Red blood cell glutathione peroxidase activity in female nulligravid and pregnant rats
© Gallo and Martino; licensee BioMed Central Ltd. 2009
Received: 17 August 2008
Accepted: 26 January 2009
Published: 26 January 2009
The alterations of the glutathione peroxidase enzyme complex system occur in physiological conditions such as aging and oxidative stress consequent to strenuous exercise.
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 nulligravid 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.
The aim of the present research is to evaluate the contribution of the enzymatic antioxidant glutathione peroxidase (GP) by optimization of the spectrophotometric method of Paglia and Valentine  so that it can be applied to blood samples from several animals differing from sex, age and species. After evaluating the usefulness of the method, it was thus, applied to study the effect of the different levels of oxidative stress consequent to pregnancy.
GP (that is PDB 1GP1, according to Protein Data Bank, and EC 22.214.171.124, according to the Enzyme Commission numbering) is the main term of an enzyme family with peroxidase activity. GP, discovered in 1957 by Mills , has the function to reduce lipid hydroperoxides to their corresponding alcohols and to reduce free hydrogen peroxide in water. GP1 is found in the cytoplasm of nearly all mammalian tissues, whose preferred substrate is hydrogen peroxide. The GP reaction is:
2GSH + H2O2 → GS-SG + 2H2O
where GSH represents reduced monomeric glutathione, and GS-SG represents glutathione disulfide. Glutathione reductase (GR) then reduces the oxidized glutathione to complete the cycle:
GS-SG + NADPH + H+ → 2 GSH + NADP+.
At present, there is not sufficient information on oxidative stress and pregnancy available in literature. Ara et al.  studied peritoneal adhesions in rats. Jackson et al.  evaluated the association between oxidative stress and endometriosis and found only a weak association between thiobarbituric acid-reactive substances (nmol/ml) in serum and endometriosis in 10 pregnant females on 32 total studied. Vanderlelie and Perkins  evaluated the oxidative stress at the end of the reproduction in rats in placental and liver tissues, but they do not describe the alteration of GP activity, resulting from the coordination of tissue GSH reductase and GSSG peroxidase enzyme activities in both selenium deficiency and L-NAME oral administration.
The authors verify that GP in RBCs membranes works as a complex to show the comprehensive systemic effect of using GSH to scavenge ROS in RBCs, producing GSSG and the correspondent hydroperoxid equivalent to ROS. Only if all NADPH is oxidized used to replenish GSH stores in the membrane preparations, than the determination of NADPH oxidized by GP, represents a true measurement of the specific enzyme complex [8, 9].
Concerning the hypertension mechanisms, the oxidative stress was shown in the rostral ventrolateral medulla (RVLM)  and in kidney  and are both present in spontaneously hypertensive rats (SHR). Zhou  demonstrated that oxidative stress in pregnancy can increase, even if the exact either pro-oxidant or antioxidant status in pregnancy-induced hypertensive patients is not clear . There is no study on rat blood and on the relationship between hypertension and pregnancy.
Sixteen weeks old nulligravid female Wistar rats were housed at a constant temperature (22°C) in a 12 hours light and dark cycle environment with free access to food and drinking water. Animals were randomised in two groups (n = 6) and fed a standard diet (MilRatti Stefano Morini, S. Polo D'Enza (RE)). The animals were treated according to the european community prescriptions [EU (86/609/EEC)], then cycled and mated with fertile males at proestrous on 120 d of age, with a positive vaginal smear for sperm, the day after proestrous indicating d 0 of pregnancy. The remaining animals continued on their diets as nulligravid controls. On d 140 pregnant rats were submitted to blood sampling under ether (50 g/kg body weight) anesthesia according to the european community prescriptions [EU (86/609/EEC)] on animal care.
Processing of tissue samples and hemoglobin estimation
Blood, 100 μL of sample drawn by cardiac puncture under ether anesthesia (50 g/kg body weight) are centrifuged and washed twice with 5 mL of 0,9% NaCl. Isolated RBCs are hemolyzed by addition of 1 mL of distilled H2O. Hemoglobin concentration is determined by mixing 1 mL of Hemoglobin test (Sclavo diagnostics, Siena, Italy) with 0,1 mL of hemolysate. The absorbance at 546 nm is measured in a Shimadzu UVPC 2100 spectrophotometer (A546 × 16 = mg Hb/mL) against a blank containing water instead of hemolysate. The hemolysate is exactly diluted to 3 mg of Hb per millilitre. From this solution, 1 mL is mixed with 0.5 mL of transformation solution (4.5 mM KCN and 0.45 mM K3 [Fe(CN)6] adjusted with 0.25 M potassium dihydrogen phosphate to pH 7.0). After 5 min, transformation to cyanmethemoglobin is complete at room temperature.
Glutathione peroxidase assay
GP activity was determined by a modified method of Paglia and Valentine (1967)  Activity was determined spectrophotometrically by coupling the oxidation of glutathione and NADPH using GR. Briefly, 1 mL of assay mixture contains optimized concentrations of the following chemicals: 0,5 M K2HPO4 (pH 7.0), 2,5 mM EDTA, 0,18 U/mL GR, 100 mM glutathione and 10 mM reduced NADPH and tissue extract (0,5 mL) was added in the spectrophotometer cuvette along with 0,1 mL of 60 mM cumene hydroperoxide, a suitable substrate for GP.
the peroxilipid is reduced by GSH (reduced glutathione) to hydroxilipids (GP activity);
GSH is oxidized to GSSG (oxidized glutathione) (GP);
GSSG is reduced to GSH by NADPH (GR) as resumed in figure 3.
According to the cited figure 3, the increase of NADPH concentration from 2,5 mM  up to 10 mM also increases the velocity of GR and the GSH reserve levels, that are increased from 10 mM up to 100 mM, in such a way to always saturate GP activity. Cumene hydroperoxide concentration is also increased from 12 mM up to 60 mM so that lipid peroxidation is increased and GP activity therefore is limited only by GP levels in RBC membrane preparations. In each measurement of the enzyme activity the decrease of A366 was determined over a 2 min period. GP activity was standardised against Hb concentrations and expressed as NADPH mmol oxidized per minute per mg of hemoglobin (mmol/min/mg Hb). All chemicals were from Sigma Aldrich (St. Louis, MO).
Optimal concentrations of Glutathione peroxidase reaction medium.
Potassium phosphate, containing 2,5 mM Na2EDTA, 2,5 mM sodium azide, pH 7.0
Glutathione reductase (from baker yeast, ammonium sulfate suspension, 100–300 U/mg protein) in the same phosphate buffer;
0,18 U/ml: at 25°C
GSH (>98%) in distilled water
NADPH (~95%) in 0,1% NaHCO3 solution
Cumene hydroperoxide (>80%) in distilled water
Initial velocity of GPX complex activity in normal and gravid RBC membranes of 140 days old female rats (mean ± S.E. M. of eight indipendent experiments)
135,30 10-6 ± 1,72 10-6
3,96 10-6 ± 0,83 10-6
The described results show that, after the optimization of enzyme kinetics measurement conditions, the GP activity of pregnant rats is clearly distinct by the GP activity of female age paired nulligravid animals.
This indeed is well in agreement with the metabolic increased oxidative metabolism of pregnant animals . Moreover the hormonal rearrangement intrinsic of pregnancy  redistributes the interrelationships of metabolic pathways that sustain fetus growth and differentiation, particularly in rats, that have a short pregnancy (about 17 d).
The work was partially supported by the Italian Ministry of University (MIUR).
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