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World Journal of Nephrology & Urology

Original Article

Volume 4, Number 2, June 2015, pages 207-212

Determination of Prooxidant and Antioxidant Balance, Clinical Parameters and Nutrient Intakes in Hemodialysis Patients

The importance of oxidative stress in the pathogenesis of many diseases, including inflammatory, chronic and progressive diseases has been recently emphasized [1-4]. Oxidative stress can damage cellular components, such as lipids, proteins or DNA [2, 5]. There is evidence that suggests the increased state of oxidative stress in uremic patients on hemodialysis (HD) treatment may be related to disturbance in antioxidant mechanisms of free radicals detoxification [3, 6, 7]. The antioxidant defense system includes both endogenous and exogenous (food derived) antioxidants to protect cells against toxic effects [3, 8]. Dietary antioxidant micronutrients especially in fruits and vegetables may revive each other in an antioxidant system [3, 8]. Chronic stress in uremic patients on HD may be associated with disturbance in antioxidant defense mechanisms resulting from dietary restriction [3, 9, 10] or the direct effect of inflammation and macrophage activation caused by reactive oxygen groups during dialysis [11]. Some studies have indicated that increased oxidative stress in dialysis patients is linked to endothelial dysfunction, increased acute phase of inflammation, hyperlipidemia, and cardiovascular atherosclerotic diseases, which are the leading causes of morbidity and mortality in the HD patients [12-14]. A correct balance between oxidants and antioxidants may contribute to maintain the biological functions and reduce the risk of developing cardiovascular disease in patients with end-stage renal disease (ESRD) [3]. For the evaluation of the prooxidant-antioxidant balance (PAB), the measurement of both the oxidative damage markers and antioxidant levels is necessary. Despite the role of oxidative stress in patients on dialysis determination of PAB yet as a routine clinical laboratory methods are known. Many methods, separately measure the total antioxidant capacity and prooxidant, which is a complex and costly method [1, 2]. In this study, the oxidant and antioxidant capacity in an evaluation was measured. We also evaluated the relationship between PAB and lipid profile, C-reactive protein (CRP), and nutrient intakes in patients undergoing dialysis.

The study protocol was confirmed by the Ethics Committee of Zahedan University of Medical Sciences (2014-7-6862). Informed consent was obtained from all subjects participating in the study.

Thirty-five patients (mean age 43.1 ± 13.3 years, range 19 - 70 years) referring to Dialysis Section of Imam Ali Hospital in Zahedan who were dialyzed three times a week for at least 8 months with Kt/v ≥ 1.3, were enrolled in the study. The causes of ESRD included blood pressure (n = 16), kidney stone (n = 4), lupus erythematosus (n = 1), polycystic kidney (n = 4), glomerulonephritis (n = 4), and unknown (n = 6).

Thirty-five healthy volunteers (mean age 38.6 ± 12.5 years, range 19 - 69 years) free from any signs of kidney disease who were matched with the patients in terms of age and sex were selected as control group. Inclusion criteria for both two groups included age over 18 years, not using steroids and lipid lowering drugs, and lack of the active infection and inflammatory disease, liver disease, thyroid, cancer, as well as lack of hospitalization till 3 months before entering to study.

Dry weight after dialysis and height were measured. Body mass index (BMI) was evaluated based on the calculation of dry weight (kg)/height² (m²) [15]. Food intake was assessed by a 24-h dietary recall questionnaire for at least 2 days. All of consumed food items were recorded on the two previous days. In order to avoid eventual differences between working and not-working days, the mean values of dietary intakes were measured on one weekday and one weekend. The questionnaire was filled in a personal interview that was conducted by trained investigators and a nutritionist. The nutrient values were calculated by CompEat nutritional analysis program [16].

The blood samples were obtained from healthy subjects and before each infusion of heparin from each patient after a 12-h fast. Routine biochemical parameters including blood urea nitrogen (BUN), uric acid, creatinine, cholesterol, triglyceride, low density lipoprotein-cholesterol (LDL-C), high density lipoprotein-cholesterol (HDL-C), serum albumin and CRP concentrations were measured using the standard techniques. The serum aliquots were immediately separated and frozen at -70 °C until analysis.

The PAB was measured by a modified method by Hamidi Alamdari et al [1, 17]. PAB values were assayed simultaneously, by photometric method, using 3,3ʹ,5,5ʹ-tetramethylbenzidine (TMB) in an ELISA reader at 450 nm (with a reference 570 nm or 620 nm wavelength). This method is based on two different reactions; in the enzymatic reaction the chromogen TMB is oxidized to a color cation by peroxides, and in the chemical reaction the TMB cation is reduced to a colorless compound by antioxidants. PAB is calibrated using a mixture of hydrogen peroxide and uric acid. The values are expressed in an arbitrary HK unit, which is as hydrogen peroxide percentage in the calibration mixture and demonstrates the oxidative stress index [1, 2, 17].

