|Year : 2010 | Volume
| Issue : 5 | Page : 852-858
|Oxidative stress in hemodialysis patients receiving intravenous iron therapy and the role of N-acetylcysteine in preventing oxidative stress
G Swarnalatha1, R Ram1, Prasad Neela1, M U.R Naidu2, KV Dakshina Murty1
1 Department of Nephrology, Nizams Institute of Medical Sciences, Hyderabad, India
2 Department of Clinical Pharmacology, Nizams Institute of Medical Sciences, Hyderabad, India
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|Date of Web Publication||31-Aug-2010|
| Abstract|| |
To determine the contribution of injectable iron administered to hemodialysis (HD) patients in causing oxidative stress and the beneficial effect of N-acetylcysteine (NAC) in reducing it, we studied in a prospective, double blinded, randomized controlled, cross over trial 14 adult HD patients who were randomized into two groups; one group received NAC in a dose of 600 mgs twice daily for 10 days prior to intravenous iron therapy and the other group received placebo. Both the groups were subjected to intravenous iron therapy, 100 mg of iron sucrose in 100 mL of normal saline given over a period of one hour. Blood samples for the markers of oxidative stress were taken before and after iron therapy. After the allowance of a week of wash out period for the effect of N-acetylcysteine we crossed over the patients to the opposite regimen. We measured the lipid peroxidation marker, malondiaaldehyde (MDA), to evaluate the oxidative stress and total anti-oxidant capacity (TAC) for the antioxidant level in addition to the highly sensitive C-reactive protein (HsCRP). Non-invasive assessment of endothelial dysfunction was measured by digital plethysmography before and after intravenous iron therapy. There was an increase of MDA (21.97 + 3.65% vs 7.06 + 3.65%) and highly sensitive C-reactive protein (HsCRP) (11.19 + 24.63% vs 13.19 + 7.7%) after iron administration both in the placebo and the NAC groups. NAC reduced the baseline acute systemic generation of oxidative stress when compared to placebo, which was statistically significant with MDA (12.76 + 4.4% vs 9.37 + 4.40%: P = 0.032) but not with HsCRP though there was a declining trend (2.85 + 22.75 % vs 8.93 + 5.19%: P = 0.112). Pre-treatment with NAC reduced the endothelial dysfunction when compared to placebo, but it was not statistically significant, except for reflection index (RI). We conclude that in our HD patients NAC reduced the oxidative stress before and after the administration of intravenous iron therapy in addition to the endothelial dysfunction induced by this treatment.
|How to cite this article:|
Swarnalatha G, Ram R, Neela P, Naidu M U, Dakshina Murty K V. Oxidative stress in hemodialysis patients receiving intravenous iron therapy and the role of N-acetylcysteine in preventing oxidative stress. Saudi J Kidney Dis Transpl 2010;21:852-8
|How to cite this URL:|
Swarnalatha G, Ram R, Neela P, Naidu M U, Dakshina Murty K V. Oxidative stress in hemodialysis patients receiving intravenous iron therapy and the role of N-acetylcysteine in preventing oxidative stress. Saudi J Kidney Dis Transpl [serial online] 2010 [cited 2020 Jan 25];21:852-8. Available from: http://www.sjkdt.org/text.asp?2010/21/5/852/68879
| Introduction|| |
Oxidative stress is defined as a disruption of the equilibrium between the oxidants and the activity of antioxidant systems. The major source of oxidants is provided by the circulating polymorph nuclear (PMN) neutrophils and mono-cytes, when activated by uremic toxins or bioincompatible dialysis membranes. ,
With respect to the markers of oxidative stress, plasma level of malondialdehyde (MDA), a byproduct of lipid peroxidation that reacts with thiobarbituric acid (TBA), TBA-reacting substances (TBARS), which is elevated in both dialyzed and nondialyzed CRF patients. ,, Advanced oxidation protein products (AOPP) are a novel oxidative stress marker formed as a result of oxidation of particularly sensitive amino acid residues, present at high levels in the plasma of uremic patients.  Dityrosine, which is generated from covalent binding links between two tyrosine residues, is a selective marker of protein attack. ,, Formation of carbonyl residues represents another early maker of protein oxidation. , It is also established that thiols such as homocyteine, are able to generate reactive oxygen species (ROS). ,
Physiologic protection against oxidants involves both enzymatic antioxidant systems that include dismutase, catalase, and glutathione peroxidase and antioxidant molecules without enzymatic activities, which are also called scavengers and comprise, such as glutathione disulphide-containing tripeptide, present in all cell types and capable of scavenging H 2 O 2 , O 2 , and OH and chlorinated oxidants in addition to alpha-tocopherol (vitamin E) and ascorbic acid (vitamin C), uric acid, glucose and mannitol. 
