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BACKGROUND/OBJECTIVES: The orexigenic peptide hormone ghrelin has been implicated in the pathophysiology of obesity and type 2 diabetes mellitus through its effects on nutrient homeostasis. Ghrelin is subject to a unique post-translational acyl modification regulating its biochemical activity. SUBJECTS/METHODS: In this study we aimed to investigate the relation of acylated (AcG) as well as unacylated ghrelin (UnG) with body weight and insulin resistance in the fasting (n = 545) and post-oral glucose tolerance test (oGTT) state (n = 245) in a metabolically well characterized cohort covering a broad range of BMI (17.95 kg/m²-76.25 kg/m²). RESULTS: Fasting AcG (median 94.2 pg/ml) and UnG (median 175.3 pg/ml) were negatively and the AcG/UnG ratio was positively correlated with BMI (all p < 0.0001). Insulin sensitivity (ISI) correlated positively with AcG (p = 0.0014) and UnG (p = 0.0004) but not with the AcG/UnG ratio. In a multivariate analysis, including ISI and BMI, only BMI, but not ISI was independently associated with AcG and UnG concentrations. Significant changes of AcG and UnG concentrations were detectable after oGTT stimulation, with slight decreases after 30 min and increases after 90-120 min. Subject stratification into BMI-divergent groups revealed more pronounced AcG increases in the two groups with BMI < 40 kg/m². CONCLUSION: Our data demonstrate lower concentrations for both AcG and UnG with increasing BMI as well as an increased proportion of the biologically active, acylated form of ghrelin giving point to pharmacologic intervention in ghrelin acylation and/or increase in UnG for treatment of obesity despite decreased absolute AcG levels.
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Diabetes Mellitus Tipo 2 , Resistência à Insulina , Humanos , Grelina/metabolismo , Teste de Tolerância a Glucose , Glicemia , Obesidade , Acilação , InsulinaRESUMO
The transition metal (palladium)-catalysed asymmetric 1,4-addition of arylboronic acids to conjugated enones belong to the most important and emerging strategies for the construction of C-C bonds in an asymmetric fashion. This review covers known catalytic systems used for this transformation. For clarity, we are using the type of ligand as a sorting criterion. Finally, we attempted to create a flowchart facilitating the selection of a suitable ligand for a given combination of enone and arylboronic acid.
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Magnetic particles (MPs) have been widely used in biological applications in recent years as a carrier for various molecules. Their big advantage is in repeated use of immobilized molecules including enzymes. Acetylcholinesterase (AChE) is an enzyme playing crucial role in neurotransmission and the enzyme is targeted by various molecules like Alzheimer's drugs, pesticides and warfare agents. In this work, an electrochemical biosensor having AChE immobilized onto MPs and stabilized through glutaraldehyde (GA) molecule was proposed for assay of the neurotoxic compounds. The prepared nanoparticles were modified by pure AChE and they were used for the measurement anti-Alzheimer's drug galantamine and carbamate pesticide carbofuran with limit of detection 1.5 µM and 20 nM, respectively. All measurements were carried out using screen-printed sensor with carbon working, silver reference, and carbon auxiliary electrode. Standard Ellman's assay was used for validation measurement of both inhibitors. Part of this work was the elimination of reversible inhibitors represented by galantamine from the active site of AChE. For this purpose, we used a lower pH to get the original activity of AChE after inhibition by galantamine. We also observed decarbamylation of the AChE-carbofuran adduct. Influence of organic solvents to AChE as well as repeatability of measurement with MPs with AChE was also established.
