Lipid Peroxidation Products Formation with Various Intravenous Iron Preparations in Chronic Kidney Disease

Abstract

The role of intravenous iron in contributing to oxidative stress and endothelial dysfunction in chronic kidney disease (CKD) is debatable. The present study assessed differences in fasting plasma malondialdehyde (pMDA) levels 30 minutes before and after intravenous infusion of low molecular weight iron dextran (ID) (n = 19), iron-sucrose (IS) (n = 20), and sodium ferrigluconate complex (SFGC) (n = 20) in stage 3 and 4 CKD patients. Post-infusion pMDA levels were significantly raised with respect to baseline (p < 0.001). pMDA was significantly higher in the SFGC group vs. IS (3.02 ± 0.84 μmol/L vs. 2.82 ± 0.44 μmol/L, p = 0.034) or SFGC vs. ID (3.02 ± 0.84 μmol/L vs. 2.92 ± 0.20 μmol/L, p = 0.048). There was no difference between IS vs. ID (2.82 ± 0.44 μmol/L vs. 2.92 ± 0.20 μmol/L, p = 0.21). To conclude, all forms of parenteral iron, especially SFGC, significantly raise pMDA levels in the immediate post-transfusion period.

Keywords:

• chronic kidney disease
• comparison
• lipid peroxidation products
• intravenous iron

INTRODUCTION

Given the severity of iron deficiency in CKD patients on hemodialysis and the invariable depletion of storage iron with erythropoiesis-stimulating agents, intravenous (i.v.) iron is recommended for the treatment and prevention of iron deficiency anemia in all stages of CKD as per the NKF DOQI guidelines.[1] However, the short- and long-term safety of intravenous iron still remains unclear.[2] The short-term adverse effects are well known and include allergic, anaphylactoid, or anaphylactic reactions commonly described with iron dextran.[2] Possible long-term effects of intravenous iron include increased oxidative stress, inflammation, and renal tubular injury, which Agarwal[3] showed was due to unbound and catalytically active iron. Stocker and Keaney[4] showed CKD to be a state of accelerated atherosclerosis contributed largely by a pro-oxidant milieu. Bishu and Agarwal[2] showed that intravenous iron is an important contributor to oxidative stress and endothelial dysfunction in CKD. However, few clinical studies are available to assess the extent of lipid peroxidation products formation with different forms of parenteral iron. The present study was designed to test the hypothesis that various forms of intravenous iron at clinically used doses can generate lipid peroxidation products in the immediate period after infusion.

MATERIALS AND METHODS

Patients of CKD (stage 3 and 4) with overt or functional iron deficiency were randomized to three groups using computer-generated numbers to receive low molecular weight iron dextran (ID), sodium ferrigluconate complex in sucrose (SFGC), and iron sucrose (IS), designated as groups 1, 2, and 3, respectively. All patients were off vitamin A, E, and C and trace metals for at least two weeks before the study period. Oral iron and intravenous iron were withheld two and four weeks, respectively, before the study. Treatment protocol was as follows:

• IS: 100 mg i.v. over 15 mins

• ID: 100 mg i.v. over 30 mins

• SFGC: 125 mg i.v. over 60 mins.

Baseline clinical data, complete blood counts, iron profile, and lipid profile were recorded. Lipid peroxidation was assessed 30 mins before and 30 mins after completion of parenteral iron infusion. The study was approved by the ethics committee of our institute, and it was in accordance to the Declaration of Helsinki on human experimentation. Informed consent was obtained from all participants before the study.

Plasma MDA levels was measured by spectrophotometry.[5] Malondialdehyde, an end product of fatty acid peroxidation, reacts with thiobarbituric acid (TBA) to form a pink chromophore that has a maximum absorbance at 535 nm. Tricarboxylic acid (10%) measured to 500 μL was added to 500 μL of plasma, followed by the addition of 1 ml of TBA (0.6% in glacial acetic acid). Blank solution (all reagents except plasma) and the sample with unknown concentration were run in parallel. The mixture was boiled for 15 minutes, cooled, and then centrifuged at 1000 rpm for 5 minutes. The supernatant was then decanted off, and absorbance of the solution was measured at 535nm. Optical density (OD) of the blank reagent was subtracted from the OD of the sample.where Ab is the absorbance at 535 nm and E 535 = 1.56 × 10⁵ M⁻¹ cm⁻¹. Results were expressed in μMol/L

The results were analyzed using the SPSS version 13.0. Non-parametric tests were used for analysis of pMDA levels pre- and post-i.v. iron infusion (Wilcoxon signed rank test) and to test the difference between the three groups post-transfusion (Kruskal Wallis Test). Correlation between various biochemical parameters and pMDA was also analyzed. p < 0.05 was taken as significant.

