Biochemical Markers of Bone Metabolism in Children with Helicobacter pylori Infection
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- Ozdem, S., Akcam, M., Yilmaz, A. et al. Dig Dis Sci (2007) 52: 967. doi:10.1007/s10620-006-9292-0
We investigated the biochemical markers of bone metabolism in children with Helicobacter pylori infection. Biochemical markers of bone metabolism and serum levels of vitamin B12, ferritin and estradiol were measured in 41 H. pylori-positive (+) children (23 girls, 18 boys; aged 11.8±3 years). Serum levels of intact parathyroid hormone, ß-collagen I carboxy terminal telopeptide, total alkaline phosphatase (ALP), bone-specific ALP, N-terminal cross-links of human procollagen type I, N-mid-osteocalcin, calcium, phosphate, ferritin, and estradiol did not differ significantly between H. pylori(+) and H. pylori negative (−) children. Vitamin B12 levels were significantly decreased in H. pylori(+) compared to H. pylori(−) children. H. pylori infection was not accompanied by significant changes in markers of bone metabolism in children, although vitamin B12 levels were decreased. Further studies are required to clarify whether H. pylori infection causes time-dependent changes in bone turnover markers during the long course of this inflammatory disease.
KeywordsBone markersChildrenFerritinHelicobacter pyloriOsteoporosisVitamin B12
Osteoporosis is a systemic bone disease characterized by low bone mass and microarchitectural deterioration of bone tissue, leading to enhanced bone fragility and a resultant increase in fracture risk . It is an important cause of morbidity and mortality . Although it is well known that many systemic and local factors have an effect on bone tissue and, therefore, on osteoporosis, the etiopathogenesis of osteoporosis still remains unclear. The incidence of osteoporosis increases with age and it has become an important health problem in society, as the number of elderly people is increasing rapidly . Disorders of bone mineralization are a major health problem not only in adults, but also in children: Literature data and clinical observations draw attention to more frequent diagnoses of both secondary and idiopathic osteoporosis and osteopenia in the developmental stage of life . Secondary osteoporosis in children develops in the setting of chronic illness related to the disease itself or to its treatment. Medications, nutritional problems, hormone deficiencies, and reduction in physical activity are the other factors contributing to a decrease in bone mass and impairment of bone quality [5, 6].
The Gram-negative (−) spiral-shaped bacterium, Helicobacter pylori , is involved in many benign and malignant gastrointestinal diseases, such as peptic ulcer  and gastric cancer [9–11]. H. pylori infection of the gastric mucosa is probably one of the most common bacterial infections throughout the world. Although it is frequently acquired during childhood and lasts through adult life, most patients with H. pylori-induced gastritis remain asymptomatic throughout their lives [12, 13], with persistent host responses to its continuing presence .
H. pylori not only causes a local gastric response but also produces systemic effects: defects particularly in vitamin B12 absorption [15–17], alterations in growth hormone (GH) levels [18–20], increments in serum and mucosal levels of cytokines [21, 22], and changes in estrogen levels  have all been reported in H. pylori-positive (+) individuals. Although it is well known that all these changes have the potential to influence bone remodeling, there are limited data on the alterations in biochemical markers of bone metabolism in patients with H. pylori infection. The only study performed in male adults, that by Figura et al. , concluded that H. pylori infection may increase the risk of developing osteoporosis. On the other hand, to our knowledge, the effect of H. pylori infection on bone turnover has not yet been established in children. Therefore, in the present study, we aimed to investigate the biochemical markers of bone metabolism together with serum vitamin B12 and ferritin levels in children with H. pylori infection in order to determine whether H. pylori contributes to osteoporosis in this age group.
A total of 61 patients (36 girls, 25 boys; aged 5–18 years) who underwent upper gastrointestinal endoscopy with dyspeptic symptoms, were recruited in the study. The study was conducted between February and September 2005, at Akdeniz University Medical School. Informed consents were obtained from the parents and the local ethical committee approved the study. Exclusion criteria were the diseases and the conditions that have the potential to affect bone metabolism and/or the level of cytokines: malignancies, hypogonadism, diabetes mellitus, anorexia nervosa, liver and thyroid diseases, gastrointestinal disorders with malabsorption, treatment with glucocorticoid or antiepileptic drugs within the last 3 months, impaired renal function, vegetarian diet, and acute or chronic inflammation. Subjects with previous H. pylori eradication were also excluded.
Standing heights of the children were measured with a wall-mounted stadiometer by one of us. The height was also expressed as Z score using age- and gender-specific reference data.
