Acetylcholinesterase Activity In Whole Blood Research Articles

On-substrate Enzymatic Reaction to Determine Acetylcholinesterase Activity in Whole Blood by Paper Spray Mass Spectrometry

Currently, all assays measuring acetylcholinesterase (AChE) activity following a suspected nerve agent exposure leverage methodologies that fail to identify the agent. This limits the overall effectiveness and ability to administer proper countermeasures. As such, there is an urgent need to identify novel, rapid, and more comprehensive approaches to establish AChE activity, including identification of the toxicant. Paper spray mass spectrometry was used to monitor the activity of acetylcholinesterase, both in-solution and on modified hydrophobic paper surface. Hydrophobic paper surfaces were prepared using vaporized trichloro(3,3,3-trifluoropropyl)silane. In both approaches, mixtures of diluted human whole blood with and without VX were mixed with a non-endogenous AChE specific substrate, 1,1-dimethyl-4-acetylthiomethylpiperidinium (MATP+). Formation of the cleaved MATP+ product was monitored over time and compared to MATP+ to determine relative AChE activity. This on-substrate assay was effective at determining AChE activity and identifying the toxicant; however, determination of AChE activity in-solution proceeded at a slower rate. The on-substrate assay serves as a pioneering example of an enzymatic reaction occurring on the surface of a paper spray ionization ticket. This work broadens the range of applications relating to paper spray ionization-based clinical diagnostic assays.Graphical ᅟ

Quercetin treatment regulates the Na+,K+-ATPase activity, peripheral cholinergic enzymes, and oxidative stress in a rat model of demyelination

Quercetin is reported to exert a plethora of health benefits through many different mechanisms of action. This versatility and presence in the human diet has attracted the attention of the scientific community, resulting in a huge output of in vitro and in vivo (preclinical) studies. Therefore, we hypothesized that quercetin can protect Na+,K+-ATPase activity in the central nervous system, reestablish the peripheral cholinesterases activities, and reduce oxidative stress during demyelination events in rats. In line with this expectation, our study aims to find out how quercetin acts on the Na+,K+-ATPase activity in the central nervous system, peripheral cholinesterases, and stress oxidative markers in an experimental model of demyelinating disease. Wistar rats were divided into 4 groups: vehicle, quercetin, ethidium bromide (EB), and EB plus quercetin groups. The animals were treated once a day with vehicle (ethanol 20%) or quercetin 50 mg/kg for 7 (demyelination phase, by gavage) or 21 days (remyelination phase) after EB (0.1%, 10 μL) injection (intrapontine).The encephalon was removed, and the pons, hypothalamus, cerebral cortex, hippocampus, striatum, and cerebellum were dissected to verify the Na+,K+-ATPase activity. Our results showed that quercetin protected against reduction in Na+,K+-ATPase in the pons and cerebellum in the demyelination phase, and it increased the activity of this enzyme in the remyelination phase. During the demyelination, quercetin promoted the increase in acetylcholinesterase activity in whole blood and lymphocytes induced by EB, and it reduced the increase in acetylcholinesterase activity in lymphocytes in the remyelination phase. On day 7, EB increased the superoxide dismutase and decreased catalase activities, as well as increased the thiobarbituric acid–reactive substance levels. Taken together, these results indicated that quercetin regulates the Na+,K+-ATPase activity, affects the alterations of redox state, and participates in the reestablishment of peripheral cholinergic activity during demyelinating and remyelination events.

Relationship Between Pathological Findings and Cholinesterase Activity and Nitric Oxide Levels in Cattle Infected Naturally by Eurytrema coelomaticum

The aim of this study was to evaluate the role of butyrylcholinesterase (BChE) (in the serum and pancreas), acetylcholinesterase (AChE) (in the whole blood and pancreas) and nitric oxide (NO) (in the serum and pancreas) in cattle infected naturally by Eurytrema coelomaticum. Fifty-one cattle were studied, including 33 infected by E. coelomaticum and 18 uninfected animals. Significantly greater AChE activity was found in the pancreas of infected animals (P<0.01); however, these cattle had lower AChE activity in whole blood. BChE activity was greater in the sera of infected animals (P=0.05), but was less in pancreatic samples. NO levels were significantly higher in the sera (P<0.05) and pancreas (P<0.001) of infected cattle compared with uninfected animals. A positive correlation was found between AChE activity in the pancreas and parasite load, but there was negative correlation between pancreatic BChE activity and parasitic load. Expression of AChE, BChE and NO is therefore linked to the inflammation caused by E. coelomaticum in cattle.

