INTRODUCTION:

Anemia affects a quarter of the world's population, accounting for 8.8% of the global burden of disease. There are different types of anemia¹.

Iron deficiency is the predominant cause of anemia across countries -; and anemia resulting from iron-restricted erythropoiesis occurs through several mechanisms. In iron deficiency, depleted iron stores are due to the imbalance between iron absorption and use². The persistence of a negative balance leads to microcytic and hypochromic anemia.

Adequate iron filling and iron deficiency cause management to solve the problem. In contrast, functional iron deficiency is due to poor iron release in the circulatory system of intestinal cells, macrophages, or liver cells. Erythrocytes are restricted iron. Anemia develops despite the presence of sufficient amounts of iron, and red blood cells may appear normal or pellets³.

Sickle cell anemia (SCA) arises from the homozygous nucleotide polymorphisms in the sixth codon of the b-globin gene on chromosome⁴ and leads to the hemoglobin molecules forming polymers under oxygen-depleting conditions. In turn, they transform biconcave red blood cells (RBCs) from into a sickle shape. These RBCs are also dehydrated due to leakage K+ and loss of water, resulting in a short lifespan of these cells. This RBC-dried sickle slows down blood flow and mediates the binding of the white blood cells to the endothelial lining of the blood vessels, leading to clogged microvascular vessels, referred to as vasoocclusion and leads to painful sickle cell crises. When the blood flow is re-established, it increases blood thinning and inflammation⁵.

One of the parameters used to monitor these patients is the condition of the iron in the body, and it has been assumed that the increase of iron in the body can affect the diagnosis and management of these patients. However, this notion of iron levels remains controversial, and we do not have categorical data to provide clinicians with clear guidelines for using this parameter in their clinics to make decisions about blood transfers, iron supplements, or heavy metal removal⁶.

Since SCA is a disease affecting a large number of people in the developing world, the cost of its management is very high. This disease is characterized by anemia and immune disorders, like free radical generation; moreover, the balance of minerals and antioxidants is essential in maintaining membrane integrity of red cell and its function⁷. Minerals such as copper, zinc, iron, chromium, magnesium, selenium, and vanadium, as well as vitamin compounds are of great benefit in reducing oxidative stress associated with RBCs’ membranes because protection of the red cell membrane from free radical-mediated oxidative stress is essential to the management of SCA⁸. In addition, the fact that the researches about trace elements in SCA and iron deficiency anemia (IDA) are very rare in Iraq, if not missing, makes this study of significant importance.

The main aim of this study is to determine the levels of such metals as copper, zinc, magnesium, and selenium and also physiological parameters such as ferritin, Hb, and vitamin D levels; PCV and reticular cells percentage; and RBCs and WBCs count in SCA patients and compare them with IDA patients.

MATERIAL AND METHODS:

In this study, 31 SCA patients, 30 IDA patients, and 28 healthy people (male and female) from the Center of Genetic Diseases, Thi-Qar, Iraq were included (age range: 2-45 years). The blood types were recorded from the profiles of all patients. 5ml blood was collected from the antecubital vein of all participants. 1µl blood was mixed with 1µl methylene bleu in the tube and incubated for 15 min at 37°C. Then it was used to make blood smear onto slide to reticulum cells and RBCs were counted by light microscopy. To find the percentage of reticulum cells, the following equation was applied:

Reticulum cell account/ RBCs account * 100

The rest of the blood sample was divided into two parts. One part was put in EDTA-coated tubes to be used in the hematology tests by hematology analyzer AR-6400 (ARI Medical Technology Co., Ltd.). The second part was put in a dry sterilized test tube which was centrifuged for 5 min at 3000rpm to obtain the serum needed to determine the following items:

1. Ferritin level by an enzyme linked assay method using a kit supplied by Biomerieux (France), measured automatically with Minividas, Biomerieux (France).

2. Vitamin D levels by immunoassay analysis using a kit from Roch (Germany), measured automatically with Cobas e 411, Roch (Germany)

3. Serum copper, zinc, magnesium, and selenium by flame atomic absorption spectrophotometer using a direct method as described by Kaneko (1999)⁹.

Statistical analysis:

The results were computed as mean±standard error or %. In all statistical analysis, only P≤0.05 was considered to be significant. Differences among groups were analyzed by one-way analysis of variance (ANOVA) or Chi-square test. The correlation coefficient was performed by Pearson correlation coefficient test.

