Comparison of methods used for the determination of cholinesterase activity in whole blood

Cholinesterases (ChEs) are classified as either acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) based on their substrate and inhibitor specificity. Organophosphate and carbamate compounds commonly represented by herbicides, pesticides, and nerve gases irreversibly inhibit ChEs. Therefore, exposure to organophosphates and carbamates is normally assessed by measuring ChE activity in blood. There are two approaches for measuring AChE and BChE activity present in whole blood: (1) separating blood into erythrocytes, which contain only AChE, and plasma which contains only BChE, to measure their activity individually, or (2) use a BChE-specific inhibitor to measure the activity of AChE in whole blood. A number of studies have reported the use of different inhibitors for the simultaneous measurement of AChE and BChE activities. However, the inhibitors used for completely inhibiting BChE activity also inhibited AChE activity leading to errors in reported values. The goal of this study was to find the most accurate and simple method for the simultaneous determination of AChE and BChE activity in animal whole blood. Solutions containing human AChE and BChE in various proportions were prepared and AChE and BChE activities were measured using three reported methods. Results demonstrate that ethopropazine and (-) huperzine A appear to be the most specific ChE inhibitors. Preliminary results with human and animal whole blood suggest that 20muM ethopropazine and 500nM (-) huperzine A can be used for measuring AChE and BChE activities across species.     

Benzene-1,2-, 1,3-, and 1,4-di-N-substituted carbamates as conformationally constrained inhibitors of acetylcholinesterase

Benzene-1,2-, 1,3-, and 1,4-di-N-substituted carbamates (1-15) are synthesized as the conformationally constrained inhibitors of acetylcholinesterase and mimic gauche, eclipsed, and anti-conformations of acetylcholine, respectively. All carbamates 1-15 are characterized as the pseudo substrate inhibitors of acetylcholinesterase. For a series of geometric isomers, the inhibitory potencies are as follows: benzene-1,4-di-N-substituted carbamate (para compound) > benzene-1,3-di-N-substituted carbamate (meta compound) > benzene-1,2-di-N-substituted carbamate (ortho compound). Therefore, benzene-1,4-di-N-substituted carbamates (para compounds), with the angle of 180 degrees between two C(benzene)-O bonds, mimic the preferable anti C-O/C-N conformers of acetylcholine for the choline ethylene backbone in the acetylcholinesterase catalysis.     

A novel enzyme immunoassay based on potentiometric measurement of molecular adsorption events by an extended-gate field-effect transistor sensor

We developed a novel enzyme immunoassay based on a potentiometric measurement of molecular adsorption events by using an extended-gate field-effect transistor (FET) sensor. The adsorbing rate of a thiol compound on a gold surface was found to depend on the concentration of the compound. To construct an electrochemical enzyme immunoassay system by using the sensor, the enzyme chemistry of acetylcholinesterase (AChE) to generate a thiol compound was used and combined with the enzyme-linked immunosorbent assays (ELISA). After the AChE-catalyzed reaction, the amount of the antigen was obtained by detecting the adsorbing rate of the generated thiol compound on the gold electrode using the FET sensor. The measurement stability was also found to improve when a high frequency voltage of 10 kHz or more was superimposed to the reference electrode. The signal corresponding to a range between 1 and 250 pg/mL of Interleukin 1β was obtained by the FET sensor when a voltage of 1 MHz was superimposed onto the reference electrode. The FET sensor based ELISA used in this measurement technique can successfully detect Interleukin 1β at concentrations as low as 1 pg/mL.     

Pesticide detection with a liposome-based nano-biosensor

Monitoring of the organophosphorus pesticides dichlorvos and paraoxon at very low levels has been achieved with liposome-based nano-biosensors. The enzyme acetylcholinesterase was effectively stabilized within the internal nano-environment of the liposomes. Within the liposomes, the pH sensitive fluorescent indicator pyranine was also immobilized for the optical transduction of the enzymatic activity. Increasing amounts of pesticides lead to the decrease of the enzymatic activity for the hydrolysis of the acetylcholine and thus to a decrease in the fluorescent signal of the pH indicator. The decrease of the liposome biosensors signal is relative to the concentration of dichlorvos and paraoxon down to 10⁻¹⁰ M levels. This biosensor system has been applied successfully to the detection of total toxicity in drinking water samples. Also a colorimetric screening device for pesticide analysis has been evaluated.     

Tacrine derivatives-acetylcholinesterase interaction: 1H NMR relaxation study

Two acetylcholinesterase (AChE) inhibitors structurally related to Tacrine, 6-methoxytacrine (1a) and 9-heptylamino-6-methoxytacrine (1b), and their interaction with Electrophorus Electricus AChE were investigated. The complete assignment of the 1H and 13C NMR spectra of 1a and 1b was performed by mono-dimensional and homo- and hetero-correlated two-dimensional NMR experiments. This study was undertaken to elucidate the interaction modes between AChE and 1a and 1b in solution, using NMR. The interaction between the two inhibitors and AChE was studied by the analysis of the motional parameters non-selective and selective spin-lattice relaxation times, thereby allowing the motional state of 1a and 1b, both free and bound with AChE, to be defined. The relaxation data pointed out the ligands molecular moiety most involved in the binding with AChE. The relevant ligand/enzyme interaction constants were also evaluated for both compounds and resulted to be 859 and 5412M(-1) for 1a and1b, respectively.     

