Eponymous plot of Richard J. Kitz and Irwin B. Wilson in biochemistry.

Irwin B. Wilson and anesthesiologist Richard J. Kitz found the enzyme acetylcholinesterase to be inactivated in two steps by covalently acting molecules resembling acetylcholine in structure. Such molecules rapidly and reversibly bind to the active site of the enzyme. Next, the reversible complex undergoes covalent fixation at a characteristic rate. The Kitz-Wilson phenomenon applies to many cases of time-dependent enzyme inhibition. Experimental data are commonly graphed in linear fashion on "Kitz-Wilson plots". Kitz also contributed to a gas chromatography-mass spectrometry assay for acetylcholine that was suitable for the nonbiological detection of that neurotransmitter in mammalian brain.

Electrocatalytic microelectrode detectors for choline and acetylcholine following separation by capillary electrophoresis.

Two electrocatalytic enzyme modified microelectrode systems were employed as end-column amperometric detectors of choline (Ch) and acetylcholine (ACh) following separation by capillary electrophoresis (CE). Horseradish peroxidase cross-linked in an osmium based redox polymer hydrogel (HRP-Os) was physically adsorbed on Au microelectrodes followed by chemical cross-linking of the enzymes acetylcholinesterase (AChE) and choline oxidase (ChO). An alternative approach utilized the deposition of the transition metal catalyst, Prussian Blue (PB), on Pt microelectrodes as the electrocatalyst. Utilizing butyrylcholine (BuCh) as an internal standard, the HRP-Os/AChE-ChO and PB/AChE-ChO electrodes exhibited excellent linear responses from 2-2000 microM and 10-2000 microM, respectively, for both Ch and ACh. Detection limits of 0.1 microM or 38 amol were determined for the HRP-Os/AChE-ChO electrode. The limit of detection for ACh and Ch at the PB/AChE-ChO electrode was 5 microM or 9.5 fmol. The electrodes were operated at potentials of +0.10 and -0.10 V vs Ag/AgCl (3 M NaCl), respectively, and thus minimized the potential response from oxidizable interferences. In addition, both electrocatalytic electrodes showed good operational stability for more than 70 h. The enhanced detection capability of the HRP-Os/AChE-ChO and PB/AChE-ChO electrodes in combination with efficient CE separation of Ch and ACh provides a new sensitive and selective strategy for monitoring and quantifying these cholinergic biomarkers in biological fluids.

Single cells as biosensors for chemical separations.

A biosensor system based on the response of living cells was demonstrated that can detect specific components of a complex mixture fractionated by a microcolumn separation technique. This system uses ligand-receptor binding and signal-transduction pathways to biochemically amplify the presence of an analyte after electrophoretic separation. The transduced signal was measured by means of two approaches: (i) fluorescence determination of intracellular calcium concentrations in one or more rat PC-12 cells and (ii) measurement of transmembrane current in a Xenopus laevis oocyte microinjected with messenger RNA that encodes a specific receptor. This analysis system has the potential to identify biologically active ligands present in a complex mixture with exceptional sensitivity and selectivity.

Voltammetric measurements of neurotransmitter-acetylcholine through metallic nanoparticles embedded 2-D material.

The most generally spread neurotransmitter acetylcholine (Ach) is used as a chemical messenger assisting in conveying signals transversely through the nerve synapse. Herein, two enzymes acetylcholinesterase and choline oxidase were covalently immobilized over the gold nanoparticles (AuNPs) embedded graphene oxide (GO; 2D carbon material) nanocomposite modified ITO coated glass plate. The synergetic and unique properties of AuNPs and GO present in nanocomposite are used to detect the ultra-small concentration of analyte, Ach. The prepared nanocomposites were characterized using different techniques i.e. TEM, XRD, SEM, FTIR, UV-Vis and Raman Spectroscopy. All the electrochemical measurements were performed using 3 electrodes integrated electrochemical system by introducing Ach through varying its concentration from 100 pM to 1000 nM. Cyclic voltammetry curves for different concentrations of Ach indicate the facile charge transfer process over the working electrode. Square wave voltammetry curves indicate the good sensing measurements of the modified electrode at the fixed potential. The limit of detection was found to be as low as 100 pM. In addition to these, selectivity of the electrode towards Ach molecule was confirmed by measuring the response towards other interfering agents. Besides this, the present nano interface is capable of detecting Ach in biological fluid such as serum.

Analysis of acetylcholine, choline and butyrobetaine in human liver tissues by hydrophilic interaction liquid chromatography-tandem mass spectrometry.

