Whole Blood Acetylcholinesterase Research Articles

A near-infrared light triggered fluormetric biosensor for sensitive detection of acetylcholinesterase activity based on NaErF4: 0.5 % Ho3+@NaYF4 upconversion nano-probe

Acetylcholinesterase (AChE), as an important neurotransmitter, is widely present in the peripheral and central nervous systems. The aberrant expression of AChE could cause diverse neurodegenerative diseases. Herein, we developed a facile and interference-free fluorimetric biosensing platform for highly sensitive AChE activity determination based on a NaErF4: 0.5 % Ho3+@NaYF4 nano-probe. This nano-probe exhibits a unique property of emitting bright monochromic red (650 nm) upconversion (UC) emission under multiband (~808, ~980, and ~1530 nm) near-infrared (NIR) excitations. The principle of this detection relies on the quenching of the strong monochromic red UC emission by oxidization products of 3,3′,5,5′-tetramethylbenzidine generated through AChE-modulated cascade reactions. This system shows a great sensing performance with a detection limit (LOD) of 0.0019 mU mL−1 for AChE, as well as good specificity and stability. Furthermore, we validated the potential of the nano-probe in biological samples by determination of AChE in whole blood with a LOD of 0.0027 mU mL−1, indicating the potential application of our proposed platform for monitoring the progression of AChE-related disease.

Effects of oral administration of copper capsules on helminth control in lactating dairy sheep: An effective alternative to replace conventional antiparasitics during lactation

Two experiments were performed to determine whether oral administration of copper oxide capsules controlled helminthic infections in Lacaune sheep without acute collateral effects on animal health. In experiment 1, 48 multiparous lactating sheep (60.1 ± 8.5 kg) were stratified according to initial number of eggs (Haemonchus contortus) per gram of feces (EPG) and were assigned randomly to 1 of two treatments (24 sheep/treatment): no oral administration (control) or oral administration of two copper capsules (treated; approximately 58 mg copper/kg body weight). Blood and fecal samples were collected on days 0, 15 and 45. Animals treated with copper capsules showed lower of EPG, eosinophils, acetylcholinesterase (AChE) in whole blood, and lower butyrylcholinesterase (BChE) activity in serum. Treated sheep had higher erythrocyte numbers, hemoglobin concentrations, hematocrit, and lymphocyte numbers. In experiment 2, 12 male lambs negative for helminths and coccidia were assigned randomly to one of two treatments (six lambs/treatment): control or treated (one copper capsule; approximately 58 mg copper/kg body weight); the experiment was designed to determine whether the results of experiment 1 were due to treatment or parasitism. Blood samples were collected on days 0, 5, 10 and 15 and fecal samples were collected on days 0, 7 and 15. Treated animals showed greater concentrations of lymphocytes; however, treatment had no effect on other hemogram variables, AChE and BChE activities, or levels of alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase, creatinine, urea, albumin, total protein, and reactive oxygen species. These data suggest that copper capsules in dairy sheep efficiently controlled H. contortus infections. Treatment was not harmful to lambs during the first 15 days, i.e. there were no signs of acute toxicity.

A ratiometric fluorescence probe based on carbon dots for discriminative and highly sensitive detection of acetylcholinesterase and butyrylcholinesterase in human whole blood

A ratiometric fluorescence probe based on carbon dots (CDs) was developed for discriminative and highly sensitive detection of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity in human whole blood. When o-phenylenediamine (OPD) was oxidized by Cu2+, the product 2,3-diaminophenazine (oxOPD) could effectively quench the fluorescence of CDs at 460 nm due to the inner filter effect and gave rise to a new emission peak at 570 nm. The AChE or BChE catalyzed hydrolysis reaction of acetylthiocholine or butyrylthiocholine to generate thiocholine, whose sulfhydryl group strongly captured Cu2+ to inhibit the oxidization of OPD, thus effectively preserving the natural fluorescence emission of CDs. The resulting fluorescence intensity ratio served as the signal output of the probe for cholinesterases (ChEs) activity sensing. The activities of AChE and BChE were determined to range from 0.2 to 14.0 U L−1 and from 0.1 to 5.0 U L−1, with detection limits of 0.1 U L−1 and 0.04 U L−1, respectively. Additionally, the IC50 of tacrine and ethopropazine for the inhibition of AChE and BChE were estimated to be 29.8 nM and 132.6 nM, respectively. Moreover, the probe was successfully applied to the discriminative determination of AChE and BChE in human whole blood without any pretreatment. These results suggested that the proposed strategy provided a discriminative, sensitive and robust analytical platform for ChEs clinical diagnostics and drug screening.

The percutaneous toxicokinetics of VX in a damaged skin porcine model and the evaluation of WoundStat™ as a topical decontaminant.

This study used a damaged skin, porcine model to evaluate the in vivo efficacy of WoundStat™ for the decontamination of superficial, nerve agent-contaminated wounds. Anaesthetized animals were randomly assigned to either control (n=7), no decontamination (n=12) or WoundStat™ (n=12) treatment groups. Pigs were exposed to a 5× LD50 dose of neat, radiolabelled S-[2-(diisopropylamino)ethyl]-O-ethyl methyl-phosphonothioate (VX; or equivalent volume of sterile saline for the control group) via an area of superficially damaged skin on the ear. WoundStat™ was applied at 30seconds post-exposure to assigned animals. The VX contaminant (or saline) and decontaminant remained in place for the duration of the study (up to 6hours). Physiological parameters and signs of intoxication were recorded during the exposure period. Skin and organ samples were taken post mortem for 14 C-VX distribution analyses. Blood samples were taken periodically for toxicokinetic and whole-blood acetylcholinesterase (AChE) activity analyses. VX exposure was accompanied by a rapid decrease in AChE activity in all animals, regardless of decontamination. However, decontamination significantly improved survival rate and time and reduced the severity of signs of intoxication. In addition, the distribution of 14 C-VX in key internal organs and post mortem blood samples was significantly lower in the WoundStat™ treatment group. This study demonstrates that WoundStat™ may be a suitable medical countermeasure for increasing both survival rate and time following VX exposure. The results also suggest that AChE activity is not a useful prognostic indicator.

