Histochemical Identification of Motor Fascicles Using Cholinesterase Staining in Rats Using a Mixed Nerve Model.

BACKGROUND: Inappropriate matching of motor and sensory fibers after nerve repair or grafting can lead to nerve recovery failure. Identifying the motor and sensory fascicles enables surgeons to match them accurately and correctly align nerve stumps, which is crucial for neural regeneration. Very few methods have been reported to differentiate between the sensory and motor nerve fascicles, and the replicability of these techniques remains unestablished. In this study, we aimed to assess the accuracy of axonal cholinesterase (CE) histochemical staining in distinguishing motor and sensory nerve fibers. METHODS: The femoral and sciatic nerves were harvested from rats. The specimens were immediately cut, frozen in isopentane, and cooled with liquid nitrogen. Nerve serial cross-sections were processed for hematoxylin and eosin staining, followed by CE histochemistry. The staining protocol solutions included acetylthiocholine iodide, phosphate buffer, cobalt sulfate hydrate, potassium phosphate monobasic, sulfuric acid, sodium bicarbonate, glutaraldehyde, and ammonium sulfide. RESULTS: Cross-sections of nerves containing efferent and afferent nerve fibers in segregated fascicles showed that CE activity was confined to motor neurons. A histochemical study revealed that motor fibers with high cholinesterase activity can be differentiated from sensory fibers. The motor branches of the femoral and sciatic nerves showed specific axonal staining, whereas the sensory branch did not show any specific staining. CONCLUSION: CE histochemical staining is a useful technique for distinguishing between motor and sensory nerve fibers. It can be potentially useful in improving the outcomes of nerve grafts or extremity allotransplantation surgery.

Spatial distribution differences of cholinesterase in healthy Chinese under the influence of geographical environmental factors.

The main targets of this were to screen the factors that may influence the distribution of cholinesterase (CHE) reference value in healthy people, and further explored the geographical distribution differences of CHE reference value in China. In this study, we collected the CHE data of 17,601 healthy people from 173 cities in China to analyse the correlation between CHE and 22 geography secondary indexes through spearman regression analysis. Six indexes with significant correlation were extracted, and a ridge regression model was built, and the country's urban CHE reference value of healthy Chinese was predicted. By using the disjunctive kriging method, we obtained the geographical distribution of CHE reference values for healthy people in China. The reference value of CHE for healthy Chinese was significantly correlated with the 6 secondary indexes, namely, latitude (°), altitude (m), annual average temperature (°C), annual average relative humidity (%) and annual precipitation (mm), and topsoil sand gravel percentage (% wt). The geographical distribution of CHE values of healthy Chinese showed a trend of being higher in southeast China and lower in northwest. This study lays a foundation for further research on the mechanism of different influencing factors on the reference value of CHE index. A ridge regression model composed of significant influencing factors has been established to provide the basis for formulating reference criteria for the treatment factors of the liver damage diseases and liver cancer using CHE reference values in different regions.

Point-of-care measured serum cholinesterase activity predicts patient outcome following severe burns.

Risk stratification is of utmost importance in burn therapy. However, suitable bedside biomarkers to evaluate the emerging inflammatory response following burn injuries are missing. Serum cholinesterase (butyrylcholinesterase, BChE) has been shown to be a clinically relevant biomarker in acute inflammatory diseases including burns. In this observational cohort study BChE activity was measured by using point-of-care testing (POCT), a novel method in acute burn care. POCT measurements were performed at emergency room admission (ERA) of 35 patients and repeated 12, 24 and 48 h later. All patients or their legal designees gave informed consent. Patients with burn injuries showed sustained BChE activity reduction following hospital admission. BChE activity correlated negatively with burn injury severity, organ failure severity and intensive care unit resource requirements. BChE activity measured at ERA and 12 h later identified survivors and predicted 28-day patient outcome with noninferior efficacy compared to the abbreviated burn severity index (ABSI) scoring. Finally, POCT-measured BChE activity might complement ABSI scoring and possibly improve early risk stratification in acute burn care therapy.

Mass Spectrometric Enzyme Histochemistry Method Developed for Visualizing In Situ Cholinesterase Activity in Mus musculus and Drosophila melanogaster.

