Study the Correlation of Red Blood Cell Indices and Electrolytes Imbalance in Anemic Patients in Ibb City, Yemen

Introduction

Anemia normally results from various etiologies, including decreased red blood cell (RBC) production (bone marrow diseases, chronic kidney disease, and autoimmune diseases), blood loss (bleeding or increased RBC destruction (haemolysis), poor RBC maturation, and nutritional deficiencies (iron, folate, or vitamin B12).^1–4

Electrolytes are electrical molecules required for metabolic homeostasis and the body’s vital reactions. Electrolytes support cell membrane functions, muscle contraction, neuron impulses, bone structure, hormone action, maintaining fluid balance, and regulating pH levels (acid-base balance).^5,6 Similarly, an imbalance of electrolytes happens when it is level abnormally higher or lower than the normal value as a result of a variety of physiological and pathological factors, including nutritional situation, acid-base imbalances, medications, acute sickness, or other co-morbid diseases such as renal disorder.^6–8 Anemia is a hematic disorder in which hemoglobin (Hb) levels are under normal values determined by a large and diverse global population.^9,10 Anemia is a global public health problem that are effects 60% of the population in low, and middle-income countries.⁹

Electrolytes such as potassium (K⁺), sodium (Na⁺), chloride (Cl⁻), and bicarbonate (HCO₃) comprise approximately 1% of the plasma in the circulatory system. They play a central role in blood clotting. The electrolytes balance is essential for maintaining the RBC’s shape and function and exchanging O₂/CO₂ between RBC and tissues.^11,12 Moreover, the channels of calcium (Ca²⁺)-sensitive potassium (K⁺) regulate RBC apoptosis. Sodium potassium ATPase (Na-K ATPase) regulates intracellular and extracellular RBC cationic homeostasis by binding to the cell membrane.^11,13,14 Na-K ATPase-bound membrane plays a vital role in maintaining the circulatory system by modifying membrane proteins, leading to the exchange of two ions of K⁺with three ions of Na⁺,^11,14 which directly affects the serum K⁺and Na⁺.^11,15 Nonetheless, there is little data on the association between RBC indices and electrolyte levels. The study objective was to study the correlation between RBC indices and electrolyte levels in anemia, Ibb City, Yemen.

Materials and Methods

Study Population

The population source consisted of 200 anemic patients (cases) and 200 healthy persons (control) who visited several hospitals in Ibb City, Yemen (26^th July 2023 to 30^th March 2024). The healthy control group was selected to match the cases in terms of gender, and the clinical baseline for both cases and controls remained homogeneous and free from confounding comorbidities. Amulticenter prospective case-control study conducted in these hospitals: Al-Thaurah General Hospital, Al-Manar Hospital, Al-Noor Hospital, Al-Badr Hospital, Al-Hekma Specialized Clinic, Saeed Thabet Nasher Specialized Centre and Dr. Fares Almareh Medical Centre. Anemic patients were newly diagnosed anemic treatment-naïve patients and evaluated based on Hb values (Hb<12 g% in females and <13 g% in males) according to the World Health Organization (WHO) definition.^12,14

Participants were assessed physically by physicians, and a medical questionnaire was administered to both study groups to detect exclusion criteria. These criteria included diseases of the heart, liver, and kidneys, as well as fever, diarrhea, vomiting, diabetes, bleeding disorders, allergic conditions, skin rashes, high blood pressure, edema, inflammatory disorders (such as tonsillitis, arthritis, or appendicitis). Additionally, individuals taking aspirin or medications for diuretics, hypertension, or diabetes, as well as those who had received or been transfused blood within the last 12 months, were also excluded. For women, the exclusions included those who were pregnant, breastfeeding, experiencing a miscarriage, menstruating, or had menstruation lasting more than seven days.

Sampling Methods

Participants were interviewed using a questionnaire translated into Arabic, the native language of Yemen. The collected data included participant number, age, weight, gender, residence, education status, socioeconomic status, and other relevant information. Approximately 5 mL of blood was collected from anemic patients and healthy individuals, then divided into two tubes: 2.5 mL was mixed with anticoagulant (EDTA-K3) (Jiangsu Xinkang Medical Instrument Co., Ltd, Jiangsu, China) for the measurement of red blood cell (RBC) indices directly, while the remaining 2.5 mL was placed in test tubes without anticoagulant, and then then the serum was isolated directly by centrifugation and analysis of electrolytes (Na⁺, Ca²⁺, Mg²⁺, K²⁺, and Cl⁻).

