Skip Navigation LinksHome > May 2013 - Volume 56 - Issue 5 > Plasma Ferritin and Indices of Oxidative Stress in Helicobac...
Journal of Pediatric Gastroenterology & Nutrition:
doi: 10.1097/MPG.0b013e3182845a29
Original Articles: Gastroenterology

Plasma Ferritin and Indices of Oxidative Stress in Helicobacter pylori Infection Among Schoolchildren

Soundaravally, R.*; Pukazhvandthen, P.*; Zachariah, Bobby*; Hamide, Abdoul

Free Access
Article Outline
Collapse Box

Author Information

*Department of Biochemistry

Department of Medicine, Jawaharlal Institute of Postgraduate Medical Education & Research, Puducherry, India.

Address correspondence and reprint requests to Bobby Zachariah, PhD, Department of Biochemistry, JIPMER, Puducherry 605006, India (e-mail:

Received 16 February, 2012

Accepted 17 October, 2012

The authors report no conflicts of interest.

Collapse Box


Objective: Iron deficiency is one of the most common causes for anemia in Indian children. The present study was performed to evaluate the prooxidant status and ferritin levels in Helicobacter pylori–infected schoolchildren.

Methods: The present study included healthy controls (control group), H pylori–infected children (group I), and H pylori–uninfected children with iron deficiency (group II). Group I was further subdivided into group Ia and Ib, depending on the presence or absence of iron deficiency, respectively. Serum levels of protein carbonyls, malondialdehyde (MDA), ferritin, total protein, and albumin were evaluated and compared among study groups.

Results: In H pylori–infected schoolchildren, serum MDA and protein carbonyls were significantly increased and ferritin levels were significantly decreased compared with the controls and group II. In group I, irrespective of presence and absence of iron deficiency, MDA and protein carbonyls were significantly increased compared with group II. In anemic H pylori–infected schoolchildren, levels of serum total protein were significantly decreased compared with healthy controls. In H pylori–infected patients, MDA was found to have a significant negative correlation with ferritin levels and total protein by partial correlation analysis.

Conclusions: An increased level of oxidative stress was found in H pylori–infected schoolchildren. Furthermore, the findings from the present study indicate that prolonged oxidative stress may enhance protein degradation in children.

Iron deficiency is one of the most common causes for anemia in Indian children (1). In recent decades, there has been an increased awareness of gastrointestinal conditions resulting in iron deficiency without evidence of abnormal blood loss or gastrointestinal symptomatology (2).

Seroepidemiological studies suggest an association between Helicobacter pylori infection and iron deficiency (3,4). Serum ferritin is considered a sensitive marker of body iron status (5). H pylori infection with or without coexisting autoimmune gastritis has been implicated as an important cause of iron deficiency in patients refractory to oral iron treatment and a favorable response to H pylori eradication (6,7). Children have low iron stores because of their increased iron requirement for growth. In the presence of H pylori infection, they probably develop iron deficiency faster than the adults (8).

H pylori infection has been shown to be associated with generation of reactive oxygen species (ROS) in the gastric mucosa (9). ROS generation has been considered an important phenomenon that is known to contribute to a great variety of deleterious reactions in the aerobic cells (10,11). Emerging evidence has indicated that ROS makes a significant contribution to the pathogenesis of H pylori infection (9). Previous studies have also shown increased free-radical production and reduced gastric vitamin C levels in H pylori infection (12,13). Severe oxidative stress could manifest as diminished levels of vitamin C, which is essential for the absorption of iron in its reduced form (14).

Even though there is evidence indicating the presence of redox imbalance in H pylori–infected patients, to our knowledge no previous report exists to define the levels of markers of oxidative stress and ferritin in H pylori–infected schoolchildren with and without anemia. To this end, indicative parameters of lipid peroxidation and protein oxidation together with plasma ferritin were evaluated.

Back to Top | Article Outline


The present study was approved by the human ethics committee and institute research committee of the Jawaharlal Institute of Postgraduate Medical Education & Research. Students in the age range of 13 to 16 years were enrolled for the present study. The present study included healthy controls (control group), H pylori–infected children (group I), and H pylori–uninfected children with iron deficiency (group II). Group I was further subdivided into group Ia and Ib, depending on the presence or absence of iron deficiency, respectively. The mean ages of group Ia, Ib, and II were 14, 14, and 15 years, respectively, and were matched with healthy controls (mean 15 years). The male to female ratio of healthy controls, group Ia, group Ib, and group II were 1:1.2, 1:1.3, 1:1.1, and 1:1.1, respectively.

