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Low serum hepcidin levels provide a physiologic response to iron demand in patients with iron deficiency (ID). Based on a discovery of suppressed hepcidin expression by a cytokine named growth differentiation factor 15 (GDF15), it was hypothesized that GDF15 may suppress hepcidin expression in humans with ID due to blood loss.
To test this hypothesis, GDF15 and hepcidin levels were measured in peripheral blood from subjects with iron-deficient erythropoiesis before and after iron supplementation.
Iron variables and hepcidin levels were significantly suppressed in iron-deficient blood donors compared to healthy volunteers. However, ID was not associated with elevated serum levels of GDF15. Instead, iron-deficient subjects’ GDF15 levels were slightly lower than those measured in the control group of subjects (307 ± 90 and 386 ± 104 pg/mL, respectively). Additionally, GDF15 levels were not significantly altered by iron repletion.
ID due to blood loss is not associated with a significant change in serum levels of GDF15.
Iron deficiency (ID) affects billions of persons worldwide, and anemia develops when iron stores become insufficient to maintain normal erythropoiesis. Iron homeostasis in mammals is largely maintained by expression of a small hepatic protein named hepcidin.1 Hepcidin itself is regulated at the transcriptional and posttranscriptional levels by multiple extracellular signals related to the iron supply and the erythropoietic demands of the host, as well as by inflammatory states. ID results in a significant reduction in serum hepcidin as measured by mass spectroscopy2 and more recently by hepcidin immunoassay.3 Even in the absence of ID, anemia-related hypoxia4 and erythropoietin production inhibit hepcidin expression.5 In addition, in anemic states characterized by ineffective erythropoiesis, such as thalassemia and congenital dyserythropoiesis,6 inappropriate hepcidin down regulation results in severe iron overload. It was recently demonstrated that human erythroblasts secrete a cytokine named growth differentiation factor 15 (GDF15) that contributes to the inhibition of hepcidin in pathologic conditions.7 As a p53 target gene,8 it was proposed that GDF15 is secreted from apoptotic erythroblasts in clinical settings of ineffective erythropoiesis. However, GDF15 involvement in iron regulation in other clinical settings is largely unknown.
All studies were performed after human subjects review and approval by a National Institutes of Health institutional review board. Study populations included healthy volunteers (n = 10) and volunteer blood donors with ID secondary to blood donation (n = 10). Venous blood from four subjects with ID anemia who underwent treatment with oral iron (ferrous sulfate [325 mg] three times daily for at least 30 days) was also studied.
The GDF15 enzyme-linked immunosorbent assay (ELISA) was performed on cryopreserved venous blood samples with use of an ELISA for human GDF15 (DuoSet, R&D Systems, Minneapolis, MN) following the manufacturer’s protocol. Hepcidin measurement was performed by an ELISA-based assay system (Intrinsic Lifescience, La Jolla, CA). All other assays were performed by the Clinical Center Hospital of the National Institutes of Health as standard clinical assays.
Replicate data are expressed as means ± SD with comparisons among groups performed using t test.
ID was defined as a serum ferritin level less than 10 ng/mL and a transferrin saturation less than 15%. Transferrin saturation, ferritin, hepcidin, and GDF15 levels were determined using venous blood from 10 blood donors with ID, as well as 10 healthy volunteers who had no evidence of ID (control group). Clinical and laboratory data in these subjects are shown in Tables S1A and S1B (available as supporting information in the online version of this paper). Serum iron, ferritin, and transferrin saturation were significantly lower (p < 0.05) in the ID group compared to controls. The hematologic variables were consistent with irondeficient erythropoiesis with reductions in plasma hemoglobin, red blood cell counts, and mean corpuscular volumes. Hepcidin levels were also significantly reduced to levels at or below the limit of detection (<5 ng/mL) in the ID samples compared to the control group (46 ± 41 ng/mL, p < 0.01), and transferrin levels were elevated. The reduced hepcidin levels are consistent with previously reported results using the same method of hepcidin measurement.3 Based on the consistently low level of serum hepcidin detected in the donor population studied here, it is predicted that hepcidin levels may eventually be useful for the identification of blood donor subpopulations who require iron supplementation.
