Title: Hepcidin in anemia of chronic heart failure
Abstract: Anemia is a common finding among patients with chronic heart failure (HF). Although comorbidities, such as kidney failure, might contribute to the pathogenesis of anemia, many patients with HF do not have any other obvious etiology for their anemia. We investigated whether anemia in HF is associated with an elevation in hepcidin concentration. We used time-of-flight mass spectrometry to measure hepcidin concentration in urine and serum samples of patients with HF and in control subjects. We found that the concentration of hepcidin was lower in urine samples of patients with HF compared with those of control subjects. Serum hepcidin was also reduced in HF but was not significantly lower than that in controls. There were no significant differences between hepcidin levels in patients with HF and anemia compared with patients with HF and normal hemoglobin level. We concluded that hepcidin probably does not play a major role in pathogenesis of anemia in patients with chronic HF. Between 20% and 40% of patients with heart failure (HF) have anemia [1, 2]. There are several possible confounding factors that might contribute to the high frequency of anemia among patients with HF, including concurrent kidney failure. However, because of an upregulation of several inflammatory cytokines in HF [3], it is a reasonable hypothesis that anemia in HF can be an anemia of inflammation. Over the past 8 years, hepcidin has been identified as the molecule contributing to anemia of inflammation [4, 5]. Initially discovered as an antimicrobial molecule [6, 7], hepcidin has an important role in regulating iron metabolism, particularly during infection and inflammation [8-10]. Hepcidin is mainly synthesized in the liver and released to the plasma. During an acute inflammatory response, hepcidin concentration in plasma increases several folds in a short period of time, which results in a rapid decline in the plasma iron concentration [4]. Hepcidin decreases export of iron absorbed from intestinal mucosa to blood and also decreases release of iron from macrophages recycling iron from senescent erythrocytes [9]. This results in a deprivation of erythroid progenitor cells from necessary iron for erythropoiesis. If the stimulus for production of hepcidin continues, such as in a chronic inflammatory condition, abnormal erythropoiesis would result in chronic anemia. We hypothesized that elevation in serum hepcidin mediates anemia in patients with HF. To study this hypothesis, we measured hepcidin in urine and serum samples of anemic and nonanemic patients with HF. Additionally, we measured hepcidin in a control group consisted of individuals without any clinical evidence of HF, who have been evaluated in various outpatient clinics. Baseline characteristics of patients with HF and control subjects are summarized in Table I. We studied 36 patients with HF and anemia, 61 patients with HF and no anemia, and 38 control subjects. Patients in the anemic group had lower hemoglobin level (11.64 ± 0.19 g/dL) compared with those in the nonanemic group (14.25 ± 0.15 g/dL) or control subjects (14.14 ± 0.27 g/dL). There was no significant difference in the serum concentration of ferritin or creatinine among the three groups. We conducted a multivariate analysis to detect the effect of age, sex, presence of coronary artery disease, history of coronary artery bypass graft surgery, hypertension, diabetes mellitus, New York Heart Association class, hemoglobin concentration, serum ferritin, left ventricle ejection fraction (LVEF), and the etiology of HF (ischemic vs. nonischemic) on serum and urine hepcidin concentration in patients with HF. Among these factors, only serum ferritin affected hepcidin concentration in both serum (P < 0.001) and urine (P < 0.002). Importantly, in anemic and nonanemic HF patients, there were no significant independent interactions between hepcidin and hemoglobin, LVEF, or etiology of HF (all P > 0.05). Over all, patients with HF had a lower urine hepcidin compared with those of control subjects (0.9 ± 0.2 and 2.0 ± 0.5, respectively, P = 0.002) (Fig. 1). There was a similar trend in the serum hepcidin concentrations of HF patients and controls, which did not reach a statistical significance (4.0 ± 0.4 and 5.3 ± 0.6, respectively, P = 0.054). Hepcidin and HF. Bar graphs demonstrate serum and urine hepcidin