==== Front Ann Saudi MedAnn Saudi MedAnnals of Saudi Medicine0256-49470975-4466King Faisal Specialist Hospital and Research Centre 1652187010.5144/0256-4947.2006.17asm-1-17Original ArticleZinc and antioxidant vitamin deficiency in patients with severe sickle cell anemia Hasanato Rana M.W. From the Department of Medical Biochemistry, College of Medicine and King Khalid University Hospital, King Saud University, Riyadh, Saudi ArabiaCorrespondence: Rana M. W. Hasanato, MD, P.O. Box 66533, Riyadh 11586, Saudi Arabia, Fax: +966-1-4671032, akalmomen@yahoo.comJan-Feb 2006 26 1 17 21 01 11 2005 Copyright © 2006, Annals of Saudi Medicine2006This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.BACKGROUND Patients with severe sickle cell anemia (SCA) have a higher potential for oxidative damage due to chronic redox imbalance in red blood cells that often leads to hemolysis, endothelial injury and recurrent vaso-occlusive episodes. This study evaluated the plasma levels of vitamins A, C and E as indicators of antioxidant status. In addition, serum levels of zinc and copper were also estimated. PATIENTS AND METHODS Twenty-five adult patients with severe sickle cell anemia (12 males and 13 females aged 29.72±12.94 years) and 25 matched controls were studied. Plasma levels of vitamins A, C and E were measured by HPLC technique. Serum zinc and copper levels were measured by atomic absorption spectrometry. RESULTS There was a significant decrease in plasma levels of vitamins A, C and E and in serum levels of zinc in patients with SCA as compared with controls (P<0.0001). Serum copper levels were signficantly elevated compared with controls (P<0.0001). CONCLUSION These findings emphasize the significant deficiencies of the antioxidant vitamins A, C and E and the trace element zinc along with the significant elevation of serum copper in patients with severe sickle cell disease. Further studies are needed to find out whether supplementation of antioxidant vitamins and zinc may ameliorate some sickle cell disease complications. ==== Body Patients with sickle cell anemia suffer from many complications, including growth retardation (decreased height and weight compared to their peers), chronic hemolytic anemia, recurrent and painful vaso-occlusive episodes, acute chest syndrome and impaired immune function. Although the exact reasons are not well established, the literature indicates that low levels of zinc, folic acid, and vitamins A, C and E could be contributing factors.1–6 Sickle cell anemia is a hereditary disorder with a high potential for oxidative damage due to a chronic redox imbalance in red cells that often results in continuous generation of reactive oxygen species (ROS) and clinical manifestations of mild to severe hemolysis.7–8 The production of ROS can be grossly amplified in response to a variety of pathophysiological conditions such as hypoxia, inflammation, infection, dehydration and deficiency in antioxidant vitamins.9,10 Our study was designed to evaluate levels of vitamins A, C and E as indicators of antioxidants status in patients with sickle cell anemia. In addition, levels of the trace elements zinc and copper were determined since zinc deficiency is known to occur in patients with sickle cell anemia and may contribute to immune impairment and growth retardation. The copper level is commonly reciprocal to the zinc level (i.e. a zinc deficiency is associated with a copper excess), which may contribute to free radical production and oxidative damage.11,13 Patients and Methods Twenty-five adult patients with severe sickle cell anemia (12 males and 13 females aged 29.72±12.94 years) and 25 normal healthy adults (age- and sex-matched, 17 males and 8 females aged 29.0±7.56 years) (Table 1) were enrolled in the study. Exclusion criteria were age younger than 15 years, the presence of β- or α-thalassemia