Plasma Butyrylcholinesterase Research Articles

Human Plasma Butyrylcholinesterase Hydrolyzes Atropine: Kinetic and Molecular Modeling Studies.

The participation of butyrylcholinesterase (BChE) in the degradation of atropine has been recurrently addressed for more than 70 years. However, no conclusive answer has been provided for the human enzyme so far. In the present work, a steady-state kinetic analysis performed by spectrophotometry showed that highly purified human plasma BChE tetramer slowly hydrolyzes atropine at pH 7.0 and 25 °C. The affinity of atropine for the enzyme is weak, and the observed kinetic rates versus the atropine concentration was of the first order: the maximum atropine concentration in essays was much less than Km. Thus, the bimolecular rate constant was found to be kcat/Km = 7.7 × 104 M-1 min-1. Rough estimates of catalytic parameters provided slow kcat < 40 min-1 and high Km = 0.3-3.3 mM. Then, using a specific organophosphoryl agent, echothiophate, the time-dependent irreversible inhibition profiles of BChE for hydrolysis of atropine and the standard substrate butyrylthiocholine (BTC) were investigated. This established that both substrates are hydrolyzed at the same site, i.e., S198, as for all substrates of this enzyme. Lastly, molecular docking provided evidence that both atropine isomers bind to the active center of BChE. However, free energy perturbations yielded by the Bennett Acceptance Ratio method suggest that the L-atropine isomer is the most reactive enantiomer. In conclusion, the results provided evidence that plasma BChE slowly hydrolyzes atropine but should have no significant role in its metabolism under current conditions of medical use and even under administration of the highest possible doses of this antimuscarinic drug.

Acute poisoning by chlorpyrifos differentially impacts survival and cardiorespiratory function in normotensive and hypertensive rats

Hypertension is the most important and well-known risk factor for cardiovascular disease (CVD). Recently, acute organophosphate (OP) poisoning has also been pointed as a CVD risk factor. Despite this evidence, no studies have contrasted the acute toxicosis and cardiovascular (CV) effects of OP poisoning under conditions of normotension and hypertension. In this work, adult male normotensive Wistar and Spontaneously Hypertensive rats (SHR) were intraperitoneally injected with saline or chlorpyrifos (CPF), an OP compound, monitored for acute toxicosis signs and 24-h survival. After poisoning, blood pressure, heart rate and ventilation were recorded, the Bezold-Jarisch Reflex (BJR), the Chemoreflex (CR) were chemically activated, as well as the cardiac autonomic tone (AUT) was assessed. Erythrocyte and brainstem acetylcholinesterase and plasmatic butyrylcholinesterase (BuChE) activities were measured as well as lipid peroxidation, advanced oxidation protein products (AOPP), nitrite/nitrate levels, expression of catalase, TNFα and angiotensin-I converting enzyme (ACE-1) within the brainstem. CPF induced a much more pronounced acute toxicosis and 33 % lethality in SHR. CPF poisoning impaired ventilation in SHR, the BJR reflex responses in Wistar rats, and the chemoreflex tachypneic response in both strains. CPF inhibited activity of cholinesterases in both strains, increased AOPP and nitrite/nitrate levels and expression of TNFα and ACE-1 in the brainstem of Wistar rats. Interestingly, SHR presented a reduced intrinsic BuChE activity, an important bioscavenger. Our findings show that, CPF at sublethal doses in normotensive rats lead to lethality and much more pronounced acute toxicity signs in the SHR. We also showed that cardiorespiratory reflexes were differentially impacted after CPF poisoning in both strains and that the cardiorespiratory disfunction seems to be associated with interference in cholinergic transmission, oxidative stress and inflammation. These results points to an increased susceptibility to acute toxicosis in hypertension, which may impose a significant risk to vulnerable populations.

Structure-Dependent Mechanism of Organophosphate Release from Albumin and Butyrylcholinesterase Adducts When Exposed to Fluoride Ion: A Comprehensive In Silico Study.

The most favorable targets for retrospectively determining human exposure to organophosphorus pesticides, insecticides, retardants, and other industrial organophosphates (OPs) are adducts of OPs with blood plasma butyrylcholinesterase (BChE) and human serum albumin (HSA). One of the methods for determining OP exposure is the reactivation of modified BChE using a concentrated solution of KF in an acidic medium. It is known that under the action of fluoride ion, OPs or their fluoroanhydrides can be released not only from BChE adducts but also from the adducts with albumin; however, the contribution of albumin to the total pool of released OPs after plasma treatment with KF has not yet been studied. The efficiency of OP release can be affected by many factors associated with the experimental technique, but first, the structure of the adduct must be taken into account. We report a comparative analysis of the structure and conformation of organophosphorus adducts on HSA and BChE using molecular modeling methods and the mechanism of OP release after fluoride ion exposure. The conformational analysis of the organophosphorus adducts on HSA and BChE was performed, and the interaction of fluoride ions with modified proteins was studied by molecular dynamics simulation. The geometric and energy characteristics of the studied adducts and their complexes with fluoride ion were calculated using molecular mechanics and semiempirical approaches. The structural features of modified HSA and BChE that can affect the efficiency of OP release after fluoride ion exposure were revealed. Using the proposed approach, the expediency of using KF for establishing exposure to different OPs, depending on their structure, can be assessed.

