Lipid Peroxidation, Antioxidant Enzymes and Glutathione Levels in Human Erythrocytes Exposed to Colloidal Iron Hydroxide in Vitro

The free form of the iron ion is one of the strongest oxidizing agents in the cellular environment. The effect of iron at different concentrations (0, 1, 5, 10, 50, and 100 µM Fe 3+) on the normal human red blood cell (RBC) antioxidant system was evaluated in vitro by measuring total (GSH) and oxidized (GSSG) glutathione levels, and superoxide dis-mutase (SOD), catalase, glutathione peroxidase (GSH-Px) and reduc-tase (GSH-Rd) activities. Membrane lipid peroxidation was assessed by measuring thiobarbituric acid reactive substance (TBARS). The RBC were incubated with colloidal iron hydroxide and phosphate-buffered saline, pH 7.45, at 37 o C, for 60 min. For each assay, the results for the control group were: a) GSH = 3.52 ± 0.27 µM/g Hb; b) GSSG = 0.17 ± 0.03 µM/g Hb; c) GSH-Px = 19.60 ± 1.96 IU/g Hb; d) GSH-Rd = 3.13 ± 0.17 IU/g Hb; e) catalase = 394.9 ± 22.8 IU/g Hb; f) SOD = 5981 ± 375 IU/g Hb. The addition of 1 to 100 µM Fe 3+ had no effect on the parameters analyzed. No change in TBARS levels was detected at any of the iron concentrations studied. Oxidative stress, measured by GSH kinetics over time, occurs when the RBC are incubated with colloidal iron hydroxide at concentrations higher than 10 µM of Fe 3+. Overall, these results show that the intact human RBC is prone to oxidative stress when exposed to Fe 3+ and that the RBC has a potent antioxidant system that can minimize the potential damage caused by acute exposure to a colloidal iron hydroxide in vitro.


Introduction
Iron deficiency anemia is the most common cause of chronic anemia throughout the world, mainly in developing countries.In addition to blood loss and iron malabsorption, anemia may be caused by inadequate diet (1), blood withdrawn by diagnostic phlebotomy in hospitalized patients (2), hookworm infestation (3), and chronic inflamma-tory disease (4).Once the diagnosis of iron deficiency anemia is confirmed and the possible causes are identified and treated, replacement of iron stores is indicated.Most patients respond favorably to oral iron preparations.However, when oral supplementation is not possible or fails, the use of parenteral iron is indicated (5).Side effects, which are usually mild, may occur in 25% of patients receiving parenteral iron treatment (6).
On the other hand, iron ions may cause cellular damage by themselves.Several disease states are due to an imbalance between the activities of an oxidant agent and the antioxidant system within the cell (7).One of the strongest oxidizing agents is the free form of iron, Fe 3+ .Under normal conditions, however, the availability of Fe 3+ for catalyzing free radical reaction in vivo is extremely limited (8,9).It has been shown that oxidation of hemoglobin and lipid peroxidation of human red blood cell (RBC) membranes occur due to iron overload (10).The RBC are intrinsically prone to oxidative stress because they are exposed to high oxygen tension, and have a characteristic structural composition with polyunsaturated fatty acid in the membrane, besides the presence of hemoglobin-bound iron (11,12).However, membranes and cytoplasmic compartments of RBC have an efficient antioxidant mechanism that maintains their integrity.A detoxifying system consisting of reduced glutathione (GSH), superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px) and vitamin E prevents oxidative damage.In addition, there is also a system consisting of NADPH-dependent methemoglobin reductase, ascorbic acid, and glutathione reductase (GSH-Rd), whose main role is the repairing of damage that follows oxidative stress (13).Colloidal iron hydroxide is used for parenteral treatment of severe iron deficiency anemia and its action upon the RBC is not completely established.Furthermore, we are not aware of a comprehensive evaluation of the consequences of acute iron overload on the RBC antioxidant system and its related enzymes in the literature.Thus, the aim of the present study was to determine the response of erythrocytes exposed to different concentrations of colloidal iron hydroxide.