The results were expressed as mean ± SD and mean ± SEM as appropriate. Comparisons between two groups were analyzed by t-test or Mann-Whitney U-test. Pearson and Spearman correlation coefficients are used for assessment of parametric and non-parametric correlations. All statistical analysis was performed using SPSS software (version 18 for windows, Chicago, USA). A P value < 0.05 was considered significant.

Demographic and clinical characteristics of participants are shown in Table 1. A significant difference was observed in BMI between HD patients and control subjects (P < 0.001).There was no significant difference in age and blood pressure levels between two groups.

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Table 1. Demographic and Clinical Characteristics of Studied Groups

Biochemical parameters are demonstrated in Table 2. The levels of BUN, serum creatinine (P < 0.0001), uric acid (P < 0.01) and CRP (P < 0.01) were markedly increased, and serum levels of albumin (P < 0.01) were significantly reduced in HD patients when compared to healthy controls. There was not any significant difference between two groups in other variables.

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Table 2. Biochemical Parameters in the Studied Groups

The PAB data analysis showed that PAB values in HD patients (88.2 ± 33) were significantly higher compared with control group (68.4 ± 23.3) (P < 0.01) (Fig. 1).

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Figure 1. Serum levels of PAB in the studied groups.

As shown in Table 3 [18, 19], intake of antioxidant vitamins including vitamins A, C and E (P < 0.01), and minerals including calcium (P < 0.01), phosphorous, potassium, zinc (P < 0.0001) and iron (P < 0.05) was significantly lower than control group.

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Table 3. Nutrient Intakes in the Studied Groups [18, 19]

A significant positive correlation was demonstrated between PAB value and serum levels of creatinine (r = 0.38, P < 0.001), BUN (r = 0.42, P < 0.0001) and CRP (r = 0.34, P < 0.001) in HD patients (Fig. 2). An inverse correlation between PAB value and intake of vitamins A (r = -0.28, P < 0.05) and C (r = -0.31, P < 0.01) was found (Fig. 3).

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Figure 2. Correlation between PAB values and serum creatinine, BUN and CRP in HD patients.

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Figure 3. Correlation between PAB and vitamins A and C in HD patients.

No statistically significant relationship was found between PAB value with dialysis duration, age, BMI, lipid profile and other micronutrients.

To date, many methods have been performed to assay separately the prooxidant and antioxidant capacities, which are costly and complex. PAB assay using simple and rapid tests can help to identify patients at risk and prevent or reduce of complications resulting from oxidative stress. In our previous studies [6, 20], we indicated a defective antioxidant defense during the process of HD, due to the reduction of antioxidants and elevated lipid peroxidation in patients on HD in agreement with the other studies [7, 9, 12-14, 21]. However, a study has shown decrease in the serum levels of MDA compared to control group [22]. While in some studies, antioxidants activity [23] and lipid peroxidation have no change [24].

In the present study, we assayed simultaneously PAB assay values in HD patients. The findings demonstrated that PAB values were markedly higher in HD patients than healthy controls. There is evidence that both inflammation markers including CRP and tumor necrosis factor-α (TNF-α), and oxidative stress elevate the risk of chronic disease in uremic patients [9]. A significant positive correlation between PAB and serum levels of creatinine, BUN and CRP suggests that increasing the toxic nitrogen substances and inflammation in uremic patients undergoing HD can contribute to the oxidative stress, which plays an important role in the endothelial tissue injury and high risk of atherogenesis [3, 6, 12].

Moreover, the evidence suggests that dialysis therapy is also one of the causes of increased oxidative stress in uremic patients rather than the disease itself [25]. It could be attributed to the direct contact of blood with dialysis membrane [11], and/or the loss of the hydro-soluble vitamins through the dialysis membrane, which may contribute markedly to a reduced antioxidant power of plasma [3, 8, 26]. On the other hand, analysis of dietary intakes in our study showed that the dialysis patients consumed lower nutrients compared to controls, in particular intake of natural sources of antioxidant vitamins including A, E and C was significantly lower than controls as reported in our previous report [3]. A significant negative correlation between the PAB values and vitamins A and C intake was also found. It may be partly due to the inadequate intake of natural vitamins from fresh fruits and vegetables, nuts and seeds due to restriction of potassium and phosphate in these patients [3, 13]. Thus, efforts should be made to ensure that these patients receive optimum antioxidant vitamins. It seems that antioxidant therapy as a way to treat these patients should be considered.

The findings show that HD patients are exposed to oxidative stress. Since little information exits on the PAB assay in the HD patients, the high values of PAB demonstrated in our study need to be confirmed by further studies.

Acknowledgement

We are grateful to Mrs Shadi Amini, staff and nurses of Dialysis Section in Zahedan Imam Ali Hospital for their kind cooperation. We thank the patients and healthy subjects who willingly participated in the study.

Funding Source

This work was supported by the Research Deputy of Zahedan University of Medical Sciences, Zahedan, Iran, No. 6862.

Conflict of Interest

None.

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World Journal of Nephrology and Urology is published by Elmer Press Inc.