Cholinesterase activities determination in frozen blood samples: an                 improvement to the occupational monitoring in developing countries

Oliveira-Silva, Jefferson José; Alves, Sérgio Rabello; Inácio, Alan Ferreira; Meyer, Armando; Sarcinelli, Paula de Novaes; Mattos, Rita de Cássia Oliveira da Costa; Ferreira, Márcio; Cunha, Julia Claro da; Moreira, Josino Costa

doi:10.1191/096032700678827762

Cited by 36 publications

(38 citation statements)

References 4 publications

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“…52 To obtain the active AchE, the erythrocytes were separated by centrifugation of a 1.8 mL blood sample, ruptured by freezing the cells in phosphate buffer (20 mmol L -1 , pH 7.6) at -10 °C and then centrifuged to separate the hemoglobin. Finally, the membranes were treated with a lyophilizing phosphate buffer and subsequently centrifuged to yield 500 mL of clean AChE solution.…”

Section: Resultsmentioning

confidence: 99%

“…The cholinesterase activities were calculated using standard curves of L-cysteine, according to the equation below, where E is the enzyme activity in nmols min -1 mL -1 , Abs is the absorbance, 2.96 and 2.11 are the linear coefficient and the slope of the curve, respectively, experimentally obtained for L-cysteine, and 20 is the dilution factor. 52 …”

Section: Ache Activity Measurementmentioning

confidence: 99%

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The effect of neutral oximes on the reactivation of human acetylcholinesterase inhibited with paraoxon

Ribeiro¹,

Prates²,

Alves³

et al. 2012

J. Braz. Chem. Soc.

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Importantes agentes para defesa contra armas de guerra química são reativadores da acetilcolinesterase humana (huAChE) inibida por compostos organofosforados neurotóxicos (OP), e precisam de uma permeabilidade razoável pela barreira hematoencefálica (HB). Neste trabalho, oximas neutras, que penetram HB melhor do que as oximas catiônicas atualmente utilizadas como agentes de defesa, foram testadas como reativadores da huAChE inibida com paraoxon usando o método de Ellman modificado e pralidoxima (2-PAM) como padrão positivo. A oxima neutra mais ativa foi a 2-tiofenoaldoxima, que reativou 93% da huAChE inibida, sendo 12% mais eficiente do que a 2-PAM. Os resultados mostraram que oximas neutras simples possuem potencial para atuarem como antídotos para intoxicação com OPs neurotóxicos, sugerindo maior investigação no desenvolvimento de agentes de defesa neutros.Important defense agents against chemical warfare weapons, which are reactivators of human acetylcholinesterase (huAChE) inhibited by neurotoxic organophosphorus compounds (OP), need a reasonable permeation of the hematoencephalic barrier (HB). In this work, neutral oximes, which permeate HB better than the cationic oximes currently used as defense agents, were tested as reactivators of huAChE inhibited with paraoxon using the modified Ellman test with pralidoxime (2-PAM) as positive standard. The most active neutral oxime was (thiophen-2-yl)aldoxime, which reactivated 93% of the inhibited huAChE and was 12% more effective than 2-PAM. The results showed that simple neutral oximes have potential to function as antidotes for intoxication with neurotoxic OPs, suggesting further research on the development of neutral defense agents.Keywords: neutral oximes, huAChE reactivation, paraoxon, chemical warfare, (thiophen-2-yl)aldoxime IntroductionIntoxication with neurotoxic organophosphorus compounds is a serious problem caused by some pesticides used in agriculture, such as paraoxon (1) and malathion (2) ( Figure 1) and chemical warfare agents. [1][2][3][4][5][6][7][8][9][10][11] According to the World Health Organization, there are about 220,000 intoxications with organophosphorus pesticides throughout the world every year, leading to approximately 20,000 deaths. 1,[12][13][14] The most dangerous neurotoxic compounds are the chemical warfare agents soman (3), sarin (4), tabun (5) and VX (6) ( Figure 1). Though their use is prohibited by the United Nations, these agents have been employed as weapons by terrorist groups and by countries that did not sign the Chemical Weapon Convention administered by the Organization for the Prohibition of Chemical Weapons (OPCW).The principal target for the action of neurotoxic organophosphorus compounds is the enzyme acetylcholinesterase (AChE), a serine hydrolase that controls the nerve impulses at the cholinergic receptors in the central and peripheral nervous systems. 1,15-20 As shown in Ribeiro et al. 1217 Vol. 23, No. 7, 2012 Figure 2, the function of AChE (7) is the hydrolysis of the nerve impulse transmitter acetylcholine...

