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.     

Improved method for the routine determination of acetylcholine and choline in brain microdialysate using a horseradish peroxidase column as the immobilized enzyme reactor

A modified microbore high-performance liquid chromatography-immobilized enzyme reactor-electrochemical detection system for acetylcholine (ACh) and choline (Ch) was developed. The system used the horseradish peroxidase and a solution mediator ferrocene to convert the analyte into an oxidized ferrocene species which was detected electrochemically by reduction at 0 mV. There was an excellent linear relationship between the concentration of ACh/Ch and the peak height over the range of 1-5000 nmol/l. The limit of detection for ACh was 2 fmol/5 microl (S/N=3:1). Compared with the common method recommended by Bioanalytical System Inc. (BAS), this method exhibits a 200-fold improvement in the detection limit. The ACh and Ch levels in rat brain microdialysate were examined.     

Recovery of acetylcholine release and choline acetyltransferase activity after freezing-induced degeneration of rat soleus muscle

In order to study the effect of synaptic contact on the amounts of choline acetyltransferase (ChAT) and acetylcholine (ACh) in the nerve terminals and on their ability to release ACh, a freeze—thaw procedure was developed as a means to induce long lasting degeneration of rat soleus muscle. It was found that 4 days after the freeze—thaw procedure the preparation did not contract upon direct electric stimulation and the level of creatine kinase (CK) was below detection. The preparation contained about 15% of the ChAT activity and 15% of the ACh content of the controls. The ACh release evoked by 50 mM KCl was 25% of controls, but it was, when expressed as a fraction of the ACh content, about twice as high as that in control muscles. At day 12, the preparation still did not contract and the level of CK was less than 5% of controls. The ChAT activity and the ACh content were 40%) and 20% of controls, respectively. However, no release of ACh could be evoked by 50 mM KCl. At days 28 and 58 the preparation contracted upon stimulation of the nerve; the CK activity had recovered to about 20% and the ACh content to 40%, while the ChAT activity did not increase above 40%. The KCl–evoked ACh release had recovered to 20—30% of controls. The results indicate that freezing destroyed muscle cells and most intramuscular nerve branches. Subsequent regeneration of muscle fibres was slow, probably because freezing had killed many satellite cells in the muscle. Because the ChAT activity at day 12 had recovered when CK was almost absent and the preparation failed to contract, we conclude that there was expression of ChAT activity in ‘nerve terminals’ which do not make contact with regenerated muscle cells, although little if any ACh was released from these sites. ©1998 Elsevier Science Ltd. All rights reserved.     

Determination of acetylcholine by on-line microdialysis coupled with pre- and post-microbore column enzyme reactors with electrochemical detection

A sensitive procedure consisting of a pre- and post-microbore column reactor sequence of a LC-electrochemical detection system coupled with on-line microdialysis system is described in the present study to measure endogenous acetylcholine concentration in freely moving rats. The pre-column packed, with immobilized choline oxidase and catalase, was used to remove choline, whereas the post-column, packed with immobilized acetylcholine oxidase and choline oxidase, was used to measure acetylcholine selectively. The detection limit of acetylcholine was found to be 5 fmol/μl (50 fmol/10 μl). The usefulness of the described methodology was evaluated by examining the change in the striatal acetylcholine concentration of freely moving rats after physostigmine (0.5 mg/kg, s.c.) administration.     

High-affinity choline uptake and acetylcholine-metabolizing enzymes in CNS white matter. A quantitative study

The presence of nicotinic and muscarinic receptors suggests the occurrence of cholinergic neurotransmission in white matter; however no quantitative information exists on acetylcholine formation and breakdown in white matter. We compared white structures of pig brain (fimbria, corpus callosum, pyramidal tracts, and occipital white matter) to gray structures (temporal, parietal and cerebellar cortices, hippocampus, and caudate) and found that sodium-dependent, high-affinity choline uptake in white structures was 25–31% of that in hippocampus. White matter choline acetyltransferase activity was 10–50% of the hippocampal value; the highest activity was found in fimbria. Acetylcholine esterase activity in white structures was 20–25% of that in hippocampus. The caudate, which is rich in cholinergic interneurons, gave values for all three parameters that were 2.8–4 times higher than in hippocampus. The results suggest a certain capacity for cholinergic neurotransmission in central nervous white matter. The white matter activity of pyruvate dehydrogenase, which provides acetyl-CoA for acetylcholine synthesis, ranged between 33 and 50% of the hippocampal activity; the activity in the caudate was similar to that in hippocampus and the other gray structures, which was true also for other enzymes of glucose metabolism: hexokinase, phosphoglucomutase, and glucose-6-phosphate dehydrogenase. Acetylcholine esterase activity in white matter was inhibited by the nerve agent soman, which may help explain the reported deleterious effect of soman on white matter. Further, this finding suggests that acetylcholine esterase inhibitors used in Alzheimer's disease may have an effect in white matter.     