trait, G6PD deficiency, regular blood transfusion, treatment with hydroxyurea, use of vitamin and trace element supplements other than folic acid, illness other than sickle cell manifestations, or pregnancy. The severity index of sickle cell anemia was considered high if the patient was having four or more severe painful episodes per year that required hospital admission, in addition to a history of other major sickle cell complications such as severe anemia (Hb < 7.0 g/dL), avascular necrosis of the hip joint, acute chest syndrome, priapism or hepatosplenic sequestration. The patients were recruited from the Hematology Clinic at King Khalid University Hospital and informed consent was taken from each patient. A careful history and thorough clinical examination were carried out. Fasting venous blood samples were collected under complete aseptic conditions for complete blood count. A peripheral blood film examination confirmed the presence of sickle cells. Confirmation of diagnosis was by hemoglobin electrophoresis with HbSS > 95% in the absence of HbA and a normal HbA2 (<3.5%). Blood samples for determination of the antioxidant vitamins A, C and E and trace elements zinc and copper were collected as follows: 5 mL of whole blood were collected into EDTA-containing, light-protected tubes for vitamins A and E measurement; 5 mL of whole blood were collected into lithium heparin tubes for vitamin C measurement; 5 mL of whole blood were collected into plain tubes for zinc and copper measurement. The EDTA tubes, the heparinized tubes and the plain tubes were centrifuged at 5400g and the plasma and serum were separated into clean, properly labeled tubes. The plasma samples were immediately used for the measurement of vitamins A, C and E using HPLC technique. The serum samples were used for zinc and copper measurement. Determination of vitamins A and E in plasma was performed according to the method described by Comstock et al (1993)14 and the determination of vitamin C was performed according to the method of Rumelin et al (1999).15 Serum copper and zinc were measured by atomic absorption spectrometry according to the method described by Evenson (1994).16 The data were analyzed using SPSS programme version 10.1. Student t test was used to compare the results between patients with sickle cell anemia and the normal control group. Results Plasma levels of vitamin A, C and E and serum zinc level were significantly lower in patients with severe sickle cell anemia as compared with the normal control group (P<0.0001) (Figures 1, 2, 3). Serum copper on the other hand, was significantly higher in patients with sickle cell anemia as compared with the normal controls (P<0.0001) (Figure 3). Discussion Our results indicate that there are significantly lower plasma levels of the antioxidant vitamins (A, C and E), lower serum levels of zinc and significantly higher serum levels of copper in patients with severe sickle cell anemia. Since patients with sickle cell anemia are under continuous oxidative stress due to sickle cell redox imbalance,17 a deficiency in antioxidant vitamins and trace elements may contribute to the severity of sickle cell manifestations, which could be aggravated further by elevated copper, which is a well known pro-oxidant.18,19 Despite a normal diet, patients with sickle cell disease may have inadequate intake of certain elements such as zinc because of chronic pain, reduced appetite and hemolysis. Another reason for reduced zinc and antioxidant vitamin levels could be increased demand and consumption. A third reason is increased urinary excretion due to impaired renal concentration and hypoxanthinuria.22–24 Other data in the literature suggest that regular supplementation of sickle cell patients with these vitamins and trace elements may ameliorate some of the sickle cell manifestations such as vaso-occlusive