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Section: Resultsmentioning

confidence: 99%

Section: Ache Activity Measurementmentioning

confidence: 99%

The effect of neutral oximes on the reactivation of human acetylcholinesterase inhibited with paraoxon

Ribeiro¹,

Prates²,

Alves³

et al. 2012

J. Braz. Chem. Soc.

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“…O plasma foi descartado e aos tubos foram adicionados 4,50 mL de tampão de lise (NaH2PO4/Na2HPO4 20,00 mM -pH 7.60). Após adição do tampão os tubos foram congelados a -6 °C e mantidos nesta temperatura por 24 horas (Oliveira-Silva et al, 2000).…”

Section: Obtenção Da Ache Presente Na Proteína Ghost Não Inibida E Inunclassified

“…Em seguida, a amostra foi submetida a duas centrifugações (5000 rpm por 15 minutos), sendo que o sobrenadante era descartado ao fim de cada uma delas e adicionado 4,50 mL do tampão de análise. Por fim, ao retirar o último sobrenadante, adicionouse 500,00 μL de tampão de análise e obteve-se a (Hemoglobinfree erythrocyte ghosts) proteína ghost inibida (Oliveira-Silva et al, 2000).…”

Section: Obtenção Da Ache Presente Na Proteína Ghost Não Inibida E Inunclassified

“…O emprego da AChE presente nos eritrócitos fantasmas livres de hemoglobina (Hemoglobin-free erythrocyte ghosts) é uma alternativa atraente, pois o ensaio é realizado na enzima humana a um baixo custo. Oliveira-Silva et al (2000) empregaram o etil-paraoxon para inibir a AChE sanguínea em concentrações variadas (0,0625 ± 50,0 µg/mL de sangue). O diclorvós (2,2-diclorovinil dimetil fosfato) é um organofosforado, inibidor direto das colinesterases, com amplo uso no ambiente doméstico, no combate a endemias e pragas agrícolas (Sotiropouloua et al, 2005).…”

Section: Introductionunclassified

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Avaliação da atividade de aldeídos aromáticos e suas oximas frente à enzima acetilcolinesterase eritrocitária humana

