Helicobacter ISSN 1523-5378 doi: 10.1111/hel.12170
The Relationship Between Iron Deficiency in Patients with Helicobacter Pylori-Infected Nodular Gastritis and the Serum Prohepcidin Level Yuichi Sato,* Osamu Yoneyama,* Masaki Azumaya,* Manabu Takeuchi,* Syun-ya Sasaki,* Junji Yokoyama,* Kazuhiko Shioji,* Yusuke Kawauchi,* Satoru Hashimoto,* Yuuki Nishigaki,* Masaaki Kobayashi,† Kazuhito Sugimura,* Terasu Honma,* Rintaro Narisawa† and Yutaka Aoyagi* *Department of Gastroenterology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan, †Division of endoscopy, Niigata University Medical and Dental Hospital, Niigata, Japan
Keywords Hepcidin, HP-related iron-deficiency anemia, hyperplastic polyp, hypochlorhydria, nodular gastritis. Reprint requests to: Yuichi Sato, MD, Department of Gastroenterology, Niigata University Graduate School of Medical and Dental Sciences, Asahimachi-dori, Niigata 951-8121, Japan. E-mail: [email protected]
Abstract Background and aims: Helicobacter pylori (H. pylori) is recognized as a causative agent for unexplained iron-deficiency anemia (IDA). We evaluated many background factors influencing an iron-deficiency state in adult patients with various H. pylori-infected upper gastrointestinal tract diseases. Method: Study 1: H. pylori-infected 121 patients (nodular gastritis (NG) (n = 19), duodenal ulcer (DU) (n = 30), or gastric ulcer (GU) (n = 47), or gastric hyperplastic polyp (GHP) (n = 25)) were enrolled. The RBC count and hemoglobin, iron, ferritin, pepsinogen (PG) I, PG II, gastrin, and anti-H. pylori antibody (Ab) levels in the serum were measured. Study 2: H. pyloriinfected 105 patients (NG, n = 19; DU, n = 43; GU, n = 32; GHP, n = 11) and non-H. pylori-infected individuals (n = 35) were examined for the levels of prohepcidin, ferritin, and iron in the serum. In addition, we measured the data before and after the H. pylori eradication. Results: In the patients with GHP and NG, hypoferritinemia was observed in comparison with the GU and DU patients. In the GHP patients, low levels of PG I, a decreased PG I/II ratio, and hypergastrinemia were observed. The levels of serum prohepcidin in the patients with H. pylori-associated disease were higher than those in the uninfected adults. In the patients with NG, the serum prohepcidin levels were higher than those in the other H. pyloriinfected patient groups and decreased after the eradication. Conclusion: H. pylori-related iron-deficiency state might be associated with several factors, such as hypochlorhydria and hepcidin, in patients with GHP or NG.
Helicobacter pylori (H. pylori) is recognized as a causative agent for various gastroduodenal diseases, such as chronic gastritis, duodenal ulcer (DU), gastric ulcer (GU), gastric cancer, gastric mucosa-associated lymphoid tissue lymphoma [1], gastric hyperplastic polyp (GHP) [2], and nodular gastritis (NG) [3]. Furthermore, H. pylori infection has been implicated in some extradigestive diseases, including iron-deficiency anemia (IDA), idiopathic thrombocytopenia purpura, and vitamin B12 deficiency [4]. There has been many research about the association between H. pylori and iron deficiency to date, and three separate meta-analyses were
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recently published that showed an association between H. pylori infection and IDA [5–7]. H. pylori is related to unexplained IDA in the absence of gastrointestinal blood loss in adult and children in the studies. Therefore, the Maastricht IV/Florence consensus recommends the eradication of H. pylori in H. pylori-positive patients with IDA (grade of recommendation, A) [4], and other guidelines also suggest eradicating H pylori in patients with IDA [8–10]. The mechanism by which H. pylori infection contributes to IDA remains unclear; however, several mechanisms have been postulated [11,12]. First, H. pylori
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infection may progress to pangastritis and atrophic gastritis, leading to hypochlorhydria or achlorhydria, resulting in iron malabsorption. Moreover, H. pylori infection can reduce the gastric juice ascorbic acid (vitamin C) level, which is a potent promoter of nonheme iron absorption. Next, H. pylori infection may increase the uptake of iron by bacteria, because iron is an essential bacterial growth factor [12–14]. H. pylori has a lactoferrin binding protein in the outer membrane, and it has been speculated that H. pylori increases the lactoferrin uptake from transferrin, and the bacterial iron stores are subsequently lost in the feces after the bacteria are destroyed [12]. Other reports have revealed that some H. pylori strains have an advantage in iron acquisition [13,14]. Moreover, recent studies have showed that hepcidin, which acts as a one of the major regulators of systemic iron homeostasis and downregulates duodenal iron absorption, is related to the IDA associated with H. pylori infection [15–17]. However, IDA does not develop in all H. pyloriinfected patients. Therefore, we evaluated the iron-deficiency status and many background factors influencing iron deficiency in adult patients with various H. pyloriassociated gastroduodenal diseases, including DU, GU, GHP, and NG. Furthermore, we focused on hepcidin which is an antimicrobial peptide and a central regulator of iron metabolism. We measured and compared the serum levels of prohepcidin, the precursor of hepcidin, between the patients with these diseases and nonH. pylori-infected people, and evaluated the prohepcidin levels before and after the eradication of H. pylori in patients with GU, DU, and NG.
Methods
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antibody (Ab) levels. The red blood cells counts and Hb level were determined using an automated electronic counter (Sysmex XE-5000; Sysmex corporation, Kobe, Japan). The serum iron levels were determined using a bathophenanthroline method. The levels of serum ferritin were measured using an EIA kit (ST AIA-PACK FER; Tosoh Bioscience, Tokyo, Japan). The fasting levels of serum gastrin and PG I and II were measured using an RIA kit (Dinabot Company, Tokyo, Japan). The estimation of gastric atrophy was based on the serum PG I and II levels and the I/II ratio. The PG I and I/II ratio are wellrecognized markers of fundic gland atrophy [18]. Serum IgG Abs against H. pylori were identified using a specific ELISA kit (HM-CAP; Enteric Products INC., Westbury, NY, USA). This assay system is reported to have 100% specificity and 96% sensitivity in Japan in comparison with the urea breath test [19]. During the esophago-gastro-endoscopy, biopsy specimens were obtained from the gastric antrum and corpus and were used for bacterial culture of H. pylori. The growth of H. pylori from antral and corpus biopsies was graded as none, scant, moderate, or abundant growth corresponding to the H. pylori colonies found in 0, ≤ 1/3, 1/3–2/3, and ≥2/3 of the culture plate, respectively, and was converted to numerical scores (none = 0, scant = 1, moderate = 2, and abundant = 3). H. pylori infection was confirmed by bacterial culture, or serum IgG Ab against H. pylori. The normal range of RBC count, Hb, and the levels of iron and ferritin in the serum were as follows: RBC count, 400– 552 9 104/lL (for male) or 378–499 9 104/lL (for female); Hb, 13.2–17.2 g/dL (for male) or 10.8–14.9 g/dL (for female); serum iron level, 60–210 lg/dL (for male) or 50–170 lg/dL (for female); and serum ferritin level, 21–282 ng/mL (for male) or 5–157 ng/mL (for female).
