|Year : 2015 | Volume
| Issue : 2 | Page : 74-78
Impact of fetal hemoglobin on micronutrients in sickle cell anemia
Mathias Abiodun Emokpae, Maryam Omo Musa
Department of Medical Laboratory Science, School of Basic Medical Sciences, University of Benin, Benin City, Nigeria
|Date of Web Publication||7-Jul-2015|
Mathias Abiodun Emokpae
Department of Medical Laboratory Science, School of Basic Medical Sciences, University of Benin, Benin City
Source of Support: None, Conflict of Interest: None
Background / Objective: The presence of persistent high fetal hemoglobin (HbF) in sickle cells disease (SCD) patients may be a modulator of clinical and biochemical features. This study seeks to test the hypothesis that high level of HbF may regulate the levels of calcium, magnesium, zinc, and copper in SCD patients in a steady clinical state. Materials and Methods: Serum calcium, magnesium, zinc, and copper were assayed in 100 SCD patients in steady clinical state and 50 control subjects using the colorimetric method while blood HbF was determined by alkaline denaturation method. Results: Twenty-five percent of the study group had high (>5%) HbF, while 75% had low (<4.9%) HbF levels. HbF (P < 0.001), serum copper (P < 0.001), and calcium (P = 0.002) were significantly higher in SCD patients compared with controls, while zinc and magnesium were significantly lower (P < 0.001) in SCD patient compared with controls. Serum calcium (P = 0.01) and copper (P = 0.118) were lower in SCD patients with high (≥5%) HbF while magnesium and zinc were significantly higher (P < 0.001) in SCD patients with high HbF compare with those with low (≤4.9). HbF correlated negatively with calcium (r = −0.25, P = 0.011) and copper (r = -0.11, P = 0.287) while magnesium (r = 0.60, P = 0.001) and zinc (r = 0.57, P < 0.001) correlated positively on HbF levels. Conclusion: HbF levels may have modulated the levels of these elements in SCD patients. It is suggested that HbF may be estimated along with hemoglobin electrophoresis in diagnosis, clinical management, and predicting clinical course of SCD patients. Nutritional studies may be routinely conducted in this group of patients for better management.
Keywords: Calcium, copper, magnesium, sickle cell disease, zinc
|How to cite this article:|
Emokpae MA, Musa MO. Impact of fetal hemoglobin on micronutrients in sickle cell anemia. J Appl Hematol 2015;6:74-8
| Introduction|| |
Sickle cell disease (SCD) or sickle cell anemia (SCA) is a hereditary blood disorder characterized by red blood cells that assume an abnormal, rigid, sickle shape. It is a genetic defect in the synthesis of hemoglobin and is the best-known hemoglobinopathy in man.  It occurs as a result of the substitution of glutamic by valine at position 6 of the amino acid sequence, which leads to the formation of defective hemoglobin molecules and causes sickling of the red blood cells.  Even though SCD is present from birth, but symptoms are rare before the age of 3-6 months because of the high level of fetal hemoglobin (HbF) which is present at birth. It is of interest to note that cells are less prone to sickling in individuals who retain a high level of HbF. HbF inhibits the polymerization of the HbS owing to its high oxygen affinity. HbF (α2 γ2 ) dissociates to a dimer, which when combined with HbS, gives a tetramer that does not form a polymer, symptoms of SCD are almost completely eliminated with HbF levels above 25%; however, any increment in HbF level was observed to improve the overall survival. , The production of HbF is normally switched off soon after birth in favor of production of adult-type HbA.