A new microdialysis-electrochemical device for in vivo simultaneous determination of acetylcholine and choline in rat brain treated with N-methyl-(R)-salsolinol

Acetylcholine (ACh) and choline (Ch) play a critical role in cholinergic neurotransmission and the abnormalities in their concentrations are related to several neural diseases. Therefore, the in vivo determination of ACh and Ch is important to the research on neurodegenerative disorders. In this work, electrochemical biosensors based on poly(m-(1,3)-phenylenediamine) (pmPD) and polytyramine (PTy) modified enzyme electrodes were fabricated. The electropolymerized pmPD polymer was used to exclude interfering substances and the PTy layer facilitated the immobilization of acetylcholinesterase (AChE) and choline oxidase (ChOx). Then, ACh/Ch sensor and Ch sensor were coupled with microdialysis to produce a novel device, which provides a sensitive and selective method for simultaneous determination of ACh and Ch. This method has detection limits of 63.0 ± 3.4 nM for ACh and 25.0 ± 1.2 nM for Ch. The integrated device was successfully applied to assessing the impact of endogenous neurotoxin N-methyl-(R)-salsolinol [1(R),2-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, (R)-NMSal] on ACh and Ch concentration, which is of great benefit to understand the pathogenesis of Parkinson's disease.     

Choline and acetylcholine metabolism in rat neostriatal slices

Choline (Ch) uptake and release and acetylcholine (ACh) synthesis and release have been studied by gas chromatography mass spectrometry (GCMS) in slices of rat neostriatum in vitro to assess the effects of depolarization by 25 mM K+ and the influence of elevated concentrations of Ch in the incubation medium. During the first 60 min after preparation, 25 mM K+ increased ACh release by 182% and reduced ACh levels by 40%. The rate of ACh synthesis was unchanged. After a 1-h equilibration period, the rate of ACh synthesis was considerably less (2.41 nmol mg-1 h-1, compared to 9.78 nmol mg-1 h-1). Exposure to 25 mM K+ during the second hour increased the rate to 6.47 nmol mg-1 h-1. During the first 10 min of exposure to 25 mM K+, ACh synthesis was reduced, regardless of incubation. Increasing concentrations of external [2H4]Ch apparently favored initial rates of net ACh synthesis, since the rank order of initial net ACh synthesis rates is the same as the rank order of external [2H4] Ch concentration under both normal and depolarized conditions. However, the only significant effect of external [2H4]Ch on ACh metabolism was that it increased ACh release during the initial 10 min, when the preparation was depolarized with K+. The efflux of endogenous [2H0]Ch was increased initially (10 min) and slowed over a 60-min period by 25 mM K+, and increased when [2H4]Ch in the medium was increased. Changes in ACh synthesis and release were dependent upon the time exposure of slices to high K+, and the results suggest that Ch favors initial rates of ACh synthesis, but that Ch influences ACh release primarily under conditions of stress (i.e., depolarization).     

Effect of ethyl choline mustard on choline dehydrogenase and other enzymes of choline metabolism

The effect of ethyl choline mustard (ECMA), and effective irreversible inhibitor of choline transport, was investigated on the enzymes of choline metabolism. ECMA at concentrations of 50 microM hardly affected choline acetyltransferase and caused only a 20% inhibition of choline kinase at a concentration of 1 mM. However, the mustard was an extremely effective inhibitor of choline dehydrogenase, producing 50% inhibition at concentrations of 6 microM. The inhibition was prevented by incubation in the presence of choline or by prior reaction of the mustard with thiosulphate. Separation of the components of the ECMA solution on TLC suggested that only the compound with an aziridine ring was an effective inhibitor of choline dehydrogenase. The inhibition was resistant to the washing out of excess unreacted mustard. The rate constant of inhibition was 395 M-1 X S-1. By the use of [3H]ECMA a single polypeptide in the enzyme preparation having a MW of 67,000 was labelled. The labelling was thiosulphate-sensitive and prevented by incubation with choline. It is concluded that ECMA is an irreversible inhibitor of choline dehydrogenase. It is at least as effective an inhibitor of choline dehydrogenase as of the choline transport system, although it does not appreciably inhibit choline acetyltransferase or choline kinase in the micromolar range.     