Design and sample collection
The biopsy materials obtained during gastrointestinal endoscopy were investigated histopathologically. Diagnosis of H. pylori infection was based on the observation of bacterium and gastritis in the histopathological examination (H. pylori[+] group). The control group consisted of those with no bacilli in the histopathological exam (H. pylori[−] group).
During endoscopy, biopsies were obtained from the 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, USA). Other specimens were placed directly in 10% formalin without a paper filter for histological examination. The biopsies stained with hematoxylin and eosin and with Diff-Quick One were examined for H. pylori and gastritis. The diagnosis of gastritis was based on the histopathological findings of inflammation, activity, metaplasia, and atrophy. Assessment of gastritis was performed according to the updated Sydney System using visual analogue scales .
Biochemical parameters and methods
Blood samples collected after overnight fasting were centrifuged and stored at −80°C until the day of analysis. Serum levels of the bone resorption markers intact parathyroid hormone (iPTH) and ß-collagen I carboxy-terminal telopeptide (β-CTX), the bone formation markers total alkaline phosphatase (ALP), bone-specific ALP, N-terminal cross-links of human procollagen type I (P1NP), and N-mid-osteocalcin (OC), and the bone turnover-related markers calcium (Ca) and phosphate (P) were all determined synchronously. Serum vitamin B12, ferritin, and estradiol levels were also measured.
The levels of P, Ca, and total ALP were studied with a Roche/Hitachi Modular PP automated clinical chemistry analyzer (Roche Diagnostics GmbH, Mannheim, Germany) using the ammonium phosphomolybdate, o-cresolphthalein endpoint, and p-nitrophenyl phosphate colorimetric methods, respectively. Levels of iPTH, OC, P1NP, β-CTX, vitamin B12, ferritin, and estradiol were measured with the Roche Modular Analytics E170 immunoassay system (Roche Diagnostics GmbH) using the electrochemiluminescence immunoassay method (ECLIA). Bone specific ALP levels were determined using the isopal plus ALP isoenzyme electrophoresis kit in Beckman Coulter's Paragon electrophoresis system (Beckman Coulter Inc., Marvue, Galway, Ireland).
Statistical analysis was performed using the SPSS 11.0 for windows statistical package (SPSS). Data are presented as mean±standard deviation. The significance of the differences between the means was tested using Student's t test. In all instances, P values <0.05 were considered significant.
Demographic characteristics and body mass index in H. pylori-positive and -negative groups
H. pylori(+) group
H. pylori(−) Group
Z score for height corrected for age and sex
Body mass index (kg/m2)
Serum levels of biochemical markers of bone metabolism, vitamin B12 and ferritin, in H. pylori-positive (n=41) and -negative (n=20) groups
Total ALP (U/L)
Bone specific ALP (%)
Vitamin B12 (pg/ml)
Secondary osteoporosis in children develops in the setting of chronic illness related to the disease itself or its treatment . It has been known that H. pylori infection is frequently acquired during childhood and lasts through adult life [12, 13], with persistent host responses to its continuing presence . Therefore, taking into consideration both the chronic and the inflammatory nature of this disease, one would expect to find alterations in bone markers in H. pylori(+) patients. However, to our knowledge, the possible effects of H. pylori infection on bone turnover have not yet been studied in children. In the present study, we found that H. pylori infection was not accompanied by significant alterations in biochemical markers of bone metabolism in children. On the other hand, the only study investigating the issue in adult male patients, that by Figura et al. , concluded that the levels of urinary cross-laps (a marker of bone resorption) were increased in patients infected with H. pylori strains expressing the chromosomal insertion called CagA, compared to both uninfected and CagA-negative strain-infected patients, but there were no significant differences in other bone turnover markers in H. pylori-infected male adults, independent of CagA status. In the present study, although it was not significant, we found a marked increase in β-CTX levels in the H. pylori(+) compared to the H. pylori(−) group, which might indicate a tendency to increased bone resorption in these children.
Fıgura et al. found a significantly low level of estradiol in CagA-positive H. pylori-infected male adults compared with CagA-negative H. pylori-infected and uninfected ones, and suggested that the reduction in estradiol levels resulting from inhibition of P450 aromatase by CagA-positive H. pylori may play a role in increased bone turnover in male adults . In the present study, although the level of estradiol was significantly higher in H. pylori(+) girls than H. pylori(+) boys, we found no significant differences in biochemical markers of bone metabolism between these groups, except for the bone formation marker total ALP, which was significantly higher in H. pylori(+) boys than H. pylori(−) girls. Furthermore, in neither boys nor girls did the levels of biochemical markers of bone metabolism differ significantly between H. pylori(+) and H. pylori(−) subjects.