RBC Indices Tests

RBC indices tests were performed using the DF52 Dymind fully automatic hematological analyzer (Dymind Biotech Co., LTD, Shenzhen, China). A total of 15μL of whole blood sample with EDTA was aspirated into the open vial of the analyzer. The indices measured included hemoglobin (Hb) level, red blood cell (RBC) count, packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC).

Electrolyte Test

The blood sample was coagulated at room temperature, then centrifuged for 5 minutes at 3500 rpm to separate the serum. Electrolyte levels, including Na⁺, Ca²⁺, K²⁺, and Cl⁻ were measured using ion-selective electrode technology by the GE300 Genrui Electrolyte Analyzer (Genrui Biotech Inc., Shenzhen, China). The serum magnesium (Mg²⁺) level was determined using flame atomic absorption photometry (Quimica Clinica Aplicada S.A., Amposta, Spain). The reference limit ranges for the electrolytes are as follows: Na⁺: 135–148 mmol/L, Mg²⁺: 0.78–1.03 mmol/L, K²⁺: 3.5–5.3 mmol/L, Ca²⁺: 2.15–2.55 mmol/L, and Cl⁻: 98–107 mmol/L. Electrolyte values below and higher than the limit were classified as hypo-electrolytemia, hyper-electrolytemia, respectively.

Data Analysis

The obtained data were recorded in Microsoft Excel spreadsheet, and statistical analyses were performed using GraphPad Prism version 8.0.1 (GraphPad Inc., USA). ROUT (Q=1%) method was used to o identify outlier values. Kolmogorov–Smirnov and Shapiro–Wilk normality tests and skewness were conducted to check the normality of continuous variables. Mann–Whitney U-tests for abnormally distributed data were applied to determine the difference in medians. Unpaired t-tests were applied to compare means. The associations between electrolyte imbalance and anemia were detected using odds ratio (OR) with 95% Confidence Interval (CI), while Fisher’s exact test was used to detect statistically significant associations. Pearson’s coefficient was estimated to demonstrate a correlation between two variables. P values ≤0.05 were considered statistically significant.

Results

Sociodemographic Characteristics

This study included 200 anemic patients and 200 healthy individuals as control groups. This study included 200 anemic patients and 200 healthy individuals as control groups. Of the anemic patient group, 54 (27%) ranged from 1 year to 10 years, 49 (24.50%) were aged 11–20 years, 41 (20.50%) were aged 21–30 years, 30 (15%) were aged 31–40 years, 16 (8%) were aged 41–50 years and 10 (5%) were aged >50 years. Of the healthy control group, 34 (17%) were aged 1–10 years, 56 (23%) were aged 11–20 years, 67 (33.5%) were aged (21–30 years), 29 (14.5%) were aged 31–40 years, 9 (4.5%) were aged 41–50 years and 5 (2.5%) were aged >50 years. Regarding the weight, 51 (25.5%), 42 (21%), 75 (37.5%), and 32 (16%) of anemic patients were weighed 5–20 kgs, 21–40 kgs, 41–60 kgs, and >60 kgs, respectively. Moreover, 22 (11%), 38 (19%), 89 (44.5%), and 51 (25.5%) of the control group were weighed 5–20 kgs, 21–40 kgs, 41–60 kgs, and >60 kgs, respectively. Concerning gender, the anemic group comprised 95 females (47.5%) and 105 males (52.5%), while the control group included 99 females (49.5%) and 101 males (50.5%). Regarding residence, 124 participants (62%) from the anemic group resided in urban areas, while 76 (38%) were resided in rural areas. In control group, 183 participants (91.5%) were urban, and 17 (8.5%) resided in rural areas. Likewise, based on the daily behaviors, 7 (3.5%), 7 (3.7%), and 76 (38%) of anemic participants had smoking, tobacco chewing, and qat chewing habits. In comparison, 16 (8%), 3 (1.5%), and 75 (37.5%) of control participants had smoking, tobacco chewing, and qat chewing habits. Based on educational status, 74 (37%) and 78 (39%) of the anemic and control participants had a primary school level, respectively (Table 1). The analysis revealed significant differences in weight, residence, educational status, and smoking habits between the two groups.

