1 Division of Nephrology, Department of Medicine, Loyola University Stritch School of Medicine and Edward Hines Jr VA Medical Center, Hines, IL and 2 Indiana University School of Medicine and Richard L. Roudebush VA Medical Center, Indianapolis, IN, USA
Correspondence and offprint requests to: David J. Leehey, MD, VA Hines, 111L, Hines, IL 60141, USA. Email: dleehey{at}lumc.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods. To determine if i.v. iron causes oxidative stress [as assessed by plasma and urine malondialdehye (MDA)] and/or renal injury (as assessed by urinary albumin, total protein and enzymuria), we conducted a prospective, four-way randomized crossover, blinded end-point trial in eight patients with CKD. Two widely used doses of sodium ferric gluconate (125 mg infused over 1 h and 250 mg infused over 2 h) were given with or without the antioxidant N-acetylcysteine (NAC), resulting in four treatment doseantioxidant/placebo combinations in each patient. Transferrin saturation was measured with urea polyacrylamide gel electrophoresis, MDA by high performance liquid chromatography, and albuminuria and proteinuria by standard clinical methods. Enzymuria was assessed by measurement of N-acetyl-ß--glucosaminidase (NAG) excretion by colorimetric assay.
Results. I.v. ferric gluconate infusion at both doses 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 or enzymuria. Pre-treatment with NAC had no effect on oxidative stress or the above urinary parameters.
Conclusions. I.v. ferric gluconate caused oxidative stress (as reflected by increased MDA), but this was not associated with biochemical manifestations of acute renal injury.
Keywords: anaemia; chronic kidney failure; iron; malondialdehyde; oxidative stress; randomized controlled trial
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recently, i.v. administration of iron sucrose at FDA-approved doses in patients with CKD not yet on dialysis was reported to result in oxidative stress and transient renal injury [14]. The present study was designed to determine if an alternative i.v. iron preparation, sodium ferric gluconate, is also associated with oxidative stress and/or renal injury in CKD patients. In addition, we wished to determine the effect of pre-treatment with the antioxidant N-acetylcysteine (NAC) in these patients.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Study design
Subjects who satisfied the inclusion and exclusion criteria and signed the consent form were considered to be eligible for the study. All subjects were patients at the Hines VA Hospital and received i.v. iron treatment at the Hines out-patient centre. After obtaining baseline blood samples for measurement of serum iron, total iron binding capacity (TIBC), ferritin, urea nitrogen, creatinine, albumin, lipid profile and a complete blood count, subjects received sodium ferric gluconate (Ferrlecit, Watson Laboratories) 250 mg over 2 h as the initial dose (this is the standard protocol used in the hospital out-patient centre). After the initial dose, they received treatment with Ferrlecit at two different doses (125 mg, the FDA-approved dose, or 250 mg) with or without pre-treatment with NAC. These four iron treatments were administered in random order to each subject at intervals of 1 week. Prior to each iron infusion, patients were given four 15 ml vials containing either NAC 600 mg or sterile water and were instructed to take one vial by mouth every 12 h starting on the morning prior to their scheduled infusion and ending on the evening of the infusion. The patient and study team were blinded as to the contents of the vials.
Thus all subjects received all treatments (in random order) as follows: treatment 1 = Ferrlecit 125 mg i.v. over 1 h plus placebo (sterile water orally given every 12 h beginning 1 day prior to infusion and continuing on the day of infusion for a total of four doses); treatment 2: Ferrlecit 125 mg i.v. over 1 h plus NAC (Mucomyst, Apothecon, Inc., Princeton, NJ) 600 mg orally given every 12 h beginning 1 day prior to infusion and continuing on the day of infusion for a total of four doses; treatment 3: Ferrlecit 250 mg i.v. over 2 h plus placebo as in treatment 1; and treatment 4: Ferrlecit 250 mg i.v. over 2 h plus NAC given as in treatment 2.
During the iron infusions, patients had their vital signs monitored by the nursing staff every 30 min. Each patient was given a monitoring form on which they recorded both the nature and severity of any adverse events. For subjective complaints such as pruritus, headache, nausea, etc. that might occur during the infusion, a symptom severity scaled from 1 to 10 was used by the patient to quantify the level of discomfort and to monitor this discomfort throughout the infusion.