Cytotoxic and oxidative changes in individuals occupationally exposed to recyclable municipal solid waste

ABSTRACT aste collectors are exposed to a wide variety of bacteria, endotoxins, fungi, allergens, particulate matter, irritating inhalants, and vehicle exhaust, making them more prone to development of chronic diseases. Although several studies described the impact of occupational exposure on the overall health of waste collectors, few investigations were conducted regarding cellular and molecular changes that may occur due to exposure. The aim of this study was to assess biomarkers of oxidative stress such as levels of reactive oxygen species (ROS), lipoperoxidation, total antioxidant capacity (TAC), apoptosis, butyrylcholinesterase (BChE) activity and mitochondrial function (MitoTrackerTM Green FM and MitoTrackerTM Red) using the peripheral blood from individuals occupationally exposed to recyclable solid waste in Southern Brazil. The study included 30 waste collectors and 30 control individuals, who did not perform activities with recognized exposure to biological and chemical substances. Waste collectors were found to exhibit in peripheral blood leukocytes (PBL) higher rates of apoptosis, increased production of ROS, and reduced mitochondrial membrane potential (MMP), associated with decreased total antioxidant capacity (TAC) and elevated activity of BChE in plasma. Therefore, evidence indicates that cytotoxicity, oxidative stress, and inflammatory responses may be involved in the multiplicity of adverse health outcomes related to contaminant exposure in waste collectors. It is thus necessary to implement and/or improve occupational health programs aimed at workers as well as mandatory inspections for the use of personal protective equipment.

Cholinesterases as hemolytic and Inflammatory markers in Beta thalassemia trait patients

Beta thalassemia trait (BTT) is one of the most common monogenic disorders characterized by increased alpha and gamma chains with concomitant decrease in beta chains. These patients are clinically asymptomatic with mild anemia, subclinical inflammation and endothelial dysfunction. This study was undertaken to determine the role of cholinesterases in BTT. The study population included 30 BTT patients and 30 age and sex matched controls. Patients were labelled as BTT only if they had Hb A2 &gt;3.5 and/ HbF&gt;2 by variant analysis using cation HPLC. Percentage hemolysis, erythrocyte acetyl cholinesterase (AChE), plasma butyryl cholinesterase (BChE) and globulins were estimated by spectrophotometric methods. Intracellular adhesion molecule (ICAM-1) was evaluated by ELISA. Percentage hemolysis was significantly high in BTT patients. Both the cholinesterases were lower in BTT patients compared to controls. Decrease in BChE was statistically significant (p&lt; 0.0001). An apparent increase in plasma ICAM-1 was observed in BTT patients compared to the normal group. The increase in plasma globulins was statistically significant in BTT (p&lt;0.0001). AChE and HbA1C showed significant negative correlation with percentage hemolysis. Further, acetylcholinesterases showed a statistically significant negative correlation with ICAM-1. A negative correlation between globulins and cholinesterases in BTT, highlights the involvement of cholinesterases in inflammation.Based on our findings, we conclude that cholinesterases play a key role in inflammation and can be considered as biomarkers of hemolysis in BTT.

The relationship between the cholinergic mechanism of toxicity and oxidative stress in rats during subacute diazinon poisoning

Diazinon is an organophosphate pesticide (OP) that has significant potential for accidental and intentional poisoning of wildlife, domestic animals and humans.The aim of the study is to investigate the correlation between cholinesterase activity and oxidative stress parameters in liver and diaphragm by continuous monitoring as a function of time during prolonged use of diazinon.Wistar rats were treated orally with diazinon (55 mg/kg/day): 7, 14, 21 and 28 days. At the end of each period, blood, liver and diaphragm were collected to examine cholinesterase activity and enzymatic/non-enzymatic oxidative stress parameters: superoxide dismutase 1 (SOD1), catalase (CAT), thiobarbituric acid substances (TBARS), protein carbonyl groups.In all four time periods, there was a significant change in acetylcholinesterase (AChE) in erythrocytes and butyrylcholinesterase (BuChE) in blood plasma, CAT in liver and diaphragm and SOD1 in diaphragm. Parameters significantly altered during the cholinergic crisis included: cholinesterases and TBARS in liver and diaphragm and partially SOD1 in liver. Protein carbonyl groups in liver and diaphragm were significantly altered outside the cholinergic crisis. In the liver, there was a very strong negative correlation between BuChE and TBARS in all four time periods and BuChE and CAT on day 7. In the diaphragm, a very strong negative correlation was found between AChE and TBARS at days 7 and 14, and a very strong positive correlation between AChE and SOD1 at days 14, 21 and 28.A better understanding of the relationship between cholinergic overstimulation and oxidative stress may help to better assess health status in prolonged OPs intoxication.

