Effect of Intravenous Iron Sucrose on Oxidative Stress in Peritoneal Dialysis Patients

Abstract

Aim. Intravenous iron therapy is an accepted treatment for patients receiving hemodialysis and continuous ambulatory peritoneal dialysis (CAPD). Studies have found enhanced oxidative stress in hemodialysis patients receiving intravenous iron, but there are no clinical data for CAPD patients. The aim of the current study was to investigate the effect of 100 mg of intravenous iron-sucrose on the erythrocyte (RBC) antioxidant enzymes (namely, superoxide dismutase [SOD], catalase [CAT], and glutathione peroxidase [GSHPx]) and plasma malondialdehyde (MDA), an oxidant molecule, in CAPD patients. Methods. Twelve CAPD patients receiving maintenance intravenous iron-sucrose were recruited. After a 12-hour fast, blood samples were taken for hemoglobin, iron, ferritin, and high-sensitivity C-reactive protein (hsCRP), and for baseline activities of erythrocyte antioxidant enzymes (i.e., SOD, CAT, GSHPx) and the plasma oxidant molecule, MDA. 100 mg iron-sucrose was infused over 30 minutes. Blood samples taken during (i.e., 15 minutes after commencement of infusion) and after (i.e., at 30 minutes, 60 minutes, and 6 hours after commencement) the infusion were taken for measurement of plasma iron, ferritin, TSAT, RBC SOD, CAT, GSHPx, and plasma MDA. Results. Plasma iron and transferrin saturation elevated significantly during infusion (p < 0.05). There was no significant change in erythrocyte SOD, CAT, GSHPx, or in MDA activities. There was a reduction of GSHPx activity at the 30th minute (from 153.69 ± 66.69 to 123.68 ± 25.50 mU/mL), but it was not statistically significant. The patients were grouped according to baseline ferritin (100–400 and 400–800 ng/mL); 60th-minute MDA was significantly higher in the latter group (p < 0.05). There was no correlation between hsCRP and oxidant-antioxidant balance. No correlation was noted between RBC antioxidant enzymes or plasma oxidant molecule and ferritin levels. Conclusion. There are no acute deteriorating effects from a 100 mg of intravenous iron-sucrose in CAPD patients with optimal iron stores. This dose may be applied safely in CAPD patients.

Keywords:

• peritoneal dialysis
• iron
• oxidative stress

INTRODUCTION

Increased oxidative stress, which occurs with excessive free radical production or low antioxidant levels, is common in patients with chronic renal failure (CRF).[1] It has been suggested that the treatment of uremic patients with hemodialysis (HD) or peritoneal dialysis (PD) may particularly contribute to oxidative stress,[2] and reduced antioxidant levels in these patients with hemodialysis may result from the activation of macrophages on the surface of dialysis membranes during dialysis. The chloramine in the dialysate solution, thermal damage, and loss or deficiency of antioxidant substances could also contribute to enhanced oxidative stress in uremia.[3] Further, the heparin used in HD causes lipid peroxidation. In a previous study, we demonstrated that both standard heparin and low-molecular-weight heparin augment free fatty acids and oxidant stress.[4] However, little information is available concerning continuous ambulatory peritoneal dialysis (CAPD) patients. There is a tendency to assume higher oxidative stress status in HD than in CAPD patients.[5–7]

Intravenous (IV) iron therapy has become an accepted treatment for iron deficiency in HD and CAPD patients because oral supplementation is usually inefficient.[8] However, it is known that free iron is pro-oxidant and causes oxidative stress by generating highly reactive, cytotoxic hydroxyl radicals through the Fenton and Haberman-Weiss reactions.[9]

IV iron has been shown to increase biologic markers of oxidative stress in cell cultures, animal models, and clinical studies of HD patients.[9–11] Whether or not the effect of IV iron in CAPD patients differs from that in HD patients because of the different oxidative milieus, there is a lack of clinical studies in the literature investigating the effect of IV iron on the antioxidant enzymes or oxidant molecules in plasma and erythrocyte in CAPD patients. This is the first study investigating the effect of IV iron on oxidative stress in PD patients.

The aim of the current study was to investigate the effect of 100 mg of intravenous iron-sucrose on the erythrocyte (RBC) antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSHPx), as well as on plasma malondialdehyde (MDA), an oxidant molecule, in CAPD patients.