Material and Methods
The protocol for this study was approved by the Ethics Committee of the Botucatu Medical School of the University of the State of São Paulo (UNESP), and the procedures were conducted according to the Helsinki Declaration, as revised in 1983.All subjects gave their informed consent.
Total Hb was estimated as cyanmethemoglobin using Drabkins reagent and absorbance measurements at 546 nm (14).GSH was determined using a kinetic assay involving the continuous reduction of 5,5'-dithiobis-(2-nitrobenzoic acid) by GSH in the presence of NADPH, forming 2-nitro-5-thiobenzoic acid, which was read at 412 nm (15).GSSG was determined in hemolysate previously incubated with 4 vinyl-pyridine for 60 min at room temperature.GSH and GSSG levels are reported as µM/g Hb.Standard curve was determined daily with freshly prepared standard solution of 33 mM GSSG.
GSH-Rd activity was evaluated by monitoring NADPH oxidation at 340 nm and at 37 o C ( 16).The assay mixture contained 1 M Tris, 5 mM EDTA buffer, pH 8.0, 33 mM GSSG and 2 mM NADPH.The enzyme activity (IU/g Hb) was calculated using a millimolar extinction coefficient of 6.22 for NADPH at 340 nm.GSH-Px activity was determined by monitoring NADPH oxidation at 340 nm and at 37 o C in an assay mixture containing 1 M Tris, 5 mM EDTA buffer, pH 8.0, 0.1 M GSH, 10 IU/ml GSH-Rd, 2 mM NADPH and 7 mM tert-butyl hydroperoxide.The assay mixture for catalase activity (IU/g Hb) determination contained 1 M Tris, 5 mM EDTA buffer, pH 7.0 and 200 mM hydrogen peroxide (H 2 O 2 ).Disappearance of H 2 O 2 was monitored at 240 nm for 30 s (17).SOD activity was measured according to the method described by Beutler (18).Briefly, the reaction is dependent on the presence of superoxide anions that cause the oxidation of pyrogallol.The inhibition of pyrogallol oxidation by SOD was monitored at 420 nm, and the amount of enzyme producing 50% inhibition was defined as one unit of enzyme activity (IU/g Hb).The assay mixture contained 1 M Tris, 5 mM EDTA buffer, pH 8.0, and 10 mM pyrogallol.
Membrane lipid peroxidation was assayed by determining the interaction of thiobarbituric acid (TBA) with the breakdown product of lipid peroxidation under acid pH conditions.The pink chromophore was determined at 535 nm.The assay mixture contained 0.67% TBA, pH 2.0, and TBARS was expressed as nM/g Hb (19).
GSH (L-g-glutamyl-L-cysteinylglycine), the most prevalent intracellular thiol, is known to function as an antioxidant.Oxidative stress leads to oxidation of GSH to GSSG and formation of protein-SS-protein.Therefore, we may expect a decrease in GSH level when the cell is under oxidizing environment.In the present investigation, oxidative stress was evaluated as the changes in GSH levels over time in hemolysate from RBC incubated previously with different iron ion concentration.
To determine iron-induced oxidative stress, RBC from 14 blood donors were incubated with 0, 5, 10, and 100 µM of colloidal iron hydroxide for 1 h at 37 o C (final incubation conditions as described above).After incubation the RBC were washed three times with PBS to remove the iron excess.The RBC were hemolyzed and the hemoglobin level determined.The GSH level in the hemolysate was measured at 50-s intervals throughout 300 s.
All data are reported as mean ± SEM.One-way analysis of variance was used to compare mean values among the six irontreated groups.Comparisons of GSH levels, over time, in hemolysates of RBC incubated with different iron concentrations were made by the two-way repeated measures analysis of variance and the post hoc Tukey test.Significance was set at 5% (20).