The strong polar quaternary ammoniums, acetylcholine (ACh), choline (Ch) and butyrobetaine (BB, (3-carboxypropyl)trimethylammonium), are believed playing important roles in liver metabolism. These metabolites are at low levels and are weakly retained on reversed-phase liquid chromatographic (RP-LC) columns. Several hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) methods have been reported to analyze these compounds from different samples. However, no application to human liver tissues has been published. In this study, HILIC-MS/MS method was developed to simultaneously determine these three metabolites in human liver tissues. They were simply extracted from tissue, separated on a HILIC column, and detected by tandem MS in the mode of multiple reaction monitoring (MRM). Further studies on the recovery and repeatability based on real samples indicated the method was accurate and reliable. This method was successfully applied to measure the levels of ACh, Ch and BB in 61 human liver tissue samples including normal, hepatocellular carcinoma (HCC) and matched non-cancerous liver tissues. By comparison of Ch and ACh contents in 29 HCC with their matched non-cancerous liver tissues, it was found that ACh content increased in 11/29 HCC cases and decreased in 13/29 cases. Furthermore, the ACh/Ch ratio increased in 16/29 HCC cases, while it decreased in 8/29 cases. These results strongly indicated that there exist different patterns of ACh content in cancer tissues among HCC patients, thus highlighting the understanding of ACh and its relevant signal pathways in hepatic carcinogenesis and HCC progression.

Detection of Ca2+-induced acetylcholine released from leukemic T-cells using an amperometric microfluidic sensor.

A microfluidic structured-dual electrodes sensor comprising of a pair of screen printed carbon electrodes was fabricated to detect acetylcholine, where one of them was used for an enzyme reaction and another for a detection electrode. The former was coated with gold nanoparticles and the latter with a porous gold layer, followed by electropolymerization of 2, 2:5,2-terthiophene-3-(p-benzoic acid) (pTTBA) on both the electrodes. Then, acetylcholinesterase was covalently attached onto the reaction electrode, and hydrazine and choline oxidase were co-immobilized on the detection electrode. The layers of both modified electrodes were characterized employing voltammetry, field emission scanning electron microscopy, X-ray photoelectron spectroscopy, and quartz crystal microscopy. After the modifications of both electrode surfaces, they were precisely faced each other to form a microfluidic channel structure, where H2O2 produced from the sequential enzymatic reactions was reduced by hydrazine to obtain the analytical signal which was analyzed by the detection electrode. The microfluidic sensor at the optimized experimental conditions exhibited a wide dynamic range from 0.7nM to 1500µM with the detection limit of 0.6 ± 0.1nM based on 3s (S/N = 3). The biomedical application of the proposed sensor was evaluated by detecting acetylcholine in human plasma samples. Moreover, the Ca2+-induced acetylcholine released in leukemic T-cells was also investigated to show the in vitro detection ability of the designed microfluidic sensor. Interference due to the real component matrix were also studied and long term stability of the designed sensor was evaluated. The analytical performance of the designed sensor was also compared with commercially available ACh detection kit.

Characterization of prejunctional muscarinic autoreceptors in the guinea-pig trachea.

1. The effects of ten muscarinic antagonists on electrically evoked [3H]-acetylcholine release and muscle contraction were compared in an epithelium-free preparation of the guinea-pig trachea that had been preincubated with [3H]-choline. 2. The M3-selective antagonists UH-AH 37, 4-diphenyl-acetoxy-N-piperidine methobromide and para-fluorohexahydrosiladiphenidol were more potent in reducing the contractile response than in facilitating the evoked [3H]-acetylcholine release. Hexahydrosiladiphenidol did not discriminate between pre- and postjunctional effects. The rank order of the postjunctional potencies of the ten antagonists as well as the postjunctional pA2 values obtained for hexahydrosiladiphenidol (7.95) and AQ-RA (7.08) identified the muscular receptor as an M3 subtype. 3. The M2-selective antagonists methoctramine, AF-DX 116 and AQ-RA 741 were more potent in facilitating the evoked [3H]-acetylcholine release than in inhibiting the contractile response. The increase in release by low concentrations of methoctramine, AF-DX 116 and AQ-RA 741 was paralleled by an enhancement of the stimulation-evoked contractions. 4. Comparison of the pre- and postjunctional potencies of the M1-, M2- and M3-selective antagonists suggests that autoinhibition of acetylcholine release is mediated via an 'M2-like' receptor which differs from the cardiac type M2 receptor in its relatively high affinity for hexahydrosiladiphenidol.

Rational design of biologically important chemosensors: a novel receptor for selective recognition of acetylcholine over ammonium cations.