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Nanopartículas , Acetilcolinesterase , Técnicas Biossensoriais , Inibidores da Colinesterase , Enzimas Imobilizadas , Compostos Organofosforados , PraguicidasRESUMO
A method is described for the determination of fatty acids in dried sweat spot and plasma samples using gas chromatography with flame ionization detection. Plasma and dried sweat spot samples were obtained from a group of blood donors. The sweat was collected from each volunteer during exercise. Sweat was spotted onto collection paper containing butylated hydroxytoluene. Fatty acids were derivatized with acetyl chloride in methanol to form methyl esters of fatty acids. The fatty acids in dried sweat spot samples treated with butylated hydroxytoluene and stored at -20°C were stable for 3 months. Our results indicate that sweat contains, among fatty acids with short chain, also fatty acids with long chain and unsaturated fatty acids. Linear relationships between percentage content of selected fatty acids in dried sweat spot and plasma were observed.
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Cromatografia Gasosa , Ácidos Graxos/análise , Ionização de Chama , Suor/química , Ésteres/análise , HumanosRESUMO
The use of a cell phone as a detection system is easy, simple and does not require trained personnel, which is in contrast to standard laboratory instruments. This paper deals with immobilization of acetylcholinesterase (AChE) in a gelatin matrix, and phenol red, as an indicator of AChE activity, is used in order to establish a method that is easily compatible with a camera device. AChE splits acetylcholine into choline and acetic acid, which changes the pH of a medium, resulting in a phenol red color change. The coloration changed in presence of an AChE inhibitor. Measurements were performed on 3D-printed, tube-shaped holder, and digital photography, with subsequent analysis of red-green-blue (RGB), served for assay purposes. Calibration of AChE inhibitors, tacrine and galantamine, was performed, with limit of detection equal to 1.1 nM and 1.28 µM, respectively. Interferences were also measured, resulting in a proof-of-method stability. The method was further successfully validated for the standard Ellman's assay, and verified on murine plasma samples spiked with inhibitors.
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A new and simple analytical method is described for the determination of the IC50 values of the inhibitors of the hydrolysis of acetylcholine (ACh) or acetylthiocholine (ATCh) by cholinesterases. The method is based on monitoring the time course of the pH value during the uninhibited and inhibited reaction. It requires only a pH meter with a suitable pH measuring cell and a small thermostated stirred batch reactor. The method has been validated for twelve different types of cholinesterase inhibitors. The determined IC50 values are comparable to those obtained by independent, more complicated, and expensive methods (Ellman's and pH-stat).
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Inibidores da Colinesterase/farmacologia , Concentração de Íons de Hidrogênio , Concentração Inibidora 50RESUMO
Vitamin B12 (B12) is an essential cofactor of two important biochemical pathways, the degradation of methylmalonic acid and the synthesis of methionine from homocysteine. Methionine is an important donor of methyl groups for numerous biochemical reactions, including DNA synthesis and gene regulation. Besides hematological abnormalities (megaloblastic anemia or even pancytopenia), a deficiency in B12 may cause neurological symptoms, including symptoms resembling diabetic neuropathy. Although extensively studied, the underlining molecular mechanism for the development of diabetic peripheral neuropathy (DPN) is still unclear. Most studies have found a contribution of oxidative stress in the development of DPN. Detailed immunohistochemical investigations in sural nerve biopsies obtained from diabetic patients with DPN point to an activation of inflammatory pathways induced via elevated advanced glycation end products (AGE), ultimately resulting in increased oxidative stress. Similar results have been found in patients with B12 deficiency, indicating that the observed neural changes in patients with DPN might be caused by cellular B12 deficiency. Since novel results show that B12 exerts intrinsic antioxidative activity in vitro and in vivo, B12 may act as an intracellular, particularly as an intramitochondrial, antioxidant, independent from its classical, well-known cofactor function. These novel findings may provide a rationale for the use of B12 for the treatment of DPN, even in subclinical early states.
