Determination Of Acetylcholinesterase Activity Research Articles

Trace Amount of Bi-Doped Core-Shell Pd@Pt Mesoporous Nanospheres with Specifically Enhanced Peroxidase-Like Activity Enable Sensitive and Accurate Detection of Acetylcholinesterase and Organophosphorus Nerve Agents.

The urgent need for sensitive and accurate assays to monitor acetylcholinesterase (AChE) activity and organophosphorus pesticides (OPs) arises from the imperative to safeguard human health and protect the ecosystem. Due to its cost-effectiveness, ease of operation, and rapid response, nanozyme-based colorimetry has been widely utilized in the determination of AChE activity and OPs. However, the rational design of nanozymes with high activity and specificity remains a great challenge. Herein, trace amount of Bi-doped core-shell Pd@Pt mesoporous nanospheres (Pd@PtBi2) have been successfully synthesized, exhibiting good peroxidase-like activity and specificity. With the incorporation of trace bismuth, there is a more than 4-fold enhancement in the peroxidase-like performance of Pd@PtBi2 compared to that of Pd@Pt. Besides, no significant improvement of oxidase-like and catalase-like activities of Pd@PtBi2 was found, which prevents interference from O2 and undesirable consumption of substrate H2O2. Based on the blocking impact of thiocholine, a colorimetric detection platform utilizing Pd@PtBi2 was constructed to monitor AChE activity with sensitivity and selectivity. Given the inhibition of OPs on AChE activity, a biosensor was further developed by integrating Pd@PtBi2 with AChE to detect OPs, capitalizing on the cascade amplification strategy. The OP biosensor achieved a detection limit as low as 0.06 ng mL-1, exhibiting high sensitivity and anti-interference ability. This work is promising for the construction of nanozymes with high activity and specificity, as well as the development of nanozyme-based colorimetric biosensors.

Cladonia rangiformis Acetone Extract— New Insight into the Chemical Composition and Biological Activity

Introduction Cladonia rangiformis, also known as reindeer lichen, has been used for various remedies in folk medicine. The rich nutritional value of this lichen is reflected in the fact that it contains atranorin, which has been proven to have numerous biological activities. Objective The chemical composition, antioxidant, anticholinesterase, and antigenotoxic activities of the C. rangiformis acetone extract were investigated. The novelty of this work is that anticholinesterase activity, protective effect on human lymphocytes, and antioxidant activity by the cupric-reducing antioxidant capacity (CUPRAC) method of C. rangiformis extract were determined for the first time, as well as gas chromatography - mass spectrometry (GC-MS) profile. Methods Chemical composition was carried out by high-performance liquid chromatography (HPLC)-diode array detector and GC-MS. Antigenotoxic effect was evaluated in human lymphocytes by cytokinesis-block micronucleus assay. Total phenolic content, DPPH and ABTS radical scavenging capacities, CUPRAC, and total reducing power were determined spectrophotometrically. Determination of acetylcholinesterase activity was performed by Ellman's colorimetry assay. The statistical analysis of variance (One-way ANOVA) was performed using Origin software package version 7.0. Results HPLC method was used to identified 3 compounds with atranorin as predominant constituent (97.2%). Fumarprotocetraric and atraric acid were represented in a much smaller mutually approximate amount, 0.3 and 0.5%, respectively. Rangiformic acid was the most abundant volatile compound. Acetone extract of C. rangiformis (2 μg/mL) decreased the frequency of micronuclei by 15.5% while the radioprotectant amifostine reduced the frequency of occurrence of micronuclei by 11.4%. On the contrary, at a concentration of 1.0 mg/mL the extract exhibited an activating effect on cholinesterase (2.0%), while at a concentration of 10.0 mg/mL it showed a weak inhibitory effect (7.3%). The total phenol content was 132.71 μg gallic acid equivalents per mg of dry extract. The IC50 value for the DPPH experiment was 16.19 mg/mL and for ABTS was 11.80 mg/mL. Cupric reducing capacity and total reducing power were 17.81 μg Trolox equivalents per mg of dry extract and 0.45 μg ascorbic acid equivalents per mg of dry extract, respectively. Conclusion In addition to previously identified compounds in the acetone extract, atraric acid was identified for the first time by using HPLC method, and orcinol, β-orcinol, lauric alcohol, atranol, and rangiformic acid by using GC-MS method. The results of biological activity proved that acetone extract had antioxidant capacity and weak anticholinesterase activity. Reduction of the number of micronuclei in human lymphocytes, classified C. rangiformis as promising substances source with beneficial effects on DNA damage.

A Comparison of the Neuroprotective and Reactivating Efficacy of a Novel Bispyridinium Oxime K870 with Commonly Used Pralidoxime and the Oxime HI-6 in Tabun-Poisoned Rats.

The comparison of neuroprotective and central reactivating effects of the oxime K870 in combination with atropine with the efficacy of standard antidotal treatment in tabun-poisoned rats. The neuroprotective effects of antidotal treatment were determined in rats poisoned with tabun at a sublethal dose using a functional observational battery 2 h and 24 h after tabun administration, the tabun-induced brain damage was investigated by the histopathological evaluation and central reactivating effects of oximes was evaluated by the determination of acetylcholinesterase activity in the brain using a standard spectrophotometric method. The central reactivating efficacy of a newly developed oxime K870 roughly corresponds to the central reactivating efficacy of pralidoxime while the ability of the oxime HI-6 to reactivate tabun-inhibited acetylcholinesterase in the brain was negligible. The ability of the oxime K870 to decrease tabun-induced acute neurotoxicity was slightly higher than that of pralidoxime and similar to the oxime HI-6. These results roughly correspond to the histopathological evaluation of tabun-induced brain damage. The newly synthesized oxime K870 is not a suitable replacement for commonly used oximes in the antidotal treatment of acute tabun poisonings because its neuroprotective efficacy is only slightly higher or similar compared to studied currently used oximes.

Evaluation of the accuracy of “ChE check mobile” in measurement of acetylcholinesterase in pesticide poisoning

Background: Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are used in clinical management to confirm the diagnosis and indicate the severity of organophosphorus and carbamate poisoning. ChE check mobile is a new portable cholinesterase testing system developed in Germany. The study aims to evaluate the accuracy of ChE check mobile compared to the standard reference method and Test-mate ChE system.Methods: Patients with organophosphorus and carbamate poisoning were recruited from two general hospitals in Sri Lanka between September 2013 and November 2014. The AChE was measured using the three methods.Results: Blood samples were collected from 185 self-poisoned patients (170 organophosphorus and 15 carbamate) and 20 normal individuals. ChE check mobile correlated well with spectrophotometer readings (Pearson’s correlation coefficient 0.87) but gave higher values (Mean bias for AChE: +6.55 (95% CI: −11 to 24) U/g Hb). A similar positive bias from Test-mate results was also observed. Applying a correction factor derived from the volunteer samples (dividing by 1.353) greatly improved agreement in pesticide poisoned patients.Conclusions: ChE check mobile system allowed for rapid determination of AChE activity but gave somewhat higher AChE compared to other methods. Applying a correction factor of 1.353 provide a good agreement to both reference and Test-mate ChE machine in this setting.

Berberine protects against memory impairment and anxiogenic-like behavior in rats submitted to sporadic Alzheimer’s-like dementia: Involvement of acetylcholinesterase and cell death

The present study aimed to investigate the effects of berberine (BRB) on spatial and learning memory, anxiety, acetylcholinesterase activity and cell death in an experimental model of intracerebroventricular streptozotocin (ICV-STZ) induced sporadic Alzheimer’s-like dementia. Sixty male Wistar rats were randomly divided into six groups: control (CTR), BRB 50mg/kg (BRB 50), BRB 100mg/kg (BRB 100), streptozotocin (STZ), streptozotocin plus BRB 50mg/kg (STZ+BRB 50), and streptozotocin plus BRB 100mg/kg (STZ+BRB 100). Rats were injected with ICV-STZ (3mg/kg) or saline, and daily oral BRB treatment began on day 4 for a period of 21days. Behavioral tests were carried out on day 17, and rats were euthanized on day 24. Cell death analysis and determination of acetylcholinesterase activity was performed on the cerebral cortex and hippocampus of the brain. Administration of BRB prevented the memory loss, anxiogenic behavior, increased acetylcholinesterase activity and cell death induced by ICV-STZ. This may be explained, in part, by a protective effect of BRB on ameliorating the progression of neurodegenerative diseases, including Alzheimer’s disease, and the results of this study provide a better understanding of the effect of BRB on the brain. Thus, BRB may act as a potential neuroprotective agent.

Improvement of cognitive performance and cholinergic transmission by development of novel acridine-piperazine hybrids

s / Parkinsonism and Related Disorders 22 (2016) e149ee192 e178 3. O'Leary TP, Savoie V, Brown RE. Learning, memory and search strategies of inbred mouse strains with different visual abilities in the Barnes maze. Behavioural brain research. 2011;216:531-542. 4. Andrade C. Quantifying the ECT dose: the right unit remains elusive. The journal of ECT 2001;17:75. P 6.009. IMPROVEMENT OF COGNITIVE PERFORMANCE AND CHOLINERGIC TRANSMISSION BY DEVELOPMENT OF NOVEL ACRIDINE-PIPERAZINE HYBRIDS Anuradha Sharma, Poonam Piplani. University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, 160014, Chandigarh, India Objective: The objective of the present study was to design and synthesize some novel acridine-piperazine hybrids and evaluation of their ability to improve scopolamine induced cognitive deficit. Further, to elucidate mechanism of action using acetylcholinesterase inhibitory assay. Methods: 1. In-silico studies: Vlife, Phase and QikProp (Maestro) softwares were employed to design novel hybrids. 2. Synthesis: Alkylation of substituted aniline with o-bromobenzoic acid was done to afford substituted phenylamino benzoic acid derivatives. These intermediates were cyclized to form substituted acridone analogues, which were subsequently chlorinated in the presence of phosphorus oxychloride and then refluxed with fluoropiperazines to afford the final compounds (1-5). 3. Pharmacological Evaluation: Behavioral studies were carried out using elevated plus maze and passive avoidance tests on mice. Biochemical studies were carried out to study their acetylcholinesterase inhibitory profile using Ellman method. Results: Four point pharmacophore (ADHR) was developed using Phase to design the hybrids, which were docked at the defined cavity of enzyme to understand the possible interactions as shown in the figure (Vlife). Compounds bearing fluoro halogen substituent at 4th position of phenylpiperazine ring enhances the binding interactions. The calculated ADME properties confirmed the druglikeness using QikProp. Conclusion: The compound 1 was found to be most potent with IC50 0.33 out of all the synthesized hybrids. Rational design of novel series of potent acridine-piperazine hybrids as cognition enhacers, binding to the active site of AChE structure was disclosed which can act as a future lead in this area.

Thiocholine mediated stabilization of in situ produced CdS quantum dots: application for the detection of acetylcholinesterase activity and inhibitors

The use of acetylcholinesterase (AChE) inhibitors as chemical warfare agents or pesticides represents a strong hazard against human health. The high toxicity of these compounds arises from their ability to inhibit acetylcholinesterase from degrading acetylcholine (ACh), which could affect the physiology of the nervous system with serious or fatal consequences. Here we report a simple and fluorimetric system for a highly sensitive detection of AChE activity and inhibitors. The principle of this approach is based on the hydrolysis of acetylthiocholine (ATCh) by AChE, which yields the thiol-bearing compound thiocholine (TCh) that at trace concentrations stabilized the in situ generated CdS quantum dots (QDs). The system shows a linear relationship between the fluorescence intensity and AChE activity from 1 to 10 mU mL(-1) in buffer solution. The accuracy of the proposed system was further demonstrated through the determination of AChE activity in human serum (HS) by the standard addition method. Furthermore, this novel and highly sensitive sensing system allows the detection of 80 pM of the AChE inhibitor paraoxon and 100 nM of galanthamine. The reported methodology shows potential applications for the development of a simple and inexpensive assay for the routine quantification of AChE activity and inhibitors.

Determination of acetylcholinesterase activity by the Ellman assay: A versatile tool for in vitro research on medical countermeasures against organophosphate poisoning

Inhibition of acetylcholinesterase (AChE) is the main mechanism of action of organophosphorus compounds (OP), and AChE reactivators (oximes) are at present the only causal therapeutic approach. Being the key target of OP toxicity, AChE may serve as a valuable tool for diagnosis of OP exposure as well as for the investigation of the kinetics of interactions between OP and oximes. At present, the rapid, simple, and cheap spectrophotometric Ellman assay is widely used for diagnosis, therapeutic monitoring and in vitro kinetic investigations. Application of the assay for investigation of the interactions between AChE, inhibitors, and oximes requires the consideration of potential matrix effects (e.g. hemoglobin), side reactions (e.g. oximolysis of substrate) and other determinants (e.g. pH, temperature). By taking these factors into account, the Ellman assay allows the precise and reproducible determination of kinetic constants as a basis for the understanding of toxic OP effects and for the development of improved therapies against poisoning by OP. In addition, advanced applications of the Ellman assay, for example, in a dynamic in vitro model for the real-time activity determination of membrane-bound AChE, enables the proper investigation of relevant tissue, primarily respiratory muscle, and extends the applicability of this method.

Effects of dichlorvos and carbaryl on the activity of free and immobilized acetylcholinesterase

Acetylcholinesterase (AChE) is responsible for the rapid hydrolytic degradation of the neurotransmitter acetylcholine into inactive products choline and acetic acid. The purpose of this study was to examine the effect of carbaryl and dichlorvos on the activity of AChE. In this experimental study, 60 samples of free and immobilized form of AChE were prepared. Determination of AChE activity followed the Ellman's method with modifications. Briefly, 200 µl of the enzyme solution was combined with 400 µl of 25 mM phosphate-buffered saline, 200 µl of DTNB [5,5'-dithio-bis(2-nitrobenzoic acid)], and 200 µl of 300 μM acetylthiocholine iodide. Triplicate (1000 µl) samples were transferred to clean 1.5-ml centrifuge tubes, mixed, and held on ice until analysed and the change in absorbance was measured. For inhibition studies, substrate solutions were pre-incubated with dichlorvos and/or carbaryl. Dichlorvos and carbaryl were used at the concentrations of 100 and 500 μM. The activity was evaluated at 412 nm using Ceceil, CE 1020 spectrophotometer. Phosphate buffer (pH 7.35) was used for blanks. AChE activity was quantified as mM/ml/min. AChE activity of free form is more affected by Dichlorvos (0.09 ± 0.03 mM/ml/min) than immobilized form (0.19 ± 0.02 mM/ml/min). AChE activity of free form is more affected by carbaryl (0.11 ± 0.01 mM/ml/min) than immobilized form (0.1 ± 0.04 mM/ml/min). Comparison of mean AChE activity showed that the activity of the enzyme in presence of dichlorvos and carbaryl was significantly lower compared to controls. To calculate the significance of the difference, the t-test for paired values was applied. The results of our study indicate that dichlorvos and carbaryl cause decrease in AChE activity for both free and immobilization form of enzyme. It is therefore concluded that measuring AChE activity is a way to evaluate poisoning with carbaryl and dichlorvos.

Evaluation of 6,6′-dithionicotinic acid as alternative chromogen in a modified Ellman method—comparison in various species

For the diagnosis and therapy monitoring of intoxications with organophosphorus compounds, the determination of acetylcholinesterase (AChE) activity in whole blood is crucial. Usually, this testing is done with the colorimetric Ellman test using 412 nm wavelength and 5,5′-dithiobis-nitrobenzoic acid (DTNB) as chromogen. However, it has been described that the assay sensitivity is impaired by the Soret band of hemoglobin.In order to enable more sensitive determination of AChE activity in human, porcine, rat, and guinea pig whole blood dilutions, we tested the alternative chromogen 6,6′-dithionicotinic acid (DTNA) with an optimal absorption wavelength of 340 nm and compared it with a modified Ellman assay using a wavelength of 436 nm.DTNB resulted in a higher background absorption compared with DTNA in human and porcine blood samples, although the saturation of sulfhydryl groups was delayed with DTNA in all species. A slightly higher whole blood AChE activity was recorded in DTNB samples.In conclusion, the results of the present study with human, porcine, guinea pig, and rat whole blood do not provide evidence that using DTNA for the spectrophotometric determination of AChE activity in whole blood is superior to a modified Ellman assay using DTNB.

Sensitizing Effect of Tacrine on M-Cholinergic Receptors in Gastric Smooth Muscle of Rats

To find if tacrine exerts a sensitizing effect on the cholinergic receptors of gastric smooth muscles, and study some of the mechanisms inducing it and measure the relative intensity of tacrine's effects on contractile activity. Isometric recording of the mechanical activity of gastric smooth muscle preparations; determination of acetyl-cholinesterase activity in smooth-muscle tissue homogenates using Ellman's method. We found that the threshold concentration for tacrine not reducing the acetylcholinesterase activity and not having an effect on the smooth muscle preparations was 1 x 10(-8) mol/l. This concentration, however, significantly increased the acetylcholine-induced contraction compared with the controls, after the smooth-muscle tissue was incubated for 60 or 100 min. Treating smooth-muscle preparations with tacrine in a concentration of 5 x 10(-6) mol/l triggered a contraction induced by the drug's anti-cholinesterase activity. A secondary contraction was induced after 38.6 +/- 5.6 min. There was no secondary contraction after the control acetylcholine-induced effect. Atropine (1 x 10(-6) mol/l) inhibits this effect. Preliminary treatment of smooth muscle preparations with hexamethonium (1 x 10(-6) mol/l) did not change significantly the intensity of the first phase of tacrine-induced contraction and shifted in time the appearance of the second contractile phase. Tacrine has a sensitizing effect on M-cholinergic receptors; it occurs after a long incubation of the gastric smooth muscles with the drug and is manifested as a secondary contraction which is shifted in time and is significantly inhibited by atropine.

Different inhibition of acetylcholinesterase in selected parts of the rat brain following intoxication with VX and Russian VX

Differences between acetylcholinesterase (AChE) inhibition in the brain structures following VX and RVX exposure are not known as well as information on the possible correlation of biochemical and histochemical methods detecting AChE activity. Therefore, inhibition of AChE in different brain parts detected by histochemical and biochemical techniques was compared in rats intoxicated with VX and RVX. AChE activities in defined brain regions 30 min after treating rats with VX and Russian VX intramuscularly (1.0 × LD50) were determined by using biochemical and histochemical methods. AChE inhibition was less expressed for RVX, in comparison with VX. Frontal cortex and pontomedullar areas containing ncl. reticularis has been found as the most sensitive areas for the action of VX. For RVX, these structures were determined to be frontal cortex, dorsal septum, and hippocampus, respectively. Histochemical and biochemical results were in good correlation (Rxy = 0.8337). Determination of AChE activity in defined brain structures was a more sensitive parameter for VX or RVX exposure than the determination of AChE activity in the whole-brain homogenate. This activity represents a “mean” of the activities in different structures. Thus, AChE activity is the main parameter investigated in studies searching for target sites following nerve-agent poisoning contributing to better understanding of toxicodynamics of nerve agents.

Evaluation of cholinergic markers in Alzheimer's disease and in a model of cholinergic deficit

Cognitive deficits in neuropsychiatric disorders, such as Alzheimer's disease (AD), have been closely related to cholinergic deficits. We have compared different markers of cholinergic function to assess the best biomarker of cognitive deficits associated to cholinergic hypoactivity. In post-mortem frontal cortex from AD patients, acetylcholine (ACh) levels, cholinacetyltransferase (ChAT) and acetylcholinesterase (AChE) activity were all reduced compared to controls. Both ChAT and AChE activity showed a significant correlation with cognitive deficits. In the frontal cortex of rats with a selective cholinergic lesion, all cholinergic parameters measured (ACh levels, ChAT and AChE activities, “in vitro” and “in vivo” basal ACh release) were significantly reduced. AChE activity was associated to ChAT activity, and even more, to “in vivo” and “in vitro” basal ACh release. Quantification of AChE activity is performed by an easy and cheap method and therefore, these results suggest that determination of AChE activity may be used as an effective first step method to evaluate cholinergic deficits.

Evaluation of the Biological Effects of a Natural Extract of Chayotte (Sechium edule): A Molecular and Cellular Analysis

Natural products have been widely used by human beings. However, sometimes the biological effects of these products are not fully known. It is concerned that many natural medicines may contain potentially toxic ingredients and contaminants such as heavy metals. Red blood cells (RBC) and plasma proteins labeled with technetium-99m (99mTc) have several clinical applications and it has been reported that some natural products are capable of reducing the efficiency of this radio labeling. The aim of this work was to assess the oxidant or antioxidant action of the agents presents in the extract of Sechium edule verifying its effects on the labeling of blood elements with 99mTc and on the inhibition of the activity of acetyl cholinesterase (AchE). A freshly extract of Sechium edule was administered to Wistar rats during 15 and 60 days. After that, samples (0.5ml) of blood were incubated with stannous chloride (SnCl ) and 99mTc. The 2 blood was centrifuged and plasma (P) and RBC were isolated. P and RBC were also precipitated with trichloroacetic acid and soluble (S) and insoluble (I) fraction (F) were determined. The results have shown that the referred extract was able to reduce the radio labeling in BC to 15 days of treatment (from 98.77%± 0.45 to 90.35%± 5.04) to 60 days of treatment (from 98.77%± 0.45 to 53.53%± 0.91), in IF-BC (from 90.36%± 2.34 to 21.20%± 2.36) and in IF-P to 15 days of treatment (77.20%± 2.19 to 53.26%± 6.69) to 60 days of treatment (77.20%± 2.19 to 11.01%±3.22). The analysis of toxicology was performed by the determination of acetyl cholinesterase activity. In the light of the results the referred extract has not shown inhibitory action due to the activity of the enzyme. In the light of the results obtained we suggest that the referred extract may induce the generation of activity metabolites with oxidant properties with direct action on the labeling process.

Choline derivatives and sodium fluoride protect acetylcholinesterase against irreversible inhibition and aging by DFP and paraoxon

A light addressable potentiometric sensor was used to measure acetylcholinesterase (AChE) activity in order to evaluate the protective effects of quaternary compounds and NaF against enzyme phosphorylation and aging by two organophosphates. The use of the immobilized AChE made possible the quick removal of reagents (i.e., organophosphate, 2-pralidoxime, and protectant), thereby permitting accurate determination of AChE activity before and after phosphorylation and aging. Paraoxon was 15-fold more potent in inhibiting AChE than DFP, while the percent aging following phosphorylation by diisopropylfluorophosphate (DFP) was much higher. Sodium fluoride (NaF), the most effective protectant against phosphorylation and aging, and the quaternary ammonium compounds reduced significantly AChE inhibition by DFP and paraoxon, to similar degrees. Even though the percent AChE activity that was lost to aging was reduced by these agents, aging as a percent of phosphorylated AChE was not reduced. Thus, their major effect was in reducing the percent AChE phosphorylation, which consequently resulted in reduction of total aged AChE. The finding that quaternary ammonium compounds protect against phosphorylation is consonant with the proposed presence of the active site of AChE in an aromatic gorge.