The students with a history of peptic ulcer disease, bleeding per rectum, menstrual irregularity, or other bleeding diathesis, and nonsteroidal anti-inflammatory drug or steroid intake were excluded from the study. After taking informed consent from their parents, 5 mL of blood sample was collected, and serum was stored at −70°C until analysis. Samples were screened for anti-H pylori antibodies using commercial enzyme-linked immunosorbent assay kits (IBL Immunobiological Laboratories, Hamburg, Germany). Samples having a value ≥12 U/mL were considered positive for H pylori infection. The positive and predictive values of the kit were 96% and 80%, respectively (15). Serum ferritin level was determined by enzyme-linked immunosorbent assay using human ferritin enzyme immunoassay test kit. Serum protein and albumin were measured using Biuret and bromocresol green methods, respectively, in fully automated Ciba Corning 550 Express Plus (Ciba Corning Diagnostic, Medfield, MA).

The serum concentration of lipid peroxides was measured by thiobarbituric acid (TBA) method (16). In this method, malondialdehyde (MDA) reacts with TBA reagent under acidic conditions to give pink-colored products, which were measured at 532 nm. Protein carbonylation assay was done according to the method of Levine et al (17). The carbonylated protein reacts with 2,4-dinitrophenyhydrazine to form a colored adduct of 2,4-dinitrophenylhydrazone, which was measured at 366 nm.

Back to Top | Article Outline
Statistical Analysis

Independent Student t test/Mann-Whitney U test or 1-way analysis of variance/Kruskal-Wallis test, as appropriate, was used based on the distribution of data and number of groups. All statistical analyses were carried out for 5% level of significance. Partial correlation analysis was used to assess the possible associations between variables.

Back to Top | Article Outline


Table 1 shows all the parameters tested in groups I and II and healthy controls. Levels of serum ferritin were significantly lower in group II in comparison with group I and controls. MDA and protein carbonylation was increased significantly in both groups I and II compared with healthy controls. H pylori–positive subjects showed higher levels of MDA and protein carbonyls than H pylori–negative subjects with iron deficiency.

Table 1
Table 1
Image Tools

Table 2 shows the subgroup analysis of H pylori–positive subjects. Oxidant markers were significantly higher in both groups Ia and Ib than in controls and group II. Levels of total protein were lower in group Ia than in controls. Among H pylori–positive subjects, no change in the levels of MDA and protein carbonyls was observed between iron-deficient and non–iron-deficient group. In H pylori–infected subjects, MDA showed a significant negative correlation with ferritin (r = −0.272, P = 0.04) and total protein (r = −0.313, P = 0.02) by partial correlation (Fig. 1A and B).

Table 2
Table 2
Image Tools
Figure 1
Figure 1
Image Tools
Back to Top | Article Outline


H pylori infection is found to be extremely common in developing countries because of poor sanitation and unhealthy food habits; however, many of these infected subjects remain asymptomatic (or develop symptoms of gastritis or peptic ulcer) for a long period (18). The first case relating iron-deficiency anemia (IDA) and H pylori infection was reported by Barabino et al (19), who described a 7-year-old boy with a long history of unexplained IDA refractory to iron therapy, which was associated with H pylori infection. The anemia was reversed after H pylori eradication even without iron supplementation. Since then, accumulating evidence suggests that there is an association between H pylori and IDA (3,4).

A fall in serum ferritin level reflects declining body iron stores, which is an accepted marker of iron deficiency (5). In our subjects, the mean ferritin value was lower in the test groups than in healthy controls. Among the H pylori–seropositive subjects, the ferritin levels were significantly lower in the group defined as having iron deficiency than in the group without iron deficiency. Seo et al showed serum ferritin to be significantly lower in H pylori–seropositive children (20). This may be because of the presence of H pylori infection, which would impair iron absorption. The mechanism for the development of IDA in the absence of any obvious blood loss is being investigated.

The initiation of lipid peroxidation has often been considered the proximal cause of cell damage (21). In the present study, the levels of lipid peroxidation were higher in all study groups than in controls. The lipid peroxide levels were higher in the H pylori–negative group with iron deficiency than in the control group. Previous studies have demonstrated increased lipid peroxide levels with parallel decrease in antioxidant defense system in erythrocytes of anemic patients (22–24). The degree of oxidative stress was higher in H pylori–positive group in comparison with group without H pylori infection but with iron deficiency. This indicates that H pylori infection could be an important component in contributing to oxidative stress, in addition to iron deficiency.

