Helicobacter pylori (H. pylori) requires iron to survive and may potentially be associated with unexplained iron-deficiency anemia (IDA) [1]. The prevalence of H. pylori infection in refractory IDA is higher than in the normal population [2], and an association between low serum ferritin levels and a high prevalence of H. pylori-specific IgG has been noted [3]. H. pylori-related refractory IDA can be reversed by eradication of H. pylori [4], and some H. pylori-infected IDA patients achieve normal serum hemoglobin levels after H. pylori eradication, even without oral iron treatment [5]. Additionally, it has been shown that combined H. pylori eradication and iron replacement produces a faster response than iron therapy alone [6]. Based on these findings, eradication of H. pylori is recommended in unexplained IDA patients [7], although no association between the pathogenesis of IDA and H. pylori infection has yet been established. A key pathogenic connection must exist to explain the relationship between H. pylori infection and IDA, as not all H. pylori-infected patients develop unexplained IDA.

Hepcidin, a 25-amino-acid disulfide-rich peptide that is synthesized in the liver, acts as a systemic iron regulatory hormone and plays an important role in duodenal iron absorption [8]. Hepcidin mRNA is induced by interleukin-6 [9], and its expression is enhanced by factors such as lipopolysaccharides and Streptococcus iniae infection [10]. Hepcidin binds to cell-surface ferroportin to trigger its tyrosine phosphorylation, internalization, and ubiquitin-mediated degradation in lysosomes, resulting in the retention of iron in enterocytes and the interruption of iron release from macrophages [11]. In murine models, parenchymal iron deposition has been reported in hepcidin knockout mice [12], whereas transgenic mice that overexpress hepcidin exhibited decreased body iron levels and presented with severe IDA [13]. Additionally, H. pylori subverts the human iron regulatory mechanism in a manner that is beneficial to H. pylori, but deleterious to the host, by inducing the up-regulation of hepcidin and/or down-regulation of ferroportin [14].

Attempts to produce correctly folded synthetic hepcidin have proved difficult. Thus, prohepcidin, the 60-amino acid product of the cleavage of the signal peptide from the hepcidin precursor, is measured with a currently available commercial assay for bioactive hepcidin analysis [15]. To determine the feasibility of serum prohepcidin as a useful biomarker in IDA, it is first necessary to determine the precise molecular mechanism underlying the action of prohepcidin under various conditions. In this study, we evaluated changes in serum prohepcidin levels before and after H. pylori eradication in H. pylori-infected IDA patients to establish whether it normalized after treatment.

Of the 50 initially enrolled subjects, 12 were excluded because the cause of their IDA was established (Fig. 1). Of the 38 remaining IDA subjects, 29 subjects (76.3%) revealed H. pylori infection. Eradication was conducted in these 29 subjects, but six refused to participate in the study after the treatment. Baseline characteristics did not differ between the remaining 23 H. pylori-infected subjects (212.9 ± 88.2 ng/mL) and nine non-infected subjects (217.8 ± 56.2 ng/mL, p = 0.879, Table 1). Of 23 H. pylori-infected subjects, six patients preferred to undergo H. pylori eradication without iron replacement based on their past experiences of severe adverse effects during iron intake. Thus, only 17 H. pylori-infected IDA subjects underwent combined H. pylori eradication and iron replacement.

In this study, we measured only the serum prohepcidin level because of the difficulty inherent in measuring serum hepcidin. Although we were unable to measure hepatic iron accumulation in each case because that would involve an invasive procedure, our data suggest that serum prohepcidin levels were increased in patients with IDA regardless of the presence of H. pylori infection. To our knowledge, this is the first reported study to measure serum prohepcidin levels in IDA patients, comparing levels before and after H. pylori eradication with or without iron replacement.

Serum prohepcidin levels decreased significantly in the H. pylori-infected IDA patients after combined H. pylori eradication and oral iron therapy. Additionally, a tendency toward a decrease in serum prohepcidin levels after H. pylori eradication or iron replacement was observed. Our findings may be explained by the suggestion made in previous work that H. pylori infection results in the regulation of hepcidin, which prevents a response to oral iron and manipulates the host's iron homeostasis to ensure its survival [14,16]. Notably, six H. pylori-infected subjects who declined to take oral iron also exhibited a reduction in serum prohepcidin levels after H. pylori eradiation. It seems that iron therapy is not required in H. pylori-infected IDA patients, as it appears that the IDA can be cured merely by eradicating the H. pylori infection [5]. However, serum prohepcidin levels decreased further with combined H. pylori eradication and oral iron replacement. This is consistent with a previous study that found that combined H. pylori eradication and iron replacement produced a faster response than iron therapy alone [6].

The serum concentration of prohepcidin in Korean female college students has been reported to be 85.1 ± 6.1 ng/mL, with a range of 13.6 to 295.7 mg/mL [17]. Serum prohepcidin levels in our IDA patients were significantly higher than this value, regardless of the presence of H. pylori infection. We are unable to explain the lack of difference in serum pro-hepcidin levels between the H. pylori-infected and noninfected groups before treatment. We suggest that long-term infection with H. pylori leads to homeostasis in IDA patients because most are infected during childhood. Interestingly, serum prohepcidin levels exhibited a tendency to decrease in the noninfected IDA group after oral iron therapy, suggesting that hepcidin is an important mediator of iron homeostasis even in noninfected IDA. During long-term follow-up observation for more than 3 months, the prohepcidin levels in nine subjects decreased further after successful H. pylori eradication therapy as IDA improved with continuous oral iron replacement. This indicates that the serum prohepcidin level does not normalize immediately.

Our study has several limitations. First, no long-term follow-up data were gathered in the H. pylori-eradication only group or the iron-replacement only group. Second, we did not check intragastric acid levels in each subject, although a possibility of achlorhydria exists due to H. pylori-induced chronic pangastritis. Third, most of the subjects were premenopausal women; indeed, only two men were included in the study. Most male subjects were ruled out from unexplained IDA because they showed positive findings in upper or lower gastrointestinal endoscopy.

In summary, our findings suggest that serum prohepcidin levels are related to the severity of IDA, rather than to the presence of H. pylori infection, for the following reasons. First, basal serum prohepcidin levels were higher in our IDA patients than reported for normal healthy Koreans regardless of H. pylori infection status [17]. Second, serum prohepcidin levels decreased after iron-replacement therapy, even in the noninfected group. Third, during longterm followup after successful H. pylori eradication, serum prohepcidin levels decreased further with improvement in the IDA. Fourth, no significant difference in the reduction ratios was found among the three groups, although combined H. pylori eradication and iron replacement therapy seemed to have a synergistic effect. Fifth, either H. pylori eradication or oral iron administration resulted in decreased serum prohepcidin levels after the treatment, accompanied by IDA improvement. Taken together. Taken together, these findings indicate that the serum concentration of prohepcidin is related to the status of anemia rather than to the current status of H. pylori infection in IDA patients, indicating that serum concentration of prohepcidin is a functional measure in IDA.