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Diabetes Mellitus , Neuropatias Diabéticas , Deficiência de Vitamina B 12 , Humanos , Vitamina B 12/uso terapêutico , Neuropatias Diabéticas/etiologia , Neuropatias Diabéticas/tratamento farmacológico , Deficiência de Vitamina B 12/diagnóstico , Antioxidantes/uso terapêutico , Metionina , Vitaminas/uso terapêutico , Diabetes Mellitus/tratamento farmacológicoRESUMO
BACKGROUND: Heterozygous mutations in the glucocerebrosidase gene lead to an increased risk for and to more severe alpha-synuclein-associated pathology in Parkinson's disease. As both glucocerebrosidase and alpha-synuclein interact with fatty acids, we hypothesized that cerebrospinal fluid fatty acid levels are altered in these Parkinson's disease patients. METHODS: Cerebrospinal fluid levels of 13 fatty acids in 8 Parkinson's disease patients with a heterozygous glucocerebrosidase mutation were compared with those of 41 idiopathic Parkinson's disease patients and 30 controls using gas chromatography. RESULTS: Parkinson's disease patients with a heterozygous glucocerebrosidase mutation had lower levels of palmitoleic (P ≤ .007), oleic (P ≤ .016), linoleic (P ≤ .005), arachidonic (P ≤ .003), eicosapentaenoic (P ≤ .003) and decosahexaenoic (P ≤ .03) acids and lower levels of total fatty acids (P < .005) compared with both idiopathic Parkinson's disease patients and control subjects. CONCLUSIONS: These results suggest that abnormalities of fatty acid metabolism are specifically involved in the pathogenesis of Parkinson's disease associated with a heterozygous glucocerebrosidase mutation.
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Ácidos Graxos/líquido cefalorraquidiano , Glucosilceramidase/genética , Mutação/genética , Doença de Parkinson/líquido cefalorraquidiano , Doença de Parkinson/genética , Idoso , Idoso de 80 Anos ou mais , Feminino , Heterozigoto , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Stearoyl-CoA desaturase-1 (SCD1) has gained much interest as a future drug target to treat fatty liver and its consequences. However, there are few and inconsistent human data about expression and activity of this important enzyme. We investigated activity and expression of SCD1 and their relationships with liver fat (LF) content in human liver samples. Fifty subjects undergoing liver surgery were studied. SCD1 activity was estimated from the ratio of oleate (C18:1) to stearate (C18:0) within lipid subfractions. Furthermore, SCD1 mRNA expression and LF content were measured. Similarly to previous studies, we observed a strong positive correlation between LF content and the C18:1/C18:0 ratio in the combined fatty acid (FA) fractions (r = 0.96, P < 0.0001), which could be interpreted as higher SCD1 activity with increasing LF. However, hepatic SCD1 mRNA expression did not correlate with LF (r = 0.16, P = 0.13). To solve these conflicting data, we analyzed the FA composition of hepatic lipid subfractions. With increasing LF content the amount of FAs from the triglyceride (TG) fraction increased (r = 0.96, P < 0.0001), whereas the FAs from the phospholipid (PL) fraction remained unchanged (r = -0.17, P = 0.19). Of these two major lipid fractions, the C18:1/C18:0 ratio in TG was 16-fold higher than in PL. Supporting the SCD1 mRNA expression data, the C18:1/C18:0 ratio of the TG or PL fraction did not correlate with LF (r = 0.26, P = 0.12 and r = 0.08, P = 0.29). We provide novel information that SCD1 activity and mRNA expression appear not to be elevated in subjects with high LF content. We suggest that the FA composition of lipid subclasses, rather than of mixed lipids, should be analyzed to estimate SCD1 activity.