Cytokines possess an important role in regulation of bone resorption and formation during pathologic as well as normal bone remodeling . IL-1, IL-6, IL-11, leptin, and TNF-α were all shown to influence osteoclast function. Dysregulation of these cytokines may contribute to the development of osteoporotic bone disease [26, 27]. Cytokines may also play a role in bone homeostasis by acting on the parathyroid hormone mRNA levels [28, 29]. The inflammatory process (i.e., chronic gastritis) initiated and sustained by H. pylori infection causes increased synthesis and release of proinflammatory cytokines [22, 30, 31]. Accordingly, Guiraldes et al. showed that gastric tissue from children with H. pylori-associated gastritis contained increased concentrations of proinflammatory cytokines compared with tissue from noninfected children with normal gastric mucosa . Although we did not measure the levels of cytokines in our study, it has been reported that H. pylori infection in children is usually mild, with low lymphocyte and neutrophile infiltration [32, 33]. Therefore, if pediatric infection represents an earlier stage of the H. pylori-induced inflammatory response, then both a different immunopathology and different patterns of cytokine expression could be anticipated in children compared to adults . Although it has been reported in a relatively small number of studies that the local cytokine profiles were similar in H. pylori infection in children and adults [21, 35–38], these studies seemed to have some methodological limitations in reflecting the in vivo situation . Further studies using immunohistochemical methods to avoid the limitations in methodology are needed to clarify the status of cytokine production in children with H. pylori infection.
It has been reported that H. pylori infection is associated with growth delay and/or growth retardation in affected children [18–20]. GH and its mediator, insulin-like growth factor-I (IGF-I), provides the main conduit for growth regulation . It was reported that H. pylori infection was associated with reductions in IGF levels . IGF-I can increase type I collagen synthesis, ALP activity, and OC production in osteoblasts . In the present study, the height including the corrected values for age and sex, weight, and BMI of H. pylori(+) children were very similar to those of H. pylori(−) children, suggesting that H. pylori was not associated with growth in this age group. In accordance, Figura et al. reported no significant differences in height, weight, or IGF-I levels between uninfected and H. pylori-infected male adults . H. pylori infection may also lead to growth retardation via iron deficiency anemia [42, 43]. However, we found no significant reduction in serum ferritin levels of H. pylori(+) children compared to H. pylori(−) children.
Low serum vitamin B12 levels related to atrophic gastritis are relatively common among elderly males (2.5%) in the general population. An ongoing H. pylori infection occurs in approximately 75% of these cases . Annibale et al. demonstrated that almost two-thirds of pernicious anemia patients showed evidence of H. pylori infection . Vitamin B12 deficiency may have direct effects on bone, ALP activity increases in osteoblastic cells after stimulation with vitamin B12, and a minimum concentration of vitamin B12 is needed for osteoblasts proliferation . The levels of both OC and bone ALP, the markers of bone formation, were found to be significantly decreased in vitamin B12-deficient patients . In the present study, although there was a significant reduction in serum vitamin B12 levels in H. pylori(+) children compared to H. pylori(−) ones, it was not accompanied with significant changes in markers of bone metabolism. It might be speculated that, although significantly decreased compared with control levels, vitamin B12 levels in H. pylori(+) children may still be far above the minimum levels required for normal osteoblast proliferation,. Although the reason for the reduction in serum vitamin B12 levels in H. pylori(+) children is unclear at the moment, mechanisms such as (1) the diminished acid secretion in H. pylori-induced gastritis, leading to a failure of critical splitting of vitamin B12 from food binders, with resultant impairment in its absorption, (2) a secretory dysfunction of the intrinsic factor; and (3) decreased secretion of ascorbic acid from the gastric mucosa and increased gastric pH may all play a role [48, 49].
The main limitation of our study was no doubt the inability to determine the strains of H. pylori in infected children. However, it should be noted that the CagA status seemed to be significantly related to only one marker of bone turnover (level of urinary cross-laps), and not the others, in male adults . Furthermore, in contrast to adults, CagA may not be a universal pathogenic factor in children . Additional shortcomings were the low number of subjects included and the absence of data related to the levels of cytokines in study groups.
In summary, the findings of the present study showed that although there was a tendency to increased bone resorption, H. pylori infection was not accompanied by significant alterations in biochemical markers of bone metabolism in children. Further studies with large number of subjects are required to clarify whether H. pylori infection causes time-dependent changes in bone turnover markers during the long course of this inflammatory disease.
This study was supported by the Akdeniz University Research Projects Unit. The authors thank Dr. Levent Donmez for his contribution in data analysis.