Table 1 Sociodemographic Characteristics of Participants

Comparison of RBC Indices Between Anemic Group and Control Group

As illustrated in Table 2, the investigation revealed significant declines in Hb, RBC, MCV, HCT, MCH, and MCHC indices among anemic patients compared to healthy controls. The mean and SD of Hb, RBC, MCV, HCT, MCH, and MCHC in anemic patients were 8.369±1.888, 3.365±1.034, 77.36±11.40, 25.89±6.082, 25.63±4.764, and 32.84±2.278, respectively. Additionally, the mean and SD of Hb, RBC, MCV, HCT, MCH, and MCHC among the control group were 13.79±1.733, 5.020±0.6822, 82.97±5.937, 41.46±5.262, 27.83±1.994, and 33.32±1.384, respectively.

Table 2 Comparison of RBC Indices Between Anemic Group and Control Group

Comparison of Electrolyte Level Among Anemic and Control Groups

In anemic patients, the median and SD of sodium, potassium, chloride, calcium, and magnesium were 136.0±5.914, 3.819±0.4587, 106.3±4.886, 2.018±0.1853, and 1.896±0.4904, respectively. Median and SD of sodium, potassium, chloride, calcium, and magnesium were 136.8±4.723, 3.937±0.4392, 104.7±4.677, 2.117±0.1701, and 1.991±0.3575 in the control group, respectively. Potassium, calcium, and magnesium were significantly lower in the anemic group compared to the control group. However, the chloride was significantly higher in the anemic group, while sodium levels did not show a significant difference between the two groups (Table 3).

Table 3 Comparison of Electrolyte Level Among Anemic and Control Groups

Association of Electrolyte Imbalance with Anemia

The outcomes in Table 4 revealed that the odds ratio (OR) were showed a strong association of hypo-electrolytemia with anemia, including hypo-magnesemia (OR=4.928, 95% CI: 2.278–10.47), hypo-kalemia (OR=2.967, 95% CI: 1.551–5.581), and hypo-calcemia (OR=2.833, 95% CI: 1.887–4.236), which was significantly higher in anemic patients compared to controls. In contrast, the OR of hyponatremia did not show a significant difference between the two groups (OR =1.438, 95% CI: 0.9522–2.143). Nonetheless, hyper-electrolytemia did not demonstrate a significant difference between anemic patients and healthy controls in hyper-kalemia (OR=0000, 95% CI: 0.000–1.311), hyper-chloremia (OR=1.585, 95% CI: 1.007–2.476), and hyper-magnesemia (OR=1.536, 95% CI: 0.7678–3.278). Notably, hyper-natremia was identified in the patient group, with no existence in controls, resulting in an infinite odds ratio (OR=∞, 95% CI: 0.5262-∞).

Table 4 Association of Electrolyte Imbalance with Anemia

Correlation of RBC Indices and Electrolytes Levels Among Anemic and Control Groups

In the anemic patients, Na⁺ showed positive correlations with Hb (r=0.2219, P=0.0017), RBC (r=0.1483, P=0.0375), and HCT (r=0.2114, P=0.0029). Ca²⁺ has positive correlations with Hb (r=0.2076, P=0.0035), RBC (r=0.2320, P=0.0011), and HCT (r=0.1803, P=0.0119), while it has a negative correlation with MCV (r=−0.1397, P=0.0500). Likewise, Mg²⁺ showed positive correlations with MCV (r=0.1946, P=0.0059) and MCH (r=0.1865, P=0.0082), and a negative correlation with RBC (r=−0.1936, P=0.0061). Cl⁻ was only shown a negative correlation with Hb (r=−0.1366, P=0.0500). Furthermore, K⁺ did not show a correlation with RBC indices.

In the control group, though there was no significant correlation between K+ and Mg2+ levels and RBC indices (Hb, RBC, MCV, HCT, MCH, and MCHC), as presented in Table 5, Na+ showed positive correlations with MCV (r=0.1731, P=0.0143), Cl- showed a negative correlation with MCV (r=−0.1478, P=0.0368), and Ca2+ showed negative correlations with MCHC (r=−0.1440, P=00419).

