|Year : 2012 | Volume
| Issue : 2 | Page : 274-279
|Evaluation of lipid peroxidation and erythrocyte glutathione peroxidase and superoxide dismutase in hemodialysis patients
Farzaneh Montazerifar1, Mohammad Hashemi2, Mansour Karajibani1, Houshang Sanadgol3, Madhurima Dikshit4
1 Department of Nutrition, School of Medicine, Zahedan University of Medical Sciences, Zahedan, Iran
2 Department of Clinical Biochemistry, School of Medicine and Cellular and Molecular Research Center, Zahedan University of Medical Sciences, Zahedan, Iran
3 Department of Internal Medicine, School of Medicine, Zahedan University of Medical Sciences, Zahedan, Iran
4 Division of Biochemistry, Department of Chemistry, Pune University, Pune, India
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|Date of Web Publication||28-Feb-2012|
| Abstract|| |
Oxidative stress often occurs in chronic hemodialysis (HD). The aim of the present study was to determine plasma malondialdehyde (MDA) level for lipid peroxidation product and erythrocyte superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities as enzymatic antioxidants. Thirty-one HD patients (aged 50.3 ± 14.9 years) who were dialyzed three times per week and 31 healthy subjects (aged 47.8 ± 13.9 years) were enrolled. The results showed lower enzymatic antioxidants activity (GPx, SOD) and higher MDA levels in comparison with control subjects. In addition, SOD and GPx activities significantly decreased and MDA increased after HD. We also found that there was a significantly negative correlation between SOD and GPx with MDA. The results suggest that elevated level of plasma MDA and reduced activities of SOD and GPx can be caused oxidative stress, which may play a critical role in HD complications.
|How to cite this article:|
Montazerifar F, Hashemi M, Karajibani M, Sanadgol H, Dikshit M. Evaluation of lipid peroxidation and erythrocyte glutathione peroxidase and superoxide dismutase in hemodialysis patients. Saudi J Kidney Dis Transpl 2012;23:274-9
|How to cite this URL:|
Montazerifar F, Hashemi M, Karajibani M, Sanadgol H, Dikshit M. Evaluation of lipid peroxidation and erythrocyte glutathione peroxidase and superoxide dismutase in hemodialysis patients. Saudi J Kidney Dis Transpl [serial online] 2012 [cited 2013 May 20];23:274-9. Available from: http://www.sjkdt.org/text.asp?2012/23/2/274/93151
| Introduction|| |
Hemodialysis (HD) is the most common technique used to treat end-stage renal disease (ESRD). The presence of a chronic inflammation has been documented in ESRD patients receiving maintenance HD,  which is a life-saving procedure required for the removal of endo-toxins in patients with chronic renal failure.  However, direct contact of blood with dialysis membrane during HD can produce inflammatory reactions and oxidative stress that results in the modification of lipoprotein particles and lipid oxidation products. , Alterations in lipid metabolism and oxidative stress are recognized as important risk factors that can be prevented or reduced by optimal therapy.  Oxidative stress, an imbalance between toxic compounds such as reactive oxygen species (ROS) and antioxidant defense mechanisms,  occur when there is excessive production of free radicals or low antioxidant levels.  Free radicals can damage proteins, DNA, lipids in cell membranes, carbohydrates (hyaluronic acid), and nucleic acids.  Free radical reactions including lipid peroxidation are considered to be important factors in the pathogenesis of a variety of disease, e.g., cancer, atherosclerosis, aging and autoimmune disease. Some studies have shown that dialysis treatment is the main source of increased oxidative injury in ESRD patients rather than the disease itself. , Oxidant stress is assayed by measuring markers of the oxidative damage to poly-unsaturated fatty acids such as malondialdehyde (lipid peroxides) or by measuring of levels of anti-oxidants. Various antioxidant enzymes that detoxify free radicals include superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase. Several studies reported on levels of lipid peroxidation and anti-oxidants in HD patients with variable results due to different factors such as dialysis materials (e.g., the kind of HD membrane), , duration of HD therapy, , and trace elements disturbances. An increase in oxidative stress is proposed as a nontraditional cardiovascular risk factor in HD patients. 
