Dig Dis Sci (2007) 52:405–410 DOI 10.1007/s10620-006-9422-8

ORIGINAL ARTICLE

Serum Ferritin, Vitamin B12 , Folate, and Zinc Levels in Children Infected with Helicobacter pylori Mustafa Akcam · Sebahat Ozdem · Aygen Yilmaz · Meral Gultekin · Reha Artan

Received: 3 December 2005 / Accepted: 1 May 2006 / Published online: 9 January 2007 C Springer Science + Business Media, Inc. 2006


Abstract We sought to explore the relationship between Helicobacter pylori infection and serum ferritin, vitamin B12 , folate, and zinc status among children. Fifty patients aged 5–18 years who underwent upper gastrointestinal endoscopy because of dyspeptic symptoms, were studied, prospectively. Patients were grouped as H. pylori positive (group 1, n = 32) or H. pylori negative (group 2, n = 18) by histopathologic examination and rapid urease test. Fasting serum ferritin, vitamin B12 , folate, and zinc levels of patients were measured. Both groups were indifferent according to age, gender, height standard deviation score (HSDS ), and weight standard deviation score (WSDS ). Serum ferritin levels were 33 ± 26 and 50 ± 46 ng/mL (P = .098), vitamin B12 levels were 303 ± 135 and 393 ± 166 pg/mL (P = .042), folate levels were 9.64 ± 3.2 and 9.61 ± 2.8 ng/mL (P = .979), and zinc levels were 95 ± 48 and 87 ± 31 µg/dL (P = .538), in groups 1 and 2, respectively. Ferritin levels of 14 (43.8%) patients in group 1 and 6 (33.3%) patients in group 2 were below the normal range (P = .470). Serum vitamin B12 levels of 9 children (28%) in group 1 and 2 children (11%) in group 2 M. Akcam (
) · A. Yilmaz · R. Artan Akdeniz University, Medical School, Department of Pediatrics, Division of Pediatric Gastroenterology, Hepatology and Nutrition, Antalya, Turkey 07059 e-mail: [email protected] S. Ozdem Akdeniz University, Medical School, Department of Central Lab, Biochemistry Unit, Antalya, Turkey M. Gultekin Akdeniz University, Medical School, Department of Biochemistry, Antalya, Turkey

were below the normal range (P = .287). The findings of the present study suggest that H. pylori infection has a negative effect on serum ferritin and vitamin B12 levels in children. This negative effect on vitamin B12 levels is rather marked in contrast to that on ferritin levels. H. pylori infection has no significant effect on serum folate or zinc levels among children. Keywords Helicobacter pylori . Child . Vitamin B12 . Folate . Zinc . Micronutrient

Introduction Helicobacter pylori has been recognized as the major etiologic factor of gastritis and peptic ulcer disease in adults and children. This infection is frequently acquired during childhood and lasts into adult life, which has been linked to the development of gastric cancer [1, 2]. Because this infection particularly develops in the stomach, it naturally affects stomach functions. Although there is no direct absorption of nutrients in the stomach, it plays a very important role, such as acid secretion, for facilitated absorption of nutrients in small intestine. H. pylori also impairs the normal secretion of hydrochloric acid, provoking achlorhydria in infected patients [3, 4]. Additionally, bacteria may metabolize some nutrients that are important for its continued survival, and may cause deficiencies in this manner. As a result of its interference, H. pylori may modify the absorption of many nutrients and then compromise the nutritional status of infected patients, resulting in diverse clinical manifestations [5–8]. It has been reported to be associated with various unexpected manifestations in children and adults.

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Positive relations between H. pylori and iron deficiency were demonstrated in previous studies conducted predominantly in adults [9–15]. A positive relation between ferritin deficiency and H. pylori infection was also found in a seroepidemiologic study performed in Korean children [10]. Additionally, Kurekci et al. [15] showed that the eradication of H. pylori infection without iron treatment led to the resolution of iron deficiency in children. Fewer studies, performed in adults, also revealed positive relations between H. pylori and vitamin B12 deficiency [16–20], whereas no obvious relation was detected between H. pylori and serum vitamin B12 levels in a study performed without endoscopy and biopsy in children [21]. A positive relation was also reported between H. pylori and folate deficiency in adults [1]. On the other hand, the relation between H. pylori and serum zinc level was investigated only in 1 study conducted among adult patients with liver cirrhosis [22]. To our knowledge, the relation between H. pylori infection and serum folate and zinc levels has not been studied in children. Therefore, in the present study, we investigated the possible relations between H. pylori infection and these parameters together with serum ferritin and vitamin B12 levels in children using endoscopy and biopsy.