METHODS

Patients

Of the 42 patients in the PD unit of our nephrology department, six men and six women who had received regular CAPD for more than one year and maintenance iron therapy (100 mg iron-sucrose/month) for at least six months were enrolled in this study after informed consent had been obtained. The causes for their renal failure were hypertension (n = 4), diabetes mellitus (n = 3), chronic glomerulonephritis (n = 2), chronic pyelonephritis (n = 1), adult polycystic kidney disease (n = 1), and unknown (n = 1). Maintenance iron therapy was administered using National Kidney Foundation/Kidney Disease Outcome Quality Initiative (NKF-K/DOQI) guidelines.[12] Serum ferritin concentrations were between 100–800 ng/mL, and transferrin saturation (TSAT) was between 20–50%. Hemoglobin (Hb) levels were between 10–12 gr/dL. All subjects were treated with epoetin alpha (Eprex®, Santa Farma, Istanbul, Turkey; 2,000–4,000 U/weekly, S.C.). No patient received any antioxidant. Patients were excluded if they were smokers or demonstrated the presence of active infection during the six months prior to the study. Patients were also excluded if they had Hb < 10 g/dL or > 12 gr/dL, ferritin <100 ng/mL or > 800 ng/mL, or TSAT < 20% or > 50%.

Iron Infusion and Collection of Blood Samples

After a 12-hour fast, baseline venous blood samples were taken for analysis of hematological parameters (Hb, plasma ferritin, plasma iron, plasma total iron binding capacity), plasma high-sensitivity C-reactive protein (hsCRP), baseline activities of erythrocyte (RBC) antioxidant enzymes (i.e., superoxide dismutase [SOD], catalase [CAT], and glutathione peroxidase [GSHPx]), and plasma malondialdehyde (MDA), an oxidant molecule. A single 100 mg dose of intravenous iron sucrose (Venofer®, Abdi Ibrahim, Turkey) was infused over 30 minutes. Blood samples taken during (i.e., 15 minutes after commencement of infusion) and after (i.e., at 30 minutes, 60 minutes, and 6 hours after commencement) the infusion were taken from the other antecubital vein for measurement of plasma iron, ferritin, TSAT, SOD, CAT, GSHPx, and plasma MDA. Plasma and RBC samples were frozen immediately after centrifugation and were stored at –80°C for assays.

Laboratory Analysis

Plasma levels of iron were analyzed by the Ferene spectrophotometric method (BTS- 370 Clinical Chemistry Analyser, BioSystems S.A., Barcelona, Spain) using the Randox plasma iron kit (Randox, Crumlin, UK). Plasma total iron binding capacity (TIBC) was measured by a colorimetric, spectrophotometric method using the Randox TIBC kit. A microparticle enzyme immunoassay was performed for plasma ferritin levels. TSAT were calculated by the plasma iron/TIBC·100 formula.

Plasma hsCRP levels were measured by a turbidimetric method using a BioSystem hsCRP kit. RBC SOD activities were measured by the method of Sun et al.[13] RBC CAT levels were measured by the reaction transforming hydrogen peroxide (H₂O₂) to water (H₂O) and oxygen (O₂). RBC GSHPx levels were estimated by measurement of the oxidation rate of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), which forms by the reaction of GSHPx and GSH reductase. Plasma MDA levels were estimated by the method of Stock, Dormandy, and Jain.[14]

Statistical Analysis

Friedman variance analysis and the Wilcoxon rank test (for paired data) were used for statistical analysis; the Mann-Whitney U test was used to evaluate differences between the groups. Data are presented as mean ± SD. A p value < 0.05 is considered significant.

RESULTS

The baseline biochemical, hematological, and demographic characteristics of the patients are outlined in Table 1.

Table 1 Characteristics of the patients in the study group

Parameters	n = 12
Mean age (years)	52.5 ± 11.6
Age range (years)	(42–78)
Sex (M/F)	6/6
Duration of PD (months)	46.16 ± 29.9
Duration of PD range (months)	(21–123)
Hb (g/dL)	10.8 ± 0.8
Cr (mg/dL)	9.46 ± 2.78
hsCRP (mg/L)	6.43 ± 2.52
Albumin (g/dL)	4.23 ± 0.19
Serum iron (μg/dL)	69.42 ± 10.71
TSAT (%)	33.67 ± 6.27
Ferritin (ng/mL)	399.33 ± 208.95
Etiology of CRF
HT	4
DM	3
Chronic GN	2
Chronic PN	1
Polycystic kidney	1
Unknown	1

Data are presented as mean ± SD.

Abbrevations: Hb = hemoglobin, Cr = creatinine, hsCRP = high sensitive C-reactive protein, TSAT = transferin saturation, HT = hypertension, DM = diabetes mellitus, GN = glomerulonephritis, PN = pyelonephritis.