In vitro detoxification of cyclosarin in human blood pre-incubated ex vivo with recombinant serum paraoxonases

An ex vivo protocol was developed to assay the antidotal capacity of rePON1 variants to protect endogenous acetylcholinesterase and butyrylcholinesterase in human whole blood against OP nerve agents. This protocol permitted us to address the relationship between blood rePON1 concentrations, their kinetic parameters, and the level of protection conferred by rePON1 on the cholinesterases in human blood, following a challenge with cyclosarin (GF). The experimental data thus obtained were in good agreement with the predicted percent residual activities of blood cholinesterases calculated on the basis of the rate constants for inhibition of human acetylcholinesterase and butyrylcholinesterase by GF, the concentration of the particular rePON1 variant, and its k cat/ K m value for GF. This protocol thus provides a rapid and reliable ex vivo screening tool for identification of rePON1 bioscavenger candidates suitable for protection of humans against organophosphorus-based toxicants. The results also permitted the refinement of a mathematical model for estimating the efficacious dose of rePON1s variants required for prophylaxis in humans.

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 20 μM ethopropazine and 500 nM (−) huperzine A can be used for measuring AChE and BChE activities across species.

Effect of small doses of ethylene glycol monomethyl ether on the acetylcholinesterase and delta-aminolevulinic acid dehydratase activity in erythrocytes, blood and bone marrow of rats.

The effect of small doses of ethylene glycol monomethyl ether (EGMME) on the activity of acetylcholinesterase (ACHE) in erythrocytes and whole blood as well as on the delta-aminolevulinic acid dehydratase (ALA-D) in blood and bone marrow was studied in Wistar rats. Significant reduction in the activity of both enzymes was noted three days after ip administration of 200 mg/kg b.w. EGMME whereas seven days later the activity of both enzymes returned to the control levels. Activity of ALA-D in blood appeared to be most sensitive to EGMME, and reacted even to the lowest dose, which did not significantly alter activity of ACHE or ALA-D in bone marrow. Haematological parameters in all treated groups remained unaltered.

Acetylcholinesterase Inhibition by (+)Physostigmine and Efficacy against Lethality Induced by Soman

The optical isomer (+)Physostigmine [(+)Phy] is a very weak anticholinesterase. In a recent report, pretreatment with (+)Phy, at a dose which failed to inhibit acetylcholinesterase (AChE), and atropine provided efficacy against a lethal dose of sarin (SYNAPSE:2, 139, 1988). It was of interest to see whether (+)Phy could protect against soman at a dose which caused only marginal inhibition of the whole blood (WB) AChE in guinea pigs (GPs). (-)Phy (0.15 mg/kg, im) and (+)Phy (10.0 mg/kg, im) produced nearly 70% inhibition of WB AChE at 30 min whereas (+)Phy (0.15 mg/kg, im) caused only marginal inhibition. Groups of guinea pigs (20/group) were dosed, im, with (-)Phy (0.15 mg/kg), (+)Phy (0.15 mg/kg), (+)Phy (10.0 mg/kg) or vehicle (0.5 ml/kg) respectively in one thigh while the mild anticholinergic trihexyphenidyl (THP), 2.0 mg/kg, was injected into the other thigh of 10 animals from each of the respective groups. Thirty min after pretreatment, all animals were challenged with soman (60 micrograms/kg, sc; 2 LD50s); this dose of soman is lethal in unprotected animals. (-)Phy or (+)Phy (10 mg/kg) alone protected nearly 50% from soman lethality, and in combination with THP, all animals survived. In contrast, (+)Phy (0.15 mg/kg; alone or together with THP) was completely ineffective against a 2 LD50 challenge of soman. These data support the hypothesis that protection against soman-induced lethality is related to the degree of carbamylation of the AChE just prior to challenge.

PRELIMINARY ASSESSMENT OF RX 72601, A NEW ANTICHOLINESTERASE IN MAN: REVERSAL OF COMPETITIVE NEUROMUSCULAR BLOCKADE

1 The effect of RX 72601, neostigmine plus atropine, or saline on neuromuscular blockade with (+)-tubocurarine was studied in a double blind trial in four healthy male volunteers. 2 The methods used to assess neuromuscular function were voluntary grip strength and indirectly evoked muscle twitches. The muscarinic action was assessed by measuring intestinal motility with a pressure sensitive radio pill, and auscultation of the abdomen. Blood pressure and pulse rate were also recorded. The degree of acetylcholinesterase inhibition achieved in whole blood was determined before and at intervals after the administration of the anticholinesterases. 3 Both RX 72601 (0.66-0.83 mg) and neostigmine (2.5 mg) completely reversed the neuromuscular blockade produced by tubocurarine, but the time course of reversal differed. RX 72601 (0.66-0.83 mg) produced similar inhibition of acetylcholinesterase in whole blood to neostigmine (2.5 mg). The doses of RX 72601 used caused minimal stimulation at muscarinic sites as evidenced by the limited effect on pulse rate and intestinal activity. 4 It was concluded that RX 72601 could be safely used to reverse the effects of neuromuscular blockade without the need for premedication with atropine.