The aim of this study is to determine the levels of the components of the oxidative stress process in our HD patients.
| Materials and Methods|| |
The study group included 31 HD patients (17 males and 14 females, with a mean age 50.3 ± 14.9 years and a range from 22-75 years) from the HD center at Khatam-Al-Anbia Hospital, Zahedan Medical University, Iran. They were regularly dialyzed thrice a week, at least four hours per session, with polysulfone membrane dialyzers, (a mean duration of hemodialysis 76.1 months and a range of 7-120 months). None of the HD patients was a smoker, HCV/ HIV positive, or receiving antioxidant supplement. Thirty-one healthy volunteers (19 males and 12 females, with a mean age 47.8 ± 13.9 years and a range of 20-79 years) were selected as the control group. They had no medical problems and no cigarette or vitamin supplement consumption. The project was approved by the ethical committee of Zahedan University of Medical Sciences, and informed consent was obtained from all patients and healthy individuals.
Blood samples were taken twice from the HD patients, immediately before (pre-HD) and after (post-HD) dialysis sessions from arteriovenous fistulas (AVF), and once from the control subjects after an overnight fast in tubes with and without an anticoagulant. Samples were immediately centrifuged at 3000 rpm for 10 minutes at 4°C. Plasma and serum samples were stored at -80°C until analysis. EDTA anticoagulant tube was used for the measurement of hemoglobin and hematocrit. Heparinized whole blood was used for measurement of SOD and GPx. Serum samples were used for measurement of biochemical parameters (urea, uric acid, and creatinine) and plasma sample was used for determination of MDA.
Superoxide dismutase (SOD) activity
Erythrocyte SOD activity was determined based on the production of O -2 anions by the xanthine/xanthine oxidase system  using commercial assay kit (Randox, UK).  The specific activity of SOD was expressed in units per gram of hemoglobin (U/g Hb).
Glutathione peroxidase (GPx) activity Gpx activity was measured using commercial test kit (Randox, UK) in heparinized whole blood by the procedure of Paglia and valentine.  Briefly, glutathione peroxidase (GPx) catalyses the oxidation of glutathione (GSH) by cumene hydroperoxide. In the presence of glutathione reductase (GR) and NADPH, the oxidized glutathione (GSSG) is immediately converted to the reduced form with a concomitant oxidation of NADPH to NADP + . The decrease in absorbance at 340 nm was measured by spectrophotometer. The specific activity for GSH-Px was expressed in units per gram hemoglobin (U/g Hb). 
Malondialdehyde (MDA) assay
Lipid peroxidation in the plasma was evaluated by the spectrophotometric method based on the reaction between MDA and thiobarbituric acid (TBARS)  as described previuosly. 
| Statistical Analysis|| |
The results were expressed as mean ± standard deviation (SD). Statistical analysis of the results was performed using the statistical package software (SPSS), version 11.5 for windows. An independent sample t-test was used to compare the parameters between controls and patients, and a paired t-test was used to compare parameters before and after HD in the patients. Pearson's correlation coefficient was used to determine the relationship between variables. A P-value less than0.05 was considered statistically significant.
| Results|| |
In this study, we found that the primary causes of ESRD included diabetes (n = 5), hypertension (n = 8), diabetes and hypertension (n = 4), lupus erythematus (n = 2), severe glomerulonephritis (n = 3), chronic glomerulonephritis (n=1), kidney stones (n=2), and unknown (n=6).
Demographic data about the patients are shown in [Table 1], and the results of clinical and laboratory findings of the study patients and controls are shown in [Table 2]. Overall, hemoglobin, hematocrit levels were lower than those of the controls, whereas the levels of BUN, uric acid, creatinine were much higher and decreased after dialysis.
The erythrocyte antioxidant enzymes activities, SOD and GPx, were found decreased after HD (P<0.0001) [Figure 1], [Figure 2], but plasma MDA levels (μmol/L) were increased in comparison with those before HD and in controls (P < 0.0001) [Figure 3].
|Figure 1: Erythrocyte superoxuide dismutase (SOD) activity in patients (pre- and post- hemodialysis) and control group. The level of SOD was significantly higher in control subjects than in hemodialysis patients and significantly decreased after hemo-dialysis (**=P<0.0001).|
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|Figure 2: Erythrocyte glutathione peroxidase (GPx) activity in patients (pre- and posthemodialysis) and control group. The level of GPx was significantly higher in control subjects than in hemodialysis patients and significantly decreased after|
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|Figure 3: Plasma malondialdehyde (MDA) levels (μmol/L) in patients (pre- and posthemodialysis) and control group. The levels of MDA was significantly higher in hemodialysis patients than control subjects and significantly increased after hemodialysis (**=P<0.0001).|
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There was no significant difference between sex and age with any parameter. There was a significant negative correlation between the anti-oxidant enzymes, SOD with MDA (r = -0.47, P = 0.007), [Figure 4], and GPx with MDA (r = -0.44, P = 0.01), [Figure 5].
|Figure 4: Correlation between erythrocyte superoxide dismutase (SOD) activity and plasma malondialdehyde (MDA) levels in hemodialysis patients. There was a significant negative correlation between SOD and MDA levels in hemodialysis|
patients (r=-0.4784, P=0.0070).