Enzyme histochemistry facilitates enzyme activity visualization in situ; however, as it is a color-based method, molecular quantification is prohibitive. This study aimed to develop a semiquantitative, mass spectrometry imaging (MSI)-based enzyme histochemistry method to determine endogenous cholinesterase (ChE) activity. Using deuterium-labeled acetylcholine (ACh-d9) as a substrate to distinguish ACh-d9 and choline-d9 from endogenous acetylcholine and choline, respectively, the heterogeneous localization of de novo ChE activity was visualized using MSI, devoid of interferences from in situ factors. Furthermore, a tissue inhibitor assay involving two ChE inhibitors in the mouse brain revealed specific ChE inhibition in the corpus callosum. To the best of our knowledge, this study is the first to report a visualization method for total ChE activity in the ganglia and abdomen in Drosophila melanogaster, indicating its applicability among different animals. The present results provide novel insights into the applicability of enzyme histochemistry via MSI to the metabolism of low-molecular-weight organic compounds (i.e., "small molecules") and semiquantitative capability, suggesting that MSI enzyme histochemistry may become a powerful tool for heterogeneous tissue studies.

Plasma cholinesterase activity as an environmental impact biomarker in juvenile green turtles (Chelonia mydas) / Atividade da colinesterase plasmática como biomarcador de impacto ambiental em tartarugas verdes juvenis (Chelonia mydas)

The objective of this study was to evaluate the enzymatic activity of plasma cholinesterase in Chelonia mydas marine turtles belonging to two populations, according to their capture sites, under the absence and probable influence of anthropic effects. A total of 74 animals were used and later divided into two groups, based on the capture site. Blood samples were collected from all captured animals, which were then released into the sea at the site of capture. A descriptive statistical analysis of the plasma cholinesterase activity values and an analysis comparing these values based on the capture site were performed. Samples of heparinized plasma from animals captured at the two different sites were analyzed. Plasma cholinesterase activity ranged from 121 to 248U/L, with a mean and standard deviation of 186.1±30.68U/L. When comparing plasma cholinesterase activity values in individuals based on the capture site, a significant difference was observed. Establishing reference values for different sea turtle populations is necessary to interpret future sampling results and to allow sea turtles to be used as sentinels of ecosystem health. Future studies are needed to evaluate other populations and the activity of plasma cholinesterase in juvenile marine turtles, in relation to environmental contamination.(AU)

O objetivo desse estudo foi avaliar a atividade enzimática da colinesterase plasmática em tartarugas marinhas da espécie Chelonia mydas em duas populações de acordo com o local de captura, sob ausência e provável influência de efeito antrópico. Foi utilizado um total de 74 animais e posteriormente divididos em dois grupos de acordo com o local de captura. Foram coletadas amostras de sangue de todos os animais capturados e em seguida liberados ao mar no mesmo local. Foi realizada uma análise estatística descritiva dos valores da atividade plasmática de colinesterase do total de animais e análise comparando os valores de acordo com o local de captura. Foram analisadas amostras de plasma heparinizado de animais capturados em dois locais distintos. Os valores da atividade plasmática de colinesterase variaram de 121 a 248U/L, com média e desvio padrão de 186.1±30.7U/L. Quando comparados os valores de atividade plasmática da colinesterase nos indivíduos de acordo com o local de captura, foi observada diferença significativa. O estabelecimento de valores de referência para diferentes populações de tartarugas marinhas são necessários para interpretar os futuros resultados amostrais e permitir que as tartarugas marinhas sejam usadas como sentinelas da saúde do ecossistema. Estudos futuros são necessários para avaliar outras populações e a atividade da colinesterase plasmática de tartarugas marinhas juvenis em relação à contaminação ambiental.(AU)

Neurotoxicity of organophosphate pesticides could reduce the ability of fish to escape predation under low doses of exposure.