RESULTS

Sixty-six patients of CKD stage 3 and 4 were enlisted in the present study; final analysis was done on 59 patients [group 1 (n = 19), group 2 (n = 20), and group 3 (n = 20)]. Baseline characteristics of the study population are shown in Table 1. Patients had similar demographic and clinical characteristics, including etiology of kidney disease, weight, body mass index, use of ACE inhibitors, and lipid profile. Iron profile was done one week prior to study. No adverse events (ADE) were recorded in the IS and the SFGC group, while one patient in the ID group had an episode of vomiting after 30 minutes of infusion.

Table 1 Baseline characteristics of patients

	ID	SFGC	IS	p value
Number of patients (n)	19	20	20	NS
Age (years)	45 ± 5.6	43 ± 4.9	46 ± 5.9	NS
Males	12	14	15	NS
Weight (kgs)	58.1 ± 3.4	56.2 ± 2.6	57.2 ± 2.9	NS
BMI (kg/m^2)	20.1 ± 1.4	21.3 ± 1.3	19.2 ± 1.6	NS
Smokers	6	8	7	NS
Prior history of coronary artery disease	1	1	2	NS
Diabetic nephropathy	5	6	5	NS
Chronic glomerulonephritis	10	11	12	NS
Hypertensive nephrosclerosis	2	2	2	NS
Other causes of CKD	1	1	1	NS
Systolic blood pressure (mmHg)	141.1 ± 2.3	139.2 ± 5.4	149.4 ± 11.4	NS
Diastolic blood pressure (mmHg)	89.2 ± 3.4	90.2 ± 4.8	90.8 ± 4.2	0.045
Number of patients on ACE inhibitors and angiotensin receptor blockers	10	10	12	NS
Number of patients on epoietin	14	16	15	NS
Hemoglobin (g/dL)	10.3 ± 1.3	9.8 ± 2.4	9.9 ± 2.8	NS
Transferrin saturation (TSAT, in %)	21 ± 3.5	20.3 ± 2.3	19.7 ± 3.8	NS
Serum ferritin (ng/mL)	85.6 ± 2.4	88.4 ± 2.7	89.3 ± 3.7	NS
Serum albumin (g/dL)	3.2 ± 1.5	3.0 ± 1.9	3.1 ± 1.8	NS
Serum cholesterol (mg/dL)	189.2 ± 2.4	190.3 ± 3.5	210 ± 15.8	NS
Low-density lipoprotein (LDL) cholesterol (mg/dL)	104 ± 3.8	110 ± 2.6	105 ± 2.8	NS
Number of patients on statins	7	8	6	NS

Abbreviations: NS = nonsignificant, BMI = body mass index, ACE = angiotensin-converting enzyme, ID = iron dextran, SFGC = sodium ferrigluconate complex, IS = iron sucrose.

Plasma MDA levels were similar in all three groups prior to intravenous iron admqinistration. Table 2 shows that post-infusion levels of pMDA at 30 minutes were found to be significantly raised in all the three groups. Among the three groups, MDA levels were maximally raised in the SFGC group. Table 3 shows that the Spearman coefficients of laboratory parameters with the combined value of post-transfusion pMDA levels do not have any significant correlation.

Table 2 Plasma malondialdehyde levels among the three groups

	ID (n = 19)	SFGC (n = 20)	IS (n = 20)	ID vs. SFGC (p value)	SFGC vs. IS (p value)	ID vs. IS (p value)
Pre-infusion pMDA (μMol/L)	1.89 ± 0.21	1.74 ± 0.32	1.83 ± 0.21	NS	NS	NS
Post-infusion pMDA(μMol/L)	2.92 ± 0.20	3.12 ± 0.84	2.82 ± 0.44	0.048	0.034	NS
Pre-infusion vs. post-infusion pMDA (p value)	<0.001	<0.001	<0.001	—	—	—

*test applied Wilcoxon signed rank test.

^†test applied Kruskal Wallis test.

Abbreviations: ID = iron dextran, SFGC = sodium ferrigluconate complex, IS = iron sucrose, pMDA = plasma malondialdehyde, NS = nonsignificant (p < 0.05 is significant).

Table 3 Correlations with post-transfusion plasma malondialdehyde levels

Variable	Correlation coefficient	p value
Hemoglobin (g/dL)	0.021	NS
TSAT (%)	0.058	NS
Serum ferritin (ng/mL)	0.012	NS
Serum creatinine (mg/dL)	0.321	NS
Serum cholesterol (mg/dL)	0.125	NS

Abbreviations: TSAT = percentage transferrin saturation (%); NS = nonsignificant.

DISCUSSION

While the use of parenteral iron in treating anemia of CKD in predialysis or dialysis patients on epoetin has been firmly established,[1] fears frequently expressed but rarely allayed[2] are the risks of short-term adverse events, like anaphylaxis, and long-term toxic effects, like oxidative stress and inflammation and its effect on atherosclerosis and cardiovascular disease (CVD) in CKD. CVD is the primary cause of death in ESRD patients,[6] primarily because CKD is a pro-oxidative state that leads to accelerated atherosclerosis.[7] Himmelfarb et al.[7] showed that the central role in the pathogenesis of atherosclerosis of CKD is endothelial dysfunction, which is mediated by oxidative stress. This can be clinically assessed by measuring plasma MDA levels—the end products of peroxidation of polyunsaturated fatty acids.