crises, acute chest syndrome, recurrent infection and growth retardation.1–6 Our findings are in agreement with those of several other studies.20,21 Essien found that the plasma concentrations of the antioxidant vitamins A (retinol), C (ascorbic acid) and E (Alpha tocopherol) were significantly low in patients with sickle cell anemia and he suggested that the deficiency of these antioxidant vitamins could account for some of the observed manifestations of sickle cell disease such as increased susceptibility to infection and hemolysis.23–25 Gray et al, analyzed the dietary intake and serum level of vitamin A, folic acid, iron, red cell folate and red cell zinc of 9 patients with sickle cell anemia and 19 controls. They found that despite adequate dietary intake of proteins, vitamins and trace elements, patients with sickle cell anemia had less body weight and a significantly lower red cell zinc and serum vitamin A levels compared with controls.26 Adelekan et al found that plasma levels of the antioxidant vitamins tocopherol, retinol, carotenes and ascorbic acid were significantly lower in 22 patients with sickle cell anemia in steady state as compared with nine controls.27 Westerman et al found that serum ascorbic acid levels were significantly lower with a 36% increase in urinary excretion as compared with controls.28 Zinc deficiency in patients with sickle cell anemia (probably due to continuous hemolysis and hyperzincuria) has been documented by several studies with strong indications that zinc deficiency is associated with impaired T-helper (TH1) functions, cell-mediated immunity and reduced interleukin-2 (IL-2) production and an increased rate of bacterial infection, vasoocclusive crises, frequent hospital admissions and growth retardation.29–32 These complications were reduced with zinc supplements.33 Complications of low levels of zinc, which is a known antioxidant, are associated with elevated levels of copper, which is a well known pro-oxidant and may potentiate oxidative stress in patients with sickle cell disease.34 Kiline et al found that patients with sickle cell anemia have lower serum zinc and higher copper levels while urine showed increased zinc excretion and very low copper excretion.35 Other studies have indicated that patients with sickle cell anemia suffer from multiple deficiencies in antioxidant vitamins and trace elements, which could be attributed to several factors, including inadequate intake in the face of increased demand, consumption and excretion.36–40 Dietary supplementation with various antioxidant vitamins and trace elements has been shown to ameliorate some of the sickle cell manifestations.41–44 The implications of our study along with other studies indicate that patients with sickle cell anemia require regular supplements with antioxidant vitamins and trace elements with proper monitoring. Our study shows that plasma levels of the antioxidant vitamins (A, C and E) and serum levels of zinc are significantly lower in patients with sickle cell disease as compared with a control group. In addition, serum levels of copper were significantly higher in sickle cell anemia patients. Therefore, supplementation with antioxidant vitamins and certain trace elements such as zinc may ameliorate some of the sickle cell symptoms and improve quality of life. Further studies are needed to assess the effect of antioxidant vitamins and trace elements supplementation on these clinical manifestations of sickle cell anemia. I would like to express my sincere thanks and appreciation to Prof. Mohsen A. F. El-Hazmi and Prof. AbdulKareem M. Al-Momen for their scientific support and guidance. Prof. Mohammed S. Al-Humayyed, for his support in performing assessments of anti-oxidant vitamins. Dr. Waheed M. Al-Harrizi and Dr. Amr S.M. Mustafa for their guidance during performing trace elements