Biomarkers are frequently used in ecotoxicology as they allow to study toxicant effects happening at low concentrations of exposure. However, most sublethal studies only evaluate cellular biomarkers which lack evident ecological relevance. We used a multibiomarker approach to estimate the toxic effects of ethoprophos, an organophosphate insecticide commonly used in banana plantations, on the tropical fish Astyanax aeneus (Characidae). We measured biomarkers at sub-individual (cellular) and individual (metabolism, behavior) levels and examined relationships among these responses. A sublethal exposure to ethoprophos caused a significant (54%) reduction of brain Cholinesterase (ChE) activity, reflecting the pesticide's high neurotoxicity. However, other biomarkers like oxidative stress, biotransformation reactions, and resting metabolic rate were not affected. Exposure to ethoprophos modified antipredator behaviors such as escape response and detection avoidance (light/dark preference): exposed fish escaped slower from a simulated attack and preferred brighter areas in a novel tank. The relationship between ChE activity and reaction time suggests that pesticide-induced ChE inhibition reduces escape ability in fish. Our results provide evidence that impacts of organophosphate pesticides on fish ecological fitness can occur even with short exposures at very low concentrations.

Isorhamnetin enhanced cortico-hippocampal learning and memory capability in mice with scopolamine-induced amnesia: Role of antioxidant defense, cholinergic and BDNF signaling.

Isorhamnetin (IRN), a 3'-O-methylated metabolite of quercetin has antioxidant, anti-inflammatory and neuroprotective properties. In this study, we investigated the learning and memory enhancing effects of IRN on spatial and non-spatial learning and memory deficits induced by scopolamine (3 mg/kg, i.p; muscarinic antagonist) using the novel object recognition test (NORT) and Morris water maze (MWM) task. IRN (1, 5 or 50 mg/kg, p.o.) or vehicle was administered to male albino for 3 consecutive days, scopolamine was given 1 h after last administration on day 3. Five minutes post scopolamine administration the behavioural test of cognitive function was carried out. One hour after probe test (MWM task) on day 7, the brains were isolated to assay for oxidative stress, cholinesterase activity and brain derived neurotrophic factor (BDNF) levels in the prefrontal cortex (PFC) and hippocampus (HIPPO). IRN treatment significantly improved scopolamine-induced learning and memory impairment in behavioural tests. IRN reduced malondialdehyde and nitrite generation induced by scopolamine through increase in glutathione (GSH) level, superoxide dismutase (SOD) and catalase (CAT) activities in the prefrontal cortex and hippocampus. In addition, IRN attenuates scopolamine induced cholinesterase activity and BDNF level in the prefrontal cortex and hippocampus of mice. Findings from this study showed that IRN possesses cognition and memory enhancing properties possibly through enhancement of antioxidant defense system, cholinergic signaling and synaptic plasticity.

Parameters of oxidative stress, cholinesterase activity, Cd bioaccumulation in the brain and midgut of Lymantria dispar (Lepidoptera: Lymantriidae) caterpillars from unpolluted and polluted forests.

Cadmium (Cd) can display a variety of different effects on living organisms. The objectives of the present study were to investigate Cd bioaccumulation and differences in parameters of oxidative stress (activities of the enzymes: superoxide dismutase, catalase, glutathione reductase, ascorbate peroxidase, glutathione S-transferase, and amounts of non-enzymatic free sulfhydryl groups and total glutathione) and cholinesterase activity in larval brain and midgut tissues of the polyphagous forest insect Lymantria dispar collected from unpolluted and polluted oak forests. Fourth instar L. dispar caterpillars from the unpolluted forest had higher body mass but accumulated more Cd in comparison to caterpillars from the polluted forest. In both populations the midgut was more sensitive than the brain to the prooxidative effects of Cd. Enzyme activities and amounts of non-enzymatic parameters tended to be greater in midgut tissues than in the brain, except for cholinesterase activity. Parameters of oxidative stress had higher values in caterpillar tissues from the polluted than from the unpolluted oak forest. The observed differences between the two natural populations point to the importance of knowing the history of population exposure to environmental pollution when monitoring forest ecosystems.

Evaluation of pelvic inflammatory disease potential in cholinesterase inhibitor pesticide-exposed females.