The average plasma iron, TSAT, and ferritin levels during iron infusion are given in Table 2. With the iron infusion, the rise in plasma iron and TSAT was statistically significant (p < 0.05). The mean activities of RBC SOD, GSHPx, and CAT and plasma MDA are given in Table 3. RBC SOD, CAT, and GSHPx activities did not differ during the iron-sucrose infusion. There was a reduction of GSHPx activity at 30 min (from 153.69 ± 66.69 to 123.68 ± 25.50 mU/mL), although it was not statistically significant. Plasma MDA levels rose at 15 min (from 5.57 ± 1.18 to 6.08 ± 2.17 μmol/L); however, it too was not significant. As shown in Table 4, when the subjects were classified according to whether their baseline ferritin levels were between 100 and 400 ng/mL or between 400 and 800 ng/mL, 60 min MDA levels were significantly higher in the group having higher ferritin levels (400–800 ng/mL; p < 0.05).

Table 2 Plasma iron, TSAT, ferritin levels during the study

	Baseline	15 min	30 min	60 min	6 hour
Iron (μg/dL)	69.4 ± 10.7	861.7 ± 311.6	1097.5 ± 99.1	865.2 ± 116.5	273.3 ± 65.0
TSAT (%)	33.6 ± 6.2	256.6 ± 121.2	314.6 ± 104.2	326.4 ± 99.1	127.7 ± 31.6
Ferritin (ng/mL)	399.33 ± 208.95	406.42 ± 239.53	392.92 ± 220.29	374.42 ± 198.70	406.08 ± 232.22

Data are presented as mean ± SD.

*Statistical significant when compared with the baseline (p < 0.05).

Table 3 Plasma MDA, erythrocyte SOD, GSHPx, and CAT activities during the study

	Baseline	15 min	30 min	60 min	6 hour	p
MDA (μmol/L)	5.57 ± 1.18	6.08 ± 2.17	5.70 ± 2.43	5.65 ± 3.17	5.58 ± 2.30	NS
SOD (U/mgHb)	7.36 ± 1.43	7.15 ± 2.89	7.31 ± 1.4	7.46 ± 1.59	7.69 ± 1.56	NS
GSHPx (mU/mL)	153.69 ± 66.6	138.40 ± 32.0	123.68 ± 25.5	159.05 ± 57.0	157.62 ± 62.2	NS
CAT (U/mgHb)	104.33 ± 44.3	107.00 ± 46.8	126.58 ± 36.7	123.97 ± 54.6	112.64 ± 60.5	NS

Data are presented as mean ± SD.

*Friedman analysis of variance was performed for evaluating differences within the groups.

Table 4 The relation with the baseline ferritin and MDA (60 min.) levels

	MDA baseline	MDA 60 min
Ferritin < 400 ng/mL (n:7)	6.21 ± 2.42	4.64 ± 1.83
Ferritin ≥ 400 ng/mL (n:5)	6.90 ± 2.36	9.10 ± 2.82

Note: 60 min MDA levels are significantly higher in the second group (having higher ferritin levels).

*p = 0.034.

No correlation was noted between RBC antioxidant enzyme activities or between plasma oxidant molecule and ferritin levels, nor did we observe a correlation between hsCRP levels and plasma ferritins. No correlation was found between hsCRP and iron levels nor between antioxidant enzymes and plasma MDA during infusion.

DISCUSSION

IV iron therapy to replenish iron stores is mandatory in dialysis patients.[13] However, little information is available concerning the influence of iron therapy on the oxidative stress in uremic patients, as it is known that iron itself is a pro-oxidant molecule that promotes cytotoxic ROS formation during the Fenton and Haberman-Weiss reactions.[15]