Arylboronic acids as safe and specific human butyrylcholinesterase inhibitors

The drug treatment of Alzheimer's disease (AD) remains a significant scientific challenge, necessitating a continual search for new drug candidates. In this context, we present here, for the first time, new oxime-arylboronic acid hybrids (L1-L6) as potential anti-AD agents. These hybrids were synthesized and evaluated for their inhibitory capabilities on the activity of non-human cholinesterases [Electrophorus electricus Acetylcholinesterase (EeAChE) and Equine Butyrylcholinesterase (EqBChE)] as well as human cholinesterases [erythrocyte Acetylcholinesterase (hAChE) and plasma Butyrylcholinesterase (hBChE)]. We also assessed the safety of these compounds in a microglia cell line and analyzed the compound-enzyme interactions using molecular docking and molecular dynamics simulations. All compounds were found to be safe for the tests conducted. None of them exhibited significant inhibitory activity against hAChE or EeAChE. However, L1-L5 demonstrated inhibition of EqBChE and hBChE, with L3 being the most potent inhibitor of hBChE (IC50 = 6.41 ± 0.62 µM) and EqBChE (IC50 = 81.28 ± 4.01 µM). Additionally, L2, L3, and L5 showed 8–15 times higher potency as inhibitors of hBChE compared to EqBChE. These findings indicate that the hybrid oxime-arylboronic acids act as specific inhibitors of BChE, with a notable selectivity for hBChE, making them promising structures for the development of more potent and selective hBChE inhibitors.

A retrospective screening method for carbamate toxicant exposure based on butyrylcholinesterase adducts in human plasma with ultra-high performance liquid chromatography–tandem mass spectrometry

Carbamate pesticides are extensively used in agriculture for their inhibition to acetylcholinesterase and damages to the insects’ neural systems. Because of their toxicity, human poisoning incidents caused by carbamate pesticide exposure have occurred from time to time. What’s more, some lethally toxic carbamate toxicants known as carbamate nerve agents (CMNAs) have been supplemented in Schedule 1 of the Annex on Chemicals in the Chemical Weapons Convention (CWC) by Organisation of the Prohibition of Chemical Weapons (OPCW) from 2020. And some other carbamates, like physostigmine, have been used in clinical treatment as anticholinergic drugs and their misuse may also cause damages to the body. Similar to organophosphorus toxicants, carbamate toxicants would react with butyrylcholinesterase (BChE) in plasma when entering the human body, resulting in the BChE adducts, based on which the exposure of carbamate toxicants could be detected retrospectively. In this study, methylcarbamyl nonapeptide and dimethylcarbamyl nonapeptide from pepsin digestion of BChE adducts were identified with ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) in product ion scan mode. Carbofuran was chosen as the target to establish the detection method of carbamate toxicant exposure based on methylcarbamyl nonapeptide digested from methylcarbamyl BChE. Procainamide-gel affinity purification, pepsin digestion and UHPLC–MS/MS analysis in multiple reaction monitoring (MRM) mode were applied. Under the optimized conditions of sample preparation and UHPLC–MS/MS MRM analysis, the limits of detection (LODs) reached 10.0 ng/mL of plasma exposed to carbofuran with satisfactory specificity. The quantitation approach was established with d3-carbofuran-exposed plasma as the internal standard (IS) and the linearity range was 30.0–1.00 × 103 nmol/L (R2 >0.998) with the accuracy of 95.6%–107% and precision of ≤9% relative standard deviation (RSD). The applicability was also evaluated by N,N-dimethyl-carbamates with the LODs of 30.0 nmol/L for pirimicarb-exposed plasma based on dimethylcarbamyl nonapeptide. Because most of carbamate toxicants has methylcarbamyl or dimethylcarbamyl groups, this approach could be applied on the retrospective screening of carbamate toxicant exposure including CMNAs, carbamate pesticides or carbamate drugs. This study could provide an effective means in the fields of CWC verification, toxicological mechanism investigation and down-selection of potential treatment options.