Dig Dis Sci (2007) 52:405–410 Table 1

Patient characteristics Group 1, H. pylori Group 2, H. pylori positive (n = 32) negative (n = 18)

Age (yrs) Gender (female/male) WSDS HSDS

11.4 ± 3.0 17/15 − 0.31 ± 1.00 − 0.44 ± 1.20

10.1 ± 3.0 11/7 0.31 ± 1.20 0.15 ± 1.10

Participant characteristics are shown in Table 1. Informed consents were obtained from the parents. The hospital ethics committee approved the study protocol. Design and sample collection

Methods

Antral, corpal, and duodenal biopsies (2 from each area) collected during routine endoscopy were examined by regular histopathologic examination. One of the antral biopsy specimens was subjected to rapid urease test for H. pylori. H. pylori infection was diagnosed by both histopathologic identification of bacillus and by positive rapid urease test (group 1). When both tests were negative, it was considered as H. pylori negative (group 2). Fasting blood samples were collected at early morning between 8 and 9 am. Sera were obtained after immediate centrifugation and stored at − 80◦ C until chemical analysis.

Study population

Histopathologic examination

A total of 50 patients (28 girls, 22 boys, aged 5–18 years) who underwent upper gastrointestinal endoscopy because of dyspeptic symptoms were recruited into the study. This study was conducted between February and August 2005 at Akdeniz University Medical School, Antalya, Turkey. The patients were all from the same geographical area of the country and had similar socioeconomic status. Exclusion criteria were as follows: (1) age <5 or >18 years; (2) H. pylori-positive patients positive for only 1 the diagnostic tests (rapid urease test [Clo test] or histopathologic examination); (3) patients diagnosed with diabetes mellitus, liver disease, celiac disease, inflammatory bowel disease, or other systemic illnesses requiring continuous medication; (4) patients with a history of steroid, nonsteroidal anti-inflammatory drug, antibiotic, antacid, H2 -receptor antagonist, or proton pump inhibitor intake in the previous month; (5) previous H. pylori eradication; (6) impaired renal function; (7) use of drugs affecting plasma ferritin, vitamin B12 , folate, or zinc; (8) history of other causes of vitamin malabsorption, such as surgery of gastrointestinal tract; (9) vegetarian diet; (10) subjects who were underweight or had cachexia, malnutrition, or a history of an eating disorder; and (11) patients with gastrointestinal bleeding.

During endoscopy, 2 biopsies were obtained from each of the following: duodenum, antrum, corpus, and esophagus. One of the antral biopsy specimens was embedded in the rapid urease test (CLO test duo, Kimberly-Clark–Ballard Medical Products, Draper, UT) which consists of urea agar and pH indicator. Other specimens were placed directly in 10% formalin without a paper filter, for histologic examination. One of the pathologists, blinded to the patients’ endoscopic or clinical findings, examined the biopsies (stained with hematoxylin and eosin, and Diff-Quick) for H. pylori and gastritis. The diagnosis of gastritis was based on the histopathologic findings of inflammation, activity, metaplasia, and atrophy. The assessment of gastritis was performed according to the updated Sydney System and using visual analog scales [23].

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Chemical analysis The levels of serum ferritin, vitamin B12 and folate were measured with Roche Modular Analytics E170 Immunoassay System (Mannheim, Germany) using electrochemiluminescence immunoassay; intraassay coefficients of variation (CVs) were 3.1%, 2.8%, and 3.6%, and interassay CVs were 4.4%, 3.6%, and 4.5%, respectively. Serum zinc level was determined spectrophotometrically using a commercial

Dig Dis Sci (2007) 52:405–410 Table 2

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Reference ranges by age and gender

Test Ferritin (ng/mL) Vitamin B12 (pg/mL)

Age (yrs) 5–18 4–6 7–9 10–12 13–18

Folate (ng/mL)

4–6 7–9 10–12 13–18

Zinc (µg/dL)

1–5 6–9 10–13 14–19

Table 3 Serum ferritin, vitamin B12 , folate and zinc levels of H. pylori-positive (group 1) and -negative (group 2) patients

Gender

Reference ranges

Both F M F M F M F M F M F M F M F M Both Both F M F M

20–200 313–1410 245–1075 247–1175 271–1170 196–1020 183–1090 182–820 214–864 2.7–14 0.5–13 2.4–13 2.3–12 1.0–10 1.5–11 1.2–7.1 1.2–8.8 65–118 78–105 78–118 78–98 59–98 65–118

Ferritin (ng/mL) Vitamin B12 (pg/mL) Folate (ng/mL) Zinc (µg/dL)

