Perturbations of serum electrolyte levels in iron deficiency anemia - A comparative analysis.
Anemia is the most common blood disorder, affecting about a third of the global population. Anemia is responsible for about 800,000 deaths per year worldwide. Nutritional anemia is of more concern in the developing countries having high prevalence rate due to dietary iron deficiency (ID).  Approximately one-third of patients with anemia exhibit ID.  The annual incidence rate of ID anemia (IDA) is 7.2-13.96 per 1000 people per year. 
Iron is vital for human health and defects in iron homeostasis result in serious pathologic abnormalities such as hemochromatosis and anemia.  ID is the world's most widespread nutritional disorder, irrespective of age, gender, and socioeconomic status, affecting both industrialized and developing countries. In ID, there is insufficient iron to maintain normal physiologic functions. (5) IDA, the most common type of anemia, is an important public health problem because of its complications. 
Electrolytes such as sodium ([Na.sup.+]), potassium ([K.sup.+]), chloride ([Cl.sup.-]), and bicarbonate (HCO[3.sup.-]) Comprise about 1% of plasma in blood. They are essential for intermediary metabolism and play an important role in controlling fluid levels, nerve conduction, acid-base balance, blood clotting, and muscle contraction. The balance of electrolytes in the body is necessary for the normal functioning of cells and organs and also for the maintenance of red blood cell (RBC) shape, O2/CO2 exchange between red cells and tissues.  The calcium-sensitive [K.sup.+] channels regulate the process of erythrocyte apoptosis. 
The red cell membrane-bound [Na.sup.+][K.sup.+]ATPase play a principal role in the regulation of intra- and extra-cellular cationic homeostasis.  Regulation of these ions is primarily achieved by the Na+/K+ pump, fuelled by ATP energy of metabolism. ATPase, found in RBC membranes, activates the stored ATP energy to change the conformation of membrane proteins which exchange three [Na.sup.+] ions for two incoming K+ ions - an exchange which opposes the ions' electrochemical gradients. This example illustrates the necessity of energy use in actively transporting key cations between the body compartments to maintain vital functions of the circulatory system. 
Previous studies have stated that there is an elevation of [Na.sup.+][K.sup.+]ATPase activity in the primary anemia patients. This elevation may compensate the mechanism for adaptation of the patients with low oxygen and its physiological role in the cell. 
In IDA, the activity of red cell membrane-bound [Na.sup.+][K.sup.+]ATPase is altered, affecting level of serum sodium and potassium levels. [3,11,12] Furthermore, few studies have reported changes in serum electrolyte levels in sickle cell anemia and[11,13-18] [beta]-thalassemia major. [11,19]
Our interest in the distribution of serum electrolytes in IDA was stimulated by the observation of contradicting reports and also due to paucity of studies done relating the alterations of serum electrolyte levels in anemia. Hence, we aimed to evaluate the alterations in serum electrolyte levels in patients with IDA and comparing it with healthy subjects without anemia.
MATERIALS AND METHODS
This was a descriptive analytical cross-sectional study carried over a period from July 2016 to July 2017 in SRM Medical College Hospital and Research Centre, Institutional Ethical Clearance obtained. Totally, 300 subjects aged more than 20 years of both genders (163 with IDA and 137 without anemia) were included in this study. Their blood samples were analyzed for hemoglobin (Hb), RBC, hematocrit (HCT), mean corpuscular volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), peripheral smear, serum iron, and ferritin levels. Medical history was recorded.
The anemic patients were selected based on their Hb levels (Hb <13 g% in males and <12 g% in females) based on definition of the World Health Organization.  Moreover, those with predominantly microcytic red cell indices (MCV <76 fl), hypochromic red cell indices (MCH <27 pg/cell and MCHC <32 g/dl), and on their peripheral smear (microcytic hypochromic) were considered to have IDA which was confirmed by low serum iron (<59 [micro]g/dl in males and <37 ug/dl in females) and low serum ferritin (<15 ng/ml in males and <9 ng/ml in females). Patients suffering from kidney diseases, thyroid disorders, infectious disease, chronic systemic inflammatory disorders, and pregnant woman were excluded from the study.