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Metabolismo dos Lipídeos/fisiologia , Fígado/enzimologia , Fígado/metabolismo , RNA Mensageiro/biossíntese , Estearoil-CoA Dessaturase/metabolismo , Idoso , Biópsia , Ésteres do Colesterol/metabolismo , Ácidos Graxos/metabolismo , Feminino , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Pessoa de Meia-Idade , Fosfolipídeos/metabolismo , RNA Mensageiro/genética , Triglicerídeos/metabolismoRESUMO
Nostotrebin 6, a new polyphenolic compound with a fully substituted 2,2'-bis(cyclopent-4-en-1,3-dione) skeleton, was isolated from a methanolic extract of the cyanobacterial strain Nostoc sp. str. Lukesová 27/97. The structure of this compound was determined using X-ray crystallography and further supported by NMR, IR spectroscopy, and MS. Nostotrebin 6 is an S-parabolic I-parabolic noncompetitive inhibitor of acetylcholinesterase (IC(50) = 5.5 microM) and an S-parabolic I-parabolic mixed inhibitor of butyrylcholinesterase (IC(50) = 6.1-7.5 microM). The inhibitory potency of nostotrebin 6 was compared with that of tacrine and galanthamine.
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Inibidores da Colinesterase/química , Ciclopentanos/farmacologia , Nostoc/química , Acetilcolinesterase/efeitos dos fármacos , Animais , Butirilcolinesterase/efeitos dos fármacos , Inibidores da Colinesterase/isolamento & purificação , Inibidores da Colinesterase/farmacologia , Ciclopentanos/química , Ciclopentanos/isolamento & purificação , Humanos , Concentração Inibidora 50 , Estrutura MolecularRESUMO
X-linked Adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene resulting in the accumulation of very long chain fatty acids (VLCFA). X-ALD is the most common peroxisomal disorder with adult patients (male and female) presenting with progressive spastic paraparesis with bladder disturbance, sensory ataxia with impaired vibration sense, and leg pain. 80% of male X-ALD patients have an adrenal failure, while adrenal dysfunction is rare in women with X-ALD. The objective of this study was to define optimal serum VLCFA cutoff values in patients with X-ALD-like phenotypes for the differentiation of genetically confirmed X-ALD and Non-X-ALD individuals. Three groups were included into this study: a) X-ALD cases with confirmed ABCD1 mutations (n = 34) and two Non-X-ALD cohorts: b) Patients with abnormal serum VCLFA levels despite negative testing for ABCD1 mutations (n = 15) resulting from a total of 1,953 VLCFA tests c) Phenotypically matching patients as Non-X-ALD controls (n = 104). Receiver operating curve analysis was used to optimize VLCFA cutoff values, which differentiate patients with genetically confirmed X-ALD and Non-X-ALD individuals. The serum concentration of C26:0 was superior to C24:0 for the detection of X-ALD. The best differentiation of Non-X-ALD and X-ALD individuals was obtained with a cutoff value of < 1.0 for the C24:0/C22:0 ratio resulting in a sensitivity of 97%, a specificity of 94.1% and a positive predictive value (PPV) of 83.8% for true X-ALD. Our findings further suggested a cutoff of < 0.02 for the ratio C26:0/C22:0 leading to a sensitivity of 90.9%, a specificity of 95.0%, and a PPV of 80.6%. Pearson correlation indicated a significant positive association between total blood cholesterol and VLCFA values. Usage of serum VLCFA are economical and established biomarkers suitable for the guidance of genetic testing matching the X-ALD phenotype. We suggest using our new optimized cutoff values, especially the two ratios (C24:0/C22:0 and C26:0/C22:0), in combination with standard lipid profiles.