Group 1

Group 2

P value

33 ± 26 303 ± 135 9.64 ± 3.20 95 ± 48

50 ± 46 393 ± 166 9.61 ± 2.80 87 ± 31

.098 .042 .979 .538

95 ± 48 and 87 ± 31 µg/dL (0.538), in groups 1 and 2, respectively (Table 3). When normal values for appropriate age and gender were considered, ferritin levels of 14 (43.8%) patients in group 1 and 6 (33.3%) patients in group 2 were below normal ranges (P = .470). Serum vitamin B12 levels of 9 (28%) children in group 1 and 2 (11%) in group 2 were below normal ranges (P = .287). Serum zinc levels of 8 (25%) children in group 1 and 7 (38%) children in group 2 were below normal ranges (P = .135) (Table 4). There was no statistical significance in terms of the presence of gastric atrophy and vitamin B12 deficiency between the groups (P = .891).

Discussion

Note. F, female; M, male

kit (Audit Diagnostics, Cork, Ireland); intra- and interassay CVs were 2.9% and 4.1%, respectively. Reference ranges are given in Table 2. Statistics Statistical analysis was performed using the statistical software program SPSS for Windows, version 11.00 (SPSS Inc., Chicago, IL). Comparisons of nonparametric and parametric variables were made with Mann–Whitney U test and Student’s t-test, respectively. χ 2 tests were used for comparison of nominal data. Numeric values were given as mean values ± SD. P < .05 was considered statistically significant. Results Thirty-two of 50 children were H. pylori-positive (group 1) and 18 were H. pylori-negative (group 2). There were no statistically significant difference when groups were compared by age (11.4 ± 3 versus 10.1 ± 3 years), gender (F/M: 17/15 versus 11/7), weight (39 ± 17 versus 37 ± 14 kg), height (141 ± 20 versus 138 ± 20 cm), height standard deviation score (HSDS ), or weight standard deviation score (WSDS ). Serum ferritin levels were 33 ± 26 and 50 ± 46 ng/mL (P = .098), vitamin B12 levels were 303 ± 135 and 393 ± 166 pg/mL (P = .042), folate levels were 9.64 ± 3.2 and 9.61 ± 2.8 ng/mL (P = .979), and zinc levels were

In recent studies, a positive relation was detected between H. pylori infection and some micronutrient malnutrition in adults. Serum iron, vitamin B12 , folate, vitamin A, and vitamin C levels were found to be low in the presence of H. pylori infection [1]. On the other hand, there is no study conducted in children investigating the relation between H. pylori infection and micronutrient status. H. pylori status was determined with both serology and urea breath test in existent literature (associated with ferritin and vitamin B12 ). In the present study, H. pylori status was evaluated by both histologic examination of biopsy specimens and rapid urease test; the diagnosis of H. pylori infection was based on the positivity of both tests. Similarly, negativity of both tests signified the absence of H. pylori infection. By exclusion of patients Table 4 Distribution of age- and gender-matched H. pylori-positive (group 1) and -negative (group 2) children by low versus adequate levels of ferritin, vitamin B12 , folate, and zinc

Ferritin Low/adequate Vitamin B12 Low/adequate Folate Low/adequate Zinc Low/adequate

Group 1

Group 2

P value

14/18

6/12

.470

9/23

2/16

.287

0/32

0/18

8/32

7/18

.135

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that were positive only by 1 of these tests, suspicious conditions were ruled out. To our knowledge, the present study is the first using such a combination for diagnosis of H. pylori infection in children. H. pylori infection and ferritin Ferritin is an important marker of total body iron. While examining the relation between H. pylori infection and iron status, we used serum ferritin level as the sole marker as in the other previous studies. A strong association was found between H. pylori infection and iron deficiency in the recent studies [9–15]. However, the mechanisms by which H. pylori infection causes iron deficiency have not been well established. A plausible mechanism that may explain the development of iron deficiency in H. pylori–infected subjects might be the result of the pattern of gastritis and related effects on gastric physiology, affecting the normal process of iron absorption (increases in gastric pH lead to decreases in iron solubility, H. pylori binding proteins lead to iron protein complex in the bacterium, decreases in vitamin C secretion in gastric juice, and decreases in bioavailability of vitamin B12 and folate) [24]. The median serum ferritin levels were significantly lower in H. pylori–seropositive children than in seronegative controls (24 versus 39 ng/mL; P < .001) in a seroepidemiologic study performed in Korean children. The prevalence of iron deficiency in H. pylori–seropositive children was significantly higher (13.9%) than in seronegative children (2.8%) [10]. H. pylori–positive and -negative groups’ median values (24.8 versus 41.2 ng/mL, respectively; P = .098) were apparently different in our study. Although the values were surprisingly similar to those reported previously, we found no statistical difference between the groups in our study that might be due to differences in sizes of the groups. Similarly, we found no significant difference (P = .470) in prevalence of iron deficiency between H. pylori–positive (43.8%) and –negative (33.3%) groups. H. pylori infection and vitamin B12 The most common malabsorptive condition leading to vitamin B12 deficiency is the body’s inability to extract cobalamin from food. Food-bound cobalamin malabsorption results from conditions that impair the secretion of gastric acid and pepsin required for the release of cobalamin from proteins in food [25, 26]. Adult studies performed in H. pylori–infected patients revealed a high rate of vitamin B12 deficiency [16–20]. The mechanism of vitamin B12 malabsorption caused by H. pylori infection is unclear, but it may be due to the effect of H. pylori on gastric acid secretion. At the onset of H. pylori infection, there is reduced secretion of gastric acid [27]. After a few months, there may be either Springer