In addition to standard iron variables and hepcidin measurements, GDF15 levels were assayed on frozen serum samples. As shown in Fig. 1, serum GDF15 levels were not increased in the ID group. Mean GDF15 levels were 386 ± 104 pg/mL in the control group and 307 ± 90 pg/mL in the ID group. The slight reduction in mean GDF15 concentration in the ID group did not reach significance (p = 0.085). GDF15 studies were also performed after iron supplementation in four individuals (one blood donor and three subjects with ID due to menstrual blood loss). A summary of the clinical data on those four subjects is shown in Table S1C. For comparison purposes, hepcidin and GDF15 levels were measured after the serum ferritin and transferrin saturation reached levels of more than 10 ng/mL and more than 15%, respectively. As shown in Fig. 2, iron concentration, transferrin saturation, and serum hepcidin levels increased with iron supplements in each individual. However, serum GDF15 levels remained static in the presupplement (247 ± 101 pg/mL) and postsupplement (266 ± 103 pg/mL) samples (p = 0.81).
In this study, otherwise healthy humans with simple ID due to blood loss had significantly reduced serum hepcidin levels, but their serum GDF15 levels were not significantly different from iron-repleted controls. In patients with ineffective erythropoiesis, extremely high-level expression of GDF15 (>10,000 pg/mL) was associated with inappropriate hepcidin suppression and pathologic iron loading.6,7 It was therefore proposed that the increased levels of apoptosis among the maturing erythroblasts could provide a potential mechanism for increased GDF15 expression in those patients. As such, the high levels of GDF15 among those patients provides a pathologic mechanism for tissue iron overloading to levels that are well beyond those required to support erythropoiesis. Based on those studies, it was hypothesized that GDF15 may also down regulate hepcidin in the setting of iron-deficient erythropoiesis. Remarkably, we found no association between GDF15 and ID, a result that was of considerable interest when compared to previous reports. In a study of highintensity blood donors, GDF15 levels failed to show a correlation with ferritin concentrations.9 However, more than 10% of the superdonors in that study possessed significant elevations in their serum GDF15 levels (>1000 pg/mL), and no separate causes for GDF15 elevations were investigated. In a subsequent study, it was demonstrated that iron depletion increases GDF15 expression in cultured cells and that iron chelation caused a transient increase in GDF15 levels among human research subjects. That study also reported significant elevations in GDF15 among randomly chosen patients with ID, but other etiologies for increased GDF15 were not explored.10 Overall, the data reported here suggest that serum GDF15 is neither regulated by simple ID nor does this cytokine play a major role in hepcidin suppression due to ID in the setting of blood donation. GDF15 may thus contribute to the pathologic tissue iron overloading associated with ineffective erythropoiesis rather than the physiologic response of iron loading associated with blood loss or donation. As such, elevations in serum GDF15 levels among blood donors should not be solely attributed to ID. Instead, major GDF15 elevations in blood donors or others with low iron stores should raise suspicion or trigger the investigation of other etiologies or disease entities.
This research was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases.
SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article:
TABLE S1. (A) Clinical and laboratory parameters in the healthy volunteer control group (n = 10). (B) Clinical and laboratory parameters in the iron deficient group (n = 10). (C) Iron deficient subjects prior to and after oral iron supplementation (n = 4)
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CONFLICT OF INTEREST There are no conflicts of interest to declare by any author.
Author contributions: TT, manuscript writing, performed experiments, assembly of data; AR, blood donors recruiting; YTL, blood sample collection; YYY, collection of clinical samples and data; SFL, collection of clinical samples and data; and JLM, manuscript writing, experimental conception and design, assisted and supervised research team.