Fluorescence nanoplatform based on conjugated polymer nanoparticles-transition metal hydroxide composite for the detection of Butyrylcholinease

Butyrylcholinesterase (BChE) as a valuable biochemical indicator could evaluate liver's function in clinical diagnosis by blood. The traditional Ellman colorimetric method needs enzymatic cascades and additional substances, which greatly limits its practical application. The fluorescence strategy was broadly applied because of great selectivity, sensitivity and convenience. Here, a novel fluorescence sensing platform was built by internal filtering effect (IFE) based on conjugated polymer nanoparticles (CPNsPBOC-COOH) and transition metal hydroxides (MOOH) for the detection of BChE. In order to get an optimized detection platform, three different MOOH (FeOOH/ CoOOH/ NiOOH) were selected, and their preparation conditions, water solubility, the morphology, the absorption and the fluorescence quenching efficiency were investigated. The results showed FeOOH preparated at 120°C for 4 h have the better water solubility and size uniform, the broad absorption and the high quenching efficiency of 95.3%. At the same time, the fluorescence platform of CPNsPBOC-COOH@FeOOH is highly selective with lower interference. So the optimized fluorescence detection platform of CPNsPBOC-COOH@FeOOH (CCPNsPBOC-COOH =1 µg·mL−1, CFeOOH =60 µg·mL−1) was selected and obtained. Furthermore, the fluorescence platform of CPNsPBOC-COOH@FeOOH has a detection range of 0.02∼2.6 µg·mL−1 and a very low detection limit of 3.5 ng·mL−1, showing it very sensitive for the BChE. The determination of BChE in human serum and plasma was applied using this detection technology, and the result was satisfactory. At the same time, the detection mechanism was explored. The result data indicate that FeOOH is decomposed by thiocholine (TCh), an enzymatic digestion product from acetylcholine (ATCh) hydrolyzed by BChE, and then the fluorescence of CPNsPBOC-COOH@FeOOH platform is recovered. The Fe2+ in products can be verified by specifically reacting with [Fe (CN)6]3−.

Butyrylcholinesterase in lipid metabolism: A new outlook.

Cholinesterase enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are traditionally associated with the termination of acetylcholine mediated neural signaling. The fact that these ubiquitous enzymes are also found in tissues not involved in neurotransmission has led to search for alternative functions for these enzymes. Cholinesterases are reported to be involved in many lipid related disease states. Taking into view that lipases and cholinesterases belong to the same enzyme class and by comparing the catalytic sites, we propose a new outlook on the link between BChE and lipid metabolism. The lipogenic substrates of BChE that have recently emerged in contrast to traditional cholinesterase substrates are explained through the hydrolytic capacity of BChE for ghrelin, 4-methyumbelliferyl (4-mu) palmitate, and arachidonoylcholine and through endogenous lipid mediators such as cannabinoids like anandamide and essential fatty acids. The abundance of BChE in brain, intestine, liver, and plasma, tissues with active lipid metabolism, supports the idea that BChE may be involved in lipid hydrolysis. BChE is also regulated by various lipids such as linoleic acid, alpha-linolenic acid or dioctanoylglycerol, whereas AChE is inhibited. The finding that BChE is able to hydrolyze 4-mu palmitate at a pH where lipases are less efficient points to its role as a backup in lipolysis. In diseases such as Alzheimer, in which elevated BChE and impaired lipid levels are observed, the lipolytic activity of BChE might be involved. It is possible to suggest that fatty acids such as 4-mu palmitate, ghrelin, arachidonoylcholine, essential fatty acids, and other related lipid mediators regulate cholinesterases, which could lead to some sort of compensatory mechanism at high lipid concentrations.

Generic detection of organophosphorus nerve agent adducts to butyrylcholinesterase in plasma using liquid chromatography-tandem mass spectrometry combined with an improved procainamide-gel separation and pepsin digestion method

Organophosphorus nerve agent (OPNA) adducts to butyrylcholinesterase (BChE) can be applied to confirm exposure in humans. A sensitive method for generic detection of G- and V-series OPNA adducts to BChE in plasma was developed by combining an improved procainamide-gel separation (PGS) and pepsin digestion protocol with ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Residual matrix interferences from prior PGS purification of OPNA-BChE adducts from plasma were found to be a critical cause of significantly reduced UHPLC-MS/MS detection sensitivity. In our developed on-column PGS approach, the matrix interference was successfully removed by adding an appropriate concentration of NaCl to the washing buffer, and it could capture ≥92.5% of the BChE in plasma. The lower pH value and the longer digestion time in all previous pepsin digestion methods were found to be a key accelerated aging factor of several adducts such as tabun (GA)-, cyclohexylsarin (GF)-, and soman (GD)-BChE nonapeptide adducts, making them difficult to detect. The aging event of several OPNA-BChE nonapeptide adducts was so successfully addressed that the formic acid level in enzymatic buffer and digestion time were lowered to 0.05% (pH 2.67) and 0.5 h, respectively, and the post-digestion reaction was immediately terminated. The improved condition parameters were optimal for pepsin digestion of all types of OPNA-BChE adducts into their individual unaged nonapeptide adducts with the highest yields, expanding the applicability of the method. The method had a nearly one-fold decrease in sample preparation time through the reduction of digestion time and removal of ultrafiltration procedure after digestion. The limit of identification (LOI) were determined respectively as 0.13 ng mL−1, 0.28 ng mL−1, 0.50 ng mL−1, 0.41 ng mL−1 and 0.91 ng mL−1 for VX-, sarin (GB)-, GA-, GF-, and GD-exposed human plasma, being low exposure value compared to previously documented approaches. The approach was utilized to fully characterize the adducted (aged and unaged) BChE levels of five OPNAs in a series of their individual exposed concentration (1.00–400 nM) of plasma sample, and successfully detect OPNA exposure from all unknown plasma samples from OPCW's second and third biomedical proficiency tests. The OPNA-BChE adducts, their aged adducts, and unadducted BChE from OPNA-exposed plasma can simultaneously be measured using the method. The study provides a recommended diagnostic tool for generic verification of any OPNA exposure with high confidence by detecting its corresponding BChE adduct.