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normal or increased gastric acid production in H. pylori gastritis [28, 29]. If the gastritis progresses to gastric atrophy, the secretion of gastric acid is again reduced [7]. However, Annibale et al. [20] reported that in 57% of the patients, H. pylori–related gastritis was the only pathologic finding detected [20]. In a Turkish study, Kaptan et al. [30] demonstrated that H. pylori was positive in 77 (56%) of 137 adult patients with vitamin B12 deficiency and the eradication of this bacteria improved vitamin B12 levels in 31 (40%) of 77 infected patients. A recent study performed in adults living in southern Turkey also reported a high probability of presence of H. pylori infection together with vitamin B12 deficiency that was independent of the gastric atrophy [17]. On the other hand, the only pediatric study investigating the relation between H. pylori infection and vitamin B12 deficiency revealed no direct strong association between vitamin B12 deficiency and H. pylori infection [21]. We found a statistically significant relation between H. pylori infection and serum vitamin B12 levels (P = .042) that was independent of gastric atrophy. Although prevalence of vitamin B12 deficiency was 28% and 11% in H. pylori–positive and –negative groups, respectively, there was no statistically significant difference between the groups (P = .287). However, it should also be noted that one may expect to find a stronger relation in further large-scale studies. Because vitamin B12 stores are adequate for long times, severe deficiencies could not be detected in childhood. Untreated H. pylori infection persists through the lifespan and may cause more severe vitamin B12 deficiency among the elderly. In addition, hyperhomocysteinemia secondary to vitamin B12 deficiency may constitute a risk for severe cardiovascular and cerebrovascular diseases. H. pylori infection and folate Some studies report a negative relation between H. pylori infection and folate metabolism in adults [16, 31]. To our knowledge, there is no study in literature that investigated this relation in children. A decrease in folate absorption may take place as a consequence of an increment in pH and/or decrement in vitamin C concentration in gastric juice, a situation frequently observed in H. pylori–infected patients [20]. Contrastingly, in the present study, we found no significant difference in folate levels between H. pylori–positive and –negative patients. Furthermore, none of our patients had a significant reduction in serum folate level. H. pylori infection and zinc Zinc is an essential mineral found in almost every cell. It stimulates the activity of approximately 100 enzymes that promote biochemical reactions in the body. Zinc supports

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a healthy immune system and is needed for wound healing and DNA synthesis. It also supports normal growth and development during pregnancy, childhood, and adolescence. Zinc deficiency most often occurs when zinc intake is inadequate or poorly absorbed, when there are increased losses of zinc from the body, or when the body’s requirement for zinc increases [32–36]. Relation between H. pylori infection and zinc is not a commonly investigated topic. It was reported that a protein that strongly binds to zinc has been identified on the membrane and in the cytosol of H. pylori [37]. Because zinc is absorbed mainly in the small intestine [38], it is possible that H. pylori, by binding dietary zinc in the stomach, may contribute to serum zinc deficiency in infected individuals. The only human study investigating relation of H. pylori infection with serum zinc levels in adults suffering from liver cirrhosis concluded that there was no relation between H. pylori infection and serum zinc levels [22]. In accordance, we found no significant difference between the serum zinc levels of H. pylori–positive and –negative patients in our study. In conclusion, the present study is the first one investigating the relation of H. pylori infection with the serum levels of folate and zinc together with ferritin and vitamin B12 in children. It indicated that there was a negative but nonsignificant relation between H. pylori infection and serum ferritin levels. H. pylori infection was significantly related with the serum vitamin B12 , but not with folate and zinc levels. ¨ Acknowledgements The authors thank Kezban Ozkalay, S¸ehriban Karakaya, and Nehime Kara for their help and cooperation; and Dr Hakan G¨ulkesen for help in statistical analysis. This study was supported by Akdeniz University Research Projects Unit.

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