Evaluation of the micromethod for determination of glutathione using enzymatic cycling and Ellman's reagent

Eyer, Peter; Podhradský, Dušan

doi:10.1016/0003-2697(86)90061-8

Cited by 175 publications

(80 citation statements)

References 27 publications

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“…The concentration was estimated using the fluorescence yield factor for the glutathione bimane derivative. j Reduced and oxidized glutathione were also measured according to Eyer & Podhradsky (1986) as described in Methods.…”

Section: Efect Of Gsh Deficiency On Metabolic Intermediates Of Sulphumentioning

confidence: 99%

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Glutathione as an endogenous sulphur source in the yeast Saccharomyces cerevisiae

Elskens

Jaspers

Penninckx

1991

Journal of General Microbiology

106

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Glutathione-deficient mutants (@A) of the yeast Saccharomyces cerevisiae, impaired in the first step of glutathione (GSH) biosynthesis were studied with respect to the regulation of enzymes involved in GSH catabolism and cysteine biosynthesis. Striking differences were observed in the content of the sulphur amino acids when gshA mutants were compared to wild-type strains growing on the same minimal medium. Furthermore, all mutants examined showed a derepression of y-glutamyltranspeptidase (y-GT), the enzyme initiating GSH degradation. However, y-cystathionase and cysteine synthase were unaffected by the GSH deficiency as long as the nutrient sulphate source was not exhausted. The results suggest that the mutants are probably not impaired in the sulphate assimilation pathway, but that the y-glutamyl cycle could play a leading role in the regulation of the sulphur fluxes. Studies of enzyme regulation showed that the derepression of y-GT observed in thegshA strains was most probably due to an alteration of the thiol status. The effectors governing the biosynthesis of cysteine synthase and ycystathionase seemed different from those playing a role in y-GT regulation and it was only under conditions of total sulphate deprivation that all these enzymes were derepressed. As a consequence the endogenous pool of GSH was used in the synthesis of cysteine. GSH might, therefore, fulfil the role of a storage compound.

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Section: Efect Of Gsh Deficiency On Metabolic Intermediates Of Sulphumentioning

confidence: 99%

“…Aliquots were passed through a Beckman 120 c amino acid analyser. Reduced and oxidized glutathione (GSSG) were also estimated according to Eyer & Podhradsky (1986) as indicated in the text.…”

Section: Introductionmentioning

confidence: 99%

Glutathione as an endogenous sulphur source in the yeast Saccharomyces cerevisiae

Elskens

Jaspers

Penninckx

1991

Journal of General Microbiology

106

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“…The mixed disulfide, GSTNB that is concomitantly produced, is reduced by GR to recycle the GSH and produce more TNB. The rate of TNB production is directly proportional to this recycling reaction, which in turn is directly proportional to the concentration of GSH in sample (Eyer & Podhradský, 1986).…”

Section: Measurement Of Reduced Glutathionementioning

confidence: 99%

Influence ofEmblica officinalisaqueous extract on growth and antioxidant defense system of human hepatoma cell line (HepG2)

Shivananjappa

Joshi

2011

Pharmaceutical Biology

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“…O uso de complexantes da GSH como N-etilmaleimida (NEM) 7 em sangue total para prevenir a oxidação da GSH a GSSG, a hemólise e a desproteinização ácida da amostra são procedimentos pré-analíticos comuns que antecedem a quantificação da GSH no sangue total ou eritrócito 11 . Concentrações alteradas de GSH por artefatos têm sido associadas à acidificação da amostra na presença de oxiemoglobina 12 , da mesma forma que o uso de agentes complexantes da GSH, como NEM 13,14 , no procedimento pré-analítico. Desta forma, para o desenvolvimento de um método confiável, os procedimentos analítico e pré-analíti-co devem ser otimizados e validados.…”

Section: Introductionunclassified

Influência de desproteinizantes ácidos na quantificação da glutationa reduzida eritrocitária por CLAE-UV

Schott¹,

Charão²,

Valentin³

et al. 2007

Quím. Nova

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INFLUENCE OF DEPROTEINIZING ACIDS IN ERYTHROCYTIC REDUCED GLUTATHIONE QUANTIFICATION BY HPLC-UV. Large differences in reduced glutathione (GSH) levels have been found in different investigations, also in healthy people. GSH oxidation in vitro has been associated with sample acidification in the presence of oxihemoglobin. In this work, the influence of different acids on GSH determination utilizing HPLC with UV detection was evaluated. The results showed that metaphosphoric acid and sulfosalicylic acid were inadequate for analysis, because metaphosphoric acid showed to be inefficient for deproteinization and with sulfosalicylic acid loss of GSH was observed. Trichloroacetic acid did not effect GSH quantification, since the deproteinized form was immediately derivatized with 5, 5´-dithio-bis (2-nitrobenzoic) acid. Methods with TCA deproteinization presented linear results from 0.5 to 3.0 mM. The correlation coefficient between aqueous curves and GSH spiked RBC exceeded 0.99. Precision calculations showed CV lower than 10% and bias within ± 10% for concentrations of 0.5; 1.5 and 3.0 mM GSH. The recovery was higher than 94%. Moreover, GSH blood concentrations were independent of hemoglobin concentrations.Keywords: GSH; erythrocytes; deproteinizing acids. INTRODUÇÃOA glutationa, um tripeptídeo contendo cisteína, desempenha importantes funções em células humanas, especialmente como agente antioxidante. A forma reduzida da glutationa (GSH) mantém os grupos tióis (-SH) das proteínas, reduz ligações dissulfetos (-S-S-) induzidas pelo estresse oxidativo, neutraliza radicais livres e detoxifica eletrófilos 1,2 . Por isso, a concentração intracelular da GSH é um indicador da capacidade da célula em manter sua homeostase, através da neutralização de agentes oxidantes. Na presença de peróxidos, a GSH intracelular é convertida a glutationa oxidada (GSSG) pela glutationa peroxidase, a qual catalisa a redução destes peróxidos 3 . A redução da GSSG a GSH é mediada pela enzima glutationa redutase, cujo substrato é o NADPH 3 . A glicose-6-fosfato desidrogenase, como fonte de NADPH, garante a redução da GSSG e a manutenção de uma constante da reserva de GSH, especialmente no eritrócito 4 . O nível de glutationa em eritrócitos está principalmente em sua forma reduzida (99,5%) e a concentração de GSSG em eritrócitos é descrita como baixa porque esta atravessa facilmente a membrana celular 5 . Na avaliação do estresse oxidativo, o uso de eritrócitos é aconselhável, pois durante seu ciclo vital entram em contato com as mais diversas estruturas orgânicas. Os eritrócitos perfazem 40% do volume do sangue e as espécies reativas do oxigênio que saem da célula vermelha têm potencial para causar dano a outros componentes celulares. Em contrapartida, a GSH em eritrócitos é responsável pela homeostase contra o dano oxidativo da própria célula, bem como de todas as outras células do organismo 6 . Um método de referência para determinação de GSH ainda não foi definido 7,8 . No decorrer dos anos, vários métodos foram introduzidos para determinação ...

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Evaluation of the micromethod for determination of glutathione using enzymatic cycling and Ellman's reagent

Cited by 175 publications

References 27 publications

Glutathione as an endogenous sulphur source in the yeast Saccharomyces cerevisiae

Glutathione as an endogenous sulphur source in the yeast Saccharomyces cerevisiae

Influence ofEmblica officinalisaqueous extract on growth and antioxidant defense system of human hepatoma cell line (HepG2)

Influência de desproteinizantes ácidos na quantificação da glutationa reduzida eritrocitária por CLAE-UV

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