RESULTS:

The basic clinical manifestations of SCA and IDA patients are shown in Table 1. The age of IDA group (28±1.41 years) was significantly higher than that of SCA group (22±1.78 years), while no significant difference was found in the gender between the two groups. Meanwhile, the frequency of blood type showed a significant difference. In both groups, the highest frequency was found in the O blood type (61% in the SCA and 37% in the IDA groups) and the lowest in the B blood type (3% and 10%, respectively).

Table 1: Clinical manifestations of SCA and IDA patients

Character		SCA (n=31)	IDA (n=30)	Significance
Age (years)	Range	2-45	2-45	P=0.021
Age (years)	Mean±SE	22 ±1.78	28±1.41	P=0.021
Gender n (%)	♂	19 (61%)	13 (43%)	P=0.543
Gender n (%)	♀	12 (39%)	17 (57%)	P=0.543
Blood Group n (%)	A	5 (16%)	8 (27%)	P˂0.0001
	B	1 (3%)	3 (10%)
	AB	6 (19%)	8 (27%)
	O	19 (61%)	11 (37%)

The results of estimated parameters in all groups showed that RBC count, Hb level, vitamin D level, and PCV were significantly lower in both anemia groups compared to the control. In addition, the lowest level was found in the SCA group. However, other parameters, including ferritin level, percentage of reticular cells, and WBC count were significantly higher in the SCA group, and significantly lower in IDA group, compared to the control (Table 2).

Copper and selenium levels were significantly higher in the SCA (117.8±0.15 and 30.93±0.21, respectively) and IDA patients (110.3±0.13 and 20.09±0.18, respectively) compared to control (98.7±0.21 and 4.94±0.14, respectively). The highest levels of copper and selenium were found in SCA group (Figure 1A and C).

Conversely, zinc and magnesium levels were significantly lower in the SCA (0.27±0.002 and 3.15±0.09, respectively) and IDA patients (0.46±0.005 and 1.2±0.002, respectively) compared to control (1.64±0.003 and 10.43±0.09, respectively). While the lowest zinc level was found in the SCA patients, the lowest magnesium level was found in the IDA ones (Figure 1BandD).

Figure 1: Levels of metals in the anemia patients and control groups.

(1) copper (Cu), (1) zinc (Zn), (1) selenium (1), and (1) magnesium (Mg).

*Significant differences between anemia patients and control groups,

# Significant difference between anemia groups

DISCUSSION:

Sickle cell disease (SCD) is one of the most common genetic disorders of hemoglobin in the world, with child mortality rates rising worldwide (50-80% of children born with SCD die before 5 years old)¹⁰. The life expectancy of people with SCD increased from 20 years in 1970 to nearly 40 years in 2005¹¹. It is believed that this increase in life expectancy is due to neonatal screening and preventive penicillin and vaccinations¹². A sickle cell is a chromosomal disorder transmitted as an autosomal recessive disease. It is not an X-linked disorder, so the ratio of males to females is 1:1¹⁰. Many researchers report that iron deficiency is linked to exhaustion, reduced overall tightness of work capacity, and poor concentration. It negatively affects learning, cognitive function, behavior, attention, and regular activities of young students^13,14 and may also lead to truancy. The world health organization (WHO) identifies iron deficiency as a major public health problem and an indicator of malnutrition and health; anemia has the highest prevalence in Southeast Asia, Eastern Mediterranean, and African regions¹⁵

The age of IDA group was higher than that of the SCA group, while no significant difference was found in the gander of the two groups. The frequency of blood type also showed a significant difference. In both groups, the highest frequency was found in the O blood type and the lowest in the B blood type.

The abnormal hemoglobin variation of ABO blood types varies from race to race. 42% of Western Europeans have type A, 9% of type B, 3% of type AB, and 46% of type O. However, some Eastern Europeans have a higher rate of up to 40%. The American Native Americans are exclusively O¹⁶. American black populations generally have different blood types (A, B, AB, and O of 27%, 20%, 4%, and 49%, respectively)¹⁷. Some studies in Nigeria investigated the frequency of ABO blood types among different ethnic groups/tribes. Worllidge¹⁸ reported the frequencies between Yuropa and Hausa as follows: 21% had type A, 17% had type B, 2% had type AB, and 58% had type O. All previous reports correspond to the frequencies obtained in this study and confirm that the abnormal hemoglobin frequency is higher in group O than other blood types.