Study 1 We examined 19 patients (six male and 13 female; mean age, 38.0 years; age range, 19–63 years) with NG, 30 patients (19 male and 11 female; mean age, 42.9 years; age range, 20–68 years) with DU, 47 patients (32 male and 15 female; mean age, 55.2 years; age range, 21– 78 years) with GU, and 25 patients (12 male and 13 female; mean age, 61.4 years; age range, 19–83 years) with GHP. The exclusion criteria were as follows: [1] chronic inflammatory disease, recent bleeding, liver diseases, or malignancy; [2] previous eradication therapy or the use of a proton-pump inhibitor, antibiotic, or H2 receptor antagonist; and [3] a history of blood transfusion or iron supplement therapy. A blood sample was obtained from each of the enrolled patients in the fasting state to assess the red blood cell count (RBC) and the hemoglobin (Hb), serum iron, serum ferritin, serum pepsinogen (PG) I, PGII, serum gastrin, and anti-H. pylori
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Statistical analysis The significance of the differences among the groups in terms of the age, RBC count, Hb, the levels of iron, ferritin, PG I, PG II, and gastrin in the serum, and the grading of H. pylori colonies was determined using nonparametric ANOVA (Kruskal–Wallis one-way ANOVA). We used Fisher’s exact test to analyze the differences in the sex distribution among the groups. Differences of p < .05 were considered to be significant. Analyses were performed using SPSS 20.0 for Windows (IBM Japan Inc., Tokyo, Japan).
Study 2 We examined 19 patients (six male and 13 female) with NG, 43 patients (27 male and 16 female) with DU, 32 patients (17 male and 15 female,) with GU, 11 patients
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(five male and six female) with GHP, and 35 individuals not infected H. pylori (11 male and 24 female). The exclusion criteria were the same as in Study 1. No patients were repeatedly enrolled between study 1 and 2. A blood samples was obtained from each patient in the fasting state to assess the prohepcidin, ferritin, and iron levels. The levels of serum prohepcidin were determined by an enzyme-linked immunosorbent assay based on a competitive principle (DRG International Inc., Mountainside, NJ, USA) according to the manufacturer’s protocol. Of these patients, 11 patients with NG, 27 patients with DU, and 20 patients with GU received H. pylori eradication treatment including amoxicillin 750 mg and clarithromycin 200 mg, and a proton-pump inhibitor (lansoprazole at 20 mg or rabeprazole at 10 mg) given twice a day for seven days. Two or three months after eradication, a 13C urea breath test was carried out; if it was positive, a second eradication was performed with amoxicillin 750 mg, metronidazole 250 mg, and a proton-pump inhibitor (lansoprazole at 20 mg or rabeprazole at 10 mg) twice a day for one week. The 13C urea breath test was performed again two or three months after eradication. Blood samples were collected after six to 12 months after the eradication of H. pylori to assess the serum prohepcidin levels. Statistical analysis The significance of differences among the groups in the levels of prohepcidin in the serum was determined using nonparametric ANOVA (Kruskal–Wallis one-way ANOVA). We used Fisher’s exact test to analyze the differences in the sex distribution among the groups. The
correlation between the levels of prohepcidin and ferritin or between the levels of prohepcidin and the iron in the serum was calculated using Spearman’s rank correlation test. The significance of the differences in the levels of prohepcidin before and after H. pylori eradication was determined using Wilcoxons matched-pairs signed-rank sum test. Differences with a value of p < .05 were considered to be significant. Analyses were performed using SPSS 20.0 for Windows (IBM Japan Inc., Tokyo, Japan). In Studies 1 and 2, all procedures and biopsies were performed with the informed consent of the patients in accordance with the Helsinki Declaration, and the study was approved by the ethics committee of Niigata University School of Medicine.
Results Study 1 Table 1 shows the clinical data and characteristics of the patients divided by the disease. The patients in the NG group were significantly younger than those in the GU and GHP groups. The NG group showed a significant female dominance in comparison with the DU and GU groups. There was no significant difference in the RBC count between the groups. However, Hb level in the NG group was significantly lower than that in the DU group, and there was a trend toward a lower level of Hb in the NG group in comparison with that in the GU group. In the GHP group, the Hb level was lower than that in the GU group. There were no significant differences in the levels of serum iron among these
Table 1 The clinical characteristics of patients with nodular gastritis (NG), duodenal ulcer (DU), gastric ulcer (GU), or gastric hyperplastic polyps (GHP) in study 1
Mean age (range) Male: Female Helicobacter pylori infection rate (%) RBC (9 104/lL)a Number of below normal range (%) Hemoglobin(g/dL)a Number of below normal range (%) Iron (lg/dL)a Number of below normal range (%) Ferritin (ng/mL)a Number of below normal range (%)
Nodular gastritis (NG) (n = 19)
Duodenal ulcer (DU) (n = 30)
Gastric ulcer (GU) (n = 47)
Gastric hyperplastic polyp (GHP) (n = 25)
38.0 (19–63)† 6: 13‡ 100 (19/19) 440.5 14.8 1 (5.3%) 12.0 0.8§ 5 (26.3%) 78.3 11.9 7 (36.8%) 37.8 10.0 4 (21.1%)
42.9 (20–68) 19: 11 100 (30/30) 472.8 10.4 2 (6.7%) 13.9 0.4 4 (13.3%) 84.6 5.9 7 (23.3%) 62.3 18.4 5 (16.7%)
55.2 (21–78) 32: 15 100 (47/47) 437.0 9.7 6 (12.7%) 13.6 0.3 6 (12.7%) 96.0 10.7 10 (21.3%) 89.0 15.2 5 (10.6%)
61.4 (19–83) 12: 13 92 (23/25) 444.9 5.8 3 (12.0%) 13.0 0.4 5 (20.0%) 78.0 10.0 6 (24.0%) 41.0 8.3¶ 3 (12.0%)
Results are expressed as mean SEM, RBC, red blood cell count. p < .01, versus GU, GHP; ‡p < .05, versus DU, GU; §p < .05 versus DU, p = .0624 versus GU; ¶p < .05 versus GU.
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groups; however, the serum ferritin levels in the NG and GHP groups were significantly lower than that in the GU group. In the NG group, the percentages of patients with Hb below normal range or serum iron levels below normal range or ferritin levels below normal range were higher in comparison with the other groups. The data of the serum PG I, PG II, and gastrin levels, and the grading of the H. pylori colonies in the patients divided by the disease are presented in Table 2. The levels of PG I in the NG group were higher than those in the GU and GHP group, while those in the GHP group were significantly lower than those in both the NG and GU groups. The levels of PG II in the NG group were higher than those in the other three groups, while those in the GHP group were lower significantly than those in not only the NG but also the GU group. On the other hand, the fasting gastrin levels in the GHP group were lower than those in the other three groups. There were no significant differences in the scores of grading of H. pylori colonies among the NG, DU, and GU groups, in either the antrum or the corpus. However, the scores of grading of H. pylori in the GHP patients were significantly lower than those in the other three groups in the antrum and were lower than those in the DU or GU groups in the corpus.
Study 2 Table 3 shows the clinical characteristics and the serum levels of prohepcidin, iron, and ferritin in the patients with NG, DU, GU, and GHP, and in the non-H. pyloriinfected individuals. The levels of serum prohepcidin in the patients with NG were significantly higher than those in the patient with DU or GU or in the noninfected individuals. The levels of serum prohepcidin in the H. pylori-negative normal individual were significantly lower than the levels in the four infected groups. As with study 1, the percentages of patients with serum
iron levels below normal range or ferritin levels below normal range in the NG group were higher in comparison with the other groups. Figure 1 shows that there was a negative correlation between the serum levels of prohepcidin and the serum iron levels; however, there was no correlation between the serum levels of prohepcidin and serum ferritin levels in the patients with NG. Meanwhile, there was no relation between the serum levels of the prohepcidin and serum iron or ferritin levels in the patients with DU, GU, or GHP (data not shown). As shown in Fig. 2 and Table 4, after the eradication of H. pylori, the levels of serum prohepcidin were significantly reduced, and there was a trend toward an increase in the levels of serum ferritin in the NG patients. However, serum iron levels were unchanged in those patients. Meanwhile, the levels of serum prohepcidin, iron, and ferritin did not show any significant change after the H. pylori eradication in the patients with DU or GU (Table 4).