et al. 2017

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ResumoConsiderando a importância terapêutica das drogas inibidoras da acetilcolinesterase, bem como dos regeneradores da enzima, em casos de intoxicações por organofosforados. A atividade de aldeídos aromáticos e suas respectivas oximas foi avaliada frente à acetilcolinesterase eritrocitária humana. As oximas foram preparadas a partir da oximação do benzaldeído, do 4-hidroxibenzaldeído, do 4-hidroxi-3-metoxibenzaldeído e do 4-dimetialminobenzaldeído, e em seguida, o ensaio de Ellman foi empregado para avaliar o comportamento destes compostos frente à acetilcolinesterase ativa e inibida por diclorvós. Os aldeídos avaliados apresentaram atividade inibidora da acetilcolinesterase nas porcentagens de 21,33%, 53,00%, 44,00% e 62,67% respectivamente para benzaldeído, 4-hidroxibenzaldeído, 4-hidroxi-3-metoxibenzaldeído e 4-dimetilminobenzaldeído. Dentre as oximas avaliadas, apenas a dimetilaminobenzaldoxima apresentou atividade inibitória (36,67%). A benzaldoxima, a 4-hidroxibenzaldoxima e a 4-hidroxi-3-metoxibenzaldoxima mostraram-se capazes de regenerar modestamente a atividade enzimática em 9,21%, 32,83% e 8,90%, respectivamente, quando a enzima se encontrava inibida por diclorvós. Os aldeídos aromáticos testados apresentaram potencial atividade inibitória da acetilcolinesterase eritrocitária, na concentração de 100mM, sendo o maior potencial demonstrado pelo 4-dimetilamibenzaldeído, com 62,67% de inibição. As oximas, com exceção da dimetilaminobenzaldoxima, se comportaram como regeneradores fracos da enzima inibida por diclorvós, mostrando a relevância do grupo oxima para a regeneração da enzima inibida por organofosforados. Palavras-chave:Organofosforado, diclorvós, pralidoxima, teste de Ellman Evaluation of the activity of aromatic aldehydes and their oximes against human erythrocyte acetylcholinesterase enzyme AbstractConsidering the therapeutic importance of the acetylcholinesterase inhibitor drugs, as well, enzyme regenerators drugs, in cases of organophosphate poisoning. The activity of aromatic aldehydes and their oximes was evaluated against human erythrocyte acetylcholinesterase. Oximes were prepared from the oximation of benzaldehyde, 4-hydroxybenzaldehyde, 4-hydroxy-3-methoxybenzaldehyde and 4-dimethylminobenzaldehyde, and then the Ellman assay was used to evaluate the behavior of these compounds against active acetylcholinesterase and the enzyme inhibited by dichlorvos. The evaluated aldehydes showed acetylcholinesterase inhibitory activity in the percentage of 21.33%, 53.00%, 44.00% and 62.67%, respectively, for benzaldehyde, 4-hydroxybenzaldehyde, 4-hydroxy-3-methoxybenzaldehyde and 4-dimethylminobenzaldehyde. Among the oximes evaluated, only dimethylaminobenzaldoxime showed inhibitory activity (36.67%). Benzaldoxime, 4-hydroxybenzaldoxime and 4-hydroxy-3-methoxybenzaldoxime could modestly regenerate the enzymatic activity in 9.21%, 32.83% and 8.90%, respectively, when the enzyme was inhibited by dichlorvos. The aromatic aldehydes tested showed a potential inhibitory activity of erythrocyte acetylchol...

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Development and validation of a simple assay for the determination of cholinesterase activity in whole blood of laboratory animals

Naik

Liu

Saxena

2012

J of Applied Toxicology

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Current methods for measuring acetylcholinesterase (AChE) activities in whole blood use butyrylcholinesterase (BChE)‐selective inhibitors. However, the poor selectivity of these inhibitors results in the inhibition of AChE activity to some degree, leading to errors in reported values. The goal of this study was to develop and validate a simple assay for measuring AChE and BChE activities in whole blood from humans as well as experimental animals. Blood was fractionated into plasma and erythrocytes, and cholinesterase activities were titrated against ethopropazine and (−)‐huperzine A to determine the lowest concentration of ethopropazine that inhibited BChE completely without affecting AChE activity and the lowest concentration of (−)‐huperzine A that inhibited AChE completely without interfering with BChE activity. Results indicate that 20 µm ethopropazine can be successfully used for the accurate measurement of AChE activity in blood from humans as well as animals. Use of (−)‐huperzine A is not required for measuring BChE activity in normal or ‘exposed’ blood samples. The method was validated for blood from several animal species, including mice, rats, guinea pigs, dogs, minipigs, and African green, cynomolgus and rhesus monkeys. This method is superior to all reported methods, does not require the separation of erythrocyte and plasma fractions, and is suitable for measuring cholinesterase activities in fresh or frozen blood from animals that were exposed to nerve agents or those that were administered high doses of BChE. The method is simple, direct, reproducible, and reliable and can easily be adapted for high‐throughput screening of blood samples. Published 2012. This article is a US Government work and is in the public domain in the USA.

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Cholinesterase activities determination in frozen blood samples: an improvement to the occupational monitoring in developing countries

Cited by 36 publications

References 4 publications

The effect of neutral oximes on the reactivation of human acetylcholinesterase inhibited with paraoxon

The effect of neutral oximes on the reactivation of human acetylcholinesterase inhibited with paraoxon

Avaliação da atividade de aldeídos aromáticos e suas oximas frente à enzima acetilcolinesterase eritrocitária humana

Development and validation of a simple assay for the determination of cholinesterase activity in whole blood of laboratory animals

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