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Red blood cell glutathione peroxidase activity in female igravid and pregnant rats.

Gallo G, Martino G - Reprod. Biol. Endocrinol. (2009)

Bottom Line: Authors optimize the spectrophotometric method to measure glutathione peroxidase activity in rat red blood cell membranes.The optimization, when applied to age paired rats, both igravid and pregnant, shows that pregnancy induces, at seventeen d of pregnancy, an increase of both reactive oxygen substance concentration in red blood cells and membrane glutathione peroxidase activity.The glutathione peroxidase increase in erythrocyte membranes is induced by systemic oxidative stress long lasting rat pregnancy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology, University of Calabria, Rende, CS, Italy. pino72@tiscali.it

ABSTRACT

Background: The alterations of the glutathione peroxidase enzyme complex system occur in physiological conditions such as aging and oxidative stress consequent to strenuous exercise.

Methods: Authors optimize the spectrophotometric method to measure glutathione peroxidase activity in rat red blood cell membranes.

Results: The optimization, when applied to age paired rats, both igravid and pregnant, shows that pregnancy induces, at seventeen d of pregnancy, an increase of both reactive oxygen substance concentration in red blood cells and membrane glutathione peroxidase activity.

Conclusion: The glutathione peroxidase increase in erythrocyte membranes is induced by systemic oxidative stress long lasting rat pregnancy.

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GP activity as function [GSH]. Each point is the mean of three independent determinations.
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Figure 6: GP activity as function [GSH]. Each point is the mean of three independent determinations.

Mentions: The described revision of Paglia and Valentine [1] method to evaluate GP activity in RBCs membranes of mammalia must be optimized for the specific kind of samples studied. The modifications shall be devolved to obtain the stable function of studied enzyme activity under saturation conditions [15] Only in this instance the time course of absorbance of NADPH at 366 nm describes the real kinetic behaviour of GP catalized redox reaction. The principle has been applied by optimizing the concentrations of substrates and cofactors of both GR and GP activities in order to obtain that only GP is the limiting enzyme activity of our RBC enzyme complex system. GP-Activity and kinetics were measured at 13 mM, 10 mM e 7 mM GSH. (figure 6). The striking feature is that the timecourses are useful to screen between the three GSH concentrations. By using the scientific software SigmaPlot (derivative), Enzyme Kinetics, (kinetics) subroutine, and the statistic analysis on SigmaStat. To shorten the description, the use of NADPH at several concentrations is not described here, but was performed by the same statistical methods of analysis previously described for GP. Special attention should be applied to cumene peroxide concentration optimization, according to the short term stability of this reactant in laboratory environment conditions. Also the RBC membrane preparations show short term stability if not stored at 4°C and utilized within 24 hours. The NADPH absorbance data at 366 nm are interpolated according to Hofstee and Eadie [8,9] by derivative routine of SigmaPlot® scientific graph program. The optimized method was therefore applied to investigate in rats the effect of pregnancy on oxidative stress action in RBC membranes as described by GP activity evaluation. As the GP activity was previously studied only in tissues such as rat liver and placenta without optimization of substrata conditions [7], the selection of blood as the target compartment to study the effect of systemic peroxide levels has the advantage of investigating the compartment subjected to all the comprehensive effects of peroxide systemic accumulation. In fact, data show the significant increase of GP activity during pregnancy in comparison to normal age paired igravid female rats. Such a clearcut increase confirms the working hypothesis that the adaptative reaction to oxidative stress in pregnancy includes the increase of GP enzyme antioxidant activity. In figures 5 and 6 the significance limit is set at p < 0.05 of results and is accomplished by two ways Student t, using SigmaStat® scientific statistical program.


Red blood cell glutathione peroxidase activity in female igravid and pregnant rats.

Gallo G, Martino G - Reprod. Biol. Endocrinol. (2009)

GP activity as function [GSH]. Each point is the mean of three independent determinations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2657797&req=5

Figure 6: GP activity as function [GSH]. Each point is the mean of three independent determinations.
Mentions: The described revision of Paglia and Valentine [1] method to evaluate GP activity in RBCs membranes of mammalia must be optimized for the specific kind of samples studied. The modifications shall be devolved to obtain the stable function of studied enzyme activity under saturation conditions [15] Only in this instance the time course of absorbance of NADPH at 366 nm describes the real kinetic behaviour of GP catalized redox reaction. The principle has been applied by optimizing the concentrations of substrates and cofactors of both GR and GP activities in order to obtain that only GP is the limiting enzyme activity of our RBC enzyme complex system. GP-Activity and kinetics were measured at 13 mM, 10 mM e 7 mM GSH. (figure 6). The striking feature is that the timecourses are useful to screen between the three GSH concentrations. By using the scientific software SigmaPlot (derivative), Enzyme Kinetics, (kinetics) subroutine, and the statistic analysis on SigmaStat. To shorten the description, the use of NADPH at several concentrations is not described here, but was performed by the same statistical methods of analysis previously described for GP. Special attention should be applied to cumene peroxide concentration optimization, according to the short term stability of this reactant in laboratory environment conditions. Also the RBC membrane preparations show short term stability if not stored at 4°C and utilized within 24 hours. The NADPH absorbance data at 366 nm are interpolated according to Hofstee and Eadie [8,9] by derivative routine of SigmaPlot® scientific graph program. The optimized method was therefore applied to investigate in rats the effect of pregnancy on oxidative stress action in RBC membranes as described by GP activity evaluation. As the GP activity was previously studied only in tissues such as rat liver and placenta without optimization of substrata conditions [7], the selection of blood as the target compartment to study the effect of systemic peroxide levels has the advantage of investigating the compartment subjected to all the comprehensive effects of peroxide systemic accumulation. In fact, data show the significant increase of GP activity during pregnancy in comparison to normal age paired igravid female rats. Such a clearcut increase confirms the working hypothesis that the adaptative reaction to oxidative stress in pregnancy includes the increase of GP enzyme antioxidant activity. In figures 5 and 6 the significance limit is set at p < 0.05 of results and is accomplished by two ways Student t, using SigmaStat® scientific statistical program.

Bottom Line: Authors optimize the spectrophotometric method to measure glutathione peroxidase activity in rat red blood cell membranes.The optimization, when applied to age paired rats, both igravid and pregnant, shows that pregnancy induces, at seventeen d of pregnancy, an increase of both reactive oxygen substance concentration in red blood cells and membrane glutathione peroxidase activity.The glutathione peroxidase increase in erythrocyte membranes is induced by systemic oxidative stress long lasting rat pregnancy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology, University of Calabria, Rende, CS, Italy. pino72@tiscali.it

ABSTRACT

Background: The alterations of the glutathione peroxidase enzyme complex system occur in physiological conditions such as aging and oxidative stress consequent to strenuous exercise.

Methods: Authors optimize the spectrophotometric method to measure glutathione peroxidase activity in rat red blood cell membranes.

Results: The optimization, when applied to age paired rats, both igravid and pregnant, shows that pregnancy induces, at seventeen d of pregnancy, an increase of both reactive oxygen substance concentration in red blood cells and membrane glutathione peroxidase activity.

Conclusion: The glutathione peroxidase increase in erythrocyte membranes is induced by systemic oxidative stress long lasting rat pregnancy.

Show MeSH