Plasma Biomarkers of Iron Regulation, Overload, and Inflammation in Sickle Cell Disease

Transfusional iron overload has been increasingly recognized among patients with sickle cell disease (SCD) over the past two decades. We recently reported on the prevalence of iron overload among 635 adult SCD patients followed at our center and found that 80 patients (12%) had developed iron overload as a result of repeated blood transfusions. Fifty six (70%) of these subjects developed iron overload as a result of episodic, mostly unnecessary transfusions at outlying hospitals. There have been reports of association of increased morbidity and mortality among iron overloaded SCD patients; it has also been hypothesized that SCD patients tend to develop fewer complications of iron overload, compared to transfusion dependent beta thalassemia, primarily due to the chronic inflammatory state with resultant upregulation of hepcidin, and lower extra-hepatic iron loading. We studied biomarkers of iron metabolism, iron regulation, and inflammatory markers in 22 patients with SCD (SS) and iron overload (two consecutive ferritin levels of >1000 ng/ml and significant transfusion history) and compared these with 14 SCD patients without iron overload (ferritin <1000 ng/ml, and no significant transfusion history). Serum Fe, ferritin, %transferrin saturation (Tf) and total iron binding capacity, as well as high sensitivity C reactive protein (hsCRP) were performed by routine laboratory methods. Plasma concentrations of soluble transferrin receptor (sTfR), interleukin-6 (IL-6), Growth Differentiation Factor-15 (GDF-15) were measured using commercially available ELISA kits (R&D Systems, Minneapolis, USA). Plasma hepcidin was measured using a commercially available kit from DRG Diagnostics (Marburg, Germany). The results are summarized below:

As expected, ferritin and % Tf saturation were significantly higher in the iron overloaded group. Hepcidin levels were also significantly higher in cases vs. controls, indicative of appropriate upregulation of hepcidin in Fe overload. sTfR and GDF-15 levels, as well as the inflammatory markers (hsCRP and IL-6) did not differ significantly between Fe overloaded and non-iron overloaded SCD patients. The two groups did not differ significantly in terms of the measures of disease severity (number of pain crises/year and number of hospitalizations/year). We further looked at the ratio of hepcidin/ferritin, sTfR/log ferritin, GDF-15/hepcidin, and tested the correlation between GDF-15 and hepcidin levels and ferritin and hepcidin levels; the ratio of hepcidin to ferritin was not different between cases and controls (0.019 and 0.021, respectively, p=0.73). sTfR to log ferritin ratio was significantly lower in cases compared to controls (22.3 vs 33.24, p=0.0004). GDF-15/hepcidin ratio was also found to be significantly lower in cases (262.1 vs 1896.7, p=0.01). Additionally, GDF-15 and hepcidin levels correlated significantly in controls but not iron overloaded SCD patients (p=0.04 vs p=0.7). Similarly, hepcidin and ferritin levels were significantly correlated in controls (p=0.03) but not in cases (p=0.8). Our results suggest that i) hepcidin levels are appropriately upregulated in iron overloaded SCD patients, ii) inflammatory markers (hsCRP and IL-6) were not significantly different between iron overloaded and non-iron overloaded patients, suggesting that systemic inflammation is not the driving factor behind hepcidin upregulation in iron overloaded SCD patients; however, a local/paracrine effect of IL-6 on hepatocytes secondary to Fe related inflammation in the liver cannot be excluded; and iii) GDF-15 and sTfR levels are not different between cases and controls, indicating that erythropoiesis does not differ between Fe overloaded and non-iron overloaded SCD patients. The observation that the correlation between GDF-15 and hepcidin levels is lost in iron overloaded SCD patients suggests that erythropoiesis does not contribute to hepcidin regulation in these subjects. This can further be clarified by studying the role of the newly described erythroid regulator of hepcidin, erythroferrone in SCD with and without iron overload.