Our results showed that RBC count, Hb level, vitamin D level, and PCV percentage were significantly lower in both anemia groups than the control group. Also, the lowest level was observed in the SCA group. However, other parameters, including ferritin level, reticular cells, and WBC count were significantly higher in the SCA group, and significantly lower in IDA group, compared to control.

Anemia is defined as a state of low hemoglobin concentration, a decrease in the number of red blood cells circulating in the blood, or both, which can occur despite having adequate iron stores and vitamins¹⁹. The distinguishing features of pathophysiology of SCA and chronic hemolytic anemia show an increasing tendency for red blood cells to dissolve and stick to them, chronic hemolysis, shortened red cell survival, as well as low erythropoietin response. These may reduce the levels of Hb and HCT and number of erythrocytes²⁰.

The emergence of hypovitaminosis D (HVD) in the general population of sunny countries approaches 100% in some reports^21-24. This is consistent with the generally accepted notion that HVD is a pandemic health problem, inclusive of areas with year-long sunshine. The frequency of HVD ranges from 5% in Jordanians up to >97% in India²⁵. Moreover, many studies have reported a high frequency of HVD in the SCA population as opposed to the non-SCA population, in study-specific groups in sunny countries^26-29. Nevertheless, when compared to the healthy general population of the same countries, the frequency was not much different from the SCA population²⁷. In our results, the frequency of HVD was 100% in the patient groups compared to 18% in control group. Further studies are needed to investigate the reason, especially in a sunny country like Iraq.

It is well known that the sickle red blood cells slow blood flow and mediate the association of white blood cells with endothelial lining of the blood vessels, leading to clogging of microvascular vessels. When blood is re-flowing, it increases blood thinning and inflammation⁵. Previous reports indicate that SCA patients have an increase in WBCs^30-32. In addition, another study reported that the use of drugs, such as hydroxyurea, reduces the number of WBCs, thus improving the clinical outcomes of SCA patients³³. Previous reports have also shown that high WBCs appear to be a risk factor for many severe complications from SCA, severe pain rates³⁴, acute chest syndrome³⁵, and mortality³⁶.

Erythropoietin, a mainly metabolized kidney agent, stimulates erythrocytes by working on stem cells committed to induce RBC proliferation and differentiation in the bone marrow. Hypoxia in tissues is the main catalyst for the production of erythropoietin. The red cell precursors of nuclear cells in the bone marrow are called natural plasma red blood cells. RBCs that mature into the non-nuclear phase gain access to peripheral blood. Once the cells lose their nuclei, they are called RBCs. Young RBCs that contain residual RNA are called reticular cells which inter blood flow and increase in hemolytic anemia such as SCA³⁷, while the numbers of reticular cells decrease in IDA and begin to increase during the first week of iron therapy³⁸.

It is commonly observed that patients with SCA similar to other chronic hemolytic anemia patients are iron-loaded because of enhanced hemolysis³⁹. RBC transfusion has been frequently applied for decades to treat acute illness in SCD and subsequent recycling of iron resulting in increased iron stores, therefore, concerns about iron overload and its associated organ injury. Some studies revealed that serum ferritin concentrations correlate well with the number of units of blood transfused⁶. Increase in iron stores in the multi-transfused subjects reflects the amount of ferritin in plasma⁴⁰. Several other factors occurring in SCA may increase serum ferritin concentration, such as liver disease and chronic infection or inflammations⁶.

In this study, zinc concentration was significantly lower in patients with SCA compared with the control group. Several researchers (Parad and Cossack,⁴²; Parad,⁴³; Idonije et al., 2011) have linked zinc deficiency in SCD to manifestations such as growth retardation, male hypogonadism, hyperammonemia, abnormal dark adaptation, and cell-mediated immune disorder⁴¹. Prasad et al. (1975) and Prasad AS (2002)^42-43concluded that zinc supplements can help SCA patients resume normal growth; these supplements can help reduce seizures and inhibit severe pain and vomiting, which cause these patients to be hospitalized. However, biochemical evidence in Zemel et al. (2002) and Singhi et al. (2003) proved that zinc deficiency can be in plasma, RBCs, and lymphocytes in hair and granular cells⁴¹. In Addition, Parade et al. reported low activities of zinc-based enzymes such as carbon anhydrase, alkaline phosphate, and thymidine kinase; and found the higher activity of plasma RNase in SCA patients which defined zinc as inhibiting the activities of this enzyme⁴¹.

