Perspective Chapter: Iron Chelation Inhibits Reduced Glutathione (GSH) as a Prooxidant in Iron-Mediated Hemoglobin Oxidation in Purified Hemoglobin

Abstract

As a trace element, iron is required by all living. Although this crucial metal is required, maintaining its biological equilibrium in an organism is far more important than any other trace element. Excess iron plays a vital role in the generation of harmful oxygen radicals due to its catalysis of one electron redox chemistry. In disorders such as thalassemia and sickle cell anemia, this is clearly visible. In vitro experiments were carried out using pure hemoglobin (HbA) exposed to ferric (Fe3+) iron. The addition of Fe3+ (0–250 µM) caused spectrophotometric alterations in the absorption spectra (500–700 nm) of (40 µM HbA; pH 7.4). There was no HbA oxidation in the absence of Fe3+. Similarly, unlike hemolysates, the mere addition of Fe3+ to HbA exhibited negligible oxidative consequences. However, the addition of glutathione (GSH) and Fe3+ caused significant oxidation. The iron chelators (DFO desferrioxamine or Deferiprone L1) suppressed Fe3+-mediated HbA oxidation in a dose-dependent manner. The findings of this study have important significance for damage mechanisms in disorders like as thalassemia and sickle cell anemia. In addition, our findings suggest that chelating bioreactive iron within aberrant erythrocytes might be a potential therapy strategy.

1. Introduction

Iron is a vital trace element for all living cells. Despite the fact, this key metal is essential, and maintaining its biological balance in an organism is far more important than any other trace element except copper [1]. Excess iron, due to its catalysis of one electron redox chemistry, plays a key role in the formation of toxic oxygen radicals. Indeed, this is readily observed in diseases such as thalassemia and sickle cell anemia. This potentially hazardous combination of oxygen and iron within the erythrocyte is kept in check by several endogenous mechanisms. Intra-erythrocytic free iron can be a potential hazard to form free radicals in the presence of reduced glutathione. Free radicals can have an adverse effect on hemoglobin by oxidative damage [2]. Glutathione (GSH) is thought to be a prooxidant in iron-mediated hemoglobin oxidation, which can be prevented by iron chelation. Researchers picked two in vitro hemoglobin models to test this theory. RBC hemolysate, which is essentially distilled water lysed RBC, and crude pure hemoglobin (Hb-A) eluted off a Sephadex desalting column.

2. Experimental methods used in this study

A random fresh blood sample collected in EDTA tube was obtained from Hamad Medical Corporation (HMC), Doha, Qatar. The blood samples were obtained from HMC blood donor center in Doha, Qatar. Institutional review board (IRB) approval was obtained from HMC for using the donor’s blood for the purpose of research. This research has two model samples (the hemolysate and the purified hemoglobin). The principal materials were: iron (III) solution, iron chelators (deferoxamine and deferiprone), and glutathione. Dry-ice acetone bath, hot water bath, centrifuge, UV-VIS spectrophotometer (absorption spectroscope), and disposable PD-10 desalting columns kit.

2.1 Preparation of hemolysate

Plasma was isolated from the entire blood sample for the hemolysate sample preparation by centrifugation at 2000 rpm. RBCs were washed three times with normal saline, 5 ml of distilled water was put into a fresh test tube, and 20 μl of packed RBCs were added to the 5 ml distilled water tube, which was gently mixed. Distilled water promotes RBC hemolysis, which results in the creation of hemolysate.

2.2 Preparation of crude purified hemoglobin

For purified hemoglobin preparation, pRBCs tube was placed in a dry-ice acetone bath for few seconds until the PRBCs freeze and visibly seen as solid. The tube immediately was removed from the dry-ice acetone bath to thaw in the hot water bath. Sudden cooling and thawing three times cause proteins of erythrocyte’s membrane to denature and eventually lead to cell lysis. After the pRBCs are lysed, they will be placed in the PD 10 desalting column. The PD-10 desalting columns are intended for the fast removal of proteins and other big macromolecules from samples [3]. The PD-10 desalting columns are used to capture large-molecular weight chemicals and proteins, particularly glutathione (307.32 g/mol), superoxide dismutase, and catalase. Eluted purified hemoglobin (64,458 g/mol) from the PD-column will be collected in another tube. The working solution of HbA is made with 40μM HbA at pH 7.4 in water.

2.3 Hemoglobin oxidation studies

Prepare stocks of iron, glutathione, deferoxamine, and deferiprone solution and dilute them with distilled water to reach the exact quantities and concentrations required for the experiment. The absorbance spectra of hemoglobin were measured using a UV-VIS spectrophotometer, either in hemolysate or pure form.