Trace elements are essential in the body which is found in minute concentrations but their deficiency, however, can be deleterious because they are important components of enzyme systems as well as metabolic pathway, which regulate the synthesis and metabolism of larger molecules.  These trace elements include zinc, copper, selenium, manganese, chromium, cobalt, iron, iodine, magnesium. The levels of trace elements and magnesium have earlier been reported to be lower in SCD patients compared with HbAA control subjects and proteinuria impacted on the levels of these elements.  Calcium is important in bone formation and metabolism.  Bone diseases are common in SCD and some patients may present with Vitamin D deficiency and low bone mineral density.  Calcium is essential in excitation events which bring about the contraction of skeletal and cardiac muscles. Magnesium is an important component (co-factor) of more than 300 enzyme systems in the body.  A balance of serum calcium and magnesium is very important in the control of blood pressure since blood vessels need calcium to constrict and sufficient magnesium to relax and dilate. 
Zinc is a co-factor for polymerase and other enzymes in the body. It is also important in the formation of healthy hair follicles. Copper is an essential component of cytochrome c oxidase which is important in cellular respiration. It also regulates gene expression, deviation from normal value of such mineral elements such as calcium, magnesium, copper, and zinc have been reported by some authors. 
Sickle cell patients are prone to painful crisis and need more, intensive medical attention; serum calcium and trace element are reported to be reduced in SCD patients, which may be due to inadequate intake, absorption or increased loss through urine.  However, HbF is also associated with improved clinical outcome in these patients. This study seeks to test the hypothesis that high level of HbF may impact on the levels of these metals and micronutrients in SCD patients in a steady clinical state.
| Materials and methods|| |
This is a cross-sectional case-control study involving 150 participants which comprised of 100 SCD patients in steady clinical state and (50) apparently healthy individuals of HbAA genotype.
Study Area and Ethical Consideration
This study was conducted in the Department of Medical Laboratory Science, University of Benin, Benin City. The study participants were SCD patients on routine visit to Sickle Cell Disease Center, Benin City. The control subjects were staff and students of the University of Benin. The study protocol was approved by the Ethical Committee of the Edo state Ministry of Health, Benin City. All subjects who gave informed consent and met the inclusion criteria were enrolled in the study.
The subjects enrolled in the study were confirmed SCD patients and were homozygotes for sickle cell hemoglobin. They were on steady clinical state without acute illness such as vaso-occlusive crisis, acute chest syndrome, or bacterial infection, and were not transfused within the last 4 months before they were enrolled in the study.
Exclusion Criteria Include
All SCD patients with acute illness such as vaso-occlusive crisis, acute chest syndrome, or bacterial infection were excluded from the study. Subjects with other hemoglobinopathies were also excluded.
Sample Preparation and Determination
Blood samples (5 mL) were collected by venipuncture from each subject with 2 mL dispensed into ethylenediaminetetraacetic acid bottle and the remaining 3 mL emptied into a plain container which was allowed to clot at room temperature. The labeled samples collected in plain containers were spun in a bucket centrifuge at a speed of 2500 rpm for 10 min to separate serum from red cells. The separated serum was stored in a chest freezer at a temperature of −20°C before the analyses were done. The anticoagulated blood samples were used to determine HbF level by alkaline denaturation method. The measured parameters were assayed by colorimetric method. Serum zinc and copper were assayed using reagent kits supplied by Centronic, Wartenberg, Germany, while magnesium and calcium were quantitated using reagents supplied by Teco Diagnostics, Anaheim, USA and Randox Laboratories, UK, respectively.
Principle of the Assays
Zinc forms with 2-(5-Brom-2-pyridylazo)-phenol, a red chelate complex. The increase of absorbance is measured and is proportional to the concentration of total zinc in the sample.
Copper forms with 4-(3,5-Dibromo-2-pyridylazo)
- N-ethyl-N-sulfopropylaniline a chelate complex. The absorbance is measured and is proportional to the concentration of the total copper in the sample. While magnesium forms a color complex with calmagite in an alkaline medium to produce a red complex that is measured spectrophotometrically at 530 nm. EGTA serves to complex and prevent calcium interference and a surfactant eliminates the effect of protein. The color produced is proportional to the magnesium concentration.