Bioconversion and P-gp-Mediated Transport of Depot Fluphenazine Prodrugs after Intramuscular Injection

Fluphenazine (FPZ) decanoate, an ester-type prodrug formulated as a long-acting injection (LAI), is used in the treatment of schizophrenia. FPZ enanthate was also developed as an LAI formulation, but is no longer in use clinically because of the short elimination half-life of FPZ, the parent drug, after intramuscular injection. In the present study, the hydrolysis of FPZ prodrugs was evaluated in human plasma and liver to clarify the reason for this difference in elimination half-lives. FPZ prodrugs were hydrolyzed in human plasma and liver microsomes. The rate of hydrolysis of FPZ enanthate in human plasma and liver microsomes was 15-fold and 6-fold, respectively, faster than that of FPZ decanoate. Butyrylcholinesterase (BChE) and human serum albumin (HSA) present in human plasma, and two carboxylesterase (CES) isozymes, hCE1 and hCE2, expressed in ubiquitous organs including liver, were mainly responsible for the hydrolysis of FPZ prodrugs. FPZ prodrugs may not be bioconverted in human skeletal muscle at the injection site because of lack of expression of BChE and CESs in muscle. Interestingly, although FPZ was a poor substrate for human P-glycoprotein, FPZ caproate was a good substrate. In conclusion, it is suggested that the shorter elimination half-life of FPZ following administration of FPZ enanthate compared with FPZ decanoate can be attributed to the more rapid hydrolysis of FPZ enanthate by BChE, HSA and CESs.

Biochemical role of zinc in vivax malaria

Introduction and Aim: Zinc is a micronutrient that has a significant immune modulatory function, its deficiency is known to increase the risk of plasmodium infection in humans Although inflammatory processes contribute to the elimination of plasmodium, persistent inflammation is the underlying cause of malaria pathology. Cholinesterases are carboxypeptidases that are zinc dependent and are the indicators of low-grade inflammation. Plasma butyryl cholinesterase, is a biomarker of dietary status of zinc. Matrix metalloproteinases (MMP) are endopeptidases that contain zinc which take part in inflammatory reactions. They participate in remodeling of extracellular matrix and aid tissue repair. The current investigation aims to study the association of these metalloproteins with zinc in patients with vivax malaria. Materials and methods: Plasma zinc, butyrylcholinesterase (BChE) and erythrocyte acetylcholinesterase (AChE) were estimated spectrophotometrically in 100 vivax malaria patients and 50 healthy subjects. MMP9 was determined using ELISA. Results: Plasma zinc was markedly lower in malaria patients compared to healthy controls (p&lt;0.05). Both BChE and AChE decreased significantly in malaria (p&lt;0.00) compared to healthy subjects. There was an elevation of MMP9 in malaria, although the increase was statistically insignificant. Cholinesterases correlated positively with zinc in controls and malaria. MMP9 showed a significant positive correlation with zinc in malaria patients (r=0.405, p=0.05). Conclusion: The study highlights the role of zinc in the pathology of malaria as it is essential for the maintenance of several enzyme activities. Further, prompt administration of micronutrients like zinc may reduce the inflammation related morbidity in malaria.

Comparison of Biochemical, Haematological and Plasmatic Butyrylcholinesterase Parameters in Farmers and Non-Farmers, Morocco.