“There is no discernible difference in the visible area between the absorption spectra obtained from hemolysate and pure hemoglobin,” writes Horecker (1943). To view the distinctive spectrum activity of oxy-Hb, the oxy-Hb must be scanned in (500–700 nm) wavelength before adding the iron or chelators to analyze their influence on oxyhemoglobin spectra, oxy-Hb has two distinct peaks at 541 and 577 nm. The oxidation of hemoglobin was evaluated by spectrophotometric analysis (500–700 nm); the concentrations of oxy-, met-, and hemichrome hemoglobin were estimated using the Winterbourn technique.

After assessing the effect of iron on oxy-Hb in both models, hemolysate and pure Hb, the iron concentration that causes the most oxidative damage will be chosen for the next tests. Because hemolysate contains its own glutathione, we shall solely evaluate the impact of reduced glutathione on purified Hb. The prepared glutathione solution, as well as precise iron concentrations, will be added. The impact of the iron chelator will then be investigated.

Microsoft Excel was used for statistical analysis. The concentration of oxyhemoglobin was determined using the Winterbourn technique from the absorption spectra. Experiments were carried out in triplicate at each time point, and the scanning data displayed are representative of the outcomes of the experiments.

3. Results and data analysis

Figures 1 and 2 show the findings of spectrum measurements in iron-mediated oxyhemoglobin oxidation on hemolysate with regard to time in minutes. The UV-visible spectra in Figure 1A and B indicate the effect of dose-dependent iron-mediated oxidative damage on oxyhemoglobin in hemolysate during the first 10 minutes of iron addition. Figure 2 is visibly expressing the effect of iron by causing oxidative damage to the oxyhemoglobin content of hemolysate. The hemolysate is losing the normal red pigmentation of the hemoglobin into brown color due to iron effect with different concentrations concerning time (5 minutes).

Figure 1.

UV-visible spectra of dose-dependent iron-mediated oxidative damage on oxyhemoglobin in hemolysate after 10 minutes of iron addition are shown in A. The % oxyhemoglobin derived from the spectral shift with dose-dependent iron-induced hemoglobin oxidation in hemolysate is shown in B.

Figure 2.

Figure demonstrating the effect of iron on hemolysate oxyhemoglobin concentration, which causes oxidative damage. Because of the iron activity in variable levels throughout time, the hemolysate loses its normal red color and turns brown (10 minutes). (1) represents the control, (2) represents 100 μM Fe³⁺, (3) represents 175 μM Fe³⁺, and (4) represents 250 μM.

The results of the spectral measurements in iron-mediated oxyhemoglobin oxidation on purified hemoglobin only with respect to the time that was measured in minutes are shown in Figure 3. The UV-visible spectra shows the effect of dose-dependent iron-mediated oxidative damage on oxyhemoglobin in purified hemoglobin during the first minute of iron addition, where there was no visible change. Figure 4 visibly expresses the effect of iron in causing oxidative damage to the oxyhemoglobin content of purified hemoglobin. There is no visible change in the normal red pigmentation of the hemoglobin of different iron concentrations concerning time (5 minutes). Figure 5 is a graph showing the binding of iron chelators with the iron. As the iron chelators are added to the iron in the cuvette, the color changed. Also, the absorbance is changed indicating a reaction (peaks) between iron and the chelating agent. As shown, the red and black lines, where the iron is bound with chelators are showing noticeable peaks that are indicative of a binding reaction. The maximum absorbance of L1-Fe and DFO-Fe complexes is achieved at 450 to 470 nm and 430 to 460 nm, respectively [4].

Figure 3.

A: UV-visible spectra showing the effect of dose-dependent iron-mediated oxidative damage on oxyhemoglobin in purified hemoglobin after 5 minutes of iron addition. B shows there is no change in spectra after 10 minutes.

Figure 4.

Figure visibly expressing the effect of iron in causing oxidative damage to the oxyhemoglobin content of purified hemoglobin. There is no visible change in the normal red pigmentation of the hemoglobin different iron concentrations with respect to time (5 minutes). (1): the control, (2): 100 μM Fe³⁺, (3): 175 μM Fe³⁺, and (4): 250 μM Fe³⁺.

Figure 5.

The graph depicts the binding of iron chelators to iron; as the iron chelators are added to the iron in the cuvette, the color changes, suggesting a reaction (peaks) between iron and the chelating agent. The red and black lines, where the iron is bonded with chelators, display prominent peaks, indicating a binding response. L1-Fe and DFO-Fe complexes had maximal absorbance at 450 to 470 nm and 430 to 460 nm, respectively [1].

Figure 6 depicts iron-mediated hemoglobin oxidation and the inhibitory impact of DFO and L1 in hemolysate. In A, the addition of 250 µM results in a significant drop in the percentage of oxyhemoglobin, and when the iron chelator DFO is applied, the percentage of oxyhemoglobin is recovered to above 90%. Similarly, in B, a bidentate iron chelator L1 has a similar response, inhibiting iron-mediated hemoglobin oxidation [5].