Discussions We herein demonstrated that there are differences in the iron-deficiency status among patients with various H. pylori-associated gastroduodenal diseases, and iron deficiency was especially observed in NG and GHP patients. Study 1 revealed that hypoferritinemia, low Hb levels, low levels of PG I, and a decreased PG I/II ratio in the serum, as well as hypergastrinemia, were observed in GHP patients. Previous studies also showed that there were low levels of PG I, a low PG I/II ratio, and hypergastrinemia in a serologic analysis of GHP patients [20,21]. These data indicate that, in GHP patients, there is progressive gastric mucosal atrophy, and decreased gastric acid secretion, and these conditions result in hypergastrinemia. Our results supported the develop-
Table 2 The serum pepsinogen (PG) I, PG II, gastrin, and grading of Helicobacter pylori colonies in the patients with nodular gastritis (NG), duodenal ulcer (DU), gastric ulcer (GU), or gastric hyperplastic polyp (GHP) in study 1 Nodular gastritis (NG) (n = 19) Pepsinogen I (ng/mL)a Pepsinogen II (ng/mL)a Pepsinogen I/II ratio Gastrin (pg/mL)a Grading of H. pylori colonies Antrum/corpus
89.3 32.4 3.0 116.3 2.5
8.3† 3.4§ 0.3 16.5 0.3/2.0 0.7
Duodenal ulcer (DU) (n = 30) 91.7 20.6 4.7 110.2 2.7
10.4 2.2 0.3 10.6 0.2/2.2 0.3
Gastric ulcer (GU) (n = 47) 61.9 19.6 3.2 153.4 1.7
5.5 1.2 0.2 18.7 0.1/1.9 0.2
Gastric hyperplastic polyp (GHP) (n = 25) 26.4 16.0 1.6 469.5 0.4
5.1‡ 2.0¶ 0.2‡ 78.4‡ 0.1k/1.0 0.2‡
Results are expressed as mean SEM. p < .01 versus GU and GHP; ‡p < .01 versus DU, GU, and NG; §p < .01 versus DU, GU, and GHP; ¶p < .01 versus NG, p < .05 versus GU; kp < .01 versus NG, p < .05 versus DU and GU.
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Table 3 The clinical characteristics of the patients with nodular gastritis (NG), duodenal ulcer (DU), gastric ulcer (GU), gastric hyperplastic polyp (GHP), and non-Helicobacter pylori-infected individuals in study 2
Mean age (range) Male: Female Prohepcidin (ng/ml)a Iron (lg/dL)a Number of below normal range (%) Ferritin (ng/mL)a Number of below normal range (%)
Nodular gastritis (NG) (n = 19)
Duodenal ulcer (DU) (n = 43)
Gastric ulcer (GU) (n = 32)
Gastric hyperplastic polyp (GHP) (n = 11)
H. pylori uninfected individuals (n = 35)
43.0 (22–63)† 6: 13 280.3 20.7§ 77.9 9.5‖ 6 (31.6%) 50.0 10.9 5 (26.3%)
46.9 (20–71)‡ 27: 16 190.9 15.8 109.4 8.4 6 (13.9%) 91.0 15.1 4 (9.3%)
52.7 (21–78) 17: 15 211.3 16.1 92.2 11.1 6 (18.8%) 97.2 20.8 2 (6.3%)
69.9 (61–86) 5: 6 237.8 20.1 106.5 11.7 1 (9.1%) 60.2 13.2 1 (9.1%)
48.2 (16–74)† 11: 24 155.6 18.0¶ 111.5 8.0 3 (8.6%) 95.3 18.9 0 (0.0%)
Results are expressed as mean SEM. p < .01 and GHP, p < .05 versus GU; ‡p < .01 versus GHP; §p < .01 versus GU, DU and uninfected individuals; ¶p < .01 versus NG, GU and GHP, p < .05 versus DU; kp < .05 versus DU and uninfected individuals.
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A
B
Figure 1 Relationship between the serum prohepcidin and serum iron (A) or serum ferritin (B) level in NG patients.
ment of these phenomena, and it is thus thought that the iron deficiency in GHP patients is caused by iron malabsorption due to the hypoacidity induced by gastric mucosal atrophy. GHP has been suggested to cause gastrointestinal blood loss due to oozing; however, AlHaddad et al. also reported that iron deficiency was observed in spite of a negative occult blood test in the GHP patients [22], which would support our results suggesting malabsorption. The colonies of H. pylori infection were low in GHP patients. H. pylori infection induces atrophy and results in the replacement of intestinal metaplasia as part of “end-stage” chronic gastritis [23]. H. pylori does not adhere to the intestinal epithelium; therefore, H. pylori is finally unable to inhabit the gastric mucosa of the patients. Our results reflect such a condition in the GHP patients’ stomachs. Furthermore, hypoferritinemia and low Hb levels were observed in NG patients in Study 1. NG is strongly
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associated with H. pylori infection [3,24] and appears to be more common in children or young people than in adults, and in female more often than in male [3,25,26]. NG is characterized by a miliary micronodule pattern with a cobblestone appearance endoscopically and is often observed in the antrum; therefore, NG is also called antral nodularity, nodular antritis, or antral gastritis. NG shows lymphoid follicles and mononuclear cell infiltration histologically, [27], and NG is therefore also called follicular gastritis. A few recent reports described the presence of IDA in NG patients [28,29]. Cardamone et al. [28] reported that three IDA patients with NG showed improvement in the iron-deficiency state after H. pylori eradication. Gulen et al. [29] described that endoscopic antral gastritis, which means NG, was observed in 21/44 (47.7%) older pediatric patients with IDA, and 19/21 patients with NG were H. pylori-positive. The patients received the eradication therapy; IDA was improved after eradication.
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Figure 2 The changes in the serum prohepcidin levels in NG patients before and after Helicobacter pylori eradication.
Gastric atrophy is rare in NG patients [3,27]; therefore, there are low levels of PG I and a decrease of the PG I/II ratio and hypergastrinemia in the serum [3,26]. Our results agree with those of previous studies and, consequently, also indicated that there was no severe atrophy and hypochlorhydria in patients with NG. Accordingly, the hypoferritinemia and anemic state in NG patients cannot be explained by iron malabsorption due to hypochlorhydria induced by gastric mucosal atrophy. Besides, the grading of H. pylori culture colonies in the NG group was relatively high in comparison with that in the GU and GHP groups. However, it was not significantly different from that in the DU group. Therefore, it is thought that the influence of the consumption of iron by H. pylori itself is relatively small. Thus, the iron-deficiency state observed in NG patients would not be explained using the past hypotheses. Accordingly, we paid attention to hepcidin. Hepcidin is a regulatory hormone of iron homeostasis and plays
an important role in duodenal iron absorption [30]. Hepcidin increases in response to inflammation and leads to a decrease in the duodenal enterocyte iron absorption and macrophage iron release by binding to ferroportin [30]. Therefore, overproduction of hepcidin has been regarded as a cause of anemia of inflammation [31]. Inflammatory cytokines such as interleukin (IL)-6, IL-1b, and bone morphogenic protein 6 all upregulate hepcidin expression [30,31]. In this study, we demonstrated that the levels of serum prohepcidin in the patients with H. pylori-associated disease were higher than those in the uninfected adults. These results suggest that the production of hepcidin would be induced by H. pylori infection. Ciacci et al. [32] have reported that the iron absorption in the duodenum is interfered in H. pylori-infected patients. Therefore, H. pylori infection may cause iron malabsorption in the duodenum via the production of the hepcidin. It is known that H. pylori infection induces the expression of interleukin IL-6 and IL-1b, which induce hepcidin production; therefore, such inflammatory cytokines in the H. pylori-infected gastric mucosa would be expected to lead to hepcidin overproduction, resulting in iron deficiency. We herein demonstrated that the serum prohepcidin levels in the patients with NG were higher than those in the other H. pylori-infected patients, and there was a negative correlation between the serum levels of prohepcidin and the serum iron levels only in the NG patients. Additionally, we found that there was a decrease in the levels of prohepcidin after the H. pylori eradication in patients with NG. Taken together, the previous results and those of our present study indicated that there is a strong relationship between iron deficiency and hepcidin production in NG. However, our study could not clarify the mechanisms underlying these findings in patients with NG. Although T helper type 2 (Th2) cytokines contribute to the formation of the lymph follicle in NG [33], H. pylori infection in children, in contrast to that in adults, results in the production of IL-10 as a Th2 cytokine [34]. A recent report
Table 4 Changes in the levels of serum prohepcidin, iron, and ferritin before and after eradication in the patients with nodular gastritis (NG), duodenal ulcer (DU), gastric ulcer (GU) in study 2
Prohepcidin (ng/ml)a Iron (lg/dL)a Ferritin (ng/mL)a
Nodular gastritis (NG) (n = 11)
Duodenal ulcer (DU) (n = 27)
Gastric ulcer (GU) (n = 20)
Before era
After era
Before era
After era
Before era
After era
348.4 19.0 85.2 29.7 20.0 6.5
257.0 32.6† 74.4 14.7 30.7 7.9
175.9 17.9 120.0 9.8 100.8 16.5
203.1 14.1 114.7 8.5 102.5 15.5
225.9 24.5 102.9 15.0 79.3 24.2
206.6 20.4 111.6 15.4 75.3 24.1
Results are expressed as mean SEM. p < .05.