The protein carbonylation was higher in the H pylori–positive group than in controls and H pylori–negative group with iron deficiency. There was no significant difference in protein carbonyl levels between the control group and H pylori–negative group with iron deficiency. This again illustrates the fact that H pylori infection could be one of the most important factors in contributing to oxidative stress.

Previous studies have reported oxidative stress and altered redox balance elicited by endogenously generated ROS as a common mechanism implicated in the pathogenesis of H pylori–associated gastritis and cancer (9,11). This is the first study carried out to evaluate the levels of plasma lipid peroxidation and protein carbonylation among schoolchildren with iron deficiency and H pylori infection.

We found a significant negative association between lipid peroxidation and ferritin levels among H pylori–infected subjects. The possible link between ferritin levels and oxidative stress in H pylori infection may be that the generated free radicals affect the iron absorption by interfering with the reduction of iron. It has been hypothesized that oxidative stress could play an important role in impairing iron absorption by diminishing the levels of vitamin C, which is essential for the absorption of iron in its reduced form (14). Oxidative stress may be one of the causes for iron deficiency. On the contrary, iron deficiency could also lead to oxidative stress, as reported in the present study; however, the exact mechanism is not clear. Probably it is required for some of the heme- and iron-containing antioxidant enzymes such as catalase and NADPH oxidase.

In the present study, the total protein levels were found to be significantly lower in the H pylori–positive group with iron deficiency than in controls. Also, we found a significant negative correlation between lipid peroxidation and serum total protein level in the H pylori–seropositive group. This decrease in protein levels could be because of the protein degradation as a result of enhanced oxidative stress–mediated carbonylation. Study by Liu and Wang (25) demonstrated enhanced protein carbonylation and their possible degradation by polyunsaturated fatty acid ester peroxidation products. This finding indicates that the prolonged oxidative stress in iron deficiency will affect the nutritional status of children. The role of oxidative stress in enhancing the state of iron deficiency in H pylori infection and protein degradation in such children needs to be further investigated.

Our study has several limitations. First, the size of study groups was small; however, it is only a preliminary study and was designed to obtain information about the possible nexus between oxidative stress and ferritin levels in schoolchildren. Second, it would have been more interesting if follow-up study has been carried out after eradication of H pylori and results were compared between successfully treated subjects and those who remain infected. Third, the H pylori infection was detected by measuring the IgG levels in blood and was not confirmed by another noninvasive method of choice. A negative serology for H pylori antibody can be used to rule out infection; however, a positive serology only determines that a patient has been exposed to H pylori at some time in the past, but not whether the patient is presently infected. Using other methods to detect H pylori could have thrown more light on the various nexuses found in the present study.

In conclusion, H pylori infection in young children is associated with oxidative stress manifested by increased lipid peroxidation and protein carbonylation. These data from the present study indicate that alteration in the levels of MDA in H pylori–infected patients may be the basis for enhanced protein degradation and anemia among schoolchildren.

Back to Top | Article Outline

The authors thank manuscript editors Drs Selvaraj Nambiar and Balasubramanian A, Department of Biochemistry, JIPMER, Pondicherry, India.

Back to Top | Article Outline


1. Pasricha SR, Black J, Muthayya S, et al. Determinants of anemia among young children in rural India. Pediatrics 2010; 126:140–149.

2. Annibale B, Capurso G, Delle Fave G. The stomach and iron deficiency anaemia: a forgotten link. Dig Liver Dis 2003; 35:288–295.

3. Milman N, Rosenstock SJ, Andersen L, et al. Serum ferritin, hemoglobin, and Helicobacter pylori infection: a seroepidemiological survey comprising 2794 Danish adults. Gasteroenterology 1998; 115:268–274.

4. Peach HG, Bath NE, Farish SJ. Helicobacter pylori infection; an added stressor on iron status of women in the community. Med J Aust 1998; 169:188–190.

5. Worwood M. Influence of disease on iron status. Proc Nutr Soc 1997; 56 (1B):409–419.

6. Annibale B, Marignani B, Monarca B, et al. Reversal of iron deficiency anemia after Helicobacter pylori eradication in patients with asymptomatic gastritis. Ann Intern Med 1999; 131:668–672.

