Glutathiones determination

Ublacker, G. A., Johnson, J. A., Siegel, F. L and Mulcahy, R. T. (1991) Influence of glutathione S-transferases on cellular glutathione determination by flow cytometry using monochlorobimane. Cancer Res. 51(7), 1783-1788. 

Erythrocyte glutathione determination in the diagnosis of glucose-6-phosphate dehydrogenase deficiency. 

In order to determine the methods of glutathione assay currently in use, a survey was made of the literature during the five-year period between 1948 and 1952. During this period approximately 300 papers on glutathione were published. It was possible to obtain about two thirds of these for study. Over half of the papers described the use of glutathione and were not concerned with assay. Glutathione determinations were made in 85 cases (Fig. 1). The iodometric titration method was used in almost half... 

Ruiz-Diaz JJJ, Torriero AAJ, Salinas E, Marchevsky EJ, Sanz MI, Raba J (2006) Enzymatic rotating biosensor for cysteine and glutathione determination in a FIA system. Talanta 68(4) 1343-1352... 

Other bioanalytical applications of systems in which the eluate of a first LC column is sampled in continuous and repetitive intervals and subjected to a second LC dimension are, for example, described by Wheatly et a/. (11) and Matsuoka et al. (12). Wheatly coupled gradient affinity LC with RPLC for the determination of the isoenzymatic- and subunit composition of glutathione 5-transferses in cytosol... 

Activities of glutamate-pyruvate transaminase (SGPT, GPT) (EC 2.6.1.2), L-y -glutamyl-transferase (y-GT) (EC 2.3.2.2) and level of triglycerides (TG) in serum, as well as levels of glutathione (GSH) and malondialdehyde (MDA) in the liver were determined. 

Emphasis is given to the critical role of metabolism, both detoxication and activation, in determining toxicity. The principal enzymes involved are described, including monooxygenases, esterases, epoxide hydrolases, glutathione-5 -transferases, and glucuronyl transferases. Attention is given to the influence of enzyme induction and enzyme inhibition on toxicity. 

Rao et. al recently published another method for the quantitation of the MB fraction. The method is based on the selective activating capacity of dithiothreitol on CR isoenzyme MB, after isoenzyme MM is activated by glutathione (51). Apparently the MB isoenzyme is not activated by glutathione" Eut is activated by dithiothreitol. The difference between CR activities obtained in the presence of glutathione alone and those obtained with both glutathione and dithiothreitol represent MB activity. The correlation is excellent (r 0.998) between the activity of MB in the isoenzyme mixture determined by this method, and the activity of isolated MB. 

Lewis, S.D., Misra, D.C. and Shafer, J.A. (1980) Determination of interactive thiol ionizations in bovine serum albumin, glutathione, and other thiols by potentiometric difference titration. Biochemistry, 19, 6129-6180. 

The major determinant of myocardial redox status is the glutathione content of the heart (Griffith and Meister, 1979). Therefore, fluctuations in myocardial glutathione status may exert a regulatory role in cellular metabolism in a comparable manner to the phosphorylation and dephosphorylation of proteins and enzymes. 

Although it is widely accepted that ischaemia/ reperfusion-induced oxidant stress is associated with a reduction of Na/K ATPase activity, it is difficult to determine which features of this process are responsible for this effect. A classical approach to this type of problem has been to determine the effect of the application of selected metabolites or agents on the activity of the enzyme of interest, an approach that has been exploited for the sarcolemmal Na/K ATPase and glutathione (Haddock et al., 1990). The application of GSH (O.l-l.OmM) induces a concentration-dependent increase in the activity of a bovine isolated ventricular Na/K ATPase preparation (determined by the ouabain-sensitive hydrolysis of ATP to release inorganic phosphate). In the presence of 1 mM GSH there was a 38% stimulation of activity compared to untreated control... 

Figure 4.9 Effect of reduced glutathione (GSH) (0.25-1.0 ihm) and oxidized glutathione (GSSG) (0.25-1.0 mM) on ouabain-sensitive Na/K ATPase activity. An isolated Na/K ATPase preparation was prepared from fresh bovine ventricular tissue. Na/K ATPase activity was determined and quantified by the ouabain-sensitive hydrolysis of ATP to yield Inorganic phosphate. The rate of inorganic phosphate production was compared prior to and following the addition of either GSH or GSSG to the Incubation mixture. The data are presented as...

The effect of cellular GSH depletion on /wa/K has also been studied using this model. Guinea-pig ventricular myocytes prepared from DEM-treated animals have been used to determine the effect of glutathione depletion on /xa/K- Myocytes prepared from DEM-treated animals showed a similar profile of glutathione content modification to that previously described in experiments using sarcolemmal homogenates. GSH levels in DEM-treated were reduced from a control value of 0.2 0.04 to 0.02 0.01 nmol/1 X10 cells. Jni/k at 0 mV was reduced from a control value of 1.1 0.12 to... 

If cellular redox state, determined by the glutathione status of the heart, plays a role in the modulation of ion transporter activity in cardiac tissue, it is important to identify possible mechanisms by which these effects are mediated. Protein S-,thiolation is a process that was originally used to describe the formation of adducts of proteins with low molecular thiols such as glutathione (Miller etal., 1990). In view of the significant alterations of cardiac glutathione status (GSH and GSSG) and ion-transporter activity during oxidant stress, the process of S-thiolation may be responsible for modifications of protein structure and function. 

Curello, S., Ceconi, C.. Cargnoni, A., Connacchian, A., Ferrari, R. and Albertini, A. (1987). Improved procedure for determining glutathione in plasma as an index of myocardial oxidative stress. Clin. Chem. 33, 1448-1449. 

Selenium is required, but levels must fall into a narrow window. Both deficiency and toxicity symptoms occur. The element is also used therapeutically in cancer treatment. It is the co-factor of the enzyme glutathione peroxidase which is thought to play an important role in oxygen toxicity. The determination of Se in blood or serum is not easy, as many incorrect, inaccurate and imprecise methods have been published (Magee and James 1994). A suggested procedure for Se in body fluids is based on GF-AAS (Thomassen et al. 1994)- For tissues SS-AAS may be used (Fler-ber 1994a). Recent developments by Turner et al. (1999) show that LC-ICP-MS is sensitive and reproducible at low levels. 

M26. Miwa, S., Luzzatto, L., Rosa, R., Paglia, D. E., Schroter, W De Flora, A., Fujii, H., Board, P. G and Beutler, E., International Committee for Standardization in Haematology Recommended methods for an additional red cell enzyme (pyrimidine 5 -nucleotidase) assay and the determination of red cell adenosine 5 -triphosphate, 2,3-diphosphoglycerate and reduced glutathione. Clin. Lab. Haematol. 11, 131-138 (1989). 

Monostori P, Wittmann G, Karg E, Turi S (2009) Determination of glutathione and glutathione disulfide in biological samples an in-dept review. J Chromatogr B 877 3331-3346... 

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