Calcium ions form a violet color complex with O-cresolphthalein complexone in an alkaline medium. The intensity of the color formed is proportional to the concentration of calcium in the sample.
The percent HbF was determined by alkaline denaturation method. The principle is based on the fact that HbF is more resistant to alkaline denaturation than any other hemoglobin. Denaturation is stopped by adding ammonium sulfate to lower the pH and precipitate the denatured hemoglobin. After filtration, the amount of unaltered hemoglobin is measured and expressed as a percentage of the total amount of hemoglobin present. Commercially available control sera were included in the assay to ensure accuracy of analyses.
The mean ± standard error of the mean was calculated and Student's t-test for unpair means was used to compare the mean of the study group and controls. Pearson correlation coefficient was calculated in order to correlate the levels of HbF with the measured variables in SCD patients. A P ≤ 0.05 was considered as significant.
| Results|| |
[Table 1] shows the mean concentrations of HbF, serum calcium, zinc, copper, magnesium in both SCD patients and control subjects. HbF (P < 0.001), serum copper (P < 0.001), and calcium (P = 0.002) were significantly higher in SCD patients compared with controls, but zinc and magnesium were significantly lower (P < 0.001) in SCD patient compared with controls.
|Table 1: The concentrations of HbF, calcium, zinc, copper, and magnesium in SCD patients and control subject (mean±SEM)|
Click here to view
[Table 2] shows that serum calcium (P = 0.01) and copper (P = 0.118) were lower in SCD patients with high (≥5%) HbF, while magnesium and zinc were significantly higher (P < 0.001) in SCD patients with high HbF compa
re with those with low (≤4.9). The difference in the levels of copper between the two groups was, however, no significant.
|Table 2: Serum calcium, copper, zinc, magnesium in SCD patients with persistent high (≥5%) HbF compared with SCD patients with low (≤4.9%) HbF concentration (mean±SEM)|
Click here to view
HbF correlated negatively with calcium (r = −0.25, P = 0.011) and copper (r = -0.11, P = 0.287) while magnesium (r = 0.60, P = 0.001) and zinc (r = 0.57, P < 0.001) correlated positively on HbF levels [Table 3].
|Table 3: Correlation of HbF with serum calcium, zinc, copper, and magnesium in SCD patients|
Click here to view
| Discussion|| |
The aim of this study was to estimate the level of serum calcium, copper, zinc, and magnesium in (SCD) patients in steady clinical state and to determine the effect of HbF on these parameters. In this study, it was observed that serum magnesium and zinc were significantly lower (P < 0.001) in SCD patients than controls, while copper (P < 0.001), calcium (P = 0.002), and HbF (P < 0.001) were significantly higher in SCD patients than controls. SCD patients with high (>5%) HbF had significantly higher (0.001) zinc and magnesium levels but lower calcium (P = 0.01) and copper (P = 0.118) levels than SCD patients with low (<4.9%) HbF. The difference in copper level in SCD with high HbF and low HbF was however not significant. HbF correlated negatively with copper (P = 0.287) and calcium (P = 0.011) in SCD patients but correlated positively (P < 0.001) with zinc and magnesium.
In this study, the mean calcium level was higher in SCD patients compared with subjects with normal hemoglobin. This observation is in agreement with that reported earlier.  Deoxygenation and reoxygenation processes modulate the movement of cations through sickle red blood cells. It was reported that deoxygenation-induced increase in Ca 2+ permeability which could be due to both the activation of a Ca 2+ channel and a transport system for cations involving interactions between polymerized HbS band 3 and other membrane components.  Endocytosis may also play some roles in the Ca 2+ uptake of deoxygenated sickle cells. These mechanisms lead to intracellular Ca 2+ accumulation accompanied by K + loss and Na + gain in SCD. This excess intracellular calcium may be responsible for the impairment of the Ca 2+ -dependent K + channel which causes efflux of K ions from the red cells (Gardos Effect).  Hemolysis of the sickle red blood cells release red blood Ca 2+ content into the plasma. Calcium also enhances the exposure of red blood cells to phosphatidylserine which is a promoter of red cell polymerization. 