Hb, RBC, HCT, MCV, MCH, and MCHC were estimated by SYSMEX XT-1800i analyzer. Serum ferritin (Bio-Rad Quanimune Ferritin IRMA, Bio-Rad lab) and serum iron (TPTZ method). Serum analysis for electrolytes was performed by ISE--direct method using Medica EasyLyte automatic analyzer.
The data are presented as mean [+ or -] standard error of the mean for continuous variables. A Student's t-test was applied for comparison of group means. Pearson's coefficient was calculated to determine correlation between two variables. P < 0.05 was considered statistically significant.
Our study results show a statistically significant difference (P = 0.0001 (**)) in mean Hb levels in patients with IDA and without anemia (10.5 [+ or -] 0.13 g/dl and 14.2 [+ or -] 0.12 g/dl, respectively). The mean RBC count, HCT, MCV, MCH, MCHC in patients with IDA, and without anemia were 3.5 [+ or -] 0.04, 31.2 [+ or -] 0.43, 72 [+ or -] 0.9, 24 [+ or -] 0.20, 29.6 [+ or -] 0.25 and 4.9 [+ or -] 0.04, 40.4 [+ or -] 0.34, 81.2 [+ or -] 0.75, 28.6 [+ or -] 0.18, and 34.1 [+ or -] 0.16, and the difference was statistically significant (P = 0.0001 (**)). The mean serum iron and ferritin levels in patients with IDA and without anemia were 32.68 [+ or -] 0.71, 10.06 [+ or -] 0.79 and 75.26 [+ or -] 0.79, and 45.19 [+ or -] 1.11 (P = 0.0001 (**)). These data are presented in Table 1.
In this study, we observed that serum sodium levels were significantly lower (P = 0.0001 (**)) and serum potassium (P = 0.0001(**)) as well as chloride levels (P < 0.05) were significantly higher in patients with IDA when compared with individuals without anemia. However, there was no significant difference in serum bicarbonate levels between patients with IDA and without anemia [Table 2].
In patients with IDA, we observed a significant positive correlation between serum sodium levels with all red cell indices except MCV and also between serum bicarbonate levels with Hb, RBC count, HCT, and MCV. We also observed a significant negative correlation between serum potassium levels with Hb, RBC count, HCT, and MCHC and also between serum chloride levels with Hb and HCT.
In individuals without anemia, we observed a significant positive correlation between serum sodium levels with MCV, but there was no significant correlation with Hb, RBC count, HCT, and MCV, and we found a significant negative correlation between serum bicarbonate levels with RBC count. There was no significant correlation between other electrolytes with red cell indices [Table 3].
We analyzed and compared serum electrolyte levels with various degree of anemia and found it was highly significant between serum sodium and potassium levels as the severity of anemia worsens when compared with chloride and bicarbonate levels [Table 4].
In this study, we observed that majority 50.4% of cases without anemia had normal serum sodium levels. Most of the patients with mild anemia had normal (53.7%) and mild hyponatremia (31.5%) and those with severe anemia had severe hyponatremia. Hence, there was a statistically significant association between severity of anemia and hyponatremia (P = 0.0001 (**)) [Table 5].
IDA and electrolyte imbalance are widely prevalent problems in the Indian population. Although few studies, both in animals  and in humans  have linked IDA with altered serum electrolyte levels, the relationship between them has long been a topic of debate in the literature. Since IDA is recognized as major public health problem, the suggested relationship between the two assumes clinical importance. Moreover, the number of published studies investigating electrolyte alterations in IDA is relatively low, and the available data of the results of these studies are inconsistent. Some of these studies show that electrolyte parameters in iron-deficient patients are higher in value than those of healthy control patients, whereas others indicate lower levels in ID. [13,18,19]
In our study, we evaluated the alterations in serum electrolyte levels in 163 patients with IDA and compared it with 137 healthy subjects without anemia. Our study has several important findings. In our study, the serum sodium levels were significantly lower in patients with IDA when compared to individuals without anemia, and also we found a significant positive correlation between serum sodium levels with all red cell indices except MCV and a statistically significant association between severity of anemia and hyponatremia which is in accordance with results of Shraf et al.  and Agoreyo and Nwanzen.  We observed that serum potassium levels were significantly high in IDA patients when compared to individuals without anemia and a significant negative correlation between serum potassium levels with Hb, RBC count, HCT, and MCHC which coincides with results of Agoreyo and Nwanzen  but contrasts with study results of Shraf et al.  who observed lower serum levels of potassium in patients with IDA. We observed a significantly higher level of serum chloride in IDA patients when compared with subjects without anemia and a significant negative correlation between serum chloride level with Hb and HCT.