The long-term use of pesticides can cause harmful consequences to both human health and the environment. In the present research, we aimed to compare biochemical, hematological, and plasmatic measurements of butyrylcholinesterase (BChE) between farmers and non-farmers. The study is cross-sectional and included 270 participants, with 135 farmers using pesticides and a control population of 135 non-farmers. The recruitment of the participants was conducted from August 2017 to the end of December 2019. Blood samples from participants were collected for the evaluation of biochemical markers of the function of the liver and determination of BChE activity. A whole blood sample with ethylenediamine tetraacetic anticoagulant (EDTA) was also taken for a complete blood count. The results showed a statistically significant (P = 0.03) decrease in mean corpuscular hemoglobin (MCH) in the cases (28.45 ± 2.94 pg) as compared with controls (29.17 ± 2.54 pg). The statistical analysis of the renal parameters between the two groups determined that the uremia value was significantly higher in cases (34 ± 12 mg/dL) when compared to the control group (29 ± 8 mg/dL) P < 0.001. The cases recorded a significant increase in aspartate aminotransferase (AST) (26.22 ± 11.59 U/L) and alanine aminotransferase (ALT) (25.63 ± 13.47 U/L) enzyme activities among cases versus controls. The results obtained showed a significantly decreased BChE activity in the group of cases exposed to pesticides (7554.52 ± 2107 U/l) compared to the unexposed control group (10135.58 ± 1909 U/l) (t-test, P < 0.001). The education of the farmers on correct practices concerning phytosanitary use has the potential of reducing their exposure to these products.

The first and the second types of diabetes mellitus, autoimmune and non-autoimmune thyroiditis differential diagnosis possibility using the calculated index based on plasma butyrylcholinesterase isoforms activity

BACKGROUND: The possibility of using a diagnostic index based on the use of blood plasma objective indicators to differentiate the diagnosis of type 1 and type 2 diabetes mellitus, autoimmune and non-autoimmune thyroiditis is an urgent task for timely decision-making on the use of a particular type of therapy, taking into account the etiology of a particular pathology. Changing the ratio of blood plasma butyrylcholinesterase isoforms activity makes it possible to identify increased excitability, to separate them among themselves depending on the genesis, and during treatment, to assess the activity of diseases and the effectiveness of therapeutic measures.&#x0D; AIM: To develop a calculated diagnostic index for the differential diagnosis of diabetes and thyroiditis autoimmune type from symptomatically similar non-autoimmune pathologies, using the ratio of the plasma pseudocholinesterase isoforms activities.&#x0D; MATERIALS AND METHODS: The study involved 188 people: 45 patients diagnosed with type 1 diabetes; 60 patients diagnosed with type 2 diabetes; 25 patients diagnosed with autoimmune thyroiditis; 25 patients with non-autoimmune thyroiditis; the control group consisted of 33 healthy volunteers. The groups were aligned by gender and age. Patients of all groups were examined by an endocrinologist and a therapeutist. The diagnoses were made in accordance with the Clinical Guidelines of the Russian Endocrinologists Association for the Diagnosis and Treatment of Autoimmune Thyroiditis in Adults, as well as the Clinical Guidelines Type 1 Diabetes Mellitus in Adults. In the blood plasma of all patients groups, the activity of pseudocholinesterase (butyrylcholinesterase) forms was determined by analysis using a modification of the Ellmans method in combination with inhibitory analysis. The index was calculated based on the ratio of the studied enzyme typical and atypical forms activities. Based on the results of data analysis for all examined groups, diagnostic intervals were determined that are characteristic for each diagnosis, as well as for conditionally healthy people. Comparison of the patients distribution by categories of diagnosis was carried out using the -square test with Yates correction in the SPSS 22 statistical software package.&#x0D; RESULTS: The results of the blood plasma analysis determined that in the diagnoses of autoimmune thyroiditis and type 1 diabetes mellitus, there was an increase in the activity of increased activity of the butyrylcholinesterase isoform, while the activity of the atypical form remains more stable. The ratio of these forms was taken as the basis for the development of the calculated diagnostic index. Diagnostic index for the control group in the range of 12; for patients with non-autoimmune thyroiditis 2,12,9; autoimmune thyroiditis 33,5; type 1 diabetes mellitus 3,65; type 2 diabetes 01. Statistical analysis of the data showed that the diagnosis based on the index obtained in this way does not inferior to the diagnostic scales in terms of sensitivity and specificity. The findings are consistent with the evolution of data supporting each diagnosis.&#x0D; CONCLUSIONS: The calculated index on the basis of butyrylcholinesterase activity is a promising, minimally invasive, fast and budgetary method, revealing a pronounced severity of occurrence in terms of symptoms, but different in etiology, type 1 diabetes mellitus and type 2 diabetes mellitus, autoimmune thyroiditis and thyroiditis of a non-autoimmune nature, which is of critical importance when choosing a therapeutic strategy and improving the quality of life of patients.

