Helicobacter pylori , a causative agent of vitamin B 12 deficiency

Background: Helicobacter pylori is one of the most common causes of peptic ulcer disease worldwide and a major cause of chronic superficial gastritis leading to atrophy of gastric glands. Methodology: A total of 60 patients suffering from gastric disease due to H. pylori infection were evaluated. Endoscopy was performed and gastric biopsies were obtained for histopathology and urease test. Blood was simultaneously collected for the determination of the levels of vitamin B12 and the MCV. Vitamin B12 levels were determined by chemiluminescent assay. Results: Our results indicate that the mean vitamin B12 level + SEM for the total population, the H.pylori infected and non-infected patients were 264.5+22.9, 207.7+21.9 and 419.7+39.8 respectively. H. pylori was found in 71.7% (43/60) of the patients tested. The level of vitamin B12 was lower than 200pg/ml (deficient) in 67.4% (29/43) of patients tested positive for H. pylori. Conclusion: H. pylori appears to be implicated in causing vitamin B12 deficiency.


Introduction
Helicobacter pylori is a fastidious gram negative microorganism with a spiral/helical shape and 5 to 7 sheathed uni-polar flagella.It requires high humidity, a microaerophilic environment, and an incubation temperature of 37 o C for growth.Although H. pylori colonies may appear after 3 to 5 days, the primary growth may take up to 7 days to appear and the colony morphology is described as translucent, small pinpoint colonies [1].H. pylori is considered to be the most common human pathogen colonizing the human gastric mucosa [1].The prevalence of H. pylori in the developed countries is 25% to 40% and is almost 100% in developing countries [2].
Studies have shown that H. pylori IgG antibodies increase with age.It is present in 40% of those aged 1 to 14 years, and has been reported in up to 60% of 18-to 24-year-old Mexicans and in 100% of 60 to 69-yearolds in Turkey [3].H. pylori causes chronic gastritis [4] and peptic ulcer disease [5] and it has been classified as a type I (definite) carcinogen since 1994.This organism has also been implicated in increasing the risk of developing gastric cancer, which is the second most frequent cause of cancer-related death [3,6].Furthermore, H. pylori infection has also been associated with an increased risk of B-cell lymphoma of gastric mucosa-associated-lymphoid-tissue (MALTlymphoma) [7,8].
Vitamin B 12 , which is a water soluble vitamin, is a complex molecule that cannot be synthesized in the human body and must be supplied in a diet containing meat and dairy products.Upon digestion, B12 is released from food and complexes with gastric intrinsic factor (IF).The B 12 -IF complex binds to specific receptors in the ileum where it is absorbed.It has important functions in DNA replication, in the synthesis of red blood, and in maintaining the myelin sheath that surrounds nerve cells.Vitamin B 12 deficiency causes pernicious anemia due to lack of production of intrinsic factor by epithelial cells in the stomach.Although many factors can contribute to the development of Vitamin B 12 deficiency, pernicious anemia is mostly attributable to conditions associated with chronic atrophic gastritis [9].
H. pylori has been suggested as an important agent in the aetiology of vitamin B 12 deficiency and pernicious anemia [10].A result of H. pylori-induced gastritis and ulcers is destruction of the parietal cells which are important for the production of intrinsic factor which is essential for vitamin B 12 absorption.H. pylori were isolated in 56% of patients with pernicious anemia and eradication of the infection has been shown to result in improved blood levels of vitamin B 12 in 40% patients [11].However, H. pylori-induced vitamin B 12 deficiency has been shown to occur in the absence of atrophic gastric mucosa [12].Thus the aim of this study was to further investigate the possible association between H. pylori infection and vitamin B 12 deficiency.

Materials and Methