The original Ellman's spectrophotometrical method for cholinesterase activity determination uses 5,5'-dithiobis-2-nitrobenzoic acid (DTNB, Ellman's reagent) as a chromogen and records the level of cholinesterase activity as an increase of absorbance at 412 nm. Although this procedure usually poses no problem, exceptions arise when the concentration of DTNB is far higher than the concentration of acetylthiocholine (ATCH). It was found that the ratio of concentrations of DTNB/ATCH is an important parameter for the ATCH hydrolysis course: high excess of DTNB decreases the hydrolysis rate resulting in a lower measured enzyme activity. Our experiments indicate that this influence of DTNB concentration can be explained by the inhibition of ATCH hydrolysis by DTNB.
Ellman's assay is the most commonly used method to measure cholinesterase activity. It is cheap, fast, and reliable, but it has limitations when used for biological samples. The problems arise from 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), which is unstable, interacts with free sulfhydryl groups in the sample, and may affect cholinesterase activity. We report that DTNB is more stable in 0.09 M Hepes with 0.05 M sodium phosphate buffer than in 0.1M sodium phosphate buffer, thereby notably reducing background. Using enzyme-linked immunosorbent assay (ELISA) to enrich tissue homogenates for cholinesterase while depleting the sample of sulfhydryl groups eliminates unwanted interactions with DTNB, making it possible to measure low cholinesterase activity in biological samples. To eliminate possible interference of DTNB with enzyme hydrolysis, we introduce a modification of the standard Ellman's assay. First, thioesters are hydrolyzed by cholinesterase to produce thiocholine in the absence of DTNB. Then, the reaction is stopped by a cholinesterase inhibitor and the produced thiocholine is revealed by DTNB and quantified at 412 nm. Indeed, this modification of Ellman's method increases butyrylcholinesterase activity by 20 to 25%. Moreover, high stability of thiocholine enables separation of the two reactions of the Ellman's method into two successive steps that may be convenient for some applications.
Assay of acetylcholinesterase (AChE) activity plays an important role in diagnostic, detection of pesticides and nerve agents, in vitro characterization of toxins and drugs including potential treatments for Alzheimer's disease. These experiments were done in order to determine whether indoxylacetate could be an adequate chromogenic reactant for AChE assay evaluation. Moreover, the results were compared to the standard Ellman's method. We calculated Michaelis constant Km (2.06 × 10(-4) mol/L for acetylthiocholine and 3.21 × 10(-3) mol/L for indoxylacetate) maximum reaction velocity V(max) (4.97 × 10(-7) kat for acetylcholine and 7.71 × 10(-8) kat for indoxylacetate) for electric eel AChE. In a second part, inhibition values were plotted for paraoxon, and reactivation efficacy was measured for some standard oxime reactivators: obidoxime, pralidoxime (2-PAM) and HI-6. Though indoxylacetate is split with lower turnover rate, this compound appears as a very attractive reactant since it does not show any chemical reactivity with oxime antidots and thiol used for the Ellman's method. Thus it can be advantageously used for accurate measurement of AChE activity. Suitability of assay for butyrylcholinesterase activity assessment is also discussed.
- Publication type
- Journal Article MeSH
The changes in cholinesterase activity in tissues were evaluated and compared with the reactivation potential of reactivators. As reactivators, (E)-1-(4-carbamoylpyridinium- 1-yl)-4-{4-[(hydroxyimino)methyl]pyridinium-1-yl} but-2-ene dibromide (K203) and common 1,3-bis{4- [(hydroxyimino)methyl]pyridinium-1-yl}-2-oxapropane dichloride (obidoxime) were used. The reactivation of both oximes was monitored in blood and blood plasma. The reactivation of tabun-inhibited acetylcholinesterase (AChE) was higher using the newly synthesized K203.
- MeSH
- Butyrylcholinesterase analysis blood MeSH
- Cholinesterase Inhibitors analysis blood MeSH
- Financing, Organized MeSH
- Colorimetry methods MeSH
- Oximes diagnostic use MeSH
- Rats, Wistar MeSH
- Reproducibility of Results MeSH
- Sarin analogs & derivatives analysis blood MeSH
- Spectrophotometry methods MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
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- Keywords
- VX agent, tabun,
- MeSH
- Acetylcholinesterase MeSH
- Biosensing Techniques MeSH
- Chemical Warfare Agents * analysis MeSH
- Colorimetry MeSH
- Dithionitrobenzoic Acid * MeSH
- Neural Networks, Computer * MeSH
- Organophosphates analysis MeSH
- Organothiophosphorus Compounds analysis MeSH
- Cholinesterase Reactivators MeSH
- Sarin analysis MeSH
- Soman analysis MeSH
A new and simple analytical method is described for the determination of the IC50 values of the inhibitors of the hydrolysis of acetylcholine (ACh) or acetylthiocholine (ATCh) by cholinesterases. The method is based on monitoring the time course of the pH value during the uninhibited and inhibited reaction. It requires only a pH meter with a suitable pH measuring cell and a small thermostated stirred batch reactor. The method has been validated for twelve different types of cholinesterase inhibitors. The determined IC50 values are comparable to those obtained by independent, more complicated, and expensive methods (Ellman's and pH-stat).
The method for automatic continual monitoring of acetylcholinesterase (AChE) activity in biological material is described. It is based on flexible system of plastic pipes mixing samples of biological material with reagents for enzyme determination; reaction product penetrates through the semipermeable membrane and it is spectrophotometrically determined (Ellman's method). It consists of sampling (either in vitro or in vivo), adding the substrate and flowing to dialyzer; reaction product (thiocholine) is dialyzed and mixed with 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB) transported to flow spectrophotometer. Flowing of all materials is realised using peristaltic pump. The method was validated: time for optimal hydratation of the cellophane membrane; type of the membrane; type of dialyzer; conditions for optimal permeation of reaction components; optimization of substrate and DTNB concentrations (linear dependence); efficacy of peristaltic pump; calibration of analytes after permeation through the membrane; excluding of the blood permeation through the membrane. Some examples of the evaluation of the effects of AChE inhibitors are described. It was demonstrated very good uniformity of peaks representing the enzyme activity (good reproducibility); time dependence of AChE inhibition caused by VX in vitro in the rat blood allowing to determine the half life of inhibition and thus, bimolecular rate constants of inhibition; reactivation of inhibited AChE by some reactivators, and continual monitoring of the activity in the whole blood in vivo in intact and VX-intoxicated rats. The method is simple and not expensive, allowing automatic determination of AChE activity in discrete or continual samples in vitro or in vivo. It will be evaluated for further research of cholinesterase inhibitors.
- MeSH
- Acetylcholinesterase blood metabolism MeSH
- Cholinesterase Inhibitors pharmacology MeSH
- Enzyme Assays instrumentation methods MeSH
- Kinetics MeSH
- Rats MeSH
- Dithionitrobenzoic Acid metabolism MeSH
- Membranes, Artificial MeSH
- Flow Injection Analysis instrumentation methods MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
- Research Support, Non-U.S. Gov't MeSH
OBJECTIVES: Celecoxib is a nonsteroidal anti-inflammatory drug inhibiting enzyme cyclooxygenase-2 (COX-2). The drug was introduced in 1990s. In the work presented here, affinity of celecoxib to enzyme acetylcholinesterase (AChE) is inferred. METHODS: Inhibition of human AChE by celecoxib was tested using standard spectrophotometric Ellman´s method and extrapolation of experimental data by Dixon plot. Interaction between AChE and celecoxib was also predicted by molecular docking using Swiss dock software. RESULTS: A non-competitive mechanism of inhibition was revealed and equilibrium inhibitory constant equal to 313±40 µmol/l was determined. Comparing to AChE, celecoxib was not proved as an inhibitor of enzyme butyrylcholinesterase (BChE). The lowest ΔG was equal to -7.78 kcal/mol. In this case, celecoxib stacked sulfonamide moiety between TYR 337 and TYR 341 of alfa anionic subsite of active site. Cation-Π interactions appears to be responsible for the inhibition. CONCLUSIONS: Though the here revealed and characterized inhibition has lower effect in real conditions than inhibition of COX-2, the inhibitory effect would be utilized in the next research and development of new AChE inhibitors.
PURPOSE OF THE STUDY The current study aimed to investigate the intra- and inter-observer reliability of the Ellman classification system in partialthickness rotator cuff tears through magnetic resonance imaging (MRI) scans instead of arthroscopic views. MATERIAL AND METHODS Pre-operative MRI scans of 45 patients, with confirmed partial-thickness rotator cuff rupture in previous arthroscopic surgeries (performed by the senior author), were obtained from Picture Archiving and Communication Systems records. The observers (n=8) were asked to categorize MRI scans according to Ellman's classification of location and grade. There were four orthopedic surgeons less experienced in rotator cuff operations in the first group and four more experienced orthopedic surgeons in the second group. They were asked to re-evaluate the MRI scans six weeks later, without access to their previous answers. Reliability evaluation was performed within and among the groups. It was also evaluated if the surgeon's experience increased the reliability of the classification. Fleiss kappa coefficient was used for the inter-observer reliability and Cohen kappa coefficient for the intra-observer reliability, and post hoc analysis was performed. RESULTS When all observers were examined in the inter-observer evaluation, it was seen that there was moderate agreement in the first location evaluation (κ=0.414); however, there was fair agreement in all other evaluations in both groups (κ=0.339- 0.383-0.337, respectively). When all observers were examined in the mean intra-observer evaluation, it was seen that there was substantial agreement in both evaluations (κ=0.795-0.721, respectively). DISCUSSION A classification system must be valid, reliable, and reproducible. It should establish a standard terminology for both surgeons and researchers. The correct identification of the tear configuration is crucial for selecting the correct repair technique. In our study, in which we evaluated the Ellman classification, which is frequently used in arthroscopic diagnosis, we investigated its intra-observer and inter-observer reliability on MRI scans. Although the mean intra-observer evaluation results were substantial agreement (κ=0.795-0.721, respectively), inter-observer evaluation results were fair agreement (κ=0.339- 0.383-0.337, respectively) except for the first location evaluation (κ=0.414). CONCLUSIONS Although intra-observer reliability was satisfactory, the Ellman system used in the classification of partial-thickness rotator cuff tears was not found to be useful by using only MRI views because of fair inter-observer reliability except for the first location evaluation, which was moderate agreement. Key words: partial, rotator cuff, tear, Ellman classification, reliability, validity.
OBJECTIVES: The aim of the study was to use methods of pharmaceutical technology, and prepare carriers in the form of pellets suitable as a filling of detection tubes for enzymatic detection of cholinesterase inhibitors. The enzymatic detection was based on enzymatic hydrolysis of acetylthiocholine iodide and the subsequent colour reaction of its hydrolysis product with Ellman's reagent. The suitable carriers should be in the form of white, regular and sufficiently mechanically resistant particles of about 1 mm allowing it to capture the enzyme during the impregnation process and ensuring its high activity for enzymatic detection. METHODS: Carriers consisting of microcrystalline cellulose, lactose, povidone, and sodium carboxymethyl cellulose were prepared using extrusion-spheronization method under three different drying conditions in either a hot air oven or a microwave oven. Subsequently, the carriers were impregnated with acetylcholinesterase and their size, shape, mechanical resistance, bulk, tapped and pycnometric density, Hausner ratio, intraparticular and total tapped porosity, and activity were measured and recorded. RESULTS: In this procedure, carriers with different physical parameters and different acetylcholinesterase activity were evaluated. It was found that higher acetylcholinesterase activity was associated not only with a higher intraparticular porosity but also with more regular particles characterized by high sphericity and low total tapped porosity. CONCLUSION: This unique finding is important for the preparation of detection tubes based on enzymatic detection which is still irreplaceable especially in the field of detection and analysis of super-toxic cholinesterase inhibitors.
- MeSH
- Acetylthiocholine analogs & derivatives metabolism MeSH
- Cellulose analysis MeSH
- Cholinesterase Inhibitors metabolism MeSH
- Dithionitrobenzoic Acid MeSH
- Lactose analysis MeSH
- Porosity MeSH
- Povidone analysis MeSH
- Carboxymethylcellulose Sodium analysis MeSH
- Sulfhydryl Reagents MeSH
- Materials Testing MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