Previous studies have stated that normal red cells have high level of intracellular potassium and low level of sodium within the extracellular environment. On the other hand, the level of potassium is low in the extracellular environment while that of sodium is high. [Na.sup.+] and [K.sup.+] ions are restricted to their compartment but can penetrate the cellular membrane through [Na.sup.+][K.sup.+]ATPase pumps. The red cell [Na.sup.+][K.sup.+]ATPase is a ubiquitous enzyme and plays a central role in the regulation of intra- and extra-cellular cationic homeostasis.  Several studies have shown variation in the erythrocyte membrane [Na.sup.+][K.sup.+]ATPase activity was modulated by the changes in the differences resulting from hematological disorders.  Furthermore, an increase in red cell membrane permeability to sodium or potassium has been described in a variety of red cell disorders. Previous studies have stated that [Na.sup.+][K.sup.+]ATPase activity is higher in the primary anemia patients. This elevation may compensate the mechanism for adaptation of the patients with low oxygen and its physiological role in the cell. 
Several researchers reported that in IDA, the activity of red cell membrane-bound [Na.sup.+][K.sup.+]ATPase is altered and due to this altered ATPase activity, serum sodium, and potassium levels are affected. [3,11,12] Salsbury et al. stated that our dietary iron absorption has been mediated by potassium voltage-gated channel subfamily E, member 2 (KCNE 2) which is single-pass integral membrane [beta] subunit of a potassium ion channel. The disruption of this regulatory subunit may also alter the levels of potassium in IDA.  This implies that IDA and serum potassium levels are interrelated with each other.
Studies have revealed that electrolyte level alterations also occur in other types of anemia like sickle cell anemia and [beta]-thalassemia. The suggested mechanisms were as follows. Rhoda et al. proposed that in sickle cells, an abnormal activation of potassium chloride ([K.sup.+] [Cl.sup.-]) cotransport system was involved in cell potassium ([K.sup.+]) loss and dehydration. Furthermore, he mentioned that potassium chloride ([K.sup.+] [Cl.sup.-]) cotransport is abnormally active in erythrocytes containing positive charged Hb such as Hb (S) (HbS).  Deoxygenation is known to increase cation permeability of sodium ([Na.sup.+]), Potassium ([K.sup.+]), and calcium (C[a.sup.2+]) in sickle cells.  Luthra and Sears proposed that in [beta]-thalassemia patients, oxidative damage induced by free globin chains has been implicated in the pathogenesis of the membrane abnormalities. He also mentioned that [Na.sup.+], [K.sup.+] pump was reduced in thalassemia-like cells, whereas it was increased in severe alpha- and beta-thalassemia cells. Thus, oxidative damage causes increased activity of [K.sup.+], [Cl.sup.-] cotransport, and hence, [K.sup.+] loss in beta-thalassemia erythrocytes.  Karim et al. suggested that an increased sodium level in [beta]-thalassemia patients may be due to renal damage resulting from iron overload. 
Strengths and Limitations
Although studies have reported alterations in serum electrolyte levels in anemia, the results of our study indicated that there was a direct correlation between electrolytes and IDA, hence revealing and adding further evidence to the fact that iron plays a pathogenic role contributing to the maintenance of electrolyte balance in our body. Few limitations of this study were small sample size and with this cross-sectional design, we could not evaluate the mechanism by which IDA affects serum electrolytes and also, we could not follow-up the electrolyte levels after iron therapy. Hence, further multicentric research with larger same size is warranted for accurate assessment of the association of anemia and electrolyte imbalance.