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has revealed that IL-10 can directly regulate hepcidin [35]; therefore, certain humoral factors, including IL-10, might be related to the hepcidin overproduction in patients with NG. However, the relationship between H. pylori infection and the hepcidin or prohepcidin levels is still controversial. The serum hepcidin or prohepcidin levels were increased in H. pylori-infected patients in previous studies [15,16], and the levels of the serum prohepcidin were decreased after H. pylori eradication [16,17]. Moreover, hepcidin showed significant negative correlations with the serum ferritin, Hb, and iron levels in H. pyloriinfected children with IDA [15]. On the contrary, there were no differences in the serum prohepcidin level or urinary hepcidin levels between H. pylori-infected and uninfected patients in other reports [17,36,37]. Interestingly, Schwarz et al. [38] described that the hepcidin mRNA expression in the gastric mucosa was also increased in H. pylori-infected patients, and it was decreased after eradication. However, neither the serum hepcidin nor prohepcidin levels was altered by H. pylori infection or eradiation, suggested that local hepcidin induction would not be sufficient to increase the serum hepcidin levels. Therefore, further studies are needed to fully elucidate the relationship between H. pylori infection and hepcidin or prohepcidin production. In conclusion, our data suggest that there are different mechanisms inducing the iron deficiency among the patients with H. pylori-associated disease. One of the causes of an iron-deficient state would be hypochlorhydria due to H. pylori-associated gastric mucosal atrophy. Another possible mechanism of iron depletion in patients with H. pylori infection is hepcidin overproduction induced by H. pylori, and the iron-deficiency state in NG patients would be strongly related to hepcidin. However, precise mechanism responsible for the production of hepcidin driven by H. pylori has been unclear, and further studies will be necessary to investigate the relationship between hepcidin and H. pylori infection. Moreover, the small number of study subjects was a limitation of the present study. Further longer follow-up after eradication for H. pylori would be needed.
Acknowledgements and Disclosures Competing interests: The authors have no competing interests.
References 1 McColl KE. Clinical practice. Helicobacter pylori infection. N Engl J Med 2010;362:1597–604.
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2 Jain R, Chetty R. Gastric hyperplastic polyps: a review. Dig Dis Sci 2009;54:1839–46. 3 Miyamoto M, Haruma K, Yoshihara M, Hiyama T, Sumioka M, Nishisaka T, Tanaka S, Chayama K. Nodular gastritis in adults is caused by Helicobacter pylori infection. Dig Dis Sci 2003;48:968–75. 4 Malfertheiner P, Megraud F, O’Morain CA, Atherton J, Axon AT, Bazzoli F, et al. Management of Helicobacter pylori infection–the Maastricht IV/Florence Consensus Report. Gut 2012;61:646–64. 5 Muhsen K, Cohen D. Helicobacter pylori infection and iron stores: a systematic review and meta-analysis. Helicobacter 2008;13:323–40. 6 Qu XH, Huang XL, Xiong P, et al. Does Helicobacter pylori infection play a role in iron deficiency anemia? A metaanalysis World J Gastroenterol 2010;16:886–96. 7 Yuan W, Li Yumin, Yang Kehu, Ma Bin, Guan Quanlin, Wang D, Yang L. Iron deficiency anemia in Helicobacter pylori infection: meta-analysis of randomized controlled trials. Scand J Gastroenterol 2010;45:665–76. 8 Fock KM, Katelaris P, Sugano K, et al. Second Asia-Pacific Consensus Guidelines for Helicobacter pylori infection. J Gastroenterol Hepatol 2009;24:1587–600. 9 Goddard AF, James MW, McIntyre AS, Scott BB. Guidelines for the management of iron deficiency anaemia. Gut 2011;60:1309–16. 10 Asaka M, Kato M, Takahashi S, Fukuda Y, Sugiyama T, Ota H, Uemura N, Murakami K, Satoh K, Sugano K. Guidelines for the management of Helicobacter pylori infection in Japan: 2009 revised edition. Helicobacter 2010;15:1–20. 11 Annibale B, Capurso G, Delle Fave G. The stomach and iron deficiency anaemia: a forgotten link. Dig Dis Sci 2003;35:288– 95. 12 Barabino A. Helicobacter pylori-related iron deficiency anemia: a review. Helicobacter 2002;7:71–5. 13 Yokota S, Konno M, Mino E, Sato K, Takahashi M, Fujii N. Enhanced Fe ion-uptake activity in Helicobacter pylori strains isolated from patients with iron-deficiency anemia. Clin Infect Dis 2008;46:e31–3. 14 Lee JH, Choe YH, Choi YO. The expression of iron-repressible outer membrane proteins in Helicobacter pylori and its association with iron deficiency anemia. Helicobacter 2009;14:36–9. 15 Azab SF, Esh AM. Serum hepcidin levels in Helicobacter pyloriinfected children with iron-deficiency anemia: a case-control study. Ann Hematol 2013;92:1477–83. 16 Ozkasap S, Yarali N, Isik P, Bay A, Kara A, Tunc B. The role of prohepcidin in anemia due to Helicobacter pylori infection. Pediatr Hematol Oncol 2013;30:425–31. 17 Lee SY, Song EY, Yun YM, Yoon SY, Cho YH, Kim SY, Lee MH. Serum prohepcidin levels in Helicobacter pylori infected patients with iron deficiency anemia. Korean J Intern Med 2010;25:195–200. 18 Samloff IM, Varis K, Ihamaki T, Siurala M, Rotter JI. Relationships among serum pepsinogen I, serum pepsinogen II, and gastric mucosal histology. A study in relatives of patients with pernicious anemia. Gastroenterology 1982;83:204–9. 19 Asaka M, Kimura T, Kudo M, Takeda H, Mitani S, Miyazaki T, Miki K, Graham DY. Relationship of Helicobacter pylori to serum pepsinogens in an asymptomatic Japanese population. Gastroenterology 1992;102:760–6. 20 Haruma K, Yoshihara M, Sumii K, Tari A, Watanabe C, Kodoi A, Kajiyama G. Gastric acid secretion, serum pepsinogen I, and
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serum gastrin in Japanese with gastric hyperplastic polyps or polypoid-type early gastric carcinoma. Scand J Gastroenterol 1993;28:633–7. Bonilla Palacios JJ, Miyazaki Y, Kanayuma S, Yasunaga Y, Matsuzawa Y. Serum gastrin, pepsinogens, parietal cell and Helicobacter pylori antibodies in patients with gastric polyps. Acta Gastroenterol Latinoam 1994;24:77–82. Al-Haddad M, Ward EM, Bouras EP, Raimondo M. Hyperplastic polyps of the gastric antrum in patients with gastrointestinal blood loss. Dig Dis Sci 2007;52:105–9. Fox JG, Wang TC. Inflammation, atrophy, and gastric cancer. J Clin Invest 2007;117:60–9. Bujanover Y, Konikoff F, Baratz M. Nodular gastritis and Helicobacter pylori. J Pediatr Gastroenterol Nutr 1990;11:41–4. Nakamura S, Mitsunaga A, Imai R, Ishikawa I, Shirato I, Shimizu S, Kishino M, Konishi H, Oi I, Shiratori K. Clinical evaluation of nodular gastritis in adults. Dig Endosc 2007;19: 74–9. Kitamura S, Yasuda M, Muguruma N, et al. Prevalence and characteristics of nodular gastritis in Japanese elderly. J Gastroenterol Hepatol 2013;28:1154–60. Nakashima R, Nagata N, Watanabe K, Kobayakawa M, Sakurai T, Akiyama J, Hoshimoto K, Shimbo T, Uemura N. Histological features of nodular gastritis and its endoscopic classification. J Dig Dis 2011;12:436–42. Cardamone M, Alex G, Harari MD, Moss WP, Oliver MR. Severe iron-deficiency anaemia in adolescents: consider Helicobacter pylori infection. J Paediatr Child Health 2008;44:647–50. Gulen H, Kasirga E, Yildirim SA, Kader S, Sahin G, Ayhan S. Diagnostic yield of upper gastrointestinal endoscopy in the evaluation of iron deficiency anemia in older children and adolescents. Pediatr Hematol Oncol 2011;28:694–701.