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Adrenoleucodistrofia/sangue , Adrenoleucodistrofia/diagnóstico , Ácidos Graxos/sangue , Transportadores de Cassetes de Ligação de ATP/genética , Adulto , Idoso , Astrócitos/patologia , Biomarcadores/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Stearoyl-coenzyme A desaturase 1 (SCD1) catalyzes the limiting step of monounsaturated fatty acid synthesis in humans and is an important player in triglyceride generation. SCD1 has been repeatedly implicated in the pathogenesis of metabolic and inflammatory diseases. Therefore it is of great importance to determine SCD1 activity in human samples. In this study we aimed to evaluate a hepatic SCD1 activity index derived from plasma VLDL triglyceride composition as a tool to estimate hepatic SCD1 expression in humans. Additionally, we further evaluated commonly used fatty acid ratios [elongase, de novo lipogenesis, and Delta5 and Delta6 desaturase] in plasma VLDL and hepatic lipid fractions. DESIGN AND METHODS: Liver biopsies and plasma samples were simultaneously collected from 15 individuals. Plasma VLDL was obtained by ultracentrifugation. Hepatic and plasma VLDL lipids were fractionated by thin-layer chromatography, and the fatty acid composition of each fraction was analyzed by gas chromatography. Hepatic SCD1 expression was determined by real-time PCR. RESULTS: Hepatic SCD1 mRNA expression was associated with the product/precursor ratios (16:1/16:0 and 18:1/18:0) of hepatic lipid fractions. The 16:1/16:0 ratio in hepatic and VLDL triglycerides as well as the 18:1/18:0 ratio in plasma VLDL were closely associated with hepatic SCD1 expression. The hepatic de novo lipogenesis index from triglycerides was associated with expression of lipogenic genes [fatty acid synthase (FASN), acetyl-Coenzyme A carboxylase alpha (ACACA), and sterol regulatory element binding transcription factor 1 (SREBP-1)] and is closely reflected by the de novo lipogenesis index in VLDL triglycerides. CONCLUSION: We demonstrated for the first time that hepatic SCD1 expression can be estimated noninvasively from routine blood samples by measuring the SCD1 activity index in fasting plasma VLDL.
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Ácidos Graxos não Esterificados/química , Lipídeos/química , Lipoproteínas VLDL/química , Fígado/metabolismo , RNA Mensageiro/biossíntese , Estearoil-CoA Dessaturase/biossíntese , Triglicerídeos/química , Acetil-CoA Carboxilase/biossíntese , Acetiltransferases/biossíntese , Acetiltransferases/genética , Idoso , Cromatografia em Camada Fina , Dessaturase de Ácido Graxo Delta-5 , Ácidos Graxos Dessaturases/biossíntese , Ácidos Graxos Dessaturases/genética , Elongases de Ácidos Graxos , Ácido Graxo Sintases/biossíntese , Ácidos Graxos não Esterificados/sangue , Feminino , Humanos , Linoleoil-CoA Desaturase/biossíntese , Linoleoil-CoA Desaturase/genética , Lipogênese , Lipoproteínas VLDL/sangue , Fígado/enzimologia , Masculino , Estearoil-CoA Dessaturase/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/biossíntese , Triglicerídeos/sangueRESUMO
The effect of 20 fatty acids in erythrocyte cell membranes on the extent of inflammatory response and cell oxidative stress was evaluated using multidimensional statistical data analysis in 54 patients suffering from ischemic heart disease undergoing percutaneous coronary intervention with coronary stent implantation using multidimensional statistical data analysis. A systemic inflammatory response was indicated by an increase of C-reactive protein (CRP), serum amyloid A (SAA) and ceruloplasmin 48 h after stent implantation and by an increase of interleukin-6 (IL-6) 24 h after intervention. The increase of malondialdehyde (MDA) after 48 h was used as a marker of cell damage by oxidative stress. Multiple linear regression revealed statistically significant relationships between concentration of some fatty acids and the magnitude of inflammatory response, or oxidative stress, after stent implantation. The most significant relationship with an increase of plasma CRP was found for myristic acid and, to a lesser extent, for oleic acid. Trans octadecenoic acid, and to a lesser extent palmitooleic and nervonic fatty acids were found in inverse correlation with the CRP increase. The increase of IL-6 showed a statistically significant correlation with myristic acid, to a lesser extent with cis-9-eicosenoic acid and to the least extent with docosahexaenoic acid, inversely with pentadecanoic, γ-linolenic and stearic acids. An increase of oxidative stress (MDA) significantly correlated only with γ-linolenic acid. Other studied markers of inflammatory response to coronary stenting were SAA and ceruloplasmin (Cp). Statistical evaluation revealed that SAA and Cp are not suitable markers for assessment relationships between inflammation and erythrocyte membrane fatty acids.