Table 5 Correlation Between RBC Indices and Electrolytes Levels Among Anemic and Control Group

Discussion

Electrolyte imbalances and anemia are common issues. Many studies indicated an association between electrolyte and anemia, particularly iron deficiency anemia.^11,14,15 Anemia is a vital public health concern; the association between electrolyte imbalances and anemia is of interest. However, published studies addressing electrolyte changes in anemic patients are inconsistent. Some studies indicate that electrolyte levels in patients with iron deficiency anemia are higher than those in healthy controls, while other studies show lower electrolyte levels in iron deficiency anemia.^14,16–18

This current study assessed the correlation between RBC indices and electrolyte levels in patients with anemia compared to healthy people without anemia. The fact that red blood cell indices (Hb, RBC, MCV, HCT, MCH, and MCHC) were significantly lower in anemic patients compared to healthy individuals, which coincides with studies conducted by Rajagopal¹⁴ and Jundi.¹⁹ A study carried out in Xi’an, China, was showed similar results to this study in Hb, RBC, HCT, MCH, and MCHC.²⁰ Levels of Mg²⁺, K⁺and Ca²⁺were significantly lower in anemic patients, consistent with the K⁺ level reported by Ashraf,²¹ Rajagopal¹⁴ and Abbas.²² Na⁺levels were decreased without a significant difference, in contrast to the results of Mansoor,²³ Rafiq,¹¹ Rajagopal¹⁴ and Nnaji.²⁴ Additionally, the Cl⁻ levels were significantly higher in anemic patients, in accordance with results of Mansoor,²³ Rafiq,¹¹ Rajagopal¹⁴ and Nnaji.²⁴ The disparity may be due to the differences in the selection and sociodemographic factors of participants.^6,8 The electrolyte imbalance in anemic patients were hypo-magnesemia, hypo-kalemia, and hypocalcemia, with odds ratios (OR) of 4.928 (95% CI: 2.278–10.47), 2.967 (95% CI: 1.551–5.581), and 2.833 (95% CI: 1.887–4.236), respectively. Additionally, in anemic group, the results showed a positive significant correlation between Na⁺ levels with Hb, RBC, and HCT, which is similar to the results of Rajagopal in Hb, RBC and HCT and disagrees with the same study in MCH, and MCHC.¹⁴ Moreover, there was a negative significant correlation between Cl⁻ levels with Hb, which corresponds to the result of study conducted by Rajagopaland et al.¹⁴ The results showed no significant correlation between K⁺ levels with RBC indices, which does not coincide with the results of Rajagopal¹⁴ and Agoreyo and Nwanzen.²⁵ The electrolyte imbalance might be associated with altered membrane integrity of RBC, leading to abnormally high red cell permeability. This leads to an efflux of K⁺ from the cell, and an influx of Na⁺causes red cell dehydration.²⁴ Potassium chloride (K-Cl cotransport) is activated abnormally to cause dehydration due to loss of free K.^11,26 The K-Cl co-transport is one of the ways through which K⁺ is lost, which is abnormally activated by low intracellular Mg²⁺.²⁴ Likewise, deoxygenation in anemia is known to elevate the permeability of Na⁺, K⁺ and Ca²⁺ through the RBC membrane.^11,27,28 Erythrocyte Ca²⁺levels may play a role in Cl⁻ homeostasis.^29,30 The Ca²⁺-sensitive K⁺ channels regulate the erythrocyte apoptosis process.^12,14

Conclusion

The study concluded a significant disparity in serum electrolytes, particularly Ca²⁺, K⁺, Mg²⁺ and Cl⁻ levels, between individuals with and without anemia. Furthermore, there were positive correlations between Na⁺ and Ca²⁺ levels with Hb, RBC, and HCT and between Mg²⁺ levels with MCV and MCH. This study also concludes that RBC indices, including Hb, RBC, MCV, HCT, MCH and MCHC were significantly lower in anemic patients compared to non-anemic individuals. The study’s limitation included evaluating electrolyte levels in anemic patients without identifying the specific types of anemia. The relationship between RBC indices and electrolytes in specialized diseases, such as cardiac or rheumatic diseases, was also not considered. It is due to thelack of differential tests, which are expensive, and the absence of funding sources. Additionally, the study did not assess electrolyte levels before and after treatment due to difficulties tracking patients. Furthermore, we recommend prospective research on the correlation of RBC indices and electrolytes in specific populations, such as societies with high rates of consanguineous marriage.