Immediately prior to each of the four treatment infusions, blood and urine samples were obtained for measurement of TSAT, plasma and urine MDA, urinary albumin, total protein and N-acetyl-ß--glucosaminidase (NAG). Blood and urine samples were repeated immediately after the infusion was completed, with the arm not utilized for iron infusion subjected to venipuncture. No subject needed to void urine during the iron infusion, and thus the post-infusion urine sample reflected all urine formed during the time of infusion. Biochemical measurements were performed at the Roudebush VA Medical Center, Indianapolis, IN, by personnel blinded as to the order of the treatment infusions.
Electrophoretic separation of transferrins
The iron forms of transferrin were separated using a TBE-urea polyacrylamide gel (6% acrylamide gels with 6 M urea) according to Williams et al. [17]. This method separates transferrin into the apotransferrin, monoferric and the diferric forms, according to their electrophoretic mobilities after all serum proteins except ß - and -globulins are precipated by 2% 6,9-diamino-2-ethoxyacridine lactate monohydrate (Sigma, St Louis, MO) [18]. Electrophoresis was performed using a Criterion mini-gel system (Biorad, Hercules, CA). Protein bands were visualized through staining with GelCode Blue stain reagent (Pierce, Rockford, IL). Densitometric analysis was performed with a Gel Logic 100 apparatus and 1D image analysis software (Kodak, Rochester, NY). The percent TSAT was calculated as follows: TSAT (%) = [diferric transferrin + (1/2 x monoferric transferrin(s))]/[apotransferrin + monoferric transferrin(s) + diferric transferrin].
Plasma and urinary malondialdehyde assay
MDA, a lipid peroxide product, is formed by ß-scission of peroxidized polyunsaturated fatty acids and was measured by derivatization with thiobarbituric acid as reported previously [19].
Urine albumin, protein, creatinine and NAG determination
Urinary albumin was determined by immunoturbidemetry and urinary protein by the pyrogallol red-molybdate method. Urine creatinine concentration was determined using an end-point spectrophotometric assay with an alkaline picrate solution (Sigma Diagnostics, St Louis, MO). Urinary NAG was measured by colorimetric assay (Roche Diagnostics Corporation, Indianapolis, IN). Other laboratory assays were performed using standard methods in the hospital laboratory.
Statistical analysis
Comparisons between pre-infusion and post-infusion values for each of the four treatments were made using paired t-test. Comparisons of pre-infusion and post-infusion values among the four treatment groups were made using analysis of variance (ANOVA). All P-values are two sided and significance set at <0.05. Data are expressed as mean±SD.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Parenteral iron preparations are clearly effective, but concerns have been raised regarding adverse events and potentially long-term toxicity. In the case of iron dextran, there is a small but serious risk of anaphylaxis, with >30 deaths reported from use of this agent [22]. Although this potentially fatal adverse effect does not appear to be a problem with newer preparations, such as ferric gluconate and iron sucrose, generation of oxidative stress with i.v. iron preparations may be a universal phenomenon. Increased in vitro superoxide production after infusion of iron sucrose has been noted in normal volunteers [23]. Herrera et al. measured plasma MDA in haemodialysis patients 1 h following iron sucrose administration and found levels to be significantly increased [24]. Lim et al. reported that iron sucrose resulted in significantly elevated levels of plasma lipid peroxide products [25]. Roob et al. found plasma MDA to peak within 30 min after iron sucrose [13]. Tovbin et al. found advanced protein oxidation products to be increased within 35 min after iron sucrose [26]. Most recently, Agarwal et al. found that iron sucrose resulted in oxidative stress (elevated plasma and urinary MDA) [14]. Iron dextran was reported to increase the esterified fraction of F2 isoprostanes 60 min after administration [12]. Oxidative stress after administration of ferric gluconate has been reported in one previous study in haemodialysis patients [27]. An increase in carbonyl stress (carbonylated fibrinogen) was noted with the 125 mg but not with the 62.5 mg dose.