Reversible AChE inhibitors in C. elegans vs. rats, mice

We are investigating whether Caenorhabditis elegans could be used as a screen for vertebrates by comparing the responses of components of its cholinergic system to well-characterized toxicants. We assessed whether C. elegans displays similar toxicity as rats and mice to reversible acetylcholinesterase (AChE) inhibitors, and sought to corroborate that the toxicity mechanism is the same. To determine relative potencies, movement-concentration curves were generated, 50th percentiles for movement were located, ranked and compared statistically to rat and mouse oral acute LD50s. The ranking was significantly correlated to rat and mouse rankings (alpha=0.05). We measured a concentration-dependent decrease in AChE activity correlating to a decrease in movement for each carbamate, suggesting that the mechanism of toxicity is the same. Finally, as seen in mammals, inhibition of AChE activity occurred before a movement decrease. The response of C. elegans to carbamate exposure shows significant correlation to rat and mouse data.     

Two immunologically distinct types of protofilaments can be identified in Natrialba magadii flagella

A rabbit antiserum to the 15-kDa acetylcholinesterase toxin neutralised the lethal effect of the 15-kDa toxin of Aeromonas hydrophila when injected into trout. However, immunisation of fish with the 15-kDa toxoid failed to induce an antibody response, and a higher molecular mass form of this toxin was purified from the extracellular products with the aim of inducing an immune response in fish. The optimal conditions for production of extracellular products by A. hydrophila strain B32 were studied to increase the concentration of this protoxin. The extracellular products were fractionated by molecular exclusion chromatography to yield a purified protoxin with an estimated molecular mass of 45 kDa by SDS-PAGE and which gave a positive reaction in Western blotting with the rabbit anti-15-kDa toxin serum. Since the 45-kDa protoxin showed lower specific acetylcholinesterase activity than the active 15-kDa toxin, the behaviour of the active site was studied using specific inhibitors. This 45-kDa protoxin was 13.3-fold less toxic than the 15-kDa toxin and induced antibody production in fish.     

Galanthamine production by Leucojum aestivum L. shoot culture in a modified bubble column bioreactor with internal sections

Shoot culture of summer snowflake (Leucojum aestivum L.) was successfully cultivated in an advanced modified glass‐column bioreactor with internal sections for production of Amaryllidaceae alkaloids. The highest amounts of dry biomass (20.8 g/L) and galanthamine (1.7 mg/L) were achieved when shoots were cultured at 22°C and 18 L/(L·h) flow rate of inlet air. At these conditions, the L. aestivum shoot culture possessed mixotrophic‐type nutrition, synthesizing the highest amounts of chlorophyll (0.24 mg/g DW (dry weight) chlorophyll A and 0.13 mg/g DW chlorophyll B). The alkaloids extract of shoot biomass showed high acetylcholinesterase inhibitory activity (IC₅₀ = 4.6 mg). The gas chromatography–mass spectrometry (GC/MS) profiling of biosynthesized alkaloids revealed that galanthamine and related compounds were presented in higher extracellular proportions while lycorine and hemanthamine‐type compounds had higher intracellular proportions. The developed modified bubble‐column bioreactor with internal sections provided conditions ensuring the growth and galanthamine production by L. aestivum shoot culture.     

Early biochemical effects of Microcystis aeruginosa extracts on juvenile rainbow trout (Oncorhynchus mykiss)

Microcystins (MC) are usually the predominant cyanotoxins associated with cyanobacterial blooms in natural surface waters. These toxins are well-known hepatotoxic agents that proceed by inhibiting protein phosphatase in aquatic biota; recent studies have also reported oxidative stress and disruption of ion regulation in aquatic organisms. In the present study, young trout (Oncorhynchus mykiss) were exposed to crude extracts of Microsystis aeruginosa for four days at 15 °C. The level of microcystins was calculated to confirm the presence of toxins in these crude extracts: 0, 0.75, 1.8 and 5 μg/L. Protein phosphatase measured in the liver increased by at least 3-fold and is significantly as a result of exposure to these sublethal concentrations of crude extract, his indicates an early defense response against protein phosphatase inhibition from cyanotoxins. This was corroborated by the decreased phosphate content in proteins found in the liver and brain. No increase in glutathione-S transferase (GST) activity was observed and lipid peroxidation was unaffected in both liver and brain tissue exposed to the cyanobacterial extracts. The data revealed that the proportion of the reduced (metal-binding) form of metallothionein (MT) decreased by two-fold relative to the control group (with a concomitant increase in the proportion of the oxidized form). The level of phosphate associated with MT increased by 1.5-fold at the highest concentration of crude extract. Acetylcholinesterase (AChE) activity in brain tissue was decreased after exposure to the highest concentration of crude extract, suggesting a slowdown in neural activity. However, no biotransformation processes or detoxification of GST was triggered. Our findings show early sign of biochemical effects of MC-LR in young trout.