It is well-established that copper is essential in the proper performance of various metal enzymes, including ceruloplasmin involved in metabolism of iron stored in the liver, making iron available for the synthesis of hemoglobin. So, the deficiency of copper causes anemia. However, it was observed that in this type of anemia, despite the high iron level in the liver, the rate of hemoglobin synthesis remains very low⁴⁴. Zinc and copper compete with each other on similar protein-binding sites. Previously, it has been observed elevated in copper level in zinc-deficient tissues⁴⁵. Therefore, it is likely that the elevation of plasma copper was secondary to zinc deficiency in our anemia subjects, which had high level of copper and low level of zinc. On the other hand, given the fact that high doses of zinc can be used for long periods in the future to treat SCA and IDA patients, this may result in a copper deficiency in such patients. This possibility must be taken into consideration by the doctors involved in the management of SCA with zinc therapy and also IDA⁴¹.

Magnesium plays a key role in cell dehydration by inhibiting transport of K⁺-Cl^-, resulting in reduced K⁺ loss and alteration of endothelial inflammation in SCA patients⁴⁶. Furthermore, magnesium deficiency is associated with increased levels of inflammatory cytokines and increased expression of endothelial adhesion molecules such as vascular cell adhesion molecule⁴⁶. In this report, the magnesium level was significantly lower in both patient groups with anemia. Previous studies have suggested that different selenium is based on the genotype of SCA, Hb-SS, or Hb-AA.^47-50. These researchers predict genetic factors as an individual-pattern difference among patients with SCA, and environmental factors may explain this difference.

Selenium also plays an important role in preventing oxidative modification of fat, reducing inflammation, and preventing plaque buildup⁵¹. In this study, selenium level was significantly higher in SCA and IDA patients than the control group. Although this is a good result, further research is needed to confirm this finding and find out the causes. There is not enough literature on trace elements in SCA and IDA patients in Iraq. The reduced or elevated levels of zinc, magnesium, copper, and selenium underline the need for their systematic review. Accordingly, regular laboratory examination of these elements is inevitable.

ACKNOWLEDGMENT:

I̕ would like to thank the Center of Genetic Diseases, Thi-Qar, Iraq and department of biology, college of science, to University of Thi-Qar, Iraq as they supported and helped me.

CONFLICT OF INTEREST:

The author has no disclosures to report.

ETHICAL CLEARANCE:

Not required.

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Received on 02.09.2019 Modified on 22.10.2019

Research J. Pharm. and Tech. 2020; 13(10):4655-4660.

DOI: 10.5958/0974-360X.2020.00819.7

Parameter Level	Normal Range	Sample value M±SE			Significance
Parameter Level	Normal Range	Control(n=28)	SCA (n=31)	IDA (n=30)	Significance
R.B.Cx10⁶ (cell/mm³)	♂=4.7-6.1 ♀=4.2-5.4	4.74±0.05	2.81±0.09	3.4±0.06	a P˂0.0001 b P˂0.0001 c P˂0.0001
Hb (g/dL)	♂=13.8-17.2 ♀=12.1-15.1	13.04±0.09	5.58±0.29	9.66±0.16	a P˂0.0001 b P˂0.0001 c P˂0.0001
Ferritin (ng/mL)	♂=18-270 ♀=18-160	214±2.19	1726±344.61	2.07±0.38	a P˂0.0001 b P˂0.0001 c P=0.0493
PCV (%)	♂=40-50 ♀=30-46	41.96±0.94	28.66±0.79	32.7±1.05	a P=0.003 b P˂0.0001 c P˂0.0001
W.B.Cx10³ (cell/mm³)	4.5-11	8.39±0.18	13.9±0.25	6.63±0.22	a P˂0.0001 b P˂0.0001 c P˂0.0001
Reticular cells (%)	0.5-2.5	1.96±0.09	2.88±0.09	0.36±0.04	a P˂0.0001 b P˂0.0001 c P˂0.0001
Vitamin D (ng/mL)	20-50	33.04±0.06	6.83±0.06	10.61±0.85	a P=0.002 b P˂0.0001 c P˂0.0001