Cholinesterase inhibitor pesticides, mainly organophosphates and carbamates, are commonly used in Egypt. Chronic exposure of males and females working in agriculture is expected. The study aimed to relate exposure to cholinesterase inhibitor pesticides to the development of pelvic inflammatory disease (PID). This is a case-control study that was conducted among 84 females. Seventy patients complained of pelvic inflammatory disease visited the outpatient Gynecology and Obstetrics Clinic. Fourteen females were not suffering from PID and were chosen as a control group. Red blood cells' cholinesterase activity was measured in blood. Cervical swaps were collected, and cultures were submitted for microbiological examination. The results showed that cholinesterase activities were significantly depressed in exposed females (6.36 ± 0.8 µmoles/min/ml red cells) when compared to non-exposed (7.5 ± 1.2 µmoles/min/ml red cells), and both were significantly depressed when compared with healthy females (9.17 ± 0.7 µmoles/min/ml red cells). The correlation coefficient (r) between previous exposure and the laboratory confirmed cervical infection was 0.31, with a P value of 0.009. The study concluded that exposure to cholinesterase inhibitor pesticides could increase the occurrence of pelvic inflammatory disease.

Reference intervals for B-esterases in gull, Larus michahellis (Nauman, 1840) from Northwest Spain: influence of age, gender, and tissue.

Over the last years, cholinesterase (ChE) and carboxylesterase (CbE) activities have been increasingly used in environmental biomonitoring to detect the exposure to anticholinesterase insecticides such as organophosphorates (OPs) and carbamates (CBs). The aim of this study was to determine ChE and CbE enzymatic activities present in liver and muscle of yellow-legged gulls (Larus michahellis), a seabird species considered suitable to monitor environmental pollution. In order to provide reference data for further biomonitoring studies, the influence of different factors, such as gender, age, sampling mode, and tissue, was considered in the present study. Our data report a statistically significant difference in CbE enzymatic activity comparing liver and muscle samples (P < 0.05) along with an age-related CbE activity in liver samples (P < 0.05). Moreover, according to our results, capture method might influence CbE and ChE activity in both liver and muscle samples (P < 0.05). These findings underline the importance to assess basal levels of ChE and CbE activity considering, among other factors, gender-, age- and organ-related differences and confirm the suitability of Larus michahellis as a sentinel species especially within an urban environment.

An improved radiosynthesis of O-(2-[18 F]fluoroethyl)-O-(p-nitrophenyl)methylphosphonate: A first-in-class cholinesterase PET tracer.

O-(2-Fluoroethyl)-O-(p-nitrophenyl) methylphosphonate 1 is an organophosphate cholinesterase inhibitor that creates a phosphonyl-serine covalent adduct at the enzyme active site blocking cholinesterase activity in vivo. The corresponding radiolabeled O-(2-[18 F]fluoroethyl)-O-(p-nitrophenyl) methylphosphonate, [18 F]1, has been previously prepared and found to be an excellent positron emission tomography imaging tracer for assessment of cholinesterases in live brain, peripheral tissues, and blood. However, the previously reported [18 F]1 tracer synthesis was slow even with microwave acceleration, required high-performance liquid chromatography separation of the tracer from impurities, and gave less optimal radiochemical yields. In this paper, we report a new synthetic approach to circumvent these shortcomings that is reliant on the facile reactivity of bis-(O,O-p-nitrophenyl) methylphosphonate, 2, with 2-fluoroethanol in the presence of DBU. The cold synthesis was successfully translated to provide a more robust radiosynthesis. Using this new strategy, the desired tracer, [18 F]1, was obtained in a non-decay-corrected radiochemical yield of 8 ± 2% (n = 7) in >99% radiochemical and >95% chemical purity with a specific activity of 3174 ± 345 Ci/mmol (EOS). This new facile radiosynthesis routinely affords highly pure quantities of [18 F]1, which will further enable tracer development of OP cholinesterase inhibitors and their evaluation in vivo.

The association of alcohol consumption with patient survival after organophosphate poisoning: a multicenter retrospective study.

