Objectives: This study was designed to determine the effects of zinc supplementation on oxidative stress in hemodialysis (HD) patients through evaluating total antioxidant capacity (TAC), whole blood glutathione peroxidase (GSH) level, superoxide dismutase (SOD) activity and malondialdehyde (MDA) level.
Design: Double-blinded randomized controlled trial.
Setting: From Oct 2006 to Dec 2007 in Tabriz Imam Khomeini Hospital.
Subjects: Sixty five HD patients were randomly enrolled into two groups.
Intervention: Patients received placebo in group A and zinc (100 mg/day) in group B for 2 months. After a washout period for 2 months, the groups were crossed over and the study was continued for additional 2 months.
Main outcome measures: Serum zinc concentration was measured using atomic absorption spectrophotometry. TAC, GSH level and SOD activity were determined by commercial ELISA kits. MDA level was measured using thiobarbituric acid method.
Results: The levels of serum zinc, TAC, GSH (p<0.00l for all), and SOD activity (p<0.00l for the group A and p=0.003 for the group B) significantly increased after zinc supplementation, while the serum level of MDA decreased after the same period (p=0.003 for the group A and p<0.00l for the group B).
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Conclusions: Zinc supplementation for two months improved the serum levels of zinc, antioxidant status, and lipid peroxidation in HD patients.
Keywords: Zinc, hemodialysis, oxidative stress, antioxidant capacity, superoxide dismutase, glutathione peroxidase, malondialdehyde, lipid peroxidation.
Zinc is an essential trace element which is required for function of more than 300 enzymes and many other biological factors including hormones, cytokines, peptides and biomembranes1. In addition, zinc plays a critical role in gene expression, protein synthesis, bone formation, growth, reproduction, wound healing, transport process, immune function and behavioral responses1.
Zinc deficiency is a worldwide problem and it is estimated that 25% of the world's population is at risk of zinc deficiency2. Lower levels of zinc were specifically reported in hemodialysis (HD) patients. Deficiency of zinc has been associated with most of uremic symptoms, including anorexia, hypogeusia, hyposmia, and disorders of the sexual and immunological functions. Studies have shown that zinc supplementation increases serum level of zinc
in HD patients 5 and improves uremic symptoms .
Furthermore, the antioxidant properties of zinc have been clearly demonstrated and, for the most part, appear to be independent of zinc metalloenzyme activity8-12. The antioxidative action of zinc can be generally divided into acute and chronic effects. The acute effect involves two mechanisms; protection of protein sulfhydryls or reduction of OH formation from H2O2 through the antagonism of redox-active and transition metals including iron and copper13. The chronic effect involves exposure of an organism to zinc on a long-term basis, resulting in induction of some other substances that are ultimate antioxidant such as metallothioneins13. So, zinc deficiency in uremic and HD patients may result in increased oxidative stress which is implicated in a number of pathologic processes like cardiovascular disorders.
Based on the antioxidative properties of zinc and its deficiency in HD patients, the present study aimed to investigate the effects of zinc supplementation on oxidative and antioxidant systems in HD patients through evaluating total antioxidant capacity (TAC), whole blood glutathione peroxidase (GSH) level, superoxide dismutase (SOD) activity and malondialdehyde (MDA) level.
We conducted a double-blind randomized controlled trial among 73 patients on maintenance HD from Oct 2006 to Dec 2007 in Tabriz Imam Khomeini Hospital. The protocol of the study compiled with the Declaration of Helsinki, and was approved by the ethics committee of Tabriz University of Medical Sciences (TUMS). An informed consent was obtained from each participant.Exclusion criteria included HD for less than 6 months, any sign of gastrointestinal disorders, smoking, being a candidate for kidney transplantation, lactation, pregnancy, consumption of glucocorticoids, antibiotics, estrogens and contraceptives (in females). Eight patients were excluded from the study; three died because of cardiac arrest, 2 received antibiotics and 3 because of zinc intolerance (nausea).
Sixty five patients (41 males and 24 females) with mean age of 52.77±12.68 years completed the study. The etiology of end-stage renal disease (ESRD) was chronic glomerulonephritis (n = 16), diabetic nephropathy (n =9), polycystic kidney disease (n = 5), chronic interstitial nephropathy (n = 5), urological problems (n = 4) and unknown (n = 26) respectively. All of the patients were on HD 3 times per week (each time for 4 hours). The dialysis schedule was not modified during the study. The dialysis was performed using cellulosic membrane to maintain a minimum Kt/V urea index of 1.2 per session.
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Patients were randomized into two groups: group A including 35 patients (22 males and 13 females), and group B with 30 patients (19 males and 11 females). Group A received 100 mg placebo (as two cornstarch capsules) per day for 2 months. At the same time, Group B received 100 mg/day elemental zinc (as two 220 mg zinc sulfates pentahydrate and cornstarch capsules, ALHAVI Co., Tehran, Iran). The placebo and zinc were withdrawn during the next 2 months and then the study was continued as a crossover design for another 2 months (group A and B received zinc and placebo, respectively).