Seizures increase acetylcholine and choline concentrations in rat brain regions

Seizures induced by three convulsant treatment produced differential effects on the concentration of acetylcholine in rat brain. Status epilepticus induced by (i) coadministration of lithium and pilocarpine caused massive increases in the concentration of acetylcholine in the cerebral cortex and hippocampus, (ii) a high dose of pilocarpine did not cause an increase of acetylcholine, and (iii) kainate increased acetylcholine, but the magnitude was lower than with the lithium/pilocarpine model. The finding that the acetylcholine concentration increases in two models of status epilepticus in the cortex and hippocampus is in direct contrast with manyin vitro reports in which excessive stimulation causes depletion of acetylcholine. The concentration of choline increased during seizures with all three models. This is likely to be due to calcium- and agonist-induced activation of phospholipase C and/or D activity causing cleavage of choline-containing lipids. The excessive acetylcholine present during status epilepticus induced by lithium and pilocarpine was responsive to pharmacological manipulation. Atropine tended to decrease acetylcholine, similar to its effects in controls. The N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801, reduced the excessive concentration of acetylcholine, especially in the cortex. Inhibition of choline uptake by hemicholinium-3 (HC-3) administered icv reduced the acetylcholine concentration in controls and when given to rats during status epilepticus. These results demonstrate that the rat brain concentrations of acetylcholine and choline can increase during status epilepticus. The accumulated acetylcholine was not in a static, inactive compartment, but was actively turning-over and was responsive to drug treatments. Excessive concentrations of acetylcholine and/or choline may play a role in seizure maintenance and in the neuronal damage and lethality associated with status epilepticus.     

Turnover Rate of Brain Acetylcholine Using HPLC Separation of the Transmitter

A simple, reliable method was developed for measuring brain acetylcholine (ACh) turnover using HPLC methodology. Mice were injected intravenously with [3H]choline ([3H]Ch), and the turnover rate of ACh was calculated from the formation of [3H]ACh. Ch and ACh were separated from phosphorylcholine and from other radioactive compounds using tetraphenylboron extraction and counterion/reverse-phase chromatography. Endogenous Ch and ACh were quantified electrochemically through hydrogen peroxide production in a postcolumn reactor containing covalently bonded ACh esterase and Ch oxidase. Labeled Ch and ACh were quantified in the same sample by collecting the chromatographic fractions for radioactive content determinations. The method is rapid, well adapted to large series, and highly reproducible, with recoveries of 72.1% for Ch and 79.3% for ACh. The turnover value in mouse cerebral hemispheres was 16.02 nmol g-1 min-1 and decreased to 9.94 nmol g-1 min-1 in mice treated with oxotremorine.     

Deficits in acetylcholine homeostasis, receptors and behaviors in choline transporter heterozygous mice

Cholinergic neurons elaborate a hemicholinium-3 (HC-3) sensitive choline transporter (CHT) that mediates presynaptic, high-affinity choline uptake (HACU) in support of acetylcholine (ACh) synthesis and release. Homozygous deletion of CHT (-/-) is lethal shortly after birth (Ferguson et al. 2004), consistent with CHT as an essential component of cholinergic signaling, but precluding functional analyses of CHT contributions in adult animals. In contrast, CHT+/- mice are viable, fertile and display normal levels of synaptosomal HACU, yet demonstrate reduced CHT protein and increased sensitivity to HC-3, suggestive of underlying cholinergic hypofunction. We find that CHT+/- mice are equivalent to CHT+/+ siblings on measures of motor co-ordination (rotarod), general activity (open field), anxiety (elevated plus maze, light/dark paradigms) and spatial learning and memory (Morris water maze). However, CHT+/- mice display impaired performance as a result of physical challenge in the treadmill paradigm, as well as reduced sensitivity to challenge with the muscarinic receptor antagonist scopolamine in the open field paradigm. These behavioral alterations are accompanied by significantly reduced brain ACh levels, elevated choline levels and brain region-specific decreased expression of M1 and M2 muscarinic acetylcholine receptors. Our studies suggest that CHT hemizygosity results in adequate baseline ACh stores, sufficient to sustain many phenotypes, but normal sensitivities to physical and/or pharmacological challenge require full cholinergic signaling capacity.     