The mean serum levels of zinc in SCD patients were lower than that of controls. This observation is consistent with previous report that there are reduced zinc levels and metalloenzymes in SCD.  A strong correlation between hypozincemia and severity of SCD crisis was reported.  Clinical severity of SCD was not however considered in this study. It was suggested that nutrient supplementation may help reduce some of the clinical manifestations of painful crisis.  This zinc deficiency has been reported as a cause of the majority of perturbations observed in SCD patients because zinc is a co-factor of polymerases and other enzymes relevant for growth and development.  The deficiency of zinc in SCD may be due to chronic hemolysis often associated with SCA because red blood cells are important storage sites for zinc. The levels of zinc in SCD patients with high HbF were higher compared to those with low HbF. This is an indication that HbF may have some protective effects against loss of zinc since HbF retards the polymerization of deoxy sickle hemoglobin. Furthermore, the observed zinc levels in SCD patients with high HbF is about the same level with that observed in subjects with normal hemoglobin.
We observed low levels of magnesium in SCD patients compared with controls and the levels in subjects with high HbF were significantly higher compared to those with low HbF. Reports on the levels of magnesium in SCD patients have been increasing with variable results. Some studies have reported normal circulating levels,  while others observed low levels.  In a study by Zehtabchi et al., low levels of magnesium were reported in a group of 74 SCD compared to levels observed in 61 subjects with normal hemoglobin. They reported that the participants with HbSS had significantly lower levels of serum magnesium compared with healthy ethnicity matched and Caucasian controls. By measuring serum magnesium and calcium, they were able to define a subset of HbSS patients with hypomagnesemia and elevated Ca 2+ /Mg 2+ ratios, who might benefit from magnesium supplementation. 
The mean serum level of copper in SCD patients was higher (P < 0.001) than that of controls. This observation conforms to that reported by Kaur et al. and Idonije et al. The clinical significance of this elevation of plasma copper is unclear but it could be as a result of increased oxidative stress as well as low antioxidant potential which trigger the release of copper from the tissues into the blood to promote repair. The elevated level of serum copper in SCD patient is also an indication that the body's natural repair processes have been activated. High copper levels may be contributing to free radical production and oxidative damage in SCD.  The levels of copper in SCD patients with high HbF was lower than those with low HbF indicating that oxidative stress and the production of free radicals may be lower in subjects with high HbF levels.
The prevalence of high HbF of 25% in this study is consistent with what had been reported previously by Jiya et al. They reported a prevalence of 29% in a group of SCD patients in Sokoto, Nigeria. High level of HbF in the red blood cell of an individual tends to impair sickling at physiologic oxygen tension and may clinically improve the general well-being of the subject. HbF is regarded as the most powerful modulator of the clinical and hematologic features of SCD especially complications most closely linked to vaso-occlusive crisis and blood viscosity were associated with HbF levels in the circulation.  In this study, the mean level of HbF observed in SCD patients was 2.40% ± 0.22%. From previous studies, it was reported that some measures of protection was conferred on some biochemical parameters in those patients with HbF levels ≥5.0% when compared to those with low (≤4.9%) level. , Hence, ≥5% was considered as high in our setting. In a study conducted in some Jamaican SCD patients in order to compare the clinical severity based on HbF levels, it was reported that packed cell volume and mean cell volume increased with increasing levels of HbF (1%, 2.5%, 3.4%, 4.6%, and 5.2%). However, differences in the incidence of painful crisis, abdominal pain, and acute chest syndrome were not apparent. ,, It was therefore suggested that the threshold level of HbF needed to prevent acute clinical events was about 20%, while the threshold to prevent organ damage was reported to be 10%. 
| Conclusion|| |
Increased HbF levels may play some roles in the modulation of micronutrients in SCD patients. It is suggested that HbF may be estimated along with hemoglobin electrophoresis in diagnosis, clinical management, and predicting clinical course of SCD patients. Furthermore, nutritional studies should be routinely conducted in this group of patients for better management.