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|Figure 5: Correlation between erythrocyte glutathione peroxidase (GPx) activity and plasma malondialdehyde (MDA) levels in hemodialysis patients. There was a significant negative correlation between GPx and MDA levels in hemodialysis|
patients (r=−0.4260, P=0.0169).
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A significant correlation between duration of HD with MDA (r = 0.42, P = 0.02), SOD (r = -0.54, P = 0.002), and GPx (r = -0.39, P = 0.03) was observed.
| Discussion|| |
Oxidative stress is present in both health and disease, and corresponding oxidative stress markers can be measured in both healthy people and those with various clinical conditions.  In renal failure, disturbances in enzymatic mechanisms of free radicals detoxification result in alteration in the antioxidant system and reactive oxygen species (ROS) attack on cell membranes and formation of lipid peroxidation products such as MDA.
There are some reports which confirm that hemodialysis can produce oxidative stress and lipid peroxidation products. ,, Our study in HD patients showed elevated MDA levels before and after HD, similar to some studies, ,,, however, in contrast with the findings of decreased MDA level after HD.  In addition, some studies have reported no differences in plasma MDA levels in HD patients from controls, , or HD decreased MDA levels. 
We believe that the finding of elevated MDA levels in our study suggest that both renal failure and dialysis process can probably cause free radical reactions and increase the oxidative stress, especially when the enzymatic antioxidant systems decrease in activity as observed in the present study.
Several studies of the erythrocyte SOD and GPx activities have reported varying changes in the pre and post-HD. The decreased erythrocyte SOD activity found in our study HD patients in comparison with controls was reported by others. ,,, However, other researchers found the SOD activity after HD less than before and the levels in controls, , while Durak et al  did not find a statistically different SOD activity of the HD patients from the controls.
Glutathione peroxidases are antioxidant enzymes that can detoxify hydrogen peroxide and lipid peroxides in the presence of reduced glutathione.  As long as this system was intact, there was no difference between HD patients and controls, but when GPx was inhibited, erythrocytes of HD patients showed a significant delay in the elimination of free radicals, which could be the reason for high MDA.  In our study, the decreased erythrocyte GPx levels before HD compared to control group was similarly found in other reports. ,,,,,,, However, in the study of Durak et al,  GPx levels were not significantly different from the control values, and also Dubey et al  reported decreased GPx levels in acute renal failure in children. As in our study, the results of some studies ,, have shown reduced erythrocyte GPx levels before HD and further reduced after HD. However, Ozden et al  demonstrated higher GPx levels in after HD than before.
Moreover, the studies performed on the effect of the different dialysis membranes on antioxidants and lipid peroxidation levels, have shown varying results. Eislet et al  showed the activity of antioxidant enzymes (GSH-Px) and SOD were lower with polysulfon (PS) membrane than with the cellulose diacetate (CDA) membrane, but the use of memranes did not result in increased MDA levels after HD. In our study, we used PS membrane dialyzers for HD, which may explain the reduced activities of the SOD and GPx. However, Malliarak et al  and Wu et al  studied different dialysis membranes and observed no significant differences in the activities of the SOD and GPx in any group during the dialysis procedure.
Patients undergoing maintenance HD have elevated markers of oxidative stress, but the reasons for this are not fully understood. It has been reported that intravenous administration of iron, which many of these patients receive, may provoke the generation of bioactive iron and enhance oxidative stress and lipid peroxidation. 
Finally, our results indicate that longer HD duration may enhance oxidative stress similar to other studies. ,
In conclusion, the results of our study suggest that long duration of dialysis caused low anti-oxidant activity in erythrocytes with subsequent increase of lipid peroxidation.
| Acknowledgement|| |
The authors would like to acknowledge the assistance of Mrs Noora and Rezaei (Clinical Biochemistry Lab, Zahedan School of Medicine). In addition, the authors thank the patients and healthy subjects who willingly participated in the study.
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Professor of Clinical Biochemistry, Department of Clinical Biochemistry, School of Medicine, Zahedan University of Medical Sciences, Zahedan
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]
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