Organophosphate (OP) intoxication remains a serious worldwide health concern, and many patients with acute OP intoxication have also consumed alcohol. Therefore, we evaluated the association of blood alcohol concentration (BAC) with mortality among patients with OP intoxication. We retrospectively reviewed records from 135 patients who were admitted to an emergency department (ED) for OP intoxication between January 2000 and December 2012. Factors that were associated with patient survival were identified via receiver operating characteristic curve, multiple logistic regression, and Kaplan-Meier survival analyses. Among 135 patients with acute OP poisoning, 112 patients survived (overall mortality rate: 17 %). The non-survivors also exhibited a significantly higher BAC, compared to the survivors [non-survivors: 192 mg/dL, interquartile range (IQR) 97-263 mg/dL vs. survivors: 80 mg/dL, IQR 0-166.75 mg/dL; p < 0.001]. A BAC cut-off value of 173 mg/dL provided an area under the curve of 0.744 [95 % confidence interval (CI) 0.661-0.815], a sensitivity of 65.2 %, and a specificity of 81.2 %. A BAC of >173 mg/dL was associated with a significantly increased risk of 6-month mortality in the multiple logistic regression model (odds ratio 4.92, 95 % CI 1.45-16.67, p = 0.001). The Cox proportional hazard model revealed that a BAC of >173 mg/dL provided a hazard ratio of 3.07 (95 % CI 1.19-7.96, p = 0.021). A BAC of >173 mg/dL is a risk factor for mortality among patients with OP intoxication.

Synthesis, physicochemical and biological studies of technetium-99m labeled tacrine derivative as a diagnostic tool for evaluation of cholinesterase level.

In the present work the synthesis and physicochemical investigations of new tacrine analogues labeled with technetium-99m are reported. All obtained novel radioconjugates showed high stability in the presence of an excess of standard amino acids cysteine or histidine, as well as in human serum. Lipophilicity (LogD values) of these compounds is within the range from 0.92 to 1.56. For the selected radioconjugate 99mTc(NS3)(CN-NH(CH2)7Tac) (LogD=1.56) the biological activity studies in the course of inhibition of acetylcholinesterase action have been performed (IC50=45.0nM, estimated by means of Ellman's method). Biodistribution studies of this compound showed its uptake in brain on the level of 0.07%ID/g and its clearance through the hepatic and renal route in comparable degree. The ascertained presence of the radioconjugate in brain indicates its possibility to cross the blood-brain barrier. Molecular modeling of 99mTc(NS3)(CN-NH(CH2)7Tac) radioconjugate showed that the main structural fragment is tacrine moiety which is responsible for most interactions within catalytic and peripheral active sites and provides the anti-acetylcholinesterase activity. The 99mTc(NS3)(CN-NH(CH2)7Tac) radioconjugate may be considered to be a diagnostic tool for patients suffering from Alzheimer's disease as well as a marker to determine the physiological condition of liver and intestines.

Development of acetophenone ligands as potential neuroimaging agents for cholinesterases.

Association of cholinesterase with ß-amyloid plaques and tau neurofibrillary tangles in Alzheimer's disease offers an opportunity to detect disease pathology during life. Achieving this requires development of radiolabelled cholinesterase ligands with high enzyme affinity. Various fluorinated acetophenone derivatives bind to acetylcholinesterase with high affinity, including 2,2,2-trifluoro-1-(3-dimethylaminophenyl)ethanone (1) and 1-(3-tert-butylphenyl)-2,2,2-trifluoroethanone (2). Such compounds also offer potential for incorporation of radioactive fluorine (18F) for Positron Emission Tomography (PET) imaging of cholinesterases in association with Alzheimer's disease pathology in the living brain. Here we describe the synthesis of two meta-substituted chlorodifluoroacetophenones using a Weinreb amide strategy and their rapid conversion to the corresponding trifluoro derivatives through nucleophilic substitution by fluoride ion, in a reaction amenable to incorporating 18F for PET imaging. In vitro kinetic analysis indicates tight binding of the trifluoro derivatives to cholinesterases. Compound 1 has a Ki value of 7nM for acetylcholinesterase and 1300nM for butyrylcholinesterase while for compound 2 these values are 0.4nM and 26nM, respectively. Tight binding of these compounds to cholinesterase encourages their development for PET imaging detection of cholinesterase associated with Alzheimer's disease pathology.

Cholinergic enzymes and inflammatory markers in rats infected by Sporothrix schenckii.