Intermittent exposure to chlorpyrifos results in cardiac hypertrophy and oxidative stress in rats

Forbidden in some countries due to its proven toxicity to humans, chlorpyrifos (CPF) still stands as an organophosphate pesticide (OP) highly used worldwide. Cardiotoxicity assessment is an unmet need in pesticide regulation and should be deeply studied through different approaches to better inform and generate an appropriate regulatory response to OP use. In the present study, we used our 4-week intermittent OP exposure model in rats to address the CPF effects on cardiac morphology allied with cardiovascular functional and biomolecular evaluation. Rats were intermittently treated with CPF at doses of 7 mg/kg and 10 mg/kg or saline (i.p.) and assessed for cardiac morphology (cardiomyocyte diameter and collagen content), cardiopulmonary Bezold-Jarisch reflex (BJR) function, cardiac autonomic tone, left ventricle (LV) contractility, cardiac expression of NADPH oxidase (Nox2), catalase (CAT), superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2) and cardiac levels of advanced oxidation protein products (AOPP) and thiobarbituric acid reactive substances (TBARS). Plasma butyrylcholinesterase (BuChE) and brainstem acetylcholinesterase (AChE) were also measured. Intermittent exposure to CPF induced cardiac hypertrophy, increasing cardiomyocyte diameter and collagen content. An impairment of cardioinhibitory BJR responses and an increase in cardiac vagal tone were also observed in CPF-treated animals without changes in LV contractility. CPF exposure increased cardiac Nox-2, CAT, SOD1, and TBARS levels and inhibited plasma BuChE and brainstem AChE activities. Our data showed that intermittent exposure to CPF induces cardiac hypertrophy together with cardiovascular reflex impairment, imbalance of autonomic tone and oxidative stress, which may bring significant cardiovascular risk to individuals exposed to OP compounds seasonally.

Characterisation of plasmatic B-esterases in bottlenose dolphins (Tursiops truncatus) and their potential as biomarkers of xenobiotic chemical exposures

A total of 164 blood samples from 16 clinically healthy bottlenose dolphins (Tursiops truncatus), were obtained from an aquarium in Spain between 2019 and 2020, as part of their preventive medicine protocol. In addition to conventional haematological and biochemical analyses, plasmatic B-esterase activities were characterised to determine the potential application of such analyses in wild counterparts. The hydrolysis rates for the substrates of acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and carboxylesterase (CE) activity in plasma were measured, the last using two commercial substrates, p-nitrophenyl acetate (pNPA) and p-nitrophenyl butyrate (pNPB). Activity rates (mean ± SEM in nmol/min/mL plasma) were (in descending order): AChE (125.6 ± 3.8), pNPB-CE (65.0 ± 2.2), pNPA-CE (49.7 ± 1.1) and BuChE (12.8 ± 1.3). These values for dolphins are reported in here for the first time in this species. Additionally, the in vitro sensitivity of two B-esterases (AChE and pNPB-CE) to chemicals of environmental concern was determined, and the protective role of plasmatic albumin assessed. Out of the B-esterases measured in plasma of dolphin, AChE activity was more responsive in vitro to pesticides, while CEs had a low response to plastic additives, likely due to the protective presence of albumin. However, the clear in vitro interaction of these environmental chemicals with purified AChE from electric eels and recombinant human hCEs (hCE1 and hCE2) and albumin, predicts their impact in other tissues that require in vivo validation. A relationship between esterase-like activities and health parameters in terrestrial mammals has already been established. Thus, B-esterase measures could be easily included in marine mammal health assessment protocols for dolphins as well, once the relationship between these measures and the animal's fitness has been established.

