Investigation of ischemia modified albumin levels in iron deficiency anemia

Introduction

Anemia is currently defined by the World Health Organization (WHO) as a hemoglobin (Hb) level lower than 13 g/dL in men and 12 g/dL in women [1]. Iron deficiency anemia is the most common type of anemia and it affects an estimated 1–2 billion people worldwide. In developing countries, over 50% of pregnant women are anemic, as are 46–66% of children under 4 years old, with one-half attributed to iron deficiency [2], [3].

Human serum albumin, a single chain of 585 amino acids, consists of three structurally homologous, largely helical domains (I, II, and III), and each domain consists of two subdomains, A and B [4]. The first three amino acids in the N-terminus, Asp-Ala-His, serves as a specific binding site for transition metals such as cobalt (II), copper (II), and nickel (II), and the most susceptible region for degradation compared with other regions of albumin. Ischemia, hypoxia, acidosis, and free radical formation can transiently alter the ability of the residues to bind free metal atoms [5], [6], [7]. On the basis of these biochemical changes, Bar-Or et al. described a rapid colorimetric assay method measuring ischemia-induced alterations of the binding capacity of human serum albumin to exogenous cobalt [8]. Ischemia-modified albumin (IMA) has been shown to be a rapidly rising and sensitive biochemical marker especially for the diagnosis of myocardial ischemia [9], [10]. Therefore, we thought that IMA measurement could be useful for the evaluation of hypoxia associated with lower levels of Hb in iron deficiency anemia. The aim of this study was to evaluate the levels of IMA in patients with established iron deficiency anemia before and after 2 months of oral iron supplementation therapy and its correlations with Hb, hematocrit (Htc), mean corpuscular volume (MCV), ferritin, iron (Fe), total iron binding capacity (TIBC) levels.

Materials and methods

In our study iron deficiency anemia was defined as hemoglobin levels lower than 12 g/dL in women as defined by the WHO. IMA, Hb, Htc, MCV, ferritin, Fe, TIBC and albumin levels were measured in 140 female iron deficiency anemia patients before and after 2×100 mg ferrous glisin sulfate (ferro sanol duodenal capsule, Adeka) treatment and in 84 healthy female controls. Iron treatments were given for 2 months and after treatment we observed all patients Hb levels were higher than 12 g/dL.

There were two groups; Group 1 was the “iron deficiency anemia group’’, Group 2; “healthy control group’’. The mean age of anemia group was 30.3±6.6 years (range; 18–44 years). Patients and control group were selected by the internal medicine department and anemic patients were otherwise healthy persons. Patients with malnutrition, obesity, cancer, alcoholism, smoking, diabetes, viral hepatitis, and other ischemic diseases were excluded from the study. Patients were newly diagnosed, menstruating womens and they didn’t receive any treatment before. The mean age of control group was 29.6+6.1 years (range; 19–42 years). All patients gave written informed consent. The study was approved by the local Ethics Committee. Blood samples were collected by venous puncture into vacutainer tubes with gel. Specimens were routinely centrifuged within 1 h of collection for 15 min at 1000 g, and aliquots of serum samples were stored at −80°C for a maximum of 4 weeks before IMA measurement.

Serum ferritin levels were measured by chemiluminescance method (Beckman Coulter, CA, USA) by using Beckman Coulter DXI 800 analyzer. Hb, Htc, MCV were analyzed by Mindray BC5800 otoanalyzer. Serum albumin, Fe, TIBC levels were measured with Olympus AU 2700 otoanalyser.

An albumin-cobalt binding test was used to define serum IMA. The decreased binding capacity of cobalt to albumin was assessed using the rapid colorimetric detection method developed by Bar-Or et al. [8]. Briefly, 200 μL of patient serum was transferred into glass tubes and 50 μL of 0.1% CoCl₂×6H₂O (Sigma-Aldrich, MO, USA) was added. After gentle shaking, the mixture was incubated for 10 min to ensure sufficient cobalt to bind to albumin. Then, 50 μL of 1.5 mg/mL dithiothreitol (DTT) (Sigma-Aldrich, MO, USA) was added as a coloring agent. After 2 min, 1 mL of 0.9% NaCl was added to stop the binding between the cobalt and albumin. A blank was prepared for every specimen. At the DTT addition step, 50 μL of distilled water was used instead of 50 μL of 1.5 mg/mL DTT to obtain a blank without DTT. The absorbances were recorded at 470 nm. [Thermo Scientific Multiskan GO (Finland) is used as a spectrophotometer]. Color formation in specimens with DTT was compared with color formation in the blank tubes, and the results were expressed as absorbance units (ABSU).

Also, the formula IMA value/individual serum albumin concentration was used to maintain the IMA rate (IMAR), in order to avoid the impact of albumin concentration differences between groups.

All data were analysed using the statistical software package SPSS Statistics version 21. Kolmogorov Smirnov test was used for to see the normality of the distributions. Data were expressed as mean±standard deviation. The Student t-test was used to evaluate the difference between groups. Regression analysis was used to evaluate the associations between IMA and Hb, Htc, MCV, ferritin, Fe, TIBC levels. Changes of IMA and IMAR after treatment were evaluated by paired samples t-test, p<0.05 was considered as statistically significant.

Results

Hb, Htc, MCV, Fe and ferritin levels were lower, TIBC levels were higher in the anemia group than the control group (p<0.05). Serum albumin levels were normal in all groups. No significant differences were observed between patients and controls with respect to age and albumin (p>0.05).