Financial Support and Sponsorship
Conflicts of Interest
There are no conflicts of interest.
| References|| |
Kato GJ, Gladwin MT, Steinberg MH. Deconstructing sickle cell disease : r0 eappraisal of the role of hemolysis in the development of clinical subphenotypes. Blood Rev 2007;21:37-47.
Ballas SK. Sickle cell anaemia : p0 rogress in pathogenesis and treatment. Drugs 2002;62:1143-72.
Atweh GF, Schechter AN. Pharmacologic induction of fetal hemoglobin : r0 aising the therapeutic bar in sickle cell disease. Curr Opin Hematol 2001;8:123-30.
Bank A. Regulation of human fetal hemoglobin : n0 ew players, new complexities. Blood 2006;107:435-43.
Endres DB, Rude RK. Disorders of bone metabolism. In: Burtis CA, Ashwood ER, Bruns DE, editors. TEITZ Fundamentals of Clinical Chemistry. Philadelphia, USA: Saunders Elselvier; 2008. p. 731.
Emokpae MA, Tijani AD. The impact of proteinuria on serum levels of trace elements in sickle cell disease patients. J Med Biochem Sci 2014;3:3-7.
Ureme SO, Ejezie FE, Ibegbulam GO, Ibeh E, Nwanya IJ. Serum calcium, inorganic phosphates and some haematological parameters in sickle cell disease in Enugu metropolis. Orient J Med 2003;15:5-8.
Adewoye AH, Chen TC, Ma Q, McMahon L, Mathieu J, Malabanan A, et al.
Sickle cell bone disease : r0 esponse to vitamin D and calcium. Am J Hematol 2008;83:271-4.
Mahyar A, Ayazi P, Pahlevan AA, Mojabi H, Sehhat MR, Javadi A. Zinc and copper status in children with Beta-thalassemia major. Iran J Pediatr 2010;20:297-302.
Touyz RM. Role of magnesium in the pathogenesis of hypertension. Mol Aspects Med 2003;24:107-36.
Rhoda MD, Apovo M, Beuzard Y, Giraud F. Calcium permeability in deoxygenated sickle cells. Br J Nutr 2000;75:2453-8.
Guyton AC, Hall JE. Textbook of Medical Physiology. 11 th
ed. Philadelphia: Elsevier Saunders; 2006. p. 511.
Weiss N, Zamponi GW. Regulation of voltage-gated calcium channels by synaptic proteins. Adv Exp Med Biol 2012;740:759-75.
Egbuta JO. Trace Elements and Metalloenzymes in Sickle Cell Disease; 2001. Available from: http://[email protected]
. [Last accessed on 2015 Mar 25].
Temiye EO, Edem SD, Mbang AO, Renner JK. Clinical study relationship between painful crisis and serum zinc level in children with sickle cell anaemia. Nutr Res Pract 2011;4:356-61.
Al-Saqladi AW, Cipolotti R, Fijnvandraat K, Brabin BJ. Growth and nutritional status of children with homozygous sickle cell disease. Ann Trop Paediatr 2008;28:165-89.
Akenami FO, Aken′Ova YA, Osifo BO. Serum zinc, copper and magnesium in sickle cell disease at Ibadan, south western Nigeria. Afr J Med Med Sci 1999;28:137-9.
Zehtabchi S, Sinert R, Rinnert S, Chang B, Heinis C, Altura RA, et al.
Serum ionized magnesium levels and ionized calcium-to-magnesium ratios in adult patients with sickle cell anemia. Am J Hematol 2004;77:215-22.
Kaur M, Dangi CB, Signh H. To study the haemoglobinopathies and ratio of copper and zinc in Sindhi community of Bhopal. Int J Pharma Bio Sci 2013;4:672-91.