PHARMACOGENETIC ASPECTS OF THE USE OF PERIOPERATIVE DRUGS IN PEDIATRICS. Review

Relevance. It is known that in children the frequency of perioperative critical events due to the introduction of drugs is more than 5%. For example fentanyl, which is used as an adjunct to surgical anesthesia, is a major factor in increasing the number of deaths from opioid overdoses. Among the areas of reducing the frequency of severe adverse reactions to perioperative drugs - taking into account the pharmacogenetic variations of the patient.&#x0D; Objective is to consider current data on pharmacogenetic aspects of pharmacokinetics and pharmacodynamics of perioperative drugs.&#x0D; Methods. Analysis of the data presented in PubMed by keywords "pharmacogenetics", "general anesthetics", "analgesics", "muscle relaxants", "children". Search depth – 7 years (2014-2020), with a retrospective deepening of some positions until 2002.&#x0D; Results. The pharmacogenetic aspects of the pharmacokinetics of perioperative drugs are related to the genes of the enzymes that metabolize them and their transporters. Current data on the prevalence of polymorphic alleles of CYP2C9 genes (provides metabolism of nonsteroidal anti-inflammatory and anticonvulsant drugs) and CYP2D6 (metabolizes opioids, antidepressants, antiemetics) in Europe and, in particular, in Ukraine were presented. Thus, the inactive allele CYP2C19 * 2 was found in 13%, while the allele of increased activity CYP2C19 * 17 - in 25% of the population of Ukraine; allele with lost CYP2D6 function (CYP2D6 * 4) - in 18.6% of Ukrainians. Homozygotes with CYP2C9 * 3 polymorphism metabolize nonsteroidal anti-inflammatory drugs much more slowly than wild-type carriers, which may lead to their accumulation and side effects. The analgesic effect of codeine occurs only after it is metabolized in the liver by CYP2D6 to morphine. Respiratory depression, apnea and death may occur in patients with excessive metabolic rate even after a single dose of codeine; however, was noted the ineffectiveness of ondansetron due to a decrease in its concentration in the blood in thesepatients. Concomitant use of midazolam and fentanyl may prolong the effect of fentanyl by competing metabolism of midazolam by the hepatic enzyme CYP3A4, especially in patients with low metabolism. Plasma butyrylcholinesterase deficiency reduces succinylcholine inactivation in 1 in 1,500 people. Changing the genotype of uridine diphosphate glycosyltransferase causes increased glucuronidation of morphine, which may lead to a decrease in its effectiveness. As an example of pharmacogenetic changes in pharmacodynamics can be considered malignant hyperthermia, which occurs due to exposure to volatile anesthetics and depolarizing muscle relaxants in mutations in the RYR1 gene. Carriers of the minor allele (G) of the 5HT2A rs6313 gene require less propofol and 40% less time to start induction of anesthesia.&#x0D; Conclusions. The efficacy and safety of perioperative drugs are largely related to the pharmacogenetic aspects of their pharmacokinetics, especially mutations in genes of enzymes of the metabolism. Mutations in the genes of volatile anesthetic receptors and depolarizing muscle relaxants when their used cause a risk of malignant hyperthermia. The application of pharmacogenetics is the need to titrate drugs that have significant variability of action depending on the genotype. It is advisable to avoid the use of drugs with a high risk of pharmacogenetic reactions, if other alternative drugs are available.

Intoxication symptoms and health conditions of family farmers associated with the use of pesticides: a study conducted in Zona da Mata Mineira, Brazil

This study aimed to evaluate the association between the use of pesticides, symptoms of intoxication and health conditions of family farmers in the Mata Mineira area, Brazil. This is a cross-sectional study with 48 family farmers from a municipality in the Zona da Mata, Minas Gerais. A semi-structured questionnaire was applied to obtain demographic, socioeconomic information and characterization of the use of pesticides. Blood samples were collected from farmers for biochemical tests and evaluation of markers of exposure to pesticides. It was found that 75% of farmers used pesticides in food production, most of these products being extremely toxic. These farmers showed changes in plasma cholinesterase, erythrocyte cholinesterase and butyrylcholinesterase, markers of exposure to pesticides. There was an association between spraying for more than 4 hours/day and reporting of acute symptoms of intoxication, as well as between the use of pesticides and the presence of chronic non-communicable diseases. Multivariate analysis indicated that non-compliance with the period of re-entry into crops after the use of pesticides remained as an explanatory variable for lung disease. We infer that acute symptoms, as well as chronic non-communicable diseases reported by family farmers, may be associated with the use of pesticides.

Halogen substituents enhance oxime nucleophilicity for reactivation of cholinesterases inhibited by nerve agents

The fluorinated bis-pyridinium oximes were designed and synthesized with the aim of increasing their nucleophilicity and potential to reactivate phosphorylated human recombinant acetylcholinesterase (AChE) and human purified plasmatic butyrylcholinesterase (BChE) in relation to chlorinated and non-halogenated oxime analogues. Compared to non-halogenated oximes, halogenated oximes showed lower pKa of the oxime group (fluorinated < chlorinated < non-halogenated) along with higher level of oximate anion formation at the physiological pH, and had a higher binding affinity of both AChE and BChE. The stability tests showed that the fluorinated oximes were stable in water, while in buffered environment di-fluorinated oximes were prone to rapid degradation, which was reflected in their lower reactivation ability. Mono-fluorinated oximes showed comparable reactivation to non-halogenated (except asoxime) and mono-chlorinated oximes in case of AChE inhibited by sarin, cyclosarin, VX, and tabun, but were less efficient than di-chlorinated ones. The same trend was observed in the reactivation of inhibited BChE. The advantage of halogen substituents in the stabilization of oxime in a position optimal for in-line nucleophilic attack were confirmed by extensive molecular modelling of pre-reactivation complexes between the analogue oximes and phosphorylated AChE and BChE. Halogen substitution was shown to provide oximes with additional beneficial properties, e.g., fluorinated oximes gained antioxidative capacity, and moreover, halogens themselves did not increase cytotoxicity of oximes. Finally, the in vivo administration of highly efficient reactivator and the most promising analogue, 3,5-di-chloro-bispyridinium oxime with trimethylene linker, provided significant protection of mice exposed to sarin and cyclosarin.