Complementary remedy of aged-related learning and memory deficits via exogenous choline acetyltransferase

The present study aimed to examine whether the aged mice with naturally occurring cognitive deficits in learning and memory would benefit from supplementation of choline acetyltransferase (ChAT), the biosynthetic enzyme for neurotransmitter acetylcholine. Delivered by protein transduction domain (PTD), ChAT could pass through the blood-brain barrier, enter the neurons, interact with heat shock protein 70kDa, and retain enzyme activity. In behavior tests, PTD-ChAT given to the aged and memory-deficient mice almost completely reversed the behavioral changes, such as impairment of memory retention in the step-through test (an index of long-term memory) and prolonged swimming time in water maze test (an index of spatial recognition memory). The results suggest a novel and potential therapeutic use of PTD-ChAT in the age-related cognitive deficits.     

Determination of choline in phloem exudates of trees

Summary Choline, a necessary compound in an artificial diet for phloem-feeding aphids, was determined quantitatively in the phloem exudates of 16 tree species. The method used was a combination of choline oxidase action and oxygen determination with an oxygen electrode. Choline was found in all species, the concentration ranging between 36 and 5340 M.     

Human choline dehydrogenase: Medical promises and biochemical challenges

Human choline dehydrogenase (CHD) is located in the inner membrane of mitochondria primarily in liver and kidney and catalyzes the oxidation of choline to glycine betaine. Its physiological role is to regulate the concentrations of choline and glycine betaine in the blood and cells. Choline is important for regulation of gene expression, the biosynthesis of lipoproteins and membrane phospholipids and for the biosynthesis of the neurotransmitter acetylcholine; glycine betaine plays important roles as a primary intracellular osmoprotectant and as methyl donor for the biosynthesis of methionine from homocysteine, a required step for the synthesis of the ubiquitous methyl donor S-adenosyl methionine. Recently, CHD has generated considerable medical attention due to its association with various human pathologies, including male infertility, homocysteinuria, breast cancer and metabolic syndrome. Despite the renewed interest, the biochemical characterization of the enzyme has lagged behind due to difficulties in the obtainment of purified, active and stable enzyme. This review article summarizes the medical relevance and the physiological roles of human CHD, highlights the biochemical knowledge on the enzyme, and provides an analysis based on the comparison of the protein sequence with that of bacterial choline oxidase, for which structural and biochemical information is available.     

Choline Uptake and Acetylcholine Synthesis in Synaptosomes: Investigations Using Two Different Labeled Variants of Choline

Abstract: Using sequential incubations in media of different K⁺ composition, we investigated the dynamics of choline (Ch) uptake and acetylcholine (ACh) synthesis in rat brain synaptosomal preparations, using two different deuterated variants of choline and a gas chromatographic-mass spectrometric (GC-MS) assay for ACh and Ch. Synaptosomes were preincubated for 10 min in a Krebs medium with or without high K⁺ and with 2 μM-[²H₉]Ch. At the end of the preincubation all variants of ACh and Ch were measured in samples of the pellet and medium. In the second incubation (4 min) samples of synaptosomes were resuspended in normal or high K⁺ solutions containing [²H₄]Ch (2 μM) and all variants of ACh and Ch were measured in the pellet and medium at the end of this period. This protocol allowed us to compare the effects of preincubation in normal or high K⁺ solution on the metabolism during a second low or high K⁺ incubation of a [²H₉]Ch pool accumulated during the preincubation period. Moreover, we were able to compare and contrast the effects of this protocol on [²H₉]Ch metabolism versus [²H₄]Ch metabolism. The most striking result we obtained was that [²H₉]Ch that had been retained by the synaptosomes after the preincubation was not acetylated during a subsequent incubation in normal or high K⁺ media. This result suggests that if an intraterminal pool of Ch is involved in ACh synthesis, the size of this pool is below the limits of detection of our assay. We have confirmed the observation that a prior depolarizing incubation results in an enhanced uptake of Ch during a second incubation in normal K⁺ Krebs. Moreover, Ch uptake is stimulated by prior incubation under depolarizing conditions relative to normal preincubation when the second incubation is in a high K⁺ solution. These results are discussed in terms of current models of the regulation of ACh synthesis in brain.     