assays. Dr. Sayed S. Al-Esawy, for typing the manuscript. Figure 1 Plasma vitamin A and E concentrations in sickle cell disease patients vs. controls. Figure 2 Plasma vitamin C concentrations in sickle cell disease patients vs. controls. Figure 3 Serum zinc and copper concentrations in sickle cell disease patients vs. controls. Table 1 Characteristics of severe sickle cell patients and normal controls. Parameter Patients (Mean ± SD) Controls (Mean ± SD) N 25 25  - Males 12 17  - Females 13 8 Age (year) 29.72 ± 12.9 29.0 ± 7.56 WBCs (× 109/L) 10.9 ±4.5 5.5 ±1.3 RBCs (× 1012/L) 3.14 ± 0.7 5.0 ± 0.5 Hemoglobin level (g/L) 88 ± 12 145 ± 15  - Hb A (%) 0 96.1 ± 0.6  - Hb S (%) 83.8 % ± 7.5 0  - Hb F (%) 12.2 % ± 7.4 1.4 ± 0.6  - Hb A2 (%) 3.0 % ± 1.2 2.6 ± 0.4 MCV (fL ) 85.8 ± 11.7 86.3 ± 4.1 MCH (pg) 28.4 ± 4.5 29.8 ±1.5 Platelets (× 109/L) 364 ± 153 239 ± 68.2 Reticulocytes (%) 6.2 ± 2.6 1.1 ± 0.5 Bilirubin (μmol/L) 50.8 ± 29 11.4 ± 2.8 Painful episodes/year 6.0 ± 4 - Infections/year 4.2 ± 2.9 1.2 ± 0.4 Acute chest syndrome/year 3 ± 2 - ==== Refs References 1 El-Hazmi MAF On the nature of sickle cell disease in the Arabian peninsula Hum Genet 1979 52 323 335 535890 2 Powars DR Natural History of sickle cell disease - the first ten years Sem Hemat 1975 12 48 50 3 Pelligrini Braga JA Kerbauy J Zinc Fisberg M Copper and iron and their interrelations in the growth of sickle cell patients Arch Latinoam Nutr 1995 45 3 198 203 9332503 4 Leonard MB Zemel BS Kawchak DA Ohene-Frempong K Stallings VA Plasma zinc status, growth and maturation in children with sickle cell disease J Pediatr 1998 132 467 71 9544903 5 Williams R George EO Wang W Nutrition assessment in children with sickle cell disease J Assoc Acad Minor Phys 1997 8 44 8 9271889 6 Finan AC Elmer MA Sasanow SR McKinney S Russell MO Gill FM Nutritional factors and growth in children with sickle cell disease Am J Dis Child 1988 142 2 237 40 3341331 7 Rank BH Carlsson J Hebbel RP Abnormal redox status of membrane-protein thiols in sickle erythrocytes J Clin Investig 1985 75 1531 7 3998148 8 Hebbel RP Eaton JW Balasingam M Steinberg MH Spontaneous oxygen radical generation by sickle erythrocytes J Clin Invest 1982 70 1253 9 6294138 9 Bunn HF Pathogenesis and treatment of sickle cell disease New Eng J Med 1997 337 762 9 9287233 10 Lachant NA Tanaka KR Antioxidants in sickle cell disease the in vitro effects of ascorbic acid Am J Med Sci 1986 292 3 10 3521279 11 Prasad AS Schoomaker EB Ortega J Brewer GJ Oberleas D Oelshlegel FJ Zinc deficiency in sickle cell disease Clin Chem 1975 21 582 7 1116294 12 Linder MC Hazegh-Azam M Copper biochemistry and molecular biology Am J Clin Nutr 1996 63 5 797S 811S 8615367 13 Natta CL Tatum VL Chow CK Antioxidant status and free-radical induced oxidative damage of sickle erythrocytes Ann N Y Acad Sci 1992 669 365 7 1444049 14 Comstock JW Alperg AJ Helzlsouer KJ Reported effect of long term freezer storage on concentration of retinol, (- carotene, and alpha-tocopherol in serum or plasma, summarized Clin Chem 1993 39 6 1075 1078 8504540 15 Rumelin A Fauth U Halmagyi M Determination of ascorbic acid in plasma and urine by high performance liquid chromatography with ultra violet detection Clin Chem Lab Med 1999 37 533 6 10418744 16 Evenson ME Photometry Textbook of clinical biochemistry 2nd ed Purcis CA Ashwood ER Philadelphia WB Saunders Co 1994 104 131 17 Aslan M Thornley-Brown D Freeman BA Reactive species in sickle cell disease Ann N Y Acad Sci 2000 899 375 91 10863554 18 Chan AC Chow CK Chiu D Interaction of antioxidants and their implication in genetic anemia Proceed Societ Exprem Biolo Med 1999 222 274 82 19 Chiu D Vichinsky E Ho SL Liu T Lubin BH Vitamin C deficiency in patients with sickle cell anemia Am J Pediatr Hematol Oncol 1990 12 3 262 7 2240472 20 Prasad AS Beck FW Kaplan J Chandrasekar PH Ortega J Fitzgerald JT Swerdlow P