Lack of Difference in Hepcidin Levels in Sickle Cell Anemia and Sickle Cell Beta Thalassemia
Authors: Shen Jessica , Wongtong Naritsara , Noubouossie Denis , Elsherif Laila , Zhou Qingning , Cai Jianwen , Ataga Kenneth I. 
Published: October 2019

Abstract
Introduction: Sickle cell disease (SCD) may be associated with iron overload following repeated RBC transfusions. In beta thalassemia major, a disease characterized by hemolysis and ineffective erythropoiesis, iron overload occurs due to both repeated RBC transfusions and hyperabsorption of iron. Hyperabsorption of iron occurs due to ineffective erythropoiesis and subsequent downregulation of hepcidin, the main regulator of systemic iron homeostasis. With the presence of a thalassemia gene, HbSβ-thalassemia patients may have an increased tendency to hyperabsorb iron compared to HbSS patients. The purpose of this exploratory study was to compare hepcidin levels in HbSS and HbSβ-thalassemia as well as the levels of GDF-15 and any associations between hepcidin and ferritin, other measures of iron balance, erythropoietin and markers of inflammation.
Methods: Eligible patients with HbSS or HbSβ-thalassemia (HbSβ0-thalassemia or HbSβ+-thalassemia) were evaluated during routine clinic visits. Hepcidin, GDF -15 and erythropoietin were analyzed using commercially available ELISA kits, while plasma cytokines were analyzed by Luminex® multi-analyte profiling technology. Complete blood counts, serum iron, transferrin, transferrin saturation and ferritin, high-sensitivity CRP and routine chemistries were performed at the clinical laboratories of UNC Hospitals. Patients' clinical data, including SCD-related clinical complications and estimates of the number of lifetime RBC transfusions, were obtained at the time of evaluation combined with reviews of their medical records. The levels of hepcidin and GDF-15 were compared in HbSS and HbSβ-thalassemia (and SCD vs. healthy controls) using Wilcoxon rank sum tests, and the association of hepcidin and laboratory covariates was evaluated using Spearman correlation coefficient. Reported p-values are for individual tests, unadjusted for multiple comparisons.
Results: Baseline demographic variables were similar in HbSS and HbSβ-thalassemia patients, but platelet counts, lactate dehydrogenase, total bilirubin, direct bilirubin, indirect bilirubin, aspartate transaminase and alkaline phosphatase were significantly higher in HbSS patients (Table 1). No significant differences were observed when hepcidin levels were compared between control subjects (N = 15) and SCD patients (N = 40) (25.2 ± 14.3 ng/mL vs. 19.5 ± 17.3 ng/mL, p = 0.13). In addition, no difference was observed in hepcidin levels (16.1 ± 11.4 ng/mL vs. 23.7 ± 22.2 ng/mL, p = 0.53) or GDF-15 levels (1635.1 ± 1357.7 pg/mL vs. 2051 ± 2385.4 pg/mL, p = 0.9) when HbSS patients were compared with HbSβ-thalassemia. Hepcidin was associated with ferritin (r= 0.57, p = 0.0001) and transferrin (r = -0.46, p = 0.0029) in SCD patients. However, no significant correlations were observed between hepcidin and erythropoietin, high sensitivity CRP, IL-6, IL-8, IL-10 and TNFα.
Conclusion: No differences in hepcidin levels were observed in SCD patients compared with healthy controls, and in HbSS patients compared with HbSβ-thalassemia patients. We confirm the association of hepcidin with ferritin, but surprisingly find no association of hepcidin with IL-6.
Table 1. Baseline Variables Variable HbSS (N = 22)Mean ± SD (or %) HbSβ-thalassemia (N = 18)Mean ± SD (or %) 95% CI for Mean Difference Age (years) 37.96 ± 12.7 36.4 ± 11.4 (-6.2,9.2) Gender (females) 15 (68.2) 12 (66.7) Weight (kg) 70.8 ± 15.1 74.7 ± 14.8 (-13.7,5.9) Transfusions 0 - 5 6 - 10 11 - 20 21 - 50 9 (23.1) 5 (12.8) 5 (12.8) 2 (5.1) 11 (28.2) 3 (7.7) 3 (7.7) 1 (2.6) White Blood Cell Count (× 109/L) 9.5 ± 3.1 9.4 ± 3.1 (-1.9,2.04) Hemoglobin(g/dL) 9.4 ± 1.7 10 ± 1.99 (-1.7,0.6) Platelet Count (× 109/L) 402.6 ± 134.9 310.1 ± 151.6 (1.8,183.1) Ferritin (ng/mL) 346.3 ± 406.9 174 ± 257.7 (-50.3,395) Reticulocyte Count (× 109/L) 233.5 ± 139.9 215.7 ± 138.9 (-71.8,107.6) Lactate Dehydrogenase (U/L) 954.3 ± 322.95 720.4 ± 174.2 (73.8,394) Total Bilirubin (mg/dL) 3.9 ± 3.1 1.4 ± 0.9 (1.2,3.9) Direct Bilirubin (mg/dL) 0.56 ± 0.5 0.3 ± 0.1 (0.04,0.48) Indirect Bilirubin (mg/dL) 3.3 ± 2.8 1.06 ± 0.8 (1.03,3.5) Aspartate Transaminase (U/L) 46.5 ± 18.8 32.8 ± 11.9 (3.4,23.9) Alanine Transaminase (U/L) 30.7 ± 13.5 28.7 ± 9.7 (-5.6,9.6) Alkaline Phosphatase (U/L) 110.6 ± 45.6 78.1 ± 24.7 (9.7,55.3) Creatinine (mg/dL) 0.67 ± 0.17 1.1 ± 1.25 (-1.07,0.18)

Disclosures
No relevant conflicts of interest to declare.