Results and Discussion
Mean ± SEM values for variables estimated at different concentrations of Fe 3+ , in the whole RBC, are shown in Table 1.No significant differences were observed among the groups treated with concentrations up to 100 µM Fe 3+ for all the variables studied.
The results show that the addition of up to 100 µM colloidal Fe 3+ to a suspension of normal human erythrocytes does not alter the glutathione redox system and associated enzymes of these cells.Therefore, it may be assumed that RBC is well equipped to neutralize the oxidative stress imposed by the colloidal iron.In this way, the RBC behaves differently from other cells exposed to the iron ion.For example, hepatocytes from patients suffering from hemochromatosis (21), isolated heart from rats treated with 6 injections of 25 µg Fe 3+ (22), rat myocardial cells incubated with 0.36 mM ferric ammonium citrate (23), and disrupted brain tissue (11) are morphologically and physiologically altered by lipid peroxidation, increased lysosomal fragility, and oxidation of GSH secondary to iron overload.We did not observe changes in lipid peroxidation or GSH in our experiment.Nevertheless, we have to point out that our experimental conditions were different from those authors.
Besides being potentially prone to oxidative stress, under some circumstances erythrocytes may spontaneously produce significant amounts of the oxidative agent superoxide (O 2 • ) and hydrogen peroxide (24).These features may facilitate and accelerate red cell membrane lipid peroxidation, as can be seen in situations such as microbleeding, where hemoglobin and hemichrome denaturation and iron release occur (11).Despite these characteristics, the observation of clinical or laboratory erythrocyte dysfunction due to oxidative damage is uncommon.RBC resistance to oxidative stress may be an evolutive characteristic of these highly specialized cells and may quite well explain why they are resistant to the damage caused by in vitro iron overload, as observed in the present study.Bartal et al. (25) found an increased malondialdehyde (MDA) and GSH depletion in red cells exposed to t-butyl hydroperoxide.Other investigators have shown increased MDA and GSH-Px activity (26) and increased superoxide dismutase (27) in red blood cells exposed to hydrogen peroxide.However, it should be pointed out that those researchers used t-butyl hydroperoxide and hydrogen peroxide, which are stronger oxidants than the ion Fe 3+ used in the present study.It is likely that the difference of oxidizing agent may explain the discrepancies observed among the studies.
All data are reported as mean ± SEM.Superscripts indicate number of samples.The groups were compared by one-way analysis of variance and the level of significance was set at 5%.
In the experiment, TBARS levels did not differ significantly among the groups exposed to the different concentrations of iron, and the possibility remains that colloidal iron hydroxide induces no measurable membrane lipid peroxidation.
The 60-min period of incubation with iron adopted in this study is similar to that used by Rice-Evans et al. (28), who found a 30% reduction in GSH levels when compared with the levels obtained at the beginning of the incubation period.These authors added H 2 O 2 and ascorbate to the system in their in vitro assays.In the present study, to simulate the acute iron overload that occurs in parenterally iron-treated patients, those reagents were not added to the medium because they might have induced oxidative lesions by themselves.Therefore, the composition of the incubation medium used by those authors may explain the differences between our results and theirs.
Significant decreases in GSH levels over time were observed in the hemolysate from RBC incubated with both 10 µM (GSH10) and 100 µM (GSH100) colloidal iron hydroxide, when compared with RBC incubated with 0 µM (GSH0) and 5 µM (GSH5).No significant difference was observed between the GSH0 and GSH5 groups (Figure 1).The decrement of GSH over time in hemolysate incubated with 10 µM colloidal iron hydroxide suggests that oxidative stress occurs above this concentration.This event does not occur at 0 or 5 µM iron concentration meaning that the oxidative stress is minimal or not present at low iron concentrations.
The present observations show that the normal human RBC has an efficient antioxidant system which is able to reduce the potential damage imposed by acute iron overload in the form of colloidal iron hydroxide.