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30 Ganz T, Nemeth E. Hepcidin and iron homeostasis. Biochim Biophys Acta 2012;9:26. 31 Weiss G. Iron metabolism in the anemia of chronic disease. Biochim Biophys Acta 2009;7:682–93. 32 Ciacci C, Sabbatini F, Cavallaro R, Castiglione F, Di Bella S, Iovino P, Palumbo A, Tortora R, Amoruso D, Mazzacca G. Helicobacter pylori impairs iron absorption in infected individuals. Dig Liver Dis 2004;36:455–60. 33 Kido M, Tanaka J, Aoki N, Iwamoto S, Nishiura H, Chiba T, Watanabe N. Helicobacter pylori promotes the production of thymic stromal lymphopoietin by gastric epithelial cells and induces dendritic cell-mediated inflammatory Th2 responses. Infect Immun 2010;78:108–14. 34 Harris PR, Wright SW, Serrano C, et al. Helicobacter pylori gastritis in children is associated with a regulatory T-cell response. Gastroenterology 2008;134:491–9. 35 Huang H, Lamikanra AA, Alkaitis MS, Thezenas ML, Ramaprasad A, Moussa E, et al. Interleukin-10 regulates hepcidin in Plasmodium falciparum malaria. PLoS One 2014;9: e88408. 36 Kim HK, Jang EC, Yeom JO, Kim SY, Cho H, Kim SS, Chae HS, Cho YS. Serum prohepcidin levels are lower in patients with atrophic gastritis. Gastroenterol Res Pract 2013;201810:28. 37 Cherian S, Forbes DA, Cook AG, Sanfilippo FM, Kemna EH, Swinkels DW, Burgner DP. An insight into the relationships between hepcidin, anemia, infections and inflammatory cytokines in pediatric refugees: a cross-sectional study. PLoS One 2008;3:24.e4030. 38 Schwarz P, Kubler JA, Strnad P, et al. Hepcidin is localised in gastric parietal cells, regulates acid secretion and is induced by Helicobacter pylori infection. Gut 2012;61:193–201.
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The Relationship Between Iron Deficiency in Patients with Helicobacter Pylori-Infected Nodular Gastritis and the Serum Prohepcidin Level Yuichi Sato,* Osamu Yoneyama,* Masaki Azumaya,* Manabu Takeuchi,* Syun-ya Sasaki,* Junji Yokoyama,* Kazuhiko Shioji,* Yusuke Kawauchi,* Satoru Hashimoto,* Yuuki Nishigaki,* Masaaki Kobayashi,† Kazuhito Sugimura,* Terasu Honma,* Rintaro Narisawa† and Yutaka Aoyagi* *Department of Gastroenterology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan, †Division of endoscopy, Niigata University Medical and Dental Hospital, Niigata, Japan
Keywords Hepcidin, HP-related iron-deficiency anemia, hyperplastic polyp, hypochlorhydria, nodular gastritis. Reprint requests to: Yuichi Sato, MD, Department of Gastroenterology, Niigata University Graduate School of Medical and Dental Sciences, Asahimachi-dori, Niigata 951-8121, Japan. E-mail: [email protected]
Abstract Background and aims: Helicobacter pylori (H. pylori) is recognized as a causative agent for unexplained iron-deficiency anemia (IDA). We evaluated many background factors influencing an iron-deficiency state in adult patients with various H. pylori-infected upper gastrointestinal tract diseases. Method: Study 1: H. pylori-infected 121 patients (nodular gastritis (NG) (n = 19), duodenal ulcer (DU) (n = 30), or gastric ulcer (GU) (n = 47), or gastric hyperplastic polyp (GHP) (n = 25)) were enrolled. The RBC count and hemoglobin, iron, ferritin, pepsinogen (PG) I, PG II, gastrin, and anti-H. pylori antibody (Ab) levels in the serum were measured. Study 2: H. pyloriinfected 105 patients (NG, n = 19; DU, n = 43; GU, n = 32; GHP, n = 11) and non-H. pylori-infected individuals (n = 35) were examined for the levels of prohepcidin, ferritin, and iron in the serum. In addition, we measured the data before and after the H. pylori eradication. Results: In the patients with GHP and NG, hypoferritinemia was observed in comparison with the GU and DU patients. In the GHP patients, low levels of PG I, a decreased PG I/II ratio, and hypergastrinemia were observed. The levels of serum prohepcidin in the patients with H. pylori-associated disease were higher than those in the uninfected adults. In the patients with NG, the serum prohepcidin levels were higher than those in the other H. pyloriinfected patient groups and decreased after the eradication. Conclusion: H. pylori-related iron-deficiency state might be associated with several factors, such as hypochlorhydria and hepcidin, in patients with GHP or NG.