The aim of this study was to evaluate the cholinesterase activity in serum, whole blood, and lymphocytes, as well as to verify its relation to immune response in rats experimentally infected by Sporothrix schenckii. For this study, 63 Wistar rats (Rattus norvegicus), male, adult were divided into three groups: the negative control group (GC: n = 21), the group infected subcutaneously (GSC: n = 21), and the group infected intraperitoneally (GIP: n = 21). The groups were divided into subgroups and the following variables were evaluated at 15, 30, and 40 days post-infection (PI): acetylcholinesterase (AChE) activity in lymphocytes and whole blood, butyrylcholinesterase (BChE) activity in serum, cytokines levels (IL-1, IL-6, TNFα, and INF-γ), immunoglobulins levels (IgA, IgG, IgM, and IgE), and protein profile by electrophoresis. Both infected groups showed increased levels of inflammatory parameters (P < 0.05) in tissue and inflammatory infiltrates. The activities of AChE in lymphocytes and BChE in serum increased (P < 0.05) significantly in animals from the GSC group on day 40 PI compared to the GC group. Regarding the GIP, there was a marked increase in the AChE activity in lymphocytes on days 30 and 40 PI, and in whole blood on days 15, 30, and 40 PI compared to GC. Furthermore, IL-10, an anti-inflammatory cytokine, was also present in high levels during chronic systemic S. schenckii infections in animals. Therefore, it is concluded that cholinesterase has an important modulatory role in the immune response during granulomatous infection by S. schenckii.

[The therapeutic value of alpha-fetoprotein and cholinesterase in patients with hepatitis B virus related acute onset chronic liver failure].

Objective: To evaluate the therapeutic value of alpha-fetoprotein (AFP) and cholinesterase (ChE) in patients with hepatitis B virus related acute onset chronic liver failure (HBV-ACLF). Methods: A case-control observation was conducted. Sixty-seven patients with HBV-ACLF admitted to Tianjin Second People's Hospital from January 2009 to October 2015 were enrolled. According to the diagnostic criteria of ACLF, the patients were divided into early, middle, and late groups, and alternatively, according to the outcome, they were divided into survival group and death group. Serum samples were collected after 0, 2, 4, 8 weeks to determine the value of AFP and ChE and analyze the value of AFP and ChE in reflecting the changes during HBV-ACLF progression. The differences in AFP and ChE between the survival group and the death group were compared. The prognostic values of AFP and ChE for HBV-ACLF patients were evaluated. Results: Among 67 patients, there were 24, 24, and 19 patients in the early, middle and late stage, respectively, and there were 0, 9, 18 deaths at 8 week. With the advance of HBV-ACLF, the levels of both AFP and ChE were decreased in the early, middle, and late stage, with the AFP value of 40.205 (14.663, 90.550), 23.445 (8.233, 64.213), 8.990 (6.120, 14.340) µg/L (F = 36.149, P = 0.000) and the ChE value of (4.217±1.408), (3.619±1.200), (2.503±1.248) kU/L, respectively (F = 19.575, P = 0.000). In the death group, the levels of serum AFP at 0, 2, 4, 8 weeks were significantly lower than those in survival group [µg/L: 21.540 (7.670, 50.470) vs. 60.680 (16.383, 146.100), 10.560 (6.170, 20.100) vs. 60.090 (27.662, 100.700), 8.750 (3.045, 10.105) vs. 51.875 (16.778, 88.833), 3.900 (2.120, 7.660) vs. 20.400 (12.950, 50.430), P < 0.05 or P < 0.01]. The levels of serum ChE at 2, 4, 8 weeks in the death group were significantly lower than those in the survival group (kU/L: 3.206±1.480 vs. 4.008±1.227, 2.893±1.478 vs. 4.140±1.236, 2.948±1.355 vs. 4.329±1.390, P < 0.05 or P < 0.01). The levels of AFP in 67 patients were 30.100 (10.100, 90.100) µg/L, and ChE was (3.685±1.382) kU/L at 2 weeks, and they showed no correlation between AFP and ChE according to the linear correlation analysis (r = 0.082, P = 0.508), suggesting that AFP and ChE could be used as two independent prognostic factors for HBV-ACLF patients. It was showed by receiver operating characteristic curve (ROC) analysis that the area under the curve of AFP (AUC) was 0.847 (P = 0.000), while the AUC of ChE was 0.681 (P = 0.012). The highest values of Youden index and the maximum effectiveness of testing were obtained when AFP and ChE reached 20.520 µg/L and 2.924 kU/L, respectively, with the sensitivity and the specificity of AFP being 85.0% and 77.8%, respectively, and of ChE being 77.5% and 59.3%, respectively. When using the value of AFP ≥ 20.520 µg/L combined with the value of ChE ≥ 2.924 kU/L, the sensitivity for predicting HBV-ACLF outcome was 65.9%, and its specificity was 91.0%. Conclusion: Both AFP and ChE were helpful in providing accurate information for the progression and prognosis of HBV-ACLF patients due to the facts that their values were less interfered by the clinical treatment and that they have higher specificity.