[structure: see text] In consideration of competition between cation-pi and hydrogen bond interaction forces, we designed a novel receptor, 1,3,5-tris(pyrrolyl)benzene, which shows high selectivity for acetylcholine (ACh). The selectivity of the receptor for ACh over other ammonium cations is demonstrated by the ion-selective electrode (ISE) method in buffer solution. The binding free energy of the receptor with ACh in chloroform solution is measured to be 3.65 kcal/mol in the presence of chloride anion by nuclear magnetic resonance spectroscopy, and that in water is estimated to be much greater ( approximately 6 kcal/mol).

Experimental autoimmune myasthenia gravis and myasthenia gravis: biochemical and immunochemical aspects.

Stucture of acetylcholine receptor protein (AChR) purified from Electrophorus electricus (eel) by affinity chromatography is described. AChR is detected in extracts from human muscle, rat muscle, and rat thymus. Rats immunized with eel AChR develop humoral antibodies, a small fraction of which recognize AChR from rat muscle. Rats immunized with AChR exhibit myasthenia, but those immunized with denatured AChR do not. Immunoglobulin fraction of antisera to eel AChR can block the activity of AChR in electroplaques. Sera from patients with myasthenia gravis contain antibodies to AChR from human muscle detectabe at an average value 300-fold the background level in sera from nonmyasthenics. Relationship of thymoma and disease intensity to antibody titer is examined. The chronic phase of EAMG appears a good model of MG, since in both cases similar concentrations of 7-S immunoglobulin against determinants on muscle AChR other than the toxin binding site are found. Assay of anti-AChR antibody in sera from MG patients using AChR from rat muscle gives titers 10%-15% of those obtained using AChR from human muscle, and using AChR from eel gives negligible titers. The assay method described for assaying antibodies against AChR from human muscle is suggested as a diagnostic test for MG.

The lipid and protein content of cholinergic synaptic vesicles from the electric organ of Torpedo marmorata purified to constant composition: implications for vesicle structure.

The lipid, protein, acetylcholine and ATP content of cholinergic synaptic vesicles isolated from the richly innervated electric organ of Torpedo marmorata and purified to constant composition has been determined. The number of vesicles present in the preparations has been estimated by quantitative electron microscopy and the mean composition of the vesicle deduced. The acetylcholine content of the purest preparations was considerably greater than that previously attained and reached a mean of 6.10 mmole/g of protein and 2.6 X 10(5) molecules/vesicle; the mean values, for all determinations, were 4.1 +/- S.E.M. 0.6 and 2.6 X 10(5) +/- S.E.M. 0.6 X 10(5) respectively. The lipid and protein content of the vesicle (about 140 and 80 ag/vesicle respectively) is relatively low, indicating a thin, lipid-rich membrane and a highly hydrated core of which not more than 1-2% can be occupied by protein. These findings are consistent with conclusions drawn from recent density determinations made at different osmotic pressures using penetrating and non-penetrating gradients.

Enhancement of acetylcholine release in the hippocampus by 6R-L-erythro-5,6,7,8-tetrahydrobiopterin is mediated by 5-hydroxytryptamine.

Recently, we reported that 6R-L-erythro-tetrahydrobiopterin (6R-BH4), a natural cofactor for L-aromatic amino acid hydroxylases, enhances in vivo release of acetylcholine (ACh) in the rat hippocampus: the enhancement was abolished after depletion of brain catecholamines and 5-hydroxytryptamine (5-HT) by pretreatment with reserpine. In the present study, we have used in vivo brain microdialysis to clarify the neuronal mechanism involved in the enhancement of ACh release by 6R-BH4. After depletion of catecholamines by pretreatment of rats with alpha-methyl-p-tyrosine, 6R-BH4 added to the perfusion fluid still induced an increase in extracellular ACh levels monitored by microdialysis as an index of ACh release. In contrast, after depletion of 5-HT by pretreatment with p-chlorophenylalanine, most of the 6R-BH4-induced enhancement was eliminated. Exogenous 5-HT and dopamine (DA) but not noradrenaline added to the perfusion fluid stimulated ACh release with 5-HT being far more potent. Intraperitoneal administration of 5-hydroxytryptophan and L-DOPA also enhanced ACh release, presumably by their conversion to 5-HT and catecholamines, respectively. Administration of 6R-BH4 increased hippocampal 5-HT release, as indicated by increased extracellular levels of the major 5-HT metabolite, 5-hydroxyindoleacetic acid. These results suggest that 6R-BH4 stimulates ACh release in the hippocampus, mainly by augmenting release of 5-HT, a potent stimulator of ACh release, and partly by augmenting release of DA.

An extract of lionfish (Pterois volitans) spine tissue contains acetylcholine and a toxin that affects neuromuscular transmission.

