DEVELOPMENT OF METHODIC OF TOTAL GLUTATHIONE AMOUNT DETERMINATION
Identifikátory výsledku
Kód výsledku v IS VaVaI
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Výsledek na webu
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DOI - Digital Object Identifier
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Alternativní jazyky
Jazyk výsledku
angličtina
Název v původním jazyce
DEVELOPMENT OF METHODIC OF TOTAL GLUTATHIONE AMOUNT DETERMINATION
Popis výsledku v původním jazyce
Glutathione plays important role in biochemical processes associated with oxidative stress in bacterial, plant and animals cells. Glutathione is occurring in the reduced (GSH) and oxidized form (GSSG) in the organism. The ratio between GSH and GSSG is considered as a marker of oxidative stress. During the sample preparation the destabilization of the GSH and GSSG forms is occurring which might be a problem for subsequent analysis. Aim of this study was the optimization and of quantitative reduction GSSG to GSH for HPLC-ED determination using tris (2-carboxyethyl) phosphin (TCEP) and ethylendiamintetracetic acid (EDTA). The efficiency of reduction of GSSG was analyzed using high pressure liquid chromatography with electrochemical detection (HPLC-ED). For GSH and GSSG determination we used gradient elution with methanol on reversed-phase column and coulometric detection. Limit of detection for GSH and GSSG was determined as 30 nM and 200 nM respectively. For qualitative reduction of GSSG to GSH we optimized the concentration of reducing agent (1, 2, 4, 6 mM), time of reaction (1, 5, 10, 15, 20, 25, 30, 40, 45, 50, 55, 60 minutes) and pH range of tested buffers (Britton-Robinson, Borate buffer, Phosphate buffer and Acetate buffer). The influence of addition of EDTA was also tested. We determined the optimal condition of the reducing reaction as 2 mM TCEP in Borate buffer (pH 9) and reaction time 15 minutes. No influence of EDTA addition on the reducing reaction was observed. The usage of the method was estimated for concentration range 60 nM - 200 mu M of GSSG.
Název v anglickém jazyce
DEVELOPMENT OF METHODIC OF TOTAL GLUTATHIONE AMOUNT DETERMINATION
Popis výsledku anglicky
Glutathione plays important role in biochemical processes associated with oxidative stress in bacterial, plant and animals cells. Glutathione is occurring in the reduced (GSH) and oxidized form (GSSG) in the organism. The ratio between GSH and GSSG is considered as a marker of oxidative stress. During the sample preparation the destabilization of the GSH and GSSG forms is occurring which might be a problem for subsequent analysis. Aim of this study was the optimization and of quantitative reduction GSSG to GSH for HPLC-ED determination using tris (2-carboxyethyl) phosphin (TCEP) and ethylendiamintetracetic acid (EDTA). The efficiency of reduction of GSSG was analyzed using high pressure liquid chromatography with electrochemical detection (HPLC-ED). For GSH and GSSG determination we used gradient elution with methanol on reversed-phase column and coulometric detection. Limit of detection for GSH and GSSG was determined as 30 nM and 200 nM respectively. For qualitative reduction of GSSG to GSH we optimized the concentration of reducing agent (1, 2, 4, 6 mM), time of reaction (1, 5, 10, 15, 20, 25, 30, 40, 45, 50, 55, 60 minutes) and pH range of tested buffers (Britton-Robinson, Borate buffer, Phosphate buffer and Acetate buffer). The influence of addition of EDTA was also tested. We determined the optimal condition of the reducing reaction as 2 mM TCEP in Borate buffer (pH 9) and reaction time 15 minutes. No influence of EDTA addition on the reducing reaction was observed. The usage of the method was estimated for concentration range 60 nM - 200 mu M of GSSG.
Klasifikace
Druh
D - Stať ve sborníku
CEP obor
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OECD FORD obor
10406 - Analytical chemistry
Návaznosti výsledku
Projekt
Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.
Návaznosti
P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)
Ostatní
Rok uplatnění
2012
Kód důvěrnosti údajů
S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů
Údaje specifické pro druh výsledku
Název statě ve sborníku
MENDELNET 2012
ISBN
978-80-7375-836-3
ISSN
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e-ISSN
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Počet stran výsledku
7
Strana od-do
1218-1224
Název nakladatele
Neuveden
Místo vydání
Neuveden
Místo konání akce
Mendel Univ, Fac Agron, Brno
Datum konání akce
21. 11. 2012
Typ akce podle státní příslušnosti
CST - Celostátní akce
Kód UT WoS článku
000366461200142