Activity of cholinesterases, pyruvate kinase and adenosine deaminase in rats experimentally infected by Fasciola hepatica: Influences of these enzymes on inflammatory response and pathological findings

The aim of this study was to investigate acetylcholinesterase (AChE) in total blood and liver tissue; butyrylcholinesterase (BChE) in serum and liver tissue; adenosine deaminase (ADA) in serum and liver tissue; and pyruvate kinase (PK) in liver tissue of rats experimentally infected by Fasciola hepatica. Animals were divided into two groups with 12 animals each, as follows: group A (uninfected) and group B (infected). Samples were collected at 20 (A1 and B1;n=6 each) and 150 (A2 and B2; n=6 each) days post-infection (PI). Infected animals showed an increase in AChE activity in whole blood and a decrease in AChE activity in liver homogenates (P<0.05) at 20 and 150 days PI. BChE and PK activities were decreased (P<0.05) in serum and liver homogenates of infected animals at 150 days PI. ADA activity was decreased in serum at 20 and 150 days PI, while in liver homogenates it was only decreased at 150 days PI (P<0.05). Aspartate aminotransferase and alanine aminotransferase activities in serum were increased (P<0.05), while concentrations of total protein and albumin were decreased (P<0.05) when compared to control. The histological analysis revealed fibrous perihepatitis and necrosis. Therefore, we conclude that the liver fluke is associated with cholinergic and purinergic dysfunctions, which in turn may influence the pathogenesis of the disease.

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.

Pharmacokinetic and Pharmacodynamic Analysis of Acetylcholinesterase Inhibition by Distigmine Bromide in Rats

Distigmine bromide (distigmine) is associated with a serious adverse reaction, cholinergic crisis, due to a marked decrease in serum acetylcholinesterase (AChE) levels. Clarifying the relationship between the plasma concentration and the inhibitory effect on AChE of distigmine is thus important for the proper use of the drug. The plasma drug concentration and AChE activity in whole blood from rats were measured simultaneously 3 min to 12 h after the oral administration of distigmine at different doses, and the data were subjected to a pharmacokinetic/pharmacodynamic (PK/PD) analysis. Clockwise hysteresis was observed between the plasma concentration of distigmine and the time course of AChE inhibition. Distigmine also displayed a delayed and sustained inhibition of blood AChE activity. We then assumed an effect compartment for the relationship between the plasma concentration of distigmine and AChE inhibition and analyzed the time course of AChE activity using a sigmoid maximum inhibitory effect model as the pharmacodynamic model. In conclusion, there is a time lag between the plasma concentration and inhibitory effect of distigmine in rats, and such a relationship could be resolved with an effect compartment model. Thus, the inhibitory effect of distigmine on AChE could be predicted by the PK/PD analysis.

Changes in acetylcholinesterase (AchE) activity in lymphocytes and whole blood in acute lymphoblastic leukemia patients

Background Acute lymphoblastic leukemia (ALL) is a type of cancer that affects lymphocytes and it is the most common form of cancer in children. Acetylcholinesterase (AChE) is well known as having non-cholinergic functions and has been detected in the blood and plasma of humans including in lymphocytes. Thus, we investigated whole blood and lymphocyte AChE activity in patients with ALL. Methods This study was performed on 72 children with ALL divided into 4 groups: newly diagnosed, remission induction, remission maintenance and out-of-treatment and one control group of 50 healthy subjects. We determined AChE activity in whole blood and lymphocytes of these patients. Results Results demonstrated that whole blood AChE activity was enhanced in the newly diagnosed group and reduced in the remission induction and remission maintenance groups in relation to the control group. For lymphocyte AChE activity we found an increase in the newly diagnosed group and a decrease in the remission induction group in relation to the control. Conclusions These results suggest that AChE activity was altered in ALL patients. This fact may be related with the essential role played by AChE in the development of hematological disease and its contribution to the regulation of immune function.