Atividade da colinesterase plasmática como biomarcador de impacto ambiental em tartarugas verdes (Chelonia mydas) no litoral do Arquipélago de Fernando de Noronha, Pernambuco / Plasma cholinesterase activity as environmental impact biomarker for green turtles (Chelonia mydas) on the coast of Fernando de Noronha, Pernambuco, Brazil

Biomarcadores podem ser usados de forma preditiva, permitindo que sejam tomadas ações de controle antes que ocorram danos ambientais irreversíveis com consequências ecológicas severas, no entanto, espécies sentinelas são necessárias para avaliação desses marcadores. As tartarugas marinhas são consideradas espécies sentinelas quando acometidas por fibropapilomas, sendo sinalizadora do desequilíbrio ambiental marinho nas suas áreas de ocorrência. Com o objetivo de propor a determinação da atividade da colinesterase plasmática em tartarugas verdes (Chelonia mydas) como biomarcador, procedeu-se a determinação da atividade enzimática em animais saudáveis e em localidade de baixo impacto antrópico (Arquipélago de Fernando de Noronha, Pernambuco, Brasil) para servir como referência para comparação com animais capturados em locais de maior impacto antrópico. Ao todo foram analisadas amostras de plasma heparinizado de 35 animais capturados. Todas as amostras analisadas apresentaram alguma atividade enzimática de colinesterase plasmática. Os valores obtidos de colinesterase variaram de 162 a 379 UI/L, com média e desvio padrão de 216,4 ± 51,4 UI/L. Nos estudos de repetibilidade e reprodutibilidade obtiveram-se coeficientes de variação menor que 5% em todas as análises, portanto a metodologia analítica utilizada se mostrou confiável. A longevidade das tartarugas marinhas da espécie C. mydas, o comportamento alimentar, juntamente com o fato de possuirem atividade enzimática detectável podem indicar essa espécie como bioindicadora de exposição a poluentes que influenciam na atividade da colinesterase plasmática.(AU)

Biomarkers can be used in a predictive manner, permitting actions control to be taken before they occur irreversible damage with severe environmental ecological consequences, however, sentinel species are needed to evaluate these markers. Sea turtles affected by fibropapillomas are considered sentinel species, signaling the marine environmental imbalance in their areas of occurrence. With the objective to use the plasma cholinesterase activity in green turtles (Chelonia mydas) as a biomarker, the measurement of enzyme activity was evaluated in healthy animals and in location of low pollution impact (Fernando de Noronha, Pernambuco, Brazil) to serve as a reference for comparison with animals trapped in places with higher pollution impact. Were analyzed heparinized plasma samples from 35 captured animals. All samples analyzed showed enzymatic activity of plasma cholinesterase. The values obtained ranged between 162-379 IU/L, with mean and standard deviation of 216.4 ± 51.4 IU/L. In studies of repeatability and reproducibility were obtained variance coefficients of less than 5% in all analyzes, so the analytical methodology proved successful. The longevity of marine turtles of the species C. mydas, feeding behavior, along with the fact possess detectable enzyme activity may indicate this species as a bioindicator of exposure to pollutants that influence plasma cholinesterase activity.(AU)

[Methods for determination of cholinesterase activity]. / Metódy stanovenia aktivity cholínesteráz.

Cholinesterases hydrolyze acetylcholine and thus they play a key role in a process of cholinergic neurotransmission. Changes in their activities are linked to many diseases (e.g Alzheimer disease, Parkinson disease, lipid disorders). Thus, it is important to determine their activity in a fast, simply and precise way. In this review, different approaches of studying cholinesterase activities (e.g pH-dependent, spectrophotometric, radiometric, histochemical methods or biosensors) are discussed. Comparisons, advantages or disadvantages of selected methods (e.g most widely used Ellman's assay, extremely sensitive Johnson Russell method or modern technique with golden nanoparticles) are presented. This review enables one to choose a suitable method for determination of cholinesterase activities with respect to laboratory equipment, type of analysis, pH, temperature scale or special conditions.