levels in all patients with HF compared with control subjects. Left, Comparison of urine hepcidin levels in patients with HF and controls. Right, Comparison of serum hepcidin levels in patients with HF and controls. Error bars represent standard error of the mean. Lower level of hepcidin in HF patients was reflected in both anemic and nonanemic subgroups. The serum hepcidin in patients with HF and anemia was significantly lower than that in control subjects (P = 0.022) (Fig. 2A). On the other hand, serum hepcidin in nonanemic HF patients was not significantly different from that of control subjects or anemic HF patients (P = 0.251 and 0.172, respectively). Urine hepcidin in both anemic and nonanemic HF patients was lower than that in control subjects (P = 0.019 and 0.003, respectively) (Fig. 2B). There was no statistically significant difference between anemic and nonanemic patients regarding their urine or serum hepcidin (P = 0.74 and 0.172, respectively). Hepcidin and anemia in HF. Bar-and-whisker plots representing hepcidin levels in patients with HF and anemia, patients with HF and no anemia, and control subjects. (A) Serum hepcidin levels (nM) and (B) urine hepcidin levels (nM/mM of creatinine). Hepcidin plays an important role in iron metabolism and in the pathogenesis of anemia of inflammation. Hepcidin mediates anemia of inflammation [8, 11, 12] by binding to and internalizing ferroportin, a membrane iron transporter responsible for exit of iron from intestine epithelial cells and macrophages, resulting in degradation of ferroportin [13, 14]. Inflammatory cytokines, such as IL-6, increase synthesis of hepcidin in the liver [15]. On the other hand, iron excess, anemia, and hypoxia decrease hepcidin synthesis and its plasma level [16, 17]. The level of hepcidin increases in various infectious or inflammatory conditions and causes rapid change in plasma iron level. Progression of HF is associated with a sustained elevation of several proinflammatory cytokines, including tumor necrosis factor-α, the interleukin (IL)-1 family, and IL-6 [18]. Among these cytokines, IL-6 has been shown to increase synthesis of hepcidin in the liver. We studied whether anemia of HF is associated with an elevation in hepcidin concentration. A previous study on anemia in patients with HF showed that a relative increase in the plasma volume rather than a decrease in the red blood cell mass was the main finding in these patients [19]. However, this study only examined patients with HF and did not include control subjects without HF. Recently, Matsumoto et al. [20] studied serum hepcidin level in patients with chronic HF. They compared serum hepcidin between 36 patients with HF and anemia, 16 patients with HF and no anemia, and 16 patients with no HF and no anemia. They found that serum hepcidin was lower in patients with HF and anemia compared with that of other groups and concluded that anemia of inflammation is a minor cause for anemia of HF. Our study confirmed this result in a larger number of patients using both urine and serum hepcidin. Anemia in patients with HF is a multifactorial disease. In this study, we investigated whether anemia in HF is associated with an elevated hepcidin concentration. We found that patients with HF and anemia had both lower urine and serum hepcidin compared with those in control subjects. It is important to mention that our control subjects are from individuals visiting different outpatient clinics at Baylor College of Medicine and might be different from a group of healthy controls. In our previous study, median of serum hepcidin among healthy individuals was found to be 4.2 nM, with a range of 0.5–13.9 nM [21], which is lower than the serum hepcidin of controls in this study. The main goal of this study was to investigate the role of hepcidin in anemia of HF, and the comparison between hepcidin concentration in HF patients with and without anemia did not show a significant difference. These findings are not consistent with the presence of a major role for hepcidin in the pathogenesis of anemia in HF patients. However, one should be cautious about interpreting our results, because several factors might affect hepcidin level in HF, some of them in opposite directions; liver synthetic defect, anemia of dilution, and elevated erythropoietin [20] would decrease hepcidin. On the other hand, inflammatory cytokines can increase the hepcidin