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Saudi Journal of Kidney Diseases and Transplantation
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ORIGINAL ARTICLE  
Year : 2012  |  Volume : 23  |  Issue : 2  |  Page : 274-279
Evaluation of lipid peroxidation and erythrocyte glutathione peroxidase and superoxide dismutase in hemodialysis patients


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 Publication28-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 2014 Nov 21];23:274-9. Available from: http://www.sjkdt.org/text.asp?2012/23/2/274/93151

   Introduction Top


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, [1] which is a life-saving procedure required for the removal of endo-toxins in patients with chronic renal failure. [2] 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. [2],[3] Alterations in lipid metabolism and oxidative stress are recognized as important risk factors that can be prevented or reduced by optimal therapy. [4] Oxidative stress, an imbalance between toxic compounds such as reactive oxygen species (ROS) and antioxidant defense mechanisms, [5] occur when there is excessive production of free radicals or low antioxidant levels. [6] Free radicals can damage proteins, DNA, lipids in cell membranes, carbohydrates (hyaluronic acid), and nucleic acids. [7] 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. [8],[9] 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), [10],[11] duration of HD therapy, [12],[13] and trace elements disturbances. An in­crease in oxidative stress is proposed as a nontraditional cardiovascular risk factor in HD patients. [14]

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 Top


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 sampling

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 [15] using com­mercial assay kit (Randox, UK). [16] 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. [17] 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). [16]

Malondialdehyde (MDA) assay

Lipid peroxidation in the plasma was evalua­ted by the spectrophotometric method based on the reaction between MDA and thiobarbituric acid (TBARS) [18] as described previuosly. [16]


   Statistical Analysis Top


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 Top


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
hemodialysis (**=P<0.0001).


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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 Top


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. [3] 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. [2],[3],[5] Our study in HD patients showed elevated MDA levels before and after HD, similar to some studies, [4],[19],[20],[22] however, in contrast with the findings of decreased MDA level after HD. [21] In addition, some studies have reported no differences in plasma MDA levels in HD patients from controls, [23],[24] or HD decreased MDA levels. [25]

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. [4],[22],[24],[26] However, other re­searchers found the SOD activity after HD less than before and the levels in controls, [27],[22] while Durak et al [28] 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. [19] 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. [5] In our study, the decreased erythrocyte GPx levels before HD compared to control group was similarly found in other reports. [4],[7],[19],[24],[25],[26],[29],[30] However, in the study of Durak et al, [28] GPx levels were not significantly different from the control values, and also Dubey et al [27] reported decreased GPx levels in acute renal failure in children. As in our study, the results of some studies [19],[22],[25] have shown reduced erythrocyte GPx levels before HD and further reduced after HD. However, Ozden et al [19] 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 [10] 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 [11] and Wu et al [30] 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. [31]

Finally, our results indicate that longer HD duration may enhance oxidative stress similar to other studies. [12],[13]

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 Top


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.

 
   References Top

1.Hashemi M, Mehrabifar H, Homayooni F, et al. Serum trypsin inhibitory capacity in hemodialysis patients. Saudi J Kidney Dis Transpl 2009;20(4): 604-7.  Back to cited text no. 1
    
2.Ziouzenkova O, Asatryan L, Tetta C, et al. Oxidative stress during ex vivo hemodialysis of blood is decreased by a novel hemolipodialysis procedure utilizing antioxidants. Free Radic Biol Med 2002; 33(2):248-58.  Back to cited text no. 2
    
3.Handelman GJ. Evaluation of oxidant stress in dialysis patients. Blood Purif 2000;18(4):343-9.  Back to cited text no. 3
    
4.Drai J, Bannier E, Chazot C, et al. Oxidants and antioxidants in long-term haemodialysis patients. Farmaco 2001;56(5-7):463-5.  Back to cited text no. 4
    
5.Galle J. Oxidative stress in chronic renal failure. Nephrol Dial Transplant 2001;16(11):2135-7.  Back to cited text no. 5
    
6.Pawlak K, Borawski J, Naumnik B, Mysliwiec M. Relationship between oxidative stress and extrinsic coagulation pathway in haemodialyzed patients. Thromb Res 2003;109(5-6):247-51.  Back to cited text no. 6
    
7.Selvaraj N, Bobby Z, Das AK, Ramesh R, Koner BC. An evaluation of level of oxidative stress and protein glycation in nondiabetic undialyzed chronic renal failure patients. Clin Chim Acta 2002;324(1-2):45-50.  Back to cited text no. 7
    
8.Morena M, Cristol JP, Canaud B. Why hemodialysis patients are in a prooxidant state? What could be done to correct the pro/antioxidant imbalance. Blood Purif 2000;18(3):191-9.  Back to cited text no. 8
    
9.Mayer B, Zitta S, Greilberger J, et al. Effect of hemodialysis on the antioxidative properties of serum. Biochim Biophys Acta 2003;1638(3):267-72.  Back to cited text no. 9
    
10.Eiselt J, Racek J, Holecek V, Krejcova I, Opatrny K. Antioxidants and malondialdehyde during hemodialysis with cellulose diacetate and polysulfone membranes. Cas Lek Cesk 1996;135 (21):691-4.  Back to cited text no. 10
    
