional financial support from The University of Toledo and the Ohio Board of Regents for the development of a microanalytical laboratory is also gratefully acknowledged. We also thank Dr. Max Funk for the use of his centrifuge, Dr. Yang Cao for assistance with synaptosomal preparations, and Dr. Susan Lunte of the University of Kansas for providing information relating to the electrode preparation procedure.

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