Effect of zinc supplementation on incidence of infection and hospital admissions in sickle cell disease Am J Hemat 1999 61 194 202 10398312 21 Gupta VL Chaubey BS Efficacy of zinc therapy in prevention of crisis in sickle cell anemia a double - blind randomized controlled clinical trial J Assoc physicians India 1995 43 7 467 9 8713219 22 Akinkugbe FM Ette SI Ascorbic acid in sickle cell disease results of a pilot therapeutic trial East Afr Med J 1983 60 683 687 6671426 23 Essien EU Plasma levels of retinol, ascorbic acid and alpha-tocopherol in sickle cell anemia Cent Afr J Med 1995 41 2 48 50 7788666 24 Stone WL Payne PH Adebonojo FO Plasma vitamin E and low plasma lipoprotein levels in sickle cell anemia patients J Assoc Acad Minor Physicians 1990 112 25 Charache S Lubin BL Reid CD Management and therapy of sickle cell disease U.S. department of health and human services, public health service. National Institute of Health. NIH publication 92 2117 26 Gray NT Bartlett JM Kolasa KM Marcuard SP Holbrook CT Horner RD Nutritional status and dietary linkage of children with sickle cell anemia AM J Pediatr Hematol Oncol 1992 14 1 57 61 1550264 27 Adelekan DA Thurnham DI Adekile AD Reduced antioxidant capacity in pediatric patients with homozygous sickle cell disease Eur J of Clin Nutr 1989 43 609 14 2606092 28 Westerman MP Zhang Y McConnell JP Chezick PA Neelam R Freels S Feldman LS Allen S Baridi R Feldman LE Fung LW Ascorbic levels in red blood cells and urine in patients with sickle cell anemia Am J Hemat 2000 65 174 5 10996838 29 Karayalcin G Lanzkowsky P Kazi AB Zinc deficiency in children with sickle cell disease Am J Pediatr Hematol Oncol 1979 12 83 4 30 Niell HB Leach BE Kraus AP Zinc metabolism in sickle cell anemia J Am Med Assoc 1979 242 2686 7 31 Prasad AS Clinical Spectrum of human zinc deficiency Prasad AS Biochemistry of zinc New York plenum Press 1993 2 19 58 32 Prasad AS Zinc deficiency in patients with sickle cell disease Am J Clin Nutr 2002 75 2 181 2 11815307 33 Prasad AS Zinc supplementation and growth in sickle cell disease Ann Intern Med 1984 100 367 71 6696358 34 Hennig B Meerarani P Toborek M McClain CJ Antioxidant-like properties of zinc in activated endothelial cells J Am Coll Nutr 1999 18 152 8 10204831 35 Kiline Y Kumi M Yilmaz B Tanyeli A A comparative study of zinc and copper values in serum erythrocytes and urine in sickle cell homozygotes and heterozygotes Acta Pediatr Scand 1991 80 873 4 36 Ndombi IO Kinoti SN Serum vitamin E and the sickling status in children with sickle cell anemia East Afr Med J 1990 67 10 720 5 2282895 37 Natta CL Stacewicz-Sapuntzakis M Bhagavan H Bowen P Low serum levels of carotenoids in sickle cell anemia Eur J Hematol 1988 41 131 5 38 Philips G Tangney CC Relationship of plasma alpha-tocopherol to index of clinical severity in individuals with sickle cell anemia Am J Hematol 1992 41 227 31 1288284 39 Ong DE Absorption of vitamin A Blomhoff R Vitamin A in Health and Disease Marcel Dekker 1994 New York 37 72 40 Jain SK Williams DM Reduced levels of plasma ascorbic acid (vitamin C) in sickle cell disease patients its possible role in the oxidant damage to sickle cells in vivo Clinica Chemica Acta 1985 149 257 61 41 Muskiet FA Muskiet FD Meiborg G Schermer JG Supplementation of patients with homozygous sickle cell disease with zinc, Alpha-tocopherol, vitamin C, soybean oil and fish oil Am J Clin Nutr 1991 54 736 44 1716847 42 Sergeant GR Galloway RE Gueri MC Oral zinc sulphate in sickle cell ulcers Lancet 1970 2 891 2 4097279 43 Sindel LJ Dishuck JF Baliga BS Mankad VN Micronutrient deficiency and neutrophil function in sickle cell disease Ann N Y Acad Sci 1990 587 70 77 2163230 44 Tangney CC Phillips G Bell RA Fernandes P Hopkins R Wu SM Selected indices of macronutrient status in adult patients with sickle cell anemia Am J Hematol 1989 32 161 6 2816908