11.Malliaraki N, Mpliamplias D, Kampa M, et al. Total and corrected antioxidant capacity in hemodialyzed patients. BMC Nephrol 2003;4:4.  Back to cited text no. 11
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12.Nguyen-Khoa T, Massy ZA, De Bandt JP, et al. Oxidative stress and haemodialysis: role of inflam­mation and duration of dialysis treatment. Nephrol Dial Transplant 2001;16(2):335-40.  Back to cited text no. 12
    
13.Ferretti G, Bacchetti T, Masciangelo S, Pallotta G. Lipid peroxidation in hemodialysis patients: Effect of vitamin C supplementation. Clin Biochem 2008;41 (6):381-6.  Back to cited text no. 13
    
14.Himmelfarb J. Oxidative stress in hemodialysis. Contrib Nephrol 2008;161:132-7.  Back to cited text no. 14
[PUBMED]  [FULLTEXT]  
15.Serdar Z, Aslan K, Dirican M, et al. Lipid and protein oxidation and antioxidant status in patients with angiographically proven coronary artery disease. Clin Biochem 2006;39(8):794-803.  Back to cited text no. 15
    
16.Karajibani M, Hashemi M, Montazerifar F, Bolouri M, Dikshit M. The status of glutathione peroxidase, superoxide dismutase, vitamins A, C, E and malon-dialdehyde in patients with cardiovascular disease in Zahedan, southeast Iran. J Nutr Sci Vitaminol 2009; 55(4):309-16.  Back to cited text no. 16
    
17.Paglia DE, Valentine WN. Studies on the quanti­tative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967;70(1): 158-69.  Back to cited text no. 17
    
18.Satoh K. Serum lipid peroxide in cerebrovascular disorders determined by a new colorimetric method. Clin Chim Acta 1978;90(1):37-43.  Back to cited text no. 18
    
19.Ozden M, Maral H, Akaydin D, Cetinalp P, Kalender B. Erythrocyte glutathione peroxidase activity, plasma malondialdehyde and erythrocyte glutathione levels in hemodialysis and CAPD patients. Clin Biochem 2002;35(4):269-73.  Back to cited text no. 19
    
20.Dasgupta A, Malhotra D, Levy H, et al. Decreased total antioxidant capacity but normal lipid hydroperoxide concentrations in sera of critically ill patients. Life Sci 1997;60(4-5):335-40.  Back to cited text no. 20
    
21.Samouilidou E, Grapsa E. Effect of dialysis on plasma total antioxidant capacity and lipid per-oxidation products in patients with end-stage renal failure. Blood Purif 2003;21(3):209-12.  Back to cited text no. 21
    
22.Gonzalez Rico M, Puchades MJ, Garcia Ramon R, et al. Effect of oxidative stress in patients with chronic renal failure. Nefrologia 2006;26(2):218-25.  Back to cited text no. 22
    
23.Erdogan C, Unlucerci Y, Turkmen A, et al. The evaluation of oxidative stress in patients with chronic renal failure. Clin Chim Acta 2002;322 (1-2):157-61.  Back to cited text no. 23
    
24.Paul JL, Sall ND, Soni T, et al. Lipid peroxidation abnormalities in hemodialyzed patients. Nephron 1993;64(1):106-9.  Back to cited text no. 24
    
25.Daschner M, Lenhartz H, Botticher D, et al. Influence of dialysis on plasma lipid peroxidation products and antioxidant levels. Kidney Int 1996; 50(4):1268-72.  Back to cited text no. 25
    
26.Zwolinska D, Grzeszczak W, Kilis-Pstrusinska K, Szprynger K, Szczepanska M. Lipid peroxidation and antioxidant enzymes in children with chronic renal failure. Pediatr Nephrol 2004;19 (8):888-92.  Back to cited text no. 26
    
27.Dubey NK, Yadav P, Dutta AK, et al. Free oxygen radicals in acute renal failure. Indian Pediatr 2000; 37(2):153-8.  Back to cited text no. 27
    
28.Durak I, Akyol O, Basesme E, Canbolat O, Kavutcu M. Reduced erythrocyte defense mechanisms against free radical toxicity in patients with chronic renal failure. Nephron 1994;66(1):76-80.  Back to cited text no. 28
    
29.Zwolinska D, Grzeszczak W, Szczepanska M, Kilis-Pstrusinska K, Szprynger K. Lipid peroxidation and antioxidant enzymes in children on maintenance dialysis. Pediatr Nephrol 2006;21 (5):705-10.  Back to cited text no. 29
    
30.Wu CC, Chen JS, Wu WM, et al. Myeloperoxidase serves as a marker of oxidative stress during single haemodialysis session using two different biocompatible dialysis membranes. Nephrol Dial Transplant 2005;20(6):1134-9.  Back to cited text no. 30
    
31.Kuo KL, Hung SC, Wei YH, Tarng DC. Intra­venous Iron Exacerbates Oxidative DNA Damage in Peripheral Blood Lymphocytes in Chronic Hemodialysis Patients. J Am Soc Nephrol 2008;19(9): 1817-26.  Back to cited text no. 31
    

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Correspondence Address:
Mohammad Hashemi
Professor of Clinical Biochemistry, Department of Clinical Biochemistry, School of Medicine, Zahedan University of Medical Sciences, Zahedan
Iran
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PMID: 22382218

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