The levels of IMA were elevated in the anemia group (0.340±0.082 ABSU) compared to control group (0.291±0.077 ABSU) (p<0.05). The laboratory results of 2 groups were shown in Table 1. After oral iron supplementation therapy IMA increased (0.392±0.080 ABSU), and was higher than the other two groups (p<0.05). Also the results of iron deficiency anemia group before and after oral iron treatment were shown in Table 2.

We made regression analysis showing IMA and Hb, Htc, MCV, Fe, TIBC, ferritin relationships in groups. We also observed significant negative correlations between IMA and Hb (r=−0.342, p=0.045), Htc (r=−0.382, p=0.024) in the iron deficiency anemia group. There were no other correlations between IMA and other parameters.

According to albumin levels of groups the IMA values were adjusted with serum IMA/albumin ratio (IMAR). Also, IMAR levels were higher in anemia patients (0.085±0.024) than the control group (0.064±0.022) significantly (p<0.05). After anemia treatment IMAR levels increased like IMA significantly (0.092±0.025) (p<0.05).

Discussion

The results of the present study indicate that IMA increases in patients with iron deficiency anemia and this could be associated with hypoxia due to low Hb levels. Also according to our findings, oral iron theraphy may be associated with oxidative stress and various iron supplementation strategies must be evaluated by clinicians.

Reactive oxygen species (ROS) resulting from conditions such as ischemia, hypoxia, acidosis, free radicals, and free iron can decrease the ability of the N-terminus of albumin to bind with transition metals [11], [12], [13]. Recent studies have reported increased levels of IMA in conditions other than ischemic heart disease including diabetes mellitus, hyperlipidemia, chronic renal disease, obesity, and others [14], [15], [16], [17], [18], [19], [20]. But there was no study investigating IMA in iron deficiency anemia patients before and after iron therapy.

The increase of IMA in anemic patients could be attributed to mild hypoxia and this hypoxia is responsible for the alteration in metal-albumin binding during anemia [13], [21]. The reduction of Hb levels could change the tissue oxygen delivery. There are also reports suggesting a role for Hb -induced variations in arterial oxygen content [22], [23].

It is logical that IMA is negatively correlated with albumin concentrations, since a serum sample with a low albumin concentration would appear to have an increased IMA level. Fewer albumin molecules would be present to bind the standardized amount of cobalt added during the cobalt binding assay, resulting in an increased amount of free cobalt. The negative correlation between the two factors was found to be strongest when hypoalbuminemia was present, a fact that could limit the utility of the cobalt binding assay. To combat this limiting factor, several authors have proposed correction formulas or ratios to normalize a sample for the albumin concentration. IMA-to-serum albumin ratio (IMAR) was calculated by dividing IMA by the concentration of albumin in g/dL [24], [25], [26], [27]. When we normalized our results, we saw that IMAR levels were higher in anemia patients than the control group significantly. After anemia treatment IMAR levels were highest like IMA.

The amount of iron must remain in balance in the body. Iron also plays a role in the occurence of oxidative damage. The oxidative free radicals during ischemia lead to chemical changes in the albumin molecule and IMA may be the earliest marker of ischemia [28].

We expected to find lower IMA levels in the anemia group after iron therapy but we found significantly higher values compared to other groups. Our hypothesis about the higher IMA values after iron therapy was that, iron is an oxidant element and oral iron supplementation may be associated with oxidative stress and may change the albumin molecule resulting higher IMA values. Roy et al. mentioned that, recent in vitro studies have demonstrated that generation of hydroxyl radicals (•OH) by the Fenton reaction was associated with a rapid rise of IMA concentration [13]. Also, the increase in the iron concentrations at the constant albumin concentration might yield increased free cobalt ions and consequently increased absorbance values [29]. Knutson et al. conclude that correction of iron deficiency with daily iron supplements results in increased lipid peroxidation in rats and these effects are mitigated by intermittent iron supplementation [30]. Perhaps, in our therapy group IMA might be lower in case of lower dosage administrations or intermittant iron supplementations. Tiwari et al. found that; oral iron supplementation with 100 mg/day as ferrous sulfate leads to oxidative imbalance in anemic women [31]. Also Kumar et al. [32] reviewed that oxidative stress worsened with anemia in most of the animal and human studies, status improved with iron in some animal and human studies, however majority of studies found it increased [33], [34], [35], [36], [37]. They concluded that there is a need of large, good quality trials assessing clinical outcomes of various iron supplementation strategies with respect to beneficial/adverse effects and oxidative stress status [38]. The exact mechanism of this still need to be elucidated. There are arguments about if the IMA can be a new marker or not in oxidative damage which is caused by elevated iron levels [39].

Also we found negative correlations between IMA and Hb, Htc in the anemia group. Şeker et al. found no significant difference between mild to severe anemia groups in patients with low Hb values due to blood loss [40]. In the study of Kurtulmuş et al. any relationship between iron levels and IMA could not determined [28]. But, Cichota et al. found significant correlations between IMA and Hb like us, in anemia patients associated to chronic kidney disease [17]. Awadallah et al. found that, iron-driven oxidative stresses in association with chronic anemia and hypoxia, are the most likely causes that lead to the modification of the N-terminus of serum albumin and thus the formation of increased levels of IMA in thalassemic patients [39]. So we thought that IMA can be demonstrative of the severity of anemia.

In summary, we have shown that IMA increase during anemia and this elevation could be associated with hypoxia and we saw that the levels of IMA were associated with the severity of anemia. For the possible oxidative stress resulting from oral iron supplementation various treatment strategies and antioxidant administration recommendations must be evaluated. To our knowledge, this is the first study investigating IMA in iron deficiency anemia patients before and after iron therapy. The limitation of this study is the small number of patients, therefore further studies are required to understand the mechanisms leading to increase of IMA in anemia.