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Estenose Coronária/terapia , Membrana Eritrocítica/imunologia , Eritrócitos/imunologia , Ácidos Graxos/metabolismo , Intervenção Coronária Percutânea , Stents , Biomarcadores/sangue , Proteína C-Reativa/análise , Estenose Coronária/sangue , Estenose Coronária/imunologia , Estudos Transversais , Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Feminino , Humanos , Interleucina-6/sangue , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo/imunologia , Proteína Amiloide A Sérica/análiseRESUMO
The pI50 index and separation coefficients of chosen 3-N,N-diethylaminophenyl-N',N'-dialkylcarbamates were determined. Index pL50 (pI50 = negative logarithm of molar concentration of inhibitor inhibiting the enzyme activity by 50%) describes the effectiveness of the inhibitor. The rate of ability of the inhibitor to pass the blood-brain barrier is usually described by the separation coefficient in a system n-octanol/water (K(ow)). Obtained results were compared with pL50 and K(ow) of Exelon, the commercially used drug against the Alzheimer's disease.
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Acetilcolinesterase/metabolismo , Carbamatos/farmacologia , Inibidores da Colinesterase/farmacologia , Acetiltiocolina/metabolismo , Alquilação , Animais , Carbamatos/química , Dioxanos/farmacologia , Electrophorus , Cinética , Modelos MolecularesRESUMO
Biperiden is a drug used in Parkinson disease treatment and it serves also as an antiseizures compound in organophosphates poisoning. It acts as antagonist of muscarinic receptor activated by acetylcholine while the enzyme acetylcholinesterase (AChE) cleaves acetylcholine in synaptic junction into choline and acetic acid. This enzyme is inhibited by various compounds; however there has not been proposed evidence about interaction with biperiden molecule. We investigated this interaction using standard Ellman's assay and experimental findings were critically completed with an in silico prediction by SwissDock docking software. Uncompetitive mechanism of action was revealed from Dixon plot and inhibition constant (Ki ) was calculated to be 1.11 mmol/l. The lowest predicted binding energy was -7.84 kcal/mol corresponding to H-bond between biperiden molecule and Tyr 341 residuum in protein structure of AChE. This interaction seems to be further stabilized by π-π interaction with Tyr 72, Trp 286, and Tyr 341. In conclusion, biperiden appears as a very weak inhibitor but it can serve as a lead structure in a pharmacological research.
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Acetilcolinesterase/metabolismo , Biperideno/uso terapêutico , Inibidores da Colinesterase/uso terapêutico , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/enzimologia , Biperideno/química , Biperideno/farmacologia , Inibidores da Colinesterase/química , Inibidores da Colinesterase/farmacologia , Ensaios Enzimáticos , Humanos , Modelos Moleculares , Especificidade por Substrato/efeitos dos fármacosRESUMO
Smartphones are widely spread and their usage does not require any trained personnel. Recently, smartphones were successfully used in analytical chemistry as a simple detection tool in some applications. This paper focuses on immobilization of acetylcholinesterase (AChE) onto commercially available pH strips with stabilization in the gelatin membrane. AChE degrades acetylcholine into choline and acetic acid which causes color change of acid-base indicator. Smartphone served as a tool for measurement of indicator color change from red to orange while inhibitors blocked this process. AChE inhibitors were measured with limits of detection, 149 nM and 22.3 nM for galanthamine and donepezil, respectively. Organic solvents were measured for method interferences. Measurement procedure was performed on 3D printed holder and digital photography was evaluated using red-green-blue (RGB) channels. The invented assay was validated to the standard Ellman's test and verified on murine plasma samples spiked with inhibitors. We consider that the assay is fully suitable for practical performance.