Before HD sessions, fasting venous blood samples were obtained prior to start of zinc and placebo, at days 0, +60, after withdrawing of intervention, and at days +120 and +180. Each sample was divided into a heparinized tube and a microtube containing disodium EDTA (1.5 mg/ml).
Body mass index (BMI) was estimated at the days 0, +60, +120, and +180 using standard formula [weight (Kg)/height (m2)]. Each patient's diet was recorded on a non-dialysis day, and dietary zinc was calculated using Food Processor IT Software (ESHA Research, Salem, OR).
Serum zinc concentration was measured by atomic absorption spectrophotometry (AAS) after a 1:5 dilution in double deionized water at 213.9 nm (CTA 2000, AAS, ChemTech Analytical Co., Bedford, UK). The reference values and cut off point for zinc deficiency in Iranian adult population and HD patients were determined by the previous studies.
TAC in serum samples were measured by spectrophotometry method (CECIL 8000, CECIL instrument, Cambridge, UK) using RANDOX TAS kit (RANDOX Lab. Ltd., Antrim, UK).
SOD activity was determined according to the method of Sun et al.18 using RANDOX RANSOD kit (RANDOX Lab. Ltd, Antrim, UK). The principle of this method was based on the inhibition of 2-(4- iodophenyl)-5-phenyltetrazolium chloride (INT) reduction by the Xanthine-Xanthine oxidase system as a superoxide generator. Activity was assessed in the ethanol phase of the RBC extract sample after 1.0 ml ethanol/chloroform mixture (5/3, v/v) was added to the same volume of RBC extract and centrifuged. A unit of SOD expressed as the enzyme amount causing 50% inhibition in INT reduction rate. Results were expressed as U/g hemoglobin (Hb).
GSH level was assayed using glutathione ELISA assay kit (Cayman Chemical Company, Michigan, USA) at 405 nm. The serum samples were deproteinated by metaphosphoric acid, and then triethanolamine was added, according to the kit's methodology. In this method the sulfhydryl group of glutathione (GSH) reacts with 5, 5i -dithiobis -2-nitrobenzoic acid (DTNB) and produces a yellow colored 5-thio-2-nitrobenzoic acid (TNB). The mixed disulfide, GSTNB (between GSH and TNB) which is concomitantly produced, is reduced by glutathione reductase to recycle the GSH and produce more TNB. The rate of TNB production is directly proportional to this recycling reaction which is in turn directly proportional to the concentration of GSH in the sample. Measuring the absorbance of TNB at 405 or 414 nm provides an accurate estimation of GSH level in the sample. By applying this kit, both GSH and the disulfide dimer (GSSG) are measured.
Serum level of lipid peroxidation was studied by evaluating MDA level as thiobarbituric acid reactive substances. Spectrofluorometric method was applied by JASCO FP-750 spectrofluorometer (Jasco International Co. Ltd., Tokyo, Japan) using tetraethoxypropane with excitation and emission spectra at 515 and 553 nm, respectively19. Hemoglobin (Hb) level was also measured by SYSMEX-KX 21 cell counter, (Sysmex Corporation of Japan, Kobe, Japan).
Statistical analyses were performed by SPSS version 13.0 for windows software package (SPSS Ins., Chicago, USA). Results are presented as mean ± standard deviation. Statistical difference between the groups, significance of changes following the periods of the study and correlations were examined using Independent Sample t-test, Paired t-test and Pearson correlation, respectively. The results were considered significant when the p value was <0.05.
The dietary zinc intake, BMI, and age were not significantly different between two groups at the beginning of the study (p=0.27, p=0.55 and p=0.34, respectively) and at the end of the study (p=0.3 6, p=0.63 and p=0.34, respectively). Twenty two of 35 patients in the group A and 21 of 30 patients in the group B were zinc deficient (lower than 80 µg/dl)17. The baseline levels of serum albumin were 3.6±0.8 and 3.6±0.5 g/dl and the baseline levels of Hb were also 9.2±2.2 and 9.0±2.5 g/dl in the group A and B, respectively.
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Table 1 shows the mean values of the serum zinc, TAC, GSH, SOD, MDA in the group A during the placebo (from day 0 to day +60) and zinc supplementation (from day +120 to day +180) periods. There were not significant differences in the serum zinc concentration, TAC and GSH levels, and SOD activity during the placebo period, while the serum MDA level was significantly increased. After the period of zinc supplementation, beside a significant decrease in the serum level of MDA, the TAC, GSH, serum zinc concentration, and the SOD activity were significantly increased.
Table 2 presents the mean values of the serum zinc, TAC, GSH, SOD, MDA in the group B during the zinc supplementation (form day 0 to day +60) and the placebo (from day +120 to day +180) periods. There were a significant decrease in the serum level of MDA and a significant increase in the serum concentration of zinc, the levels of TAC, GSH and the SOD activity after the zinc supplementation. In contrast, a significant reduction was found in the mean concentration of serum zinc and GSH level after the placebo trial period, while there were no significant differences in the serum MDA and TAC levels and the SOD activity.