Figure 6.

A: Fe³⁺-mediated hemoglobin oxidation in hemolysate. Shown is the oxyhemoglobin concentration following the addition of exogenous 250 μM Fe³⁺ and DFO in hemolysate at pH 7.4. Hemoglobin concentration was adjusted to ~ 40 μM heme. B: shows the inclusion of 250 μM Fe3+ and deferiprone L1 a bidentate iron chelator.

Figure 7 depicts the iron-mediated oxidation of pure hemoglobin, with no substantial loss of % oxyhemoglobin in purified hemoglobin solution as compared to hemolysate. However, the mixing of iron and glutathione in pure hemoglobin produced results comparable to hemolysate. Iron chelator DFO inhibits iron and glutathione-mediated hemoglobin oxidation in A, whereas iron chelator L1 inhibits comparable findings in B.

Figure 7.

Reduced glutathione (GSH) has been found to be a mediator of Fe3+-mediated hemoglobin oxidation in crude purified HbA (α2 ß2). The oxyhemoglobin content in HbA solution prepared of crude purified hemoglobin after the addition of exogenous 100 μM Fe³⁺ and 1 mM GSH is shown. In contrast to μ100 M Fe³⁺ alone, no appreciable hemoglobin oxidation occurs. A and B shows DFO and L1 at 100 μM or 1 mM reduced Fe3+ GSH-driven hemoglobin oxidation. The content of hemoglobin was adjusted to 40 M heme.

4. Discussions

Excess bio-reactive iron contributes to the formation of free radicals, which can lead to oxidative damage to the hemoglobin in our red blood cells. This oxidative damage happens as a result of conditions, such as thalassemia and other hemoglobinopathies [6]. In these illnesses, the hemoglobin moiety’s qualitative or quantitative deficiency causes iron to be ejected from the red cell into circulation [2]. Patients with thalassemia or sickle cell anemia receive blood transfusions on a regular basis as a treatment technique, which contributes to the same problem of having excess circulatory iron. This fact was examined in this study. The effect of iron on hemoglobin was investigated in vitro using two study models: hemolysate and pure hemoglobin solution. Winterbourn has previously demonstrated in high-throughput investigations that switching from oxyhemoglobin to methemoglobin leads to the loss of the peaks of typical normal hemoglobin spectra [7] and that is similar to what we have shown here.

From research findings, it is clear that iron has a great impact on the hemoglobin oxidation rate in hemolysate [8]. Upon adding iron in dose-dependent manner (100, 175, and 250 μM) the hemolysate constitution of oxyhemoglobin is reduced. It is also visibly seen that the hemolysate is losing the normal red pigmentation of the hemoglobin into brown color due to the iron effect of different concentrations to time (5 minutes). RBCs have been the most ferruginous cells in the human body, where the single circulating RBC contains ~ 20 mM iron, [9]. With mild hemolysis, loose iron in the body may cause the observed type of oxidative events in our body.

In contrast to hemolysate, iron does not affect purified hemoglobin. Even after 5 minutes, no change has been observed for the oxy-Hb absorbance spectra. Iron, being an oxidizing agent, requires a catalyst to produce free radicals, which are responsible for oxidative damage to hemoglobin. Free radicals are formed by enzymes, such as nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, nitric oxide synthase (NOS), xanthine oxidase (XO), cytochrome P450, cyclo-oxygenase (COX), and lipoxygenase [10]. As evidence, Figure 4 shows that there is no visible change in the normal red pigmentation of the hemoglobin with different iron concentrations with respect to time (5 minutes). However, when glutathione was added, there was a steep decrease in the percentage of oxyhemoglobin as a result of the pro-oxidation effect of glutathione. Similarly, it was proved from Atamna's research that reduced glutathione (GSH) can degrade heme in solution with a pH of 7 [11].

5. Conclusions

These findings might be related to the intra-erythrocytic chemical alterations seen in thalassemic individuals. When there is a quantitative deficiency in the alpha or beta chains, membrane-bound iron becomes free and can react with reduced glutathione, as we have shown. Perhaps, this may be one of the reasons, thalassemic cells further damage and hemolysis, leading to severe anemia in thalassemia [12]. However, iron chelators, such as deferoxamine (DFO) or deferiprone (L1), inhibited the GSH/Fe3+-mediated hemoglobin oxidation damage.

Acknowledgments

This study was supported by “Qatar University internal grant QUUG-CAS- DHS- 15\16-21.” The author acknowledges the support of students Anjud Khamis Al-Mohannadi, Israa Mahmoud Moursi, and laboratory technician Jaufar Pakari.

Author’s notes

The author previously worked at Qatar University.