Determination of erythrocyte-bound acetylcholinesterase activity for monitoring pyridostigmine therapy in myasthenia gravis

Monitoring of pyridostigmine therapy in patients with myasthenia gravis is not routinely performed, since the daily pyridostigmine doses are adjusted to the patient's actual clinical status rather than to pyridostigmine plasma concentrations (PPC). Moreover, PPC determination is time-consuming and needs much technical equipment. Since pyridostigmine reversible blocks acetylcholinesterase (AChE) at the neuromuscular junction, we studied the correlation between the enzyme's blood activity (erythrocyte-bound AChE) and PPC, on the one hand, and between blood AChE activity and the clinical status of the individual patient, on the other. In five previously untreated patients with myasthenia gravis blood AChE activity decreased in accordance with the actual PPC after a single oral dose of 60 mg pyridostigmine (group A). Amelioration of the clinical status corresponded to the decrease of AChE activity in the same way. In another five patients, who were on stable pyridostigmine medication for at least 1 week, AChE activity and PPC were constant during the day (group B). Since it is easier to perform than PPC, our results suggest that the determination of AChE activity may be superior to measuring PPC for monitoring cholinesterase inhibitor therapy in selected cases.

Determination of acetylcholinesterase activity by gas-liquid chromatography

1. A modified Deans' method for quantitative gas-chromatographic determination of trace components in a large volume of sample of test mixture introduced into the apparatus was developed and used. 2. In the suggested modification this method can be used for gas-chromatographic determination of microquantities of acetic acid. 3. Deans' method can be used for quantitative differential determination of acetylcholinesterase and pseudocholinesterase in the blood, based on determination of end products of hydrolysis of specific substrates.

Determination of acetylcholinesterase activity by a new chemiluminescence assay with the natural substrate.

A chemiluminescence method for determining acetylcholinesterase activity is described. It is an adaptation of the chemiluminescence assay of acetylcholine described by Israël & Lesbats [(1981) Neurochem. Int. 3, 81-90; (1981) J. Neurochem. 37, 1475-1483]. The acetylcholinesterase activity is measured by monitoring the increase in light emission produced by the accumulation of choline or by determining the amount of choline generated after a short interval. The assay is rapid and sensitive, and uses the natural substrate of the enzyme. Kinetic data obtained with this procedure for acetylcholinesterase from Torpedo and Electrophorus electric organs were comparable with those obtained by using the method of Ellman, Courtney, Andres & Featherstone [(1961) Biochem. Pharmacol. 7, 88-95]. In addition, it was shown that sodium deoxycholate totally inactivated Torpedo acetylcholinesterase but not the Electrophorus enzyme. Competitive inhibitors of acetylcholinesterase protected the enzyme from inactivation.

Quantitative histochemical determination of acetylcholinesterase activity in layers of the rat hippocampus

A technique has been suggested for quantitative determination of the activity of acetylcholinesterase (AChE) in each layer of rat hippocamp histochemical preparations. The data obtained have been used for making up the maps of layer distribution of AChE activity. The maps have been prepared of AChe activity distribution for the field CA1, CA2 and CA3 of the hippocamp of rats decapitated in summer and autumn. The map for the field CA1 was compared with those made up with the aid of other quantitative histochemistry techniques. The maps for the field CA2 have been compared in rats decapitated in spring, summer and autumn. AChE activity has been found to be greater in spring than in summer or in autumn, with the relations between the layers being unchanged throughout all the seasons.

Determination of acetylcholinesterase activity in normal and denervated sympathetic ganglia of the cat. A biochemical and histochemical comparison.

AbstractAcChE was determined in normal and preganglionically denervated sympathetic ganglia with a titrigraphic method. The superior cervical, stellate, 7th lumbar and 1st sacral ganglia showed higher AcChE activities than the remaining paravertebral ganglia. Especially low AcChE values were obtained in the prevertebral ganglia. In denervation experiments a considerable decrease in AcChE activity was noted. The remaining activity was proportional to the number of histochemically heavily stained cell bodies in the ganglia investigated. Whereas ganglia containing many such cells retained 50% of their total AcChE activity after denervation (L7 and S1), the corresponding figure for ganglia almost lacking them was about 20% (superior cervical ganglion). Thus, some cells in the sympathetic system, showing intense histochemical staining possess significantly higher AcChE activities than the remaining ones as determined biochemically. These cells have been linked to a peripheral cholinergic function. Small traces of AcChE are found in adrenergic as well as sensory ganglion cells. The data presented fit in with the classical concept about the sympathetic system being composed of predominantly adrenergic cells with a few cholinergic ones concentrated in special relay stations.