Distribution in brain and retina of four enzymes of acetyl CoA synthesis in relation to choline acetyl transferase and acetylcholine esterase

Eleven regions of mouse brain and twelve layers of monkey retina were assayed for choline acetyl transferase (ChAT), acetylcholine esterase (AChE), and 4 enzymes that synthesize acetyl CoA. The purpose was to seek evidence concerning the source of acetyl CoA for acetylcholine generation. In brain ATP citrate lyase was strongly correlated with ChAT as well as AChE (r=0.914 in both cases). Weak, but statistically significant correlation, was observed between ChAT and both cytoplasmic and mitochondrial thiolase, whereas there was a significant negative correlation between ChAT and acetyl thiokinase. In retina ChAT was essentially limited to the inner plexiform and ganglion cell layers, whereas substantial AChE activity extended as well into inner nuclear, outer plexiform and fiber layers, but no further. ATP citrate lyase activity was also highest in the inner four retinal layers, but was not strongly correlated with either ChAT or AChE (r=0.724 and 0.761, respectively). Correlation between ChAT and acetyl thiokinase was at least as strong (r=0.757), and in the six inner layers of retina, the correlation between ChAT and acetylthiokinase was very strong (r=0.932).Special issue dedicated to Dr. Lawrence Austin     

Effects of chronic (dietary) choline availability on the transport of choline across the blood-brain barrier

The effects of dietary choline availability on the transport of choline across the blood-brain barrier (BBB) were investigated using the intracarotid injection technique. Maintenance of rats on choline-deficient, basal choline, or choline-supplemented diets for 28-32 days led to respective increases in blood levels of choline and correlative increases in the velocity of transport of choline measured using a buffer injectate. When serum from these rats was included in the injectate and transport determined in control animals, there was a marked inhibition of choline transport that was related to the concentration of choline in the diets. Results suggest that the activity of the choline carrier at the BBB is antagonized by an inhibitory substance in serum whose concentration or activity may be modified by chronic alterations in circulating levels of choline and whose presence may normally regulate the velocity of choline transport.     

Modulation of acetylcholinesterase and voltage-gated Na(+) channels in choline acetyltransferase- transfected neuroblastoma clones

Neurotransmitters appear early in the developing embryo and may play a role in the regulation of neuronal differentiation. To study potential effects of acetylcholine production in neuronal differentiation, we used the FB5 subclone of N18TG2 murine neuroblastoma cells stably transfected with cDNA for choline acetyltransferase. We tested whether the forced acetylcholine production can modify the expression or the cellular localization of different neuronal markers. We studied the activity, localization, and secretion of acetylcholinesterase in view of its possible role in the modulation of the morphogenetic action of acetylcholine and of its proposed role of a regulator of neurite outgrowth. FB5 cells are characterized by a high level of acetylcholinesterase, predominantly released into the culture medium. Acetylcholinesterase secretion into the medium was lower in choline acetyltransferase-transfected clones than in nontransfected and antisense-transfected controls. Moreover, sequential extraction of acetylcholinesterase revealed that detergent-extracted, i.e., membrane-associated, activity was higher in the transfected clones expressing choline acetyltransferase activity than in both control groups. These observations suggest that a shift occurs in the utilization of acetylcholinesterase in choline acetyltransferase-transfected clones from a secretion pathway to a pathway leading to membrane localization. In addition, the choline acetyltransferase-positive clones showed higher densities of voltage-gated Na(+) channels and enhanced high-affinity choline uptake, suggesting the accomplishment of a more advanced differentiated neuronal phenotype. Finally, binding experiments demonstrated the presence of muscarinic acetylcholine receptors in all examined clones. This observation is consistent with the proposed existence of an autocrine loop, which may be important for the enhancement in the expression of neurospecific traits.