level. Chronic HF is associated with elevation of several cytokines; among them, tumor necrosis factor-α has been shown to decrease iron absorption from intestine and iron release from macrophages [22] and might contribute to anemia of HF. Once HF patients become anemic, it is likely that their anemia downregulates synthesis of hepcidin and causes lower level of hepcidin. This is a possible explanation for the lower concentration of hepcidin in anemic HF patients. The other possible explanation for our results is that, in patients with HF, elevated erythropoietin [20] might decrease hepcidin level and overrides the effect of inflammatory cytokines. This study was conducted according to the protocol for human subject study approved by the Institutional Review Board of Baylor College of Medicine. All of the subjects signed a consent form to participate in this study. Ninety-seven patients with chronic HF (LVEF less than 40% and New York Heart Association class II–IV symptoms) were recruited from hospitals affiliated with Baylor College of Medicine in Houston, Texas. According to their hemoglobin level, patients with HF were divided into anemic (hemoglobin of less than 13 g/dL for men and 12 g/dL for women) and nonanemic subgroups. Thirty eight patients without a history of HF were selected during outpatient clinic visits as the control subjects. Exclusion criteria included presence of kidney failure (serum creatinine above 1.5 mg/dL), history of cardiac transplant, and history of gastrointestinal or severe menstrual bleeding. Hepcidin comprises three isoforms that contains 20 (hepcidin-20), 22 (hepcidin-22), or 25 amino acids (hepcidin-25). Hepcidin-25 is the biologically active isoform. We performed urine and serum hepcidin-25 measurements using a combination of weak cation exchange chromatography and surface-enhanced laser desorption ionization–time-of-flight mass spectrometry, as previously reported [23, 24]. An internal standard (synthetic hepcidin-24; Peptide International) was used for quantification. Peptide spectra were generated on the time-of-flight mass spectrometry platform of a PBS IIc mass spectrometer (Purchased from Ciphergen Biosystems). This method reproducibly detect elevated hepcidin levels in inflammatory conditions such as infection and rheumatoid arthritis and decreased hepcidin level in iron-deficiency anemia [23, 24]. Serum hepcidin levels were expressed in nM, and urine levels in nM/mM creatinine (normalized to urine creatinine concentration). For serum, the intrarun coefficient of variation was 3.9% at 7.3 nM (n = 8) and 3.1% at 3.4 nM (n = 8), and the inter-run coefficient of variation was 7.5% at 4.0 nM (n = 8). Intrarun variation of hepcidin measured in urine is 3.0% both at 3.3 nM (n = 8) and 9.9 nM (n = 8). Inter-run variation for urine varies between 12.6% at 1.5 nM (n = 8) and 10.2% at 9.1 nM (n = 8). Lower limit of detection for serum was 0.5 nM and for urine was 50 pM. All values are expressed as mean ± standard error of mean. Differences in baseline characteristics between the three groups (anemic HF, nonanemic HF, and control) were tested using the χ2 test or Fisher's exact test for categorical variables and analysis of variance for continuous variables. Overall differences in biomarker levels were tested using one-way analysis of variance or Kruskal–Wallis test (for non-Gaussian variables). Tukey's test was used for post-hoc testing where appropriate. HF patients as a group were compared with control subjects using the Student's t-test or Mann–Whitney U test. Multivariate regression analysis was performed on serum and urine hepcidin using the following predictors: age, sex, presence of coronary artery disease, history of coronary artery bypass graft surgery, hypertension, diabetes mellitus, New York Heart Association class, LVEF, ferritin, hemoglobin, and etiology of HF (ischemic vs. nonischemic). All data analysis was performed using SPSS 13 (SPSS, Chicago, IL). A P-value <0.05 was considered statistically significant. Vijay Divakaran*, Sachin Mehta*, David Yao*, Saamir Hassan*, Steven Simpson*, Erwin Wiegerinck , Dorine W. Swinkels , Douglas L. Mann , Vahid Afshar-Kharghan§, * Baylor College of Medicine, Houston, Texas, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands, Washington University School of Medicine, St. Louis, Missouri, § The University of Texas M.D.Anderson Cancer Center, Houston, Texas.