The role of parenteral iron in promoting an already existing pro-oxidant and pro-inflammatory milieu of CKD is still a matter of debate. In an in vitro experiment, Zager et al.[8] found that cell cultures of proximal tubule cells, endothelial cells, and tubules when exposed to IS, SFGC, and ID resulted in a similar and massive increase in lipid peroxidation products. However, IS was most toxic to tubules and ID the least. Lim and Vaziri[9] showed that ID when administrated to CKD rats markedly increased markers of oxidative stress. Roob et al.[10] demonstrated an increase in pMDA levels and other markers of oxidative stress after 30 mins of infusing 100 mg of IS to hemodialysis patients, which was only partly prevented by prior vitamin E prophylaxis. Tovbin et al.[11] showed an increase in products of protein oxidation in hemodialysis patient after intravenous iron. Cavedar et al.,[12] however, noted a decrease and not an increase in oxidative stress and erythrocyte deformability after rapid infusions of 20 and 100 mg of IS in hemodialysis patients.

It is possible that this pro-oxidant milieu generated by intravenous iron is responsible for endothelial dysfunction in CKD. Druekke et al.[13] found that carotid intima media thickness, a marker of early atherosclerosis, correlated well with annual dose of i.v. iron, serum ferritin, and levels of oxidant mediated protein damage in ESRD patients. Similarly Rooyakkers et al.[14] showed flow mediated vasodilatation (FMD) was maximally deranged after 10 mins of a rapid push of IS and as late as one hour after a slow infusion of IS. The nadir of this drop in FMD correlated well with the time point of maximal oxygen free radicals concentration, but not with the peak concentration of free/catalytically active iron.

At present, controversy exists regarding which form of i.v. iron generates maximal oxidative stress. Unlike those by Lim et al.,[15] in vivo experiments by Rooyakkers et al.[14] could not prove that oxidative stress occurred in the presence of unbound or catalytically active iron. Agarwal et al.[3] demonstrated a significantly greater ability of non-dextran preparations to donate iron to transferrin than ID, and the propensity for free ion generation was in the order SFGC > IS > ID. In an in vivo study, Legssyers et al.[16] showed that the ability of various forms of intravenous iron to increase macrophage NO2 levels was in the order of SFGC > IS > ID > iron polymaltose. In another comparative study, Pai et al.[17] noted a significant increase in lipid peroxidation products in hemodialysis patients only with SFGC despite greater concentration of post-infusion non-transferrin-bound iron with SFGC and IS than ID. Contrary to our observations, Pai et al.[17] noted a significant positive correlation with baseline transferrin saturation above 30%, baseline transferrin (Tf) levels below 180 mg/dL, and ferritin levels above 500 ng/mL, while transferrin levels below 180 mg/dL was independently associated with a rise in pMDA levels.[17]

In a review, Wyck[18] noted that comparative studies of bioactive or labile iron activity, like the direct donation of iron to Tf, generation of oxidant stress, and alteration of intracellular iron homeostasis with IV iron agents, consistently showed an inverse relationship with the molecular weight of the iron-carbohydrate compound. All i.v. iron agents consist of a iron-oxyhydroxide core with a surrounding carbohydrate shell. Wyck[18] hypothesized that labile iron reflects the ionic iron that is first released from the surface of the iron-oxyhydroxide core. Therefore for the same total amount of core iron, surface area available for iron release increases as core radius decreases. SFGC has the smallest iron-hydroxide core radius, IS is of intermediate size, and ID or polymaltose has the largest. Hence, it is natural to expect that release of free iron, and therefore its bioactivity, will be maximal with SFGC and least with ID or polyaltose.

The present study has been limited to CKD stage 3/4 patients to avoid the confounding effects of dialyzer membrane in generating free radicals. Because we measured single-point pMDA levels, we could not analyze the time profile of release of lipid peroxidation products with different forms of parenteral iron, which is especially significant given the different kinetics of iron release in dextran versus non-dextran molecules. The simultaneous measurement of free/unbound iron was also not measured. Despite the limitations, we believe that the present study should be used as a benchmark for further trials to analyze the differential effects of dextran vs. non-dextran molecules on oxidative stress and its relationship with endothelial dysfunction, atherosclerosis, and suspected tubular toxicity. Given the growing popularity of nondextran iron as a safe form of parenteral iron, the recent observations of Pai et al.,[17] Wyck,[18] and our own results are disturbing. It is necessary, therefore, to establish clearly the risk:benefit ratio of each of these three forms of parenteral iron before definite recommendations can be made on the vexatious issue of comparative safety.

To conclude, the present study showed that all three forms of parenteral iron raise pMDA—a well-validated marker of oxidative stress—immediately post-transfusion. The levels are maximal with SFGC. It remains to be clarified if these short-term effects translate into differential long term effects on atherosclerosis and CVD in CKD patients.

DECLARATION OF INTEREST

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.