In summary, findings of the present study revealed a significant difference in serum electrolytes, especially sodium, potassium, and chloride levels between patients with IDA and without anemia.
Serum electrolyte levels are altered in patients with IDA. Based on our results, we suggest that IDA patients should be monitored closely for their electrolyte levels to avoid complications and better management. The impact of this study might have been improved by studying the electrolyte levels following iron replacement therapy.
I would like to thank our Dean Dr. A Sundaram, SRM Medical College Hospital and Research Centre, for his support throughout the study. Am thankful to all the participating people for their cooperation.
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Source of Support: Nil, Conflict of Interest: None declared.
Lavanya Rajagopal (1), Vinothkumar Ganesan (2), Saleh Mohammed Abdullah (3), Sundaram Arunachalam (1), Kumaresan Kathamuthu (4), Balaji Ramraj (5)
(1) Department of Pathology, SRM Medical College Hospital and Research Centre, Kattankulathur, Chennai, Tamil Nadu, India, (2) Department of Medical Research, SRM Medical College Hospital and Research Centre, Kattankulathur, Chennai, Tamil Nadu, India, (3) Department of Medical Laboratory, Faculty of Applied Medical Sciences, Jazan University, Jazan, Saudi Arabia, (4) Department of Pathology, Jazan University, Jazan, Saudi Arabia, (5) Department of Community Medicine, SRM Medical College Hospital and Research Centre, Kattankulathur, Chennai, Tamil Nadu, India
Correspondence to: Lavanya Rajagopal, E-mail: email@example.com
Received: September 18, 2017; Accepted: October 12, 2017
Table 1: Comparison of red cell indices between anemic IDA and not anemic individuals RBC indices Mean[+ or -]SEM t P Anemic (IDA) Not anemic n=163 n=137 HB 10.5[+ or -]0.13 14.2[+ or -]0.12 19.8 0.0001 (**) RBC count 3.5[+ or -]0.04 4.9[+ or -]0.04 21.7 0.0001 (**) HCT 31.2[+ or -]0.43 40.4[+ or -]0.34 16.2 0.0001 (**) MCV 72[+ or -]0.9 81.2[+ or -]0.75 7.7 0.0001 (**) MCH 24[+ or -]0.20 28.6[+ or -]0.18 16.3 0.0001 (**) MCHC 29.6[+ or -]0.25 34.1[+ or -]0.16 14.4 0.0001 (**) (*)Significant (P<0.05), (**)Highly significant (P=0.0001). IDA: Iron deficiency anemia, RBC: Red blood cell, HB: Hemoglobin, HCT: Hematocrit, MCV: mean corpuscular volume, MCH: Mean corpuscular hemoglobin, MCHC: Mean corpuscular hemoglobin concentration, SEM: Standard error of the mean Table 2: Comparison of electrolyte levels between anemic (IDA) and not anemic individuals Electrolyte Mean[+ or -]SEM t P Anemic (IDA) Non Anemic n=163 n=137 Sodium 130.04[+ or -]0.55 134.05[+ or -]0.37 -6.038 0.0001 (**) Potassium 4.55[+ or -]0.06 4.17[+ or -]0.05 5.167 0.0001 (**) Chloride 104.31[+ or -]0.50 102.59[+ or -]0.46 2.522 0.012 (*) Bicarbonate 23.45[+ or -]0.36 23.61[+ or -]0.35 -0.329 0.743 (*) Significant. (P<0.05), (**) Highly significant (P=0.0001). IDA: Iron deficiency anemia Table 3: Correlation between