Helicobacter pylori (H. pylori) is recognized as a causative agent for various gastroduodenal diseases, such as chronic gastritis, duodenal ulcer (DU), gastric ulcer (GU), gastric cancer, gastric mucosa-associated lymphoid tissue lymphoma [1], gastric hyperplastic polyp (GHP) [2], and nodular gastritis (NG) [3]. Furthermore, H. pylori infection has been implicated in some extradigestive diseases, including iron-deficiency anemia (IDA), idiopathic thrombocytopenia purpura, and vitamin B12 deficiency [4]. There has been many research about the association between H. pylori and iron deficiency to date, and three separate meta-analyses were
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recently published that showed an association between H. pylori infection and IDA [5–7]. H. pylori is related to unexplained IDA in the absence of gastrointestinal blood loss in adult and children in the studies. Therefore, the Maastricht IV/Florence consensus recommends the eradication of H. pylori in H. pylori-positive patients with IDA (grade of recommendation, A) [4], and other guidelines also suggest eradicating H pylori in patients with IDA [8–10]. The mechanism by which H. pylori infection contributes to IDA remains unclear; however, several mechanisms have been postulated [11,12]. First, H. pylori
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infection may progress to pangastritis and atrophic gastritis, leading to hypochlorhydria or achlorhydria, resulting in iron malabsorption. Moreover, H. pylori infection can reduce the gastric juice ascorbic acid (vitamin C) level, which is a potent promoter of nonheme iron absorption. Next, H. pylori infection may increase the uptake of iron by bacteria, because iron is an essential bacterial growth factor [12–14]. H. pylori has a lactoferrin binding protein in the outer membrane, and it has been speculated that H. pylori increases the lactoferrin uptake from transferrin, and the bacterial iron stores are subsequently lost in the feces after the bacteria are destroyed [12]. Other reports have revealed that some H. pylori strains have an advantage in iron acquisition [13,14]. Moreover, recent studies have showed that hepcidin, which acts as a one of the major regulators of systemic iron homeostasis and downregulates duodenal iron absorption, is related to the IDA associated with H. pylori infection [15–17]. However, IDA does not develop in all H. pyloriinfected patients. Therefore, we evaluated the iron-deficiency status and many background factors influencing iron deficiency in adult patients with various H. pyloriassociated gastroduodenal diseases, including DU, GU, GHP, and NG. Furthermore, we focused on hepcidin which is an antimicrobial peptide and a central regulator of iron metabolism. We measured and compared the serum levels of prohepcidin, the precursor of hepcidin, between the patients with these diseases and nonH. pylori-infected people, and evaluated the prohepcidin levels before and after the eradication of H. pylori in patients with GU, DU, and NG.
Methods
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antibody (Ab) levels. The red blood cells counts and Hb level were determined using an automated electronic counter (Sysmex XE-5000; Sysmex corporation, Kobe, Japan). The serum iron levels were determined using a bathophenanthroline method. The levels of serum ferritin were measured using an EIA kit (ST AIA-PACK FER; Tosoh Bioscience, Tokyo, Japan). The fasting levels of serum gastrin and PG I and II were measured using an RIA kit (Dinabot Company, Tokyo, Japan). The estimation of gastric atrophy was based on the serum PG I and II levels and the I/II ratio. The PG I and I/II ratio are wellrecognized markers of fundic gland atrophy [18]. Serum IgG Abs against H. pylori were identified using a specific ELISA kit (HM-CAP; Enteric Products INC., Westbury, NY, USA). This assay system is reported to have 100% specificity and 96% sensitivity in Japan in comparison with the urea breath test [19]. During the esophago-gastro-endoscopy, biopsy specimens were obtained from the gastric antrum and corpus and were used for bacterial culture of H. pylori. The growth of H. pylori from antral and corpus biopsies was graded as none, scant, moderate, or abundant growth corresponding to the H. pylori colonies found in 0, ≤ 1/3, 1/3–2/3, and ≥2/3 of the culture plate, respectively, and was converted to numerical scores (none = 0, scant = 1, moderate = 2, and abundant = 3). H. pylori infection was confirmed by bacterial culture, or serum IgG Ab against H. pylori. The normal range of RBC count, Hb, and the levels of iron and ferritin in the serum were as follows: RBC count, 400– 552 9 104/lL (for male) or 378–499 9 104/lL (for female); Hb, 13.2–17.2 g/dL (for male) or 10.8–14.9 g/dL (for female); serum iron level, 60–210 lg/dL (for male) or 50–170 lg/dL (for female); and serum ferritin level, 21–282 ng/mL (for male) or 5–157 ng/mL (for female).
Study 1 We examined 19 patients (six male and 13 female; mean age, 38.0 years; age range, 19–63 years) with NG, 30 patients (19 male and 11 female; mean age, 42.9 years; age range, 20–68 years) with DU, 47 patients (32 male and 15 female; mean age, 55.2 years; age range, 21– 78 years) with GU, and 25 patients (12 male and 13 female; mean age, 61.4 years; age range, 19–83 years) with GHP. The exclusion criteria were as follows: [1] chronic inflammatory disease, recent bleeding, liver diseases, or malignancy; [2] previous eradication therapy or the use of a proton-pump inhibitor, antibiotic, or H2 receptor antagonist; and [3] a history of blood transfusion or iron supplement therapy. A blood sample was obtained from each of the enrolled patients in the fasting state to assess the red blood cell count (RBC) and the hemoglobin (Hb), serum iron, serum ferritin, serum pepsinogen (PG) I, PGII, serum gastrin, and anti-H. pylori
2
Statistical analysis The significance of the differences among the groups in terms of the age, RBC count, Hb, the levels of iron, ferritin, PG I, PG II, and gastrin in the serum, and the grading of H. pylori colonies was determined using nonparametric ANOVA (Kruskal–Wallis one-way ANOVA). We used Fisher’s exact test to analyze the differences in the sex distribution among the groups. Differences of p < .05 were considered to be significant. Analyses were performed using SPSS 20.0 for Windows (IBM Japan Inc., Tokyo, Japan).
Study 2 We examined 19 patients (six male and 13 female) with NG, 43 patients (27 male and 16 female) with DU, 32 patients (17 male and 15 female,) with GU, 11 patients
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(five male and six female) with GHP, and 35 individuals not infected H. pylori (11 male and 24 female). The exclusion criteria were the same as in Study 1. No patients were repeatedly enrolled between study 1 and 2. A blood samples was obtained from each patient in the fasting state to assess the prohepcidin, ferritin, and iron levels. The levels of serum prohepcidin were determined by an enzyme-linked immunosorbent assay based on a competitive principle (DRG International Inc., Mountainside, NJ, USA) according to the manufacturer’s protocol. Of these patients, 11 patients with NG, 27 patients with DU, and 20 patients with GU received H. pylori eradication treatment including amoxicillin 750 mg and clarithromycin 200 mg, and a proton-pump inhibitor (lansoprazole at 20 mg or rabeprazole at 10 mg) given twice a day for seven days. Two or three months after eradication, a 13C urea breath test was carried out; if it was positive, a second eradication was performed with amoxicillin 750 mg, metronidazole 250 mg, and a proton-pump inhibitor (lansoprazole at 20 mg or rabeprazole at 10 mg) twice a day for one week. The 13C urea breath test was performed again two or three months after eradication. Blood samples were collected after six to 12 months after the eradication of H. pylori to assess the serum prohepcidin levels. Statistical analysis The significance of differences among the groups in the levels of prohepcidin in the serum was determined using nonparametric ANOVA (Kruskal–Wallis one-way ANOVA). We used Fisher’s exact test to analyze the differences in the sex distribution among the groups. The
correlation between the levels of prohepcidin and ferritin or between the levels of prohepcidin and the iron in the serum was calculated using Spearman’s rank correlation test. The significance of the differences in the levels of prohepcidin before and after H. pylori eradication was determined using Wilcoxons matched-pairs signed-rank sum test. Differences with a value of p < .05 were considered to be significant. Analyses were performed using SPSS 20.0 for Windows (IBM Japan Inc., Tokyo, Japan). In Studies 1 and 2, all procedures and biopsies were performed with the informed consent of the patients in accordance with the Helsinki Declaration, and the study was approved by the ethics committee of Niigata University School of Medicine.