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Kinetics of hydrolysis of acetylcholine and acetylthiocholine by two types of acetylcholinesterase and butyrylcholinesterase inhibited by 13 new inhibitors (5 carbamates and 8 carbazates--hydrazinium derivatives) was measured in vitro in a batch reactor at 25 degrees C, pH 8, ionic strength 0.11 M and enzyme activity 3.5 U by four nondependent analytical methods. Sevin, rivastigmin (Exelon) and galantamin (Reminyl) served as comparative inhibiting standards. Kinetics of hydrolyses inhibited by all studied carbamates, sevin, carbazates (with exceptions) and rivastigmin (with exceptions) can be simulated by the competitive inhibition model with irreversible reaction between enzyme and inhibitor. Galantamin does not fulfil this model. In positive simulations, the value of inhibition (carbamoylation) rate constant k3 was calculated, describing the reaction velocity between the given enzyme and inhibitor. Physiologically important hydrolyses of acetylcholine catalyzed by acetylcholinesterase from electric eel or bovine erythrocytes and butyrylcholinesterase from horse plasma can be most quickly inhibited by carbamoylation of the mentioned enzymes by the 3-N,N-diethylaminophenyl-N'-(1-alkyl) carbamates 4 and 5. Probably this is due to a long enough hydrocarbon aliphatic substituent (hexyl and octyl) on the amidic nitrogen atom. The tested carbazates failed as inhibitors of cholinesterases. The regeneration ability of the inhibited enzymes was not measured.
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Acetilcolinesterase/metabolismo , Inibidores da Colinesterase/química , Inibidores da Colinesterase/farmacologia , Butirilcolinesterase/metabolismo , Carbaril/farmacologia , Galantamina/farmacologia , Cinética , Relação Estrutura-AtividadeRESUMO
Kinetics and the mechanism of total in vitro hydrolyses (i.e. up to the exhaustion of substrate) of acetylcholine and acetylthiocholine by acetylcholinesterase and butyrylcholinesterase were studied in vitro in a batch reactor at 25 degrees C, pH 8 and ionic strength of 0.11 M. Every hydrolysis was monitored by 2-3 independent analytical methods. All studied types of enzymatic hydrolyses fulfilled the Michaelis-Menten reaction scheme with the irreversible second step. A table of obtained average values of rate constants and estimations of initial molar enzyme concentrations, and discussion of the results are presented.
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Acetilcolina/metabolismo , Acetilcolinesterase/metabolismo , Acetiltiocolina/metabolismo , Butirilcolinesterase/metabolismo , Acetilcolinesterase/sangue , Animais , Bovinos , Eritrócitos/enzimologia , Hidrólise , Cinética , Espectrofotometria , Especificidade por SubstratoRESUMO
BACKGROUND: The determination of amino acids can be a reliable approach for extended diagnosis of liver diseases. This is because liver disease can be a cause of impaired amino acid metabolism. Therefore, a method for the determination of serum amino acids, applicable for clinical purposes, is necessary. OBJECTIVES: The aim of this study was to find differences in the levels of selected amino acids between patients with liver disease and a control group. MATERIAL AND METHODS: Samples of peripheral venous blood were obtained from a group of patients with liver disease (n = 131, 59 women at an average age of 60 years and 72 men at an average age of 52 years) and a control group (n = 105, 47 women at an average age of 62 years and 58 men at an average age of 58 years). Before the separation, the amino acids were derivatized with naphthalene-2,3-dicarboxaldehyde. For the separation, reverse phase column was used. The effluent was monitored with a fluorescence detector. RESULTS: There were significant differences in the concentrations of some amino acids between the patients and the control group, but also between women and men. Correlations between some amino acids and markers of liver blood tests and lipid metabolism were observed. CONCLUSIONS: A simple, relatively rapid and selective HPLC method with fluorescence detection for the determination of selected amino acids in serum has been developed.