A soluble toxic extract derived from spine tissue of the lionfish (Pterois volitans) decreased heart rate and force of contraction in isolated clam and frog hearts. These actions were due to the presence of micromolar concentrations of acetylcholine in the extract. Toxicity was retained after hydrolysis of acetylcholine by exogenous acetylcholinesterase, but heart function was no longer affected. Toxin treated in this way induced muscle fibrillation in an isolated nerve-muscle preparation, followed by blockade of neuromuscular transmission. Bursts of transient depolarizations were recorded at the muscle endplate shortly after toxin addition that correlated in time with the duration of toxin-induced muscle fibrillation. These effects are thought to be due to the increased release and then depletion of acetylcholine from the nerve terminal.

Bioanalysis of picomole amounts of acetylcholine by ion-pair partition chromatography applied to rat sciatic nerve.

A method for determination of acetylcholine in small, discrete biological objects by use of ion-pair technique has been developed. Acetylcholine is extracted as an ion pair with 3,5-di-t-butyl-2-hydroxybenzenesulphonate and separated from co-extracted components by ion-pair partition chromatography with picrate as the counter ion and porous cellulose as support. The quantitative evaluation is made from the acetylcholine peak in the chromatogram obtained by ultraviolet detection. Acetylcholine has been analysed in 1 cm large pieces of rat sciatic nerve containing about 60 pmol (10 ng). The overall recovery of the method is 100 +/- 10% at the 120 pmol level of acetylcholine in a sample.

Acetylcholinesterase inhibitors from the twigs of Vaccinium oldhami Miquel.

In the course of finding Korean natural products with acetylcholinesterase (AChE) inhibitory activity, we found that a methanolic extract of the twigs of Vaccinium oldhami significantly inhibited AChE. Bioassay-guided fractionation of the methanolic extract resulted in the isolation of two compounds, taraxerol (1) and scopoletin (2), as active constituents. These compounds inhibited AChE activity in a dose-dependent manner, and the IC50 values of compounds 1 and 2 were 33.6 (79 microM) and 10.0 (52 microM) microg/mL, respectively.

Effect of Mg(2+)-ATP on acetylcholinesterase of Electrophorus electricus (L.).

The effect of Mg(2+)-ATP on purified acetylcholinesterase (AChE) from electric tissue of Electrophorus electricus (L.) was studied. The enzymatic activities were measured with acetylcholine and acetylthiocholine as substrates. The kinetic parameters Vmax, Km and Hill coefficient (nH), for acetylcholine and acetylthiocholine were modified with Mg(2+)-ATP. It was shown that acetylcholinesterase presents an apparent activation at high concentration of substrates and an inhibition in the presence of Mg(2+)-ATP at low concentration of acetylcholine and acetylthiocholine. In addition, the data suggest that Mg(2+)-ATP induced an allosteric modulation of the acetylcholinesterase obtained from Electrophorous electricus (L.), and indicate an active adenosine triphosphate participation during cholinergic activity.

[Assessment of the activity of choline acetyltransferase in heart tissues]. / Otsenka aktivnosti kholinatsetiltransferazy v tkaniakh serdtsa.

A procedure is described for the radioisotope assay of acetylcholine transferase activity (EC 2.3.1.6), which involved specific synthesis of acetylcholine in vivo. Bromine acetylcholine was used as an inhibitor of the enzyme; 14C-AcCoA was used as a substrate and product of the enzymatic reaction. 14C-acetylcholine was separated from the substrate by means of anion exchange chromatography. The procedure described was 5 times more sensitive than the methods developed by F. Fonnum (1975) and S. Tucek (1983) being similarly reproducible. The assay was tested in experiments with rats under various conditions of hyperbaric oxygenation, in simulation of myocardial infarction as well as in moderate immobilization stress. The findings suggest that estimation of the acetylcholine transferase activity may be involved in complex evaluation of the cholinergic system state in tissues, which is essential for the study of pathogenesis of their dysfunctions and development of respective approaches to eliminate these impairments.

An eco-friendly, simple, and sensitive fluorescence biosensor for the detection of choline and acetylcholine based on C-dots and the Fenton reaction.

A simple and novel method is proposed for the preparation of Carbon dots (C-dots) with excellent properties. We firstly demonstrated that the fluorescence of C-dots decreased apparently in the presence of H2O2 and Fe(2+). Based on the this finding, C-dots are successfully adopted as probes for the detection of H2O2. After the experimental conditions are optimized, the limit of detection (LOD) for H2O2 is found to be 0.1 µM. Furthermore, we established an eco-friendly, simple and sensitive biosensor for the detection of choline and acetylcholine (ACh) based on the detection of H2O2 using C-dots as probes. The detection limit for choline is 0.1 µM and the linear range is 0.1-40 µM. The detection limit for ACh is found to be 0.5 µM and the linear range is 0.5-60 µM. The excellent performance of the proposed biosensor shows that this method possesses the potential for practical application.