7. Valiyaveettil AN, Hamide A, Bobby Z, et al. Effect of anti-Helicobacter pylori therapy on outcome of iron-deficiency anemia: a randomized controlled study. Indian J Gastroenterol 2005; 24:155–157.

8. Poddar U, Yachha SK. Helicobacter pylori in children: an Indian perspective. Indian Pediatr 2007; 44:761–770.

9. Handa O, Naito Y, Yoshikawa T. Redox biology and gastric carcinogenesis: the role of Helicobacter pylori. Redox Rep 2011; 16:1–7.

10. Kohen R, Nyska A. Oxidation of biological systems: oxidative stress phenomena, antioxidants, redox reactions and methods for their quantification. Toxicol Pathol 2002; 30:620–650.

11. Selvaraj N, Bobby Z, Sridhar MG. Oxidative stress: does it play a role in the genesis of early glycated protein? Med Hypotheses 2008; 70:265–268.

12. Naito Y, Yoshikawa T. Molecular and cellular mechanisms involved in Helicobacter pylori-induced inflammation and oxidative stress. Free Radic Biol Med 2002; 33:323–336.

13. Vijayan G, Sundaram RC, Bobby Z, et al. Increased plasma malondialdehyde and fructosamine in anemic H pylori infected patients: effect of treatment. World J Gastroenterol 2007; 13:796–880.

14. Ruiz B, Rood JC, Fontham ETH, et al. Vitamin C concentration in gastric juice before and after anti Helicobacter pylori treatment. Am J Gastroenterol 1994; 89:533–539.

15. Mohammadi M, Talebkhan Y, Khalili G, et al. Advantage of using a home-made ELISA kit for detection of Helicobacter pylori infection over commercially imported kits. Indian J Med Microbiol 2008; 26:127–131.

16. Sato K. Serum lipid peroxide in CVD determined by new colorimetric method. Clin Chim Acta 1978; 90:37–43.

17. Levine RL, Williams JA, Stadtman BR, et al. Carbonyl assays for determination of oxidatively modified protein. Enzymology 1994; 233:346–357.

18. Malaty HM, Graham DY. Importance of childhood socioeconomic status on the current prevalence of Helicobacter pylori infection. Gut 1994; 35:742–745.

19. Barabino A, Dufour C, Marino CE, et al. Unexplained refractory anemia associated with Helicobacter pylori gastric infection in children; further clinical evidence. J Pediatr Gastroenterol Nutr 1999; 28:116–119.

20. Seo JK, Ko JS, Choi KD. Serum ferritin and Helicobacter pylori infection in children; a sero epidemiologic study in Korea. J Gastroenterol Hepatol 2001; 17:754–757.

21. Selvaraj N, Bobby Z, Sridhar MG. Increased glycation of hemoglobin in chronic renal failure: potential role in oxidative stress. Arch Med Res 2008; 39:277–284.

22. Kurtoglu E, Ugur A, Baltaci AK, et al. Effect of supplementation on oxidative stress and antioxidant status in iron deficiency anemia. Biol Trace Elem Res 2003; 96:117–123.

23. Isler M, Delibas N, Guclu M, et al. Superoxide dismutase and glutathione peroxidase in erythrocytes of patients with iron deficiency anemia: effects of different treatment modalities. Croat Med J 2002; 43:16–19.

24. Sundaram RC, Selvaraj N, Vijayan G, et al. Increased plasma malondialdehyde and fructosamine in iron deficiency anemia: effect of treatment. Biomed Pharmacother 2007; 61:682–685.

25. Liu W, Wang JY. Modifications of protein by polyunsaturated fatty acid ester peroxidation products. Biochim Biophys Acta 2005; 1752:93–98.

Cited By:

This article has been cited 2 time(s).

Extragastric Diseases and Helicobacter pylori
Baudron, CR; Franceschi, F; Salles, N; Gasbarrini, A
Helicobacter, 18(): 44-51.
Helicobacter pylori in Pediatrics
Alarcon, T; Martinez-Gomez, MJ; Urruzuno, P
Helicobacter, 18(): 52-57.
Back to Top | Article Outline

ferritin; H pylori; iron-deficiency anemia; oxidative stress; schoolchildren

© 2013 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology,


Article Tools



Search for Similar Articles
You may search for similar articles that contain these same keywords or you may modify the keyword list to augment your search.

Connect With Us





Visit on your smartphone. Scan this code (QR reader app required) with your phone and be taken directly to the site.