Idonije BO, Iribhogbe OI, Okogun GR. Serum trace element levels in sickle cell disease patients in an urban city in Nigeria. Nat Sci 2011;9:67-71.
Hasanato R. Zinc and antioxidant vitamin deficiency in patients with severe sickle cell anemia. Ann Saudi Med 2006;26:17-21.
Jiya NM, Matazu IK, Bilbis LS, Shehu RA. Serum levels of antioxidant vitamins in foetal haemoglobin (HbF) persistent sickle cell anaemia children in Sokoto, Nigeria. Ann Afr Med 2005;4:168-71.
Akinsheye I, Alsultan A, Solovieff N, Ngo D, Baldwin CT, Sebastiani P, et al.
Fetal hemoglobin in sickle cell anemia. Blood 2011;118:19-27.
Donaldson A, Thomas P, Serjeant BE, Serjeant GR. Foetal haemoglobin in homozygous sickle cell disease : a0 study of patients with low HBF levels. Clin Lab Haematol 2001;23:285-9.
Hedo CC, Okpala IE, Aken′ova YA. Foetal haemoglobin levels in Nigerians with sickle cell anaemia. A revisitation. Trop Geogr Med 1993;45:162-4.
Powars DR, Schroeder WA, Weiss JN, Chan LS, Azen SP. Lack of influence of fetal hemoglobin levels or erythrocyte indices on the severity of sickle cell anemia. J Clin Invest 1980;65:732-40.
Steinberg MH. Predicting clinical severity in sickle cell anaemia. Br J Haematol 2005;129:465-81.
[Table 1], [Table 2], [Table 3]
|This article has been cited by|
||Interplay between foetal haemoglobin, micronutrients and oxidative stress biomarkers in sickle cell anaemia children
| ||Ama Gyasiwaah Owusu-Poku, Daniel Gyamfi, Eliezer Togbe, Stephen Opoku, Richard Kobina Dadzie Ephraim, Jocelyn Gyapomaa Asibey, Benedict Sackey, Wina Ivy Ofori Boadu, Max Efui Annani-Akollor, Enoch Odame Anto, Otchere Addai-Mensah |
| ||Human Nutrition & Metabolism. 2022; : 200173 |
|[Pubmed] | [DOI]|
||Copper-To-Zinc Ratio as an Inflammatory Marker in Patients with Sickle Cell Disease
| ||Mathias Abiodun Emokpae,Emmanuel Bamidele Fatimehin |
| ||Sci. 2020; 2(4): 89 |
|[Pubmed] | [DOI]|
||Evaluation of Serum Electrolytes in Sickle Cell Disease Patients with Respect to Hydroxyurea Therapy
| ||Debapriya Rath,Damini S Tomar,Prafulla K Khodiar,Pradeep K Patra |
| ||Indian journal of Medical Biochemistry. 2019; 23(1): 182 |
|[Pubmed] | [DOI]|
||Some haematological parameters, copper and selenium level among children of African descent with sickle cell disease in specialist hospital Sokoto, Nigeria
| ||Osaro Erhabor,Kevin Ogar,Erhabor Tosan,Amos Dangana |
| ||Human Antibodies. 2019; : 1 |
|[Pubmed] | [DOI]|
||Copper-to-Zinc Ratio Correlates with an Inflammatory Marker in Patients with Sickle Cell Disease (Version 1)
| ||Mathias Abiodun Emokpae,Emmanuel Babatunde Fatimehin |
| ||Sci. 2019; 1(2): 34 |
|[Pubmed] | [DOI]|
||Copper-to-Zinc Ratio Correlates with an Inflammatory Marker in Patients with Sickle Cell Disease
| ||Mathias Abiodun Emokpae,Emmanuel Bamidele Fatimehin |
| ||Sci. 2019; 1(2): 55 |
|[Pubmed] | [DOI]|