The administration of iron has become accepted means of prophylaxis against the development of iron deficiency in HD patients. ,,,,,, However, both iron and erythropoietin (rhEPO) have adverse effects, including potential induction of oxidative stress. ,,,, Several lines of evidence have indicated that the ROS may be involved in the uremic toxicity of patients with end-stage renal disease and decreased antioxidant capacity, and concern has been raised about the effects of long-term iron administration, which is associated with increased plasma lipid peroxidation. ,,,,,
Animal and human data demonstrate beneficial effect of N-acetylcysteine (NAC) in models of oxidative stress such as contrast induced acute renal failure  and a variety of experimentally or clinically induced ischemia reperfusion syndrome of the heart, kidney, lungs and liver. , However, its effect in oxidative stress induce by intravenous iron therapy has not been demonstrated.
We aim in our study determine the oxidative stress and the prophylactic antioxidant effect of the NAC to iron injection in HD patients.
| Material and Methods|| |
We studied a total of 14 in- and out-patients on HD program from Nizam's Institute of Medical Sciences (NIMS). The patients included in the study were more than 18 years of age, undergoing HD with iron deficiency anemia. Anemia was defined as blood hemoglobin concentration below 10g/dL. Iron deficiency was defined using National Kidney Foundation/DOQI guidelines as having serum ferritin of < 200 ng/dL and/or serum transferrin saturation < 20%. The patients excluded from the study were those who had blood transfusion within one month of the study, anemia such that erythrocyte transfusion was imminent, demonstrated iron overload- serum ferritin > 800 mg/dL, or transferrin saturation > 50%, anemia not caused by iron deficiency, underwent surgery or suffered infection within one month of before the start of the study, acute renal failure, hypersensitivity to intravenous iron, and therapy with immunosuppressive agents.
Our study is a prospective, double blinded, randomized, controlled, cross over trial. The patients were randomized into two groups; group 1 received N-acetylcysteine in a dose of 600 mg twice daily for ten days prior to intravenous iron therapy and the other group received a placebo. Both the groups were subjected to intravenous iron therapy, 100 mg of iron sucrose in 100 mL of normal saline was administered over a period of one hour in the middle of the week dialysis after HD session. In the second phase, the same patients after the allowance of a week of wash out period for the effect of N-acetylcysteine to wane off were subjected to a cross over. Accordingly, the patients who received N-acetylcycsteine in phase 1 were switched to a placebo in phase 2 and vice versa.
Base line laboratory values of hemoglobin, urea, creatinine, creatinine clearance, serum albumin, total proteins, total cholesterol, triglycerides and viral markers were measured iron, ferritin, transferrin saturation, total iron binding capacity (TIBC), malondiaaldehyde (MDA), high sensitivity C-reactive protein (hsCRP), and antioxidant level like total anti oxidant capacity (TAC) were measured before after iron infusion in each patient.
Measurement of MDA, TAC and hsCRP
MDA is a lipid peroxidation product generated in the tissues by free radical injury. The lipid peroxidation products react with thiobarbituric acid forming a pink colored adduct on boiling, which is measured at 532 nm.  The concentration of MDA is read from a standard calibration curve plotted using TEPP (1, 1, 3, 3tetra ethoxy propane). The results are presented in nano-moles per mL.
TAC in plasma was measured by decolorization assay.  The pre-formed radical monocation of 2,2-azinobis- (3-ethylenbenzothiozoline6-sulfonicacid) (ABTS) is generated by oxidation of ABTS (Sigma Chemical Co., USA) with potassium persulfate and is reduced in the presence of hydrogen-donating antioxidants. The inhibition percentage of the ABTS radical cation formation by the added serum sample at a fixed time point is quantified as the result. Trolax (6-hydroxy-2, 5, 7, 8-tetramethychroman2-carboixylic acid, Aldrich Chemical Co, UK) a water-soluble vitamin-E analogue is used as a standard. The antioxidant capacity of the serum then is expressed in molar Trolax equivalents per L.
Polystyrene particle coated with monoclonal antibodies specific to human CRP are aggregated when mixed with samples containing CRP. These aggregated scatter a beam of light passed through the sample. The intensity of the scattered light is proportional to the concenration of the relevant protein in the sample. The result is evaluated by comparison with a standard of a known concentration. The assigned value of CRP in N Rheumatology standard SL is standardized against the international reference preparation BCR-CRM 470. ,
| Statistical Analysis|| |
The statistical analysis was carried out with Sigma graph pad software, USA Version-4. All the data was presented as mean and standard deviation. Unpaired "t" test and ANOVA were used to compare two and more than two treatment groups, respectively. Paired "t" test was used for comparing post treatment group with a baseline. All the efficacy parameters are presented as a percent change from base line. The negative sign indicates a decrease in the value from baseline. P value of <0.05 was considered statistically significant
| Results|| |
There were 13 males and only one female in the study with a mean age of 44 ± 10 years. Subjects treated with intravenous iron with or with out N-acetylcysteine were similar with respect to demographic and clinical characteristics [Table 1].