serum electrolytes and red cell indices levels among anemic (IDA) and not anemic individuals Study group Electrolyte Hb RBC r P r P Anemia (IDA) Sodium 0.427 0.0001 (**) 0.165 0.036 (*) Potassium -0.412 0.0001 (**) -0.212 0.007 (*) Chloride -0.222 0.004 (*) -0.037 0.641 Bicarbonate 0.232 0.003 (*) 0.217 0.006 (*) Not anemia Sodium -0.042 0.630 -0.135 0.116 Potassium 0.036 0.674 0.127 0.142 Chloride -0.146 0.088 -0.017 0.847 Bicarbonate -0.118 0.170 -0.179 0.037 (*) Study group Electrolyte HCT MCV r P r P Anemia (IDA) Sodium 0.406 0.0001 (**) 0.162 0.058 Potassium -0.374 0.0001 (**) -0.043 0.622 Chloride -0.210 0.007 (*) 0.078 0.365 Bicarbonate 0.262 0.001* 0.281 0.001 (*) Not anemia Sodium -0.058 0.503 0.292 0.0001 (**) Potassium 0.051 0.554 -0.136 0.083 Chloride -0.055 0.526 -0.083 0.295 Bicarbonate -0.075 0.385 -0.046 0.564 Study group Electrolyte MCH MCHC r P r P Anemia (IDA) Sodium 0.173 0.027 (*) 0.196 0.012 (*) Potassium 0.019 0.814 -0.177 0.024 (*) Chloride 0.144 0.066 -0.014 0.859 Bicarbonate 0.026 0.739 0.035 0.660 Not anemia Sodium 0.110 0.200 0.002 0.981 Potassium -0.060 0.484 0.012 0.892 Chloride -0.077 0.371 0.130 0.130 Bicarbonate 0.125 0.146 -0.050 0.559 (*) Significant. (P<0.05), (**) Highly significant (P=0.0001). IDA: Iron deficiency anemia, RBC: Red blood cell, HB: Hemoglobin, HCT: Hematocrit, MCV: Mean corpuscular volume, MCH: Mean corpuscular hemoglobin, MCHC: Mean corpuscular hemoglobin concentration Table 4: Comparison of serum electrolyte levels with grades of anemia Anemia grade n Mean[+ or -]SEM Sodium Potassium Mild 54 134.76[+ or -]4.33 4.06[+ or -]0.56 Moderate 87 128.28[+ or -]6.86 4.74[+ or -]0.70 Severe 22 125.41[+ or -]6.87 4.97[+ or -]0.66 t 25.788 23.850 P 0.0001 (**) 0.0001 (**) Anemia grade Mean[+ or -]SEM Chloride Bicarbonate Mild 102.72[+ or -]6.36 23.91[+ or -]3.70 Moderate 104.74[+ or -]6.17 24.02[+ or -]4.45 Severe 106.50[+ or -]6.95 20.05[+ or -]5.58 t 3.201 7.650 P 0.043 0.001 (*) (*) Significant.(P<0.05), (**) Highly significant (P=0.0001), SEM: Standard error of the mean Table 5: Relationship between serum sodium levels and grades of anemia Anemia grade Serum sodium levels n (%) Normal Mild hyponatremia Normal (n=137) 69 (50.4) 49 (35.8) Mild anemia (n=54) 29 (53.7) 17 (31.5) Moderate anemia (n=87) 21 (24.1) 9 (10.3) Severe anemia (n=22) 4 (18.2) 2 (9.1) Anemia grade Serum sodium levels n (%) Moderate hyponatremia Severe hyponatremia Normal (n=137) 14 (10.2) 5 (3.6) Mild anemia (n=54) 7 (13.0) 1 (1.9) Moderate anemia (n=87) 20 (23.0) 37 (42.5) Severe anemia (n=22) 3 (13.6) 13 (59.1) Chi-square: 106.958, (**) P: 0.0001, (*) Significant
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|Title Annotation:||RESEARCH ARTICLE|
|Author:||Rajagopal, Lavanya; Ganesan, Vinothkumar; Abdullah, Saleh Mohammed; Arunachalam, Sundaram; Kathamuth|
|Publication:||National Journal of Physiology, Pharmacy and Pharmacology|
|Date:||Mar 1, 2018|
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