After the period of zinc supplementation in both groups, serum concentration of zinc increased above the cut off value (80 µg/dl) and the serum zinc levels of all patients were normalized.
In the present study, zinc supplementation improved the serum concentration of zinc that has been previously shown by other authors. Increased concentration of serum zinc is consistent with Chevalier et al.5 observation that supplied the HD patients with different dosage of zinc for 90 days. Also similar results have been shown following zinc supplementation in patients with type 2 diabetes mellitus for 3-6 months20 and in normal individuals for 2-4 weeks8. Low dietary zinc intake, serum zinc concentration lower than 80 µg/dl, and its improvement by zinc supplementation, all indicated the zinc deficiency in the patients on HD. It is naturally expected that zinc deficiency is compensated through dietary zinc supplementation, as the results of above studies.
During the placebo period of the present investigation, the serum MDA level increased, while the serum GSH level decreased. Our findings are in agreement to the results of previous studies reporting higher levels of MDA and lower levels of GSH in the patients on HD or with CRF22-26. Moreover, Ozden et al.11 have reported that the serum levels of MDA increased in HD patients when erythrocyte glutathione level decreased.
In the present study, the SOD activity decreased following the placebo period, but did not reach statistical significant level. Similar reduction in the SOD activity has been shown in HD patients, patients with CRF and even in rats with zinc deficiency. Pawlak et al.28 implied that the activity of plasma copper-zinc SOD may be a simple and sensitive marker of oxidative stress in ESRD. However, Ceballos-Picot et al. have reported that the erythrocyte copper-Zinc SOD activity was not changed in uremic patients29.
The increased level of MDA in the group A along with insignificant changes in the TAC and GSH levels and the SOD activity during the placebo period, signify aggravation of oxidative stress without any change in antioxidant status. Interestingly, the MDA level in the group B have not changed following zinc withdrawal, which implies that lipid peroxidation as an indicator of oxidative stress did not aggravated. However, the decreased level of GSH in the group B during the placebo period was not reflected in the TAC level. This phenomenon may be explained by increasing the other antioxidants (which were not measured during this study) to compensate the decreased levels of GSH.
Our results showed that zinc supplementation leads to the decreased level of MDA, and the increased levels of TAC and GSH, and the SOD activity. This would mean that zinc supplementation improves antioxidant status and decreases lipid peroxidation in the HD patients. The increased levels of GSH and SOD activity possibly lead to increased TAC level and improvement of antioxidant status. Finally, improved antioxidant status resulted in decreased oxidative stress and lipid peroxidation. Decreased level of MDA in diabetic patients20 and increased GSH level and SOD activity in protein-deficient rats30 have been reported by zinc administration that is in consistent with the our results.
In an experimental study on zinc-deficient rats, zinc supplementation led to lower MDA level, higher GSH level and improved SOD activity23. The ability of zinc to improve oxidative stress may be due to its acute and/or chronic antioxidative effects. The beneficial effects of long-term administration of zinc can be linked to the induction of some other substance that serves as the ultimate antioxidant such as metallothioneins. Some authors have hypothesized that the metallothioneins are a link between cellular zinc and the redox state of the cell31. Induction of renal metallothionein in proximal tubular cells has been proposed as an endogenous antioxidant effect of zinc which may prevent oxidative stress32. Gene expression study has clearly shown that zinc leads to higher metallothionein synthesis and pretreatment by zinc inhibits cytotoxicity and apoptosis33. Collectively, it is suggested that zinc might act as a physiological signal which is mediating the response to oxidative stress.
One of the acute antioxidant effects of zinc is stabilization of sulfhydryl groups by direct binding of zinc to the sulfhydryl groups, steric hindrance and a conformational change from binding to some other sites on the proteins. In this study, a portion of improved antioxidant status is related to the GSH level and SOD activity. Since, zinc has been supplemented for a short time period in the present study, majority of its effect may be acute. It has been shown that the depletion of intracellular glutathione triggers a progress of collagen synthesis in zinc deficient-hepatic satellite cells and this depletion may be induced by the stimulation of cellular production of H2O234.
In the present study, BMI values were in the normal range and not changed following the study periods, whereas there was a significant difference in BMI between case and control groupsin some of previous studies. Furthermore, crossover design of the present study helped to control possible confounding factors. However, the possible limitation of our study is that the erythrocyte zinc level was not assessed.
In conclusion, the results demonstrate lower (less than 80 µg/dl) basal serum concentration of zinc in the HD patients and suggest monitoring of the serum zinc concentration in this population. Also, zinc supplementation improved the serum zinc concentration and increased potential to defend against oxidative stress, which means increased serum TAC and GSH levels and SOD activity, and also decreased lipid peroxidation (MDA level).
Uremic patients or patients undergoing HD may suffer from zinc deficiency and therefore, monitoring of the serum zinc is suggested in this population. Zinc supplementation, by increasing serum zinc concentration, improves antioxidant status and protects HD patients against oxidative stress and lipid peroxidation.
The Present study was supported by the Drug Applied Research Center of Tabriz University of Medical Sciences. The authors would like to thank Dr. Sona Ghorashi for her help.