Results Study 1 Table 1 shows the clinical data and characteristics of the patients divided by the disease. The patients in the NG group were significantly younger than those in the GU and GHP groups. The NG group showed a significant female dominance in comparison with the DU and GU groups. There was no significant difference in the RBC count between the groups. However, Hb level in the NG group was significantly lower than that in the DU group, and there was a trend toward a lower level of Hb in the NG group in comparison with that in the GU group. In the GHP group, the Hb level was lower than that in the GU group. There were no significant differences in the levels of serum iron among these
Table 1 The clinical characteristics of patients with nodular gastritis (NG), duodenal ulcer (DU), gastric ulcer (GU), or gastric hyperplastic polyps (GHP) in study 1
Mean age (range) Male: Female Helicobacter pylori infection rate (%) RBC (9 104/lL)a Number of below normal range (%) Hemoglobin(g/dL)a Number of below normal range (%) Iron (lg/dL)a Number of below normal range (%) Ferritin (ng/mL)a Number of below normal range (%)
Nodular gastritis (NG) (n = 19)
Duodenal ulcer (DU) (n = 30)
Gastric ulcer (GU) (n = 47)
Gastric hyperplastic polyp (GHP) (n = 25)
38.0 (19–63)† 6: 13‡ 100 (19/19) 440.5 14.8 1 (5.3%) 12.0 0.8§ 5 (26.3%) 78.3 11.9 7 (36.8%) 37.8 10.0 4 (21.1%)
42.9 (20–68) 19: 11 100 (30/30) 472.8 10.4 2 (6.7%) 13.9 0.4 4 (13.3%) 84.6 5.9 7 (23.3%) 62.3 18.4 5 (16.7%)
55.2 (21–78) 32: 15 100 (47/47) 437.0 9.7 6 (12.7%) 13.6 0.3 6 (12.7%) 96.0 10.7 10 (21.3%) 89.0 15.2 5 (10.6%)
61.4 (19–83) 12: 13 92 (23/25) 444.9 5.8 3 (12.0%) 13.0 0.4 5 (20.0%) 78.0 10.0 6 (24.0%) 41.0 8.3¶ 3 (12.0%)
Results are expressed as mean SEM, RBC, red blood cell count. p < .01, versus GU, GHP; ‡p < .05, versus DU, GU; §p < .05 versus DU, p = .0624 versus GU; ¶p < .05 versus GU.
a
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groups; however, the serum ferritin levels in the NG and GHP groups were significantly lower than that in the GU group. In the NG group, the percentages of patients with Hb below normal range or serum iron levels below normal range or ferritin levels below normal range were higher in comparison with the other groups. The data of the serum PG I, PG II, and gastrin levels, and the grading of the H. pylori colonies in the patients divided by the disease are presented in Table 2. The levels of PG I in the NG group were higher than those in the GU and GHP group, while those in the GHP group were significantly lower than those in both the NG and GU groups. The levels of PG II in the NG group were higher than those in the other three groups, while those in the GHP group were lower significantly than those in not only the NG but also the GU group. On the other hand, the fasting gastrin levels in the GHP group were lower than those in the other three groups. There were no significant differences in the scores of grading of H. pylori colonies among the NG, DU, and GU groups, in either the antrum or the corpus. However, the scores of grading of H. pylori in the GHP patients were significantly lower than those in the other three groups in the antrum and were lower than those in the DU or GU groups in the corpus.
Study 2 Table 3 shows the clinical characteristics and the serum levels of prohepcidin, iron, and ferritin in the patients with NG, DU, GU, and GHP, and in the non-H. pyloriinfected individuals. The levels of serum prohepcidin in the patients with NG were significantly higher than those in the patient with DU or GU or in the noninfected individuals. The levels of serum prohepcidin in the H. pylori-negative normal individual were significantly lower than the levels in the four infected groups. As with study 1, the percentages of patients with serum
iron levels below normal range or ferritin levels below normal range in the NG group were higher in comparison with the other groups. Figure 1 shows that there was a negative correlation between the serum levels of prohepcidin and the serum iron levels; however, there was no correlation between the serum levels of prohepcidin and serum ferritin levels in the patients with NG. Meanwhile, there was no relation between the serum levels of the prohepcidin and serum iron or ferritin levels in the patients with DU, GU, or GHP (data not shown). As shown in Fig. 2 and Table 4, after the eradication of H. pylori, the levels of serum prohepcidin were significantly reduced, and there was a trend toward an increase in the levels of serum ferritin in the NG patients. However, serum iron levels were unchanged in those patients. Meanwhile, the levels of serum prohepcidin, iron, and ferritin did not show any significant change after the H. pylori eradication in the patients with DU or GU (Table 4).
Discussions We herein demonstrated that there are differences in the iron-deficiency status among patients with various H. pylori-associated gastroduodenal diseases, and iron deficiency was especially observed in NG and GHP patients. Study 1 revealed that hypoferritinemia, low Hb levels, low levels of PG I, and a decreased PG I/II ratio in the serum, as well as hypergastrinemia, were observed in GHP patients. Previous studies also showed that there were low levels of PG I, a low PG I/II ratio, and hypergastrinemia in a serologic analysis of GHP patients [20,21]. These data indicate that, in GHP patients, there is progressive gastric mucosal atrophy, and decreased gastric acid secretion, and these conditions result in hypergastrinemia. Our results supported the develop-
Table 2 The serum pepsinogen (PG) I, PG II, gastrin, and grading of Helicobacter pylori colonies in the patients with nodular gastritis (NG), duodenal ulcer (DU), gastric ulcer (GU), or gastric hyperplastic polyp (GHP) in study 1 Nodular gastritis (NG) (n = 19) Pepsinogen I (ng/mL)a Pepsinogen II (ng/mL)a Pepsinogen I/II ratio Gastrin (pg/mL)a Grading of H. pylori colonies Antrum/corpus
89.3 32.4 3.0 116.3 2.5
8.3† 3.4§ 0.3 16.5 0.3/2.0 0.7
Duodenal ulcer (DU) (n = 30) 91.7 20.6 4.7 110.2 2.7
10.4 2.2 0.3 10.6 0.2/2.2 0.3
Gastric ulcer (GU) (n = 47) 61.9 19.6 3.2 153.4 1.7
5.5 1.2 0.2 18.7 0.1/1.9 0.2
Gastric hyperplastic polyp (GHP) (n = 25) 26.4 16.0 1.6 469.5 0.4
5.1‡ 2.0¶ 0.2‡ 78.4‡ 0.1k/1.0 0.2‡
Results are expressed as mean SEM. p < .01 versus GU and GHP; ‡p < .01 versus DU, GU, and NG; §p < .01 versus DU, GU, and GHP; ¶p < .01 versus NG, p < .05 versus GU; kp < .01 versus NG, p < .05 versus DU and GU.
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Table 3 The clinical characteristics of the patients with nodular gastritis (NG), duodenal ulcer (DU), gastric ulcer (GU), gastric hyperplastic polyp (GHP), and non-Helicobacter pylori-infected individuals in study 2
Mean age (range) Male: Female Prohepcidin (ng/ml)a Iron (lg/dL)a Number of below normal range (%) Ferritin (ng/mL)a Number of below normal range (%)
Nodular gastritis (NG) (n = 19)
Duodenal ulcer (DU) (n = 43)
Gastric ulcer (GU) (n = 32)
Gastric hyperplastic polyp (GHP) (n = 11)
H. pylori uninfected individuals (n = 35)
43.0 (22–63)† 6: 13 280.3 20.7§ 77.9 9.5‖ 6 (31.6%) 50.0 10.9 5 (26.3%)
46.9 (20–71)‡ 27: 16 190.9 15.8 109.4 8.4 6 (13.9%) 91.0 15.1 4 (9.3%)
52.7 (21–78) 17: 15 211.3 16.1 92.2 11.1 6 (18.8%) 97.2 20.8 2 (6.3%)
69.9 (61–86) 5: 6 237.8 20.1 106.5 11.7 1 (9.1%) 60.2 13.2 1 (9.1%)
48.2 (16–74)† 11: 24 155.6 18.0¶ 111.5 8.0 3 (8.6%) 95.3 18.9 0 (0.0%)
Results are expressed as mean SEM. p < .01 and GHP, p < .05 versus GU; ‡p < .01 versus GHP; §p < .01 versus GU, DU and uninfected individuals; ¶p < .01 versus NG, GU and GHP, p < .05 versus DU; kp < .05 versus DU and uninfected individuals.