In vitro studies by Zager and associates in mouse proximal tubular segments, murine renal cortical homogenates and cultured human proximal tubular cells have demonstrated oxidative stress, as determined by lipid peroxidation after exposure to iron dextran, iron gluconate, iron sucrose and an iron oligosaccharide [6]. However, iron sucrose was far more cytotoxic than the other compounds tested, as assessed by lactate dehydrogenase release, although some evidence of cellular toxicity was seen at the higher doses of iron gluconate. Cytotoxicity was protected by reduced glutathione (GSH) independent of its antioxidant effect, as MDA generation was not altered. Thus, it is apparent that oxidative stress and cell injury after iron exposure can be dissociated, at least in vitro. In a recent publication from the same group [7] involving in vivo studies in mice, similar amounts of lipid peroxidation (MDA accumulation) were noted in renal cortex with both iron gluconate and iron sucrose, yet histological changes were reported to be more evident with iron sucrose. In these studies, glomerular iron accumulation was observed predominantly in the mesangial and endothelial cells associated with endothelial cell swelling. These renal changes were not seen with iron dextran or the iron oligosaccharide.
Of note, in the study by Agarwal and associates [14], administration of iron sucrose at standard clinical doses (100 mg given i.v. over 5 min) resulted in both oxidative stress and evidence of renal injury (increased proteinuria and enzymuria), as might be predicted by the studies of Zager et al. [6,7]. Likewise, the dissociation between oxidative stress and renal cell injury with ferric gluconate in the present clinical study is consistent with the in vitro and in vivo findings with respect to this iron compound of Zager and colleagues. Differences between iron preparations have been noted by other investigators. Sengoelge et al. demonstrated greater impairment in neutrophil migration when incubated in vitro in iron sucrose compared with sodium ferric gluconate [28].
The regimen of NAC administration utilized in our study was the same regimen that has been reported to be beneficial in preventing oxidative injury due to i.v. contrast [29]. NAC is thought to exert its effect by promoting intracellular GSH synthesis. The failure of this regimen of NAC to prevent oxidative stress could be related to many factors, including inadequate dose or duration of treatment. Of note, in the study of Agarwal and associates [14], the plasma GSSG/GSH ratio was not altered by the same daily dose (1200 mg) of NAC. It is possible that NAC was not protective because it did not sufficiently prevent iron-catalysed hydroxyl radical production, although this is speculative. It is also possible that an alternative antioxidant may have been beneficial, as has been reported previously with vitamin E in haemodialysis patients receiving iron sucrose [13].
A major strength of the above study is the four-way randomized crossover design, in which the order of the experimental treatments was randomized, with end-points determined by personnel blinded as to the treatment group of the patient. The obvious limitation is the small number of patients studied. In this regard, it is possible that the small but non-significant increase in urinary NAG excretion with the 250 mg dose would be statistically significant if enough patients were studied. However, the small magnitude of the effect even if statistically significant in the absence of changes in proteinuria would be of doubtful clinical significance. Pre-infusion values for both oxidative stress parameters and urinary proteinuria/enzymuria were similar for each patient throughout the study period, indicating that any changes seen after infusion of iron were of a transient (i.e. <1 week) nature. However, we cannot be certain as to whether findings would be similar if the iron doses were administered in a different manner (e.g. slow i.v. push rather than by infusion over 12 h) or more frequently (e.g. daily). Moreover, it is possible that larger amounts or repeated courses of 1.0 g amounts of ferric gluconate might result in acute renal injury and/or exacerbation of chronic renal injury.
In summary, i.v. sodium ferric gluconate at commonly used clinical doses caused oxidative stress (as reflected by increased lipid peroxidation), but not acute renal injury. Since it appears that there may be differences between different i.v. iron preparations with regard to their ability to induce cell injury, a randomized trial comparing the efficacy and safety of clinically used iron preparations is warranted.
![]() |
Acknowledgments |
---|
Conflict of interest statement. D. J. Leehey is a member of the Speaker's Bureau of Watson Pharmaceuticals. R. Agarwal is a member of the Speaker's Bureau and a consultant for Watson. The present study was supported by the investigators. No support was requested or received from the pharmaceutical industry including Watson Pharmaceuticals.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|