There was an increase in hemoglobin, ferritin and % saturation in both the NAC and placebo groups. The percentage of the increase in the level of hemoglobin and % saturation was higher in the NAC than the placebo group, but was not statistically significant. There was a fall in the level of iron and TIBC in the NAC group [Table 2].
|Table 2 :Mean percentage change of laboratory parameter before and after iron sucrose therapy in two|
groups - iron + placebo or N-acetylcysteine (NAC).
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There was no change in the levels of the oxidative stress markers in the patients after administration of placebo; however there was a rise in the oxidative markers levels after parenteral iron administration in this group of patients [Table 3]. However, there was no change of the total antioxidant capacity either with placebo or parenteral iron therapy.
|Table 3 :Placebo premedicated group: Means of the levels of the oxidative stress parameters.|
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Among the oxidative stress markers, MDA demonstrated a statistically significant decrease after NAC and parenteral iron therapy [Table 4]. Though HsCRP showed a declining trend in its levels, which was not statistically significant, after the administration of NAC and a marginal elevation after parenteral iron compared to a placebo. Similarly, the TAC did not change significantly during both phases with and without injection of iron [Table 5].
|Table 4 :N-Acetyl cysteine (NAC) premedicated group: Means of the levels of the oxidative stress|
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|Table 5 :Percentage change in biochemical parameters in placebo vs N-acetylcysteine (NAC).|
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| Discussion|| |
In our study, iron and transferrin saturation levels were measured along with the oxidative stress markers 15 minutes after iron sucrose administration. There was increase in transferrin saturation and a decrease in the iron levels in the placebo and the NAC groups. These data support the notion that oxidative stress occur very early after iron sucrose administration, at the time point when substantial transferrin is available to bind to free iron. Moreover, there was an increase in the oxidative stress markers MDA and HsCRP after parenteral iron sucrose administration both in the placebo and the NAC groups. NAC had reduced the baseline acute systemic generation of oxidative stress when compared to placebo, which was statistically significant with MDA but not with HsCRP though there was a declining trend.
In several previous studies, ,,, uremic patients on hemodialysis were found to be exposed to high levels of ROS. Several studies have demonstrated that intravenous administration of iron sucrose in dialysis patients results in an increase in markers of lipid per oxidation , and advanced oxidative protein products. , Paik-Seong Lim  suggested that the elevated baseline ferritin levels and intravenous iron infusion exaggerate the increased oxidative stress in uremic patients on HD. Consistent with this observation, the HD patients in our study revealed a significant increase in lipid peroxidation marker MDA and decrease in total antioxidant capacity.
An imbalance between the generation and removal of ROS and free radicals may be a contributory factor for the HD-related complications. Several lines of evidence have indicated that the ROS may be involved in the uremic toxicity of patients with end-stage renal disease, and concern has been raised about the effects of long-term iron administration, which is associated with increased plasma lipid peroxidation.
Animal and human data demonstrate beneficial effect of N-acetylcysteine (NAC) in models of oxidative stress such as contrast induced acute renal failure. There are only two studies so far, which tried to know the beneficial effect of NAC in the oxidative stress induced by parenteral iron therapy in HD patients. ,
The time course of transferrin saturation has been reported by Parkkinen et al  In HD patients. They reported that transferrin saturation was between 80% and 90% at 3˝ hours following iron sucrose administration, and it returned to normal in 48 hours. Free iron was not increased unless transferrin saturation was 80% or more. Herrera et al  measured plasma MDA levels in HD patients 1 hour following iron sucrose administration and were significantly increased. Roob et al  found plasma MDA levels to peak within 30 minutes after injection of iron sucrose, and Tovbin et al  found advanced protein oxidative products to be increased within 3-5 minutes after the administration of iron sucrose.
Agarwal et al  demonstrated that there was no further increase in oxidative stress or renal damage beyond the first 30 minutes of injection of iron, despite progressive uptake of iron by transferrin. The results of the antioxidant (NAC) administration was mixed, whereas NAC reduced acute generation of oxidative stress and delayed renal generation of oxidative stress, there was no improvement in either proteinuria or enzymuria. Another pilot study has demonstrated that intravenous ferric gluconate infusion resulted in a marked increase in transferrin saturation and a significant increase in plasma MDA levels. Urinary MDA levels also increased at the higher dose of iron. There was no evidence of acute renal injury as assessed by albuminuria, proteinuria, and enzymuria. It seems that pre-treatment with NAC had no effect on oxidative stress or the above urinary parameters. 
We conclude that the HD patients in our study displayed an increase in the MDA and hsCRP levels after parenteral iron sucrose administration in both the placebo and the NAC groups. NAC reduced the baseline acute systemic generation of oxidative stress when compared to placebo and this reduced oxidative stress was maintained even after administration of intravenous iron therapy.
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Department of Nephrology, Nizams Institute of Medical Sciences, Hyderabad
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]
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