Previous studies have shown that uremic patients are exposed to higher levels of reactive oxygen species (ROS) than healthy controls.[2],[16],[17] The augmented lipid peroxidation products shown in the plasma and RBCs of CRF patients are attributed to increased ROS.[18–21] An imbalance between the production and removal of ROS is blamed as a factor accelerating complications associated with uremia and dialysis.[17] Uremic toxic metabolites are oxidant, either directly or—by defeating the functions of antioxidant enzymes—indirectly.[18],[19],[22] Although a reduction in oxidant stress may be expected during the treatment of anemia, there will also be an oxidative stimulus because of the oxidant property of iron.[23],[24] The study of Agarwal et al. showed that IV iron increases plasma MDA and the urinary excretion rate of MDA after 15–30 min of IV iron-sucrose administration in CRF patients. This was accompanied by enzymuria and an increase in proteinuria.[9] In another study with HD patients, Roob et al. demonstrated an increase in plasma MDA with a single 100-mg dose of iron-sucrose; this was reduced by 1200 IU vitamin E given 6 hours before infusion.[11] In our study, there was an increase in the 15 min MDA levels, but it was not significant. In a previous study, we investigated and compared the effect of 120 mg and 100 mg IV iron-sucrose in HD patients and demonstrated that there was no effect on plasma MDA by IV iron sucrose.[25] However, when patients of our current study were grouped based on their baseline ferritin levels, a significant difference was assessed in 60 min MDA levels between the groups. The group having higher ferritin levels (>400 ng/mL) had higher MDA levels. This may reflect the effect of iron stores on the influence of iron therapy on oxidant stress. It is well known that iron overload causes oxidant stress by the release of unbinding iron, and it has been shown that iron treatment in iron-overloaded patients, such as those with thalassemia and hemochromatosis, causes oxidative stress.[26],[27] Concerned about iron overload in HD patients, Reddi et al. investigated the effect of IV iron-dextran on oxidative stress and its correlation with baseline ferritin levels. After a maintenance therapy of 10–14 times of 100 mg doses of IV iron-dextran, they found no negative effect either in the group having a ferritin level below 325 ng/mL or in the group having a ferritin level above 325 ng/mL. Plasma and RBC antioxidant enzymes (SOD, CAT, GSHPx) and MDA activity did not differ between the two groups.[28] Our study similarly demonstrated no differences in RBC SOD, CAT, and GSHPx levels during iron infusion, even when patients were grouped based on baseline ferritin levels. Paik-Seong Lim et al. also examined the influence of baseline ferritin level and IV infusion of 100 mg iron-sucrose on the oxidative status of patients on maintenance therapy.[10] Based on patients' baseline ferritin levels (< 300, 301–600, and > 600 μg/L), it was found that plasma SOD activity was significantly reduced in the third group. Plasma MDA increased in all groups, but the increment was significantly higher in the third group as compared with the first and second. The study demonstrates a pro-oxidative effect of 100 mg iron-sucrose in HD patients; it also shows that high baseline ferritin levels influence the effect of parenteral iron treatment. This study differs from the previous ones, and reflects that HD patients treated by IV iron-sucrose may be exposed to enhanced oxidant stress.

The various results found by this limited number of studies in HD patients may be due either to the patients' heterogeneous baseline oxidative status and iron load or to different formulations and duration of iron treatment. We therefore aimed to select patients receiving similar maintenance iron treatment. Moreover, we excluded oxidative factors (e.g., infection and inflammation, iron overload, smoking) to form a homogeneous group.

Plasma iron was more than 15-fold higher after iron administration. It decreased between 60 min and 6 hours. Given that the spike in IV iron occurs in 15 min and that the half-life of iron in plasma is 5 to 6 hours, this result clearly reflect the physiological kinetics of IV iron.[29] TSAT was 326% in 30 min after the infusion and 127% at 6 hours. TSAT is a clinical marker that reflects the iron overload exceeding the binding capacity of transferrin.[13] The high TSAT levels found in our study are associated with the oversaturation of transferrin. Measurement of hsCRP, a biological marker indicating that tissue damage, necrosis, and inflammation produced in the liver is a response to a systemic stimulus, was performed to assess the baseline inflammatory status of the patients.[30] It also has a prognostic value for inflammation, and thus is used in monitoring chronic inflammatory diseases.[30] We found no correlation between baseline hsCRP and RBC SOD, CAT, GSGPx, or plasma MDA levels. Therefore, the difference in 60 min MDA levels between the group having higher ferritin and the group having lower ferritin can be attributed to the iron itself, without any relation to inflammation.

In the studies of Roob and Lim with HD patients, there was an increase of lipid peroxidation.[10],[11] The plasma MDA levels increased by IV iron-sucrose treatment. We demonstrated no significant reduction in the RBC SOD, GSHPx, and CAT activities with 100 mg IV iron-sucrose administration. Even with the oversaturation of transferrin by the iron dose and infusion rate in our study, the augmented oxidant stress demonstrated previously in some studies in HD patients was not found.[10],[11] The reason for the absence of a detoriating effect of iron treatment was probably the optimal iron stores and perhaps the low inflammation status of the patients.

In conclusion, in CAPD patients having optimal iron stores with no demonstration of active inflammation, there may be no acute deteriorating effect of a single 100 mg IV dose of iron-sucrose. However, baseline ferritin levels may influence the peroxidative effect of iron. It seems that 100 mg IV iron-sucrose may be a applied safely in CAPD patients. However, there is still need for further studies to investigate the effects of chronic use of different iron formulations in CAPD patients.