The assessment of cholinesterase from the liver of Puntius javanicus as detection of metal ions.

Crude extract of ChE from the liver of Puntius javanicus was purified using procainamide-sepharyl 6B. S-Butyrylthiocholine iodide (BTC) was selected as the specific synthetic substrate for this assay with the highest maximal velocity and lowest biomolecular constant at 53.49 µmole/min/mg and 0.23 mM, respectively, with catalytic efficiency ratio of 0.23. The optimum parameter was obtained at pH 7.5 and optimal temperature in the range of 25 to 30°C. The effect of different storage condition was assessed where ChE activity was significantly decreased after 9 days of storage at room temperature. However, ChE activity showed no significant difference when stored at 4.0, 0, and -25°C for 15 days. Screening of heavy metals shows that chromium, copper, and mercury strongly inhibited P. javanicus ChE by lowering the activity below 50%, while several pairwise combination of metal ions exhibited synergistic inhibiting effects on the enzyme which is greater than single exposure especially chromium, copper, and mercury. The results showed that P. javanicus ChE has the potential to be used as a biosensor for the detection of metal ions.

Phenolic contents, antioxidant and anticholinesterase potentials of crude extract, subsequent fractions and crude saponins from Polygonum hydropiper L.

BACKGROUND: We investigated Polygonum hydropiper L. (P. hydropiper) for phenolic contents, antioxidant, anticholinesterase activities, in an attempt to rationalize its use in neurological disorders. METHODS: Plant crude extract (Ph.Cr), its subsequent fractions: n-hexane (Ph.Hex), chloroform (Ph.Chf), ethyl acetate (Ph.EtAc), n-Butanol (Ph.Bt), aqueous (Ph.Aq) and saponins (Ph.Sp) were evaluated for 1,1-diphenyl,2-picrylhydrazyl (DPPH), 2,2-azinobis[3-ethylbenzthiazoline]-6-sulfonic acid (ABTS) free radical scavenging potential. Further, acetylcholinesterase (AChE) & butyrylcholinesterase (BChE) inhibitory activities were performed using Ellman's assay. Moreover, total phenolic contents of plant extracts were determined and expressed in mg of gallic acid equivalent per gram of dry sample (mg GAE/g dry weight). RESULTS: Among different fractions, Ph.Cr (90.82), Ph.Chf (178.16), Ph.EtAc (203.44) and Ph.Bt (153.61) exhibited high phenolic contents. All fractions showed concentration dependent DPPH scavenging activity, with Ph.EtAc 71.33% (IC50 15 µg/ml), Ph.Bt 71.40% (IC50 3 µg/ml) and Ph.Sp 71.40% (IC50 35 µg/ml) were most potent. The plant extracts exhibited high ABTS scavenging ability i.e. Ph.Bt (91.03%), Ph.EtAc (90.56%), Ph.Sp (90.84%), Ph.Aq (90.56%) with IC50<0.01 µg/ml. All fractions showed moderate to high AChE inhibitory activity as; Ph.Cr, 86.87% (IC50 330 µg/ml), Ph.Hex, 87.49% (IC50 35 µg/ml), Ph.Chf, 84.76% (IC50 55 µg/ml), Ph.Sp, 87.58% (IC50 108 µg/ml) and Ph.EtAc 79.95% (IC50 310 µg/ml) at 1 mg/ml). Furthermore the BChE inhibitory activity was most prominent in Ph.Hex 90.30% (IC50 40 µg/ml), Ph.Chf 85.94% (IC50 215 µg/ml), Ph.Aq 87.62% (IC50 3 µg/ml) and Ph.EtAc 81.01% (IC50 395 µg/ml) fractions. CONCLUSIONS: In this study, for the first time, we determined phenolic contents, isolated crude saponins, investigated antioxidant and anticholinestrase potential of P. hydropiper extracts. The results indicate that P. hydropiper is enriched with potent bioactive compounds and warrant further investigation by isolation and structural elucidation to find novel and affordable compounds for the treatment of various neurological disorders.