a
†
A
B
Figure 1 Relationship between the serum prohepcidin and serum iron (A) or serum ferritin (B) level in NG patients.
ment of these phenomena, and it is thus thought that the iron deficiency in GHP patients is caused by iron malabsorption due to the hypoacidity induced by gastric mucosal atrophy. GHP has been suggested to cause gastrointestinal blood loss due to oozing; however, AlHaddad et al. also reported that iron deficiency was observed in spite of a negative occult blood test in the GHP patients [22], which would support our results suggesting malabsorption. The colonies of H. pylori infection were low in GHP patients. H. pylori infection induces atrophy and results in the replacement of intestinal metaplasia as part of “end-stage” chronic gastritis [23]. H. pylori does not adhere to the intestinal epithelium; therefore, H. pylori is finally unable to inhabit the gastric mucosa of the patients. Our results reflect such a condition in the GHP patients’ stomachs. Furthermore, hypoferritinemia and low Hb levels were observed in NG patients in Study 1. NG is strongly
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associated with H. pylori infection [3,24] and appears to be more common in children or young people than in adults, and in female more often than in male [3,25,26]. NG is characterized by a miliary micronodule pattern with a cobblestone appearance endoscopically and is often observed in the antrum; therefore, NG is also called antral nodularity, nodular antritis, or antral gastritis. NG shows lymphoid follicles and mononuclear cell infiltration histologically, [27], and NG is therefore also called follicular gastritis. A few recent reports described the presence of IDA in NG patients [28,29]. Cardamone et al. [28] reported that three IDA patients with NG showed improvement in the iron-deficiency state after H. pylori eradication. Gulen et al. [29] described that endoscopic antral gastritis, which means NG, was observed in 21/44 (47.7%) older pediatric patients with IDA, and 19/21 patients with NG were H. pylori-positive. The patients received the eradication therapy; IDA was improved after eradication.
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Figure 2 The changes in the serum prohepcidin levels in NG patients before and after Helicobacter pylori eradication.
Gastric atrophy is rare in NG patients [3,27]; therefore, there are low levels of PG I and a decrease of the PG I/II ratio and hypergastrinemia in the serum [3,26]. Our results agree with those of previous studies and, consequently, also indicated that there was no severe atrophy and hypochlorhydria in patients with NG. Accordingly, the hypoferritinemia and anemic state in NG patients cannot be explained by iron malabsorption due to hypochlorhydria induced by gastric mucosal atrophy. Besides, the grading of H. pylori culture colonies in the NG group was relatively high in comparison with that in the GU and GHP groups. However, it was not significantly different from that in the DU group. Therefore, it is thought that the influence of the consumption of iron by H. pylori itself is relatively small. Thus, the iron-deficiency state observed in NG patients would not be explained using the past hypotheses. Accordingly, we paid attention to hepcidin. Hepcidin is a regulatory hormone of iron homeostasis and plays
an important role in duodenal iron absorption [30]. Hepcidin increases in response to inflammation and leads to a decrease in the duodenal enterocyte iron absorption and macrophage iron release by binding to ferroportin [30]. Therefore, overproduction of hepcidin has been regarded as a cause of anemia of inflammation [31]. Inflammatory cytokines such as interleukin (IL)-6, IL-1b, and bone morphogenic protein 6 all upregulate hepcidin expression [30,31]. In this study, we demonstrated that the levels of serum prohepcidin in the patients with H. pylori-associated disease were higher than those in the uninfected adults. These results suggest that the production of hepcidin would be induced by H. pylori infection. Ciacci et al. [32] have reported that the iron absorption in the duodenum is interfered in H. pylori-infected patients. Therefore, H. pylori infection may cause iron malabsorption in the duodenum via the production of the hepcidin. It is known that H. pylori infection induces the expression of interleukin IL-6 and IL-1b, which induce hepcidin production; therefore, such inflammatory cytokines in the H. pylori-infected gastric mucosa would be expected to lead to hepcidin overproduction, resulting in iron deficiency. We herein demonstrated that the serum prohepcidin levels in the patients with NG were higher than those in the other H. pylori-infected patients, and there was a negative correlation between the serum levels of prohepcidin and the serum iron levels only in the NG patients. Additionally, we found that there was a decrease in the levels of prohepcidin after the H. pylori eradication in patients with NG. Taken together, the previous results and those of our present study indicated that there is a strong relationship between iron deficiency and hepcidin production in NG. However, our study could not clarify the mechanisms underlying these findings in patients with NG. Although T helper type 2 (Th2) cytokines contribute to the formation of the lymph follicle in NG [33], H. pylori infection in children, in contrast to that in adults, results in the production of IL-10 as a Th2 cytokine [34]. A recent report
Table 4 Changes in the levels of serum prohepcidin, iron, and ferritin before and after eradication in the patients with nodular gastritis (NG), duodenal ulcer (DU), gastric ulcer (GU) in study 2
Prohepcidin (ng/ml)a Iron (lg/dL)a Ferritin (ng/mL)a
Nodular gastritis (NG) (n = 11)
Duodenal ulcer (DU) (n = 27)
Gastric ulcer (GU) (n = 20)
Before era
After era
Before era
After era
Before era
After era
348.4 19.0 85.2 29.7 20.0 6.5
257.0 32.6† 74.4 14.7 30.7 7.9
175.9 17.9 120.0 9.8 100.8 16.5
203.1 14.1 114.7 8.5 102.5 15.5
225.9 24.5 102.9 15.0 79.3 24.2
206.6 20.4 111.6 15.4 75.3 24.1
Results are expressed as mean SEM. p < .05.
a
†
6
© 2014 John Wiley & Sons Ltd, Helicobacter
Sato et al.
has revealed that IL-10 can directly regulate hepcidin [35]; therefore, certain humoral factors, including IL-10, might be related to the hepcidin overproduction in patients with NG. However, the relationship between H. pylori infection and the hepcidin or prohepcidin levels is still controversial. The serum hepcidin or prohepcidin levels were increased in H. pylori-infected patients in previous studies [15,16], and the levels of the serum prohepcidin were decreased after H. pylori eradication [16,17]. Moreover, hepcidin showed significant negative correlations with the serum ferritin, Hb, and iron levels in H. pyloriinfected children with IDA [15]. On the contrary, there were no differences in the serum prohepcidin level or urinary hepcidin levels between H. pylori-infected and uninfected patients in other reports [17,36,37]. Interestingly, Schwarz et al. [38] described that the hepcidin mRNA expression in the gastric mucosa was also increased in H. pylori-infected patients, and it was decreased after eradication. However, neither the serum hepcidin nor prohepcidin levels was altered by H. pylori infection or eradiation, suggested that local hepcidin induction would not be sufficient to increase the serum hepcidin levels. Therefore, further studies are needed to fully elucidate the relationship between H. pylori infection and hepcidin or prohepcidin production. In conclusion, our data suggest that there are different mechanisms inducing the iron deficiency among the patients with H. pylori-associated disease. One of the causes of an iron-deficient state would be hypochlorhydria due to H. pylori-associated gastric mucosal atrophy. Another possible mechanism of iron depletion in patients with H. pylori infection is hepcidin overproduction induced by H. pylori, and the iron-deficiency state in NG patients would be strongly related to hepcidin. However, precise mechanism responsible for the production of hepcidin driven by H. pylori has been unclear, and further studies will be necessary to investigate the relationship between hepcidin and H. pylori infection. Moreover, the small number of study subjects was a limitation of the present study. Further longer follow-up after eradication for H. pylori would be needed.
Acknowledgements and Disclosures Competing interests: The authors have no competing interests.
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