Androgen receptor antagonism by the organophosphate insecticide fenitrothion.

Organophosphate insecticides represent one of the most widely used classes of pesticides with high potential for human exposure in both rural and residential environments. We investigated the interaction of the organophosphothioate pesticide fenitrothion (O,O-dimethyl O-(4-nitro-m-tolyl) phosphorothioate) with the human androgen receptor (AR). Fenitrothion blocked dihydrotestosterone-dependent AR activity in a concentration-dependent and competitive manner in HepG2 human hepatoma liver cells transiently transfected with human AR and an AR-dependent luciferase reporter gene. Schild regression analysis yielded an equilibrium dissociation constant value of 2.18 x 10(-8) M. To determine the antiandrogenic potential of fenitrothion in vivo, 7-week-old castrated Sprague-Dawley rats were dosed once a day for 7 days with testosterone propionate (50 microg/day, sc) plus gavage doses of either corn oil vehicle or fenitrothion (15 or 30 mg/kg/day). An additional group of rats was given testosterone propionate and flutamide (50 mg/kg/day). Motor activity and acetylcholinesterase activity in whole blood and brain were also assessed. Both fenitrothion and the reference antiandrogen flutamide caused significant decreases in the ventral prostate, seminal vesicle, and levator ani plus bulbocavernosus muscles tissue weights. In contrast, blood acetylcholinesterase activity, a standard biomarker of organophosphate poisoning, was only inhibited at the higher dose of fenitrothion (30 mg/kg). Our results demonstrate that fenitrothion is a competitive AR antagonist, comparable in potency to the pharmaceutical antiandrogen flutamide and more potent, based on in vitro assays, than the known environmental antiandrogens linuron and p,p'-, 2,2-bis(p-hydroxyphenyl)-1,1-dichloroethylene ( p,p'-DDE).

Improved determination of acetylcholinesterase activity in human whole blood

Determination of erythrocyte acetylcholinesterase (AChE) activity is the appropriate tool for the diagnosis of organophosphate exposure and intoxication. The original colorimetric Ellman procedure is disturbed by a high hemoglobin absorption at 412 nm. In our modified method the wavelength was changed to 436 nm. This reduced the indicator absorption to 80% and the hemoglobin absorption to 25%. The signal-to-noise ratio was further enhanced by reduction of pH and substrate concentration, thus making it possible to measure 3% residual activity. AChE activity was determined in whole blood samples in the presence of the selective butyrylcholinesterase inhibitor ethopropazine. Dilution of blood samples (1:100) stops secondary reactions in the presence of inhibitor (organophosphate) and reactivator (oxime). Normalization of the AChE activity to the hemoglobin content, determined as cyanmethemoglobin, prevented dilution errors. This modified approach provides a simple way for sensitive and precise determination of AChE activity in whole blood in the presence of organophosphates even with low-tech equipment.

Neurobehavioral effects of repeated sublethal soman in primates

Juvenile male baboons were trained to perform a match-to-sample discrimination task; effects of repeated sublethal exposure to the organophosphate nerve gas, soman, upon task performance were then explored. Both acute and subchronic exposure schedules were employed, and soman potency was verified by assay of soman-induced inhibition of acetylcholinesterase activity in whole blood, plasma, and erythrocytes. A characteristic profile of behavioral effects encompassing immediate, persistent, and delayed effects was observed. Immediate dose-related effects of soman included: increases in mean session response time, increases in errors, and decreases in extra responses. Seizures were also observed at the highest dose of soman employed (5 μg/kg). The increase in mean session response time was due to intermittent lapses in responding to stimuli (attentional deficits). Both the attentional deficits and intermittent generalized seizures were also persistent effects, with both occuring randomly after acute exposure to 5 μg/kg soman. Preliminary evidence suggests that occurrence of attentional deficits was associated with the occurrence of generalized and/or focal seizures; and that these effects may reflect irreversible lesions which become more threatening to the animal with increasing time. An additional, delayed effect was a sudden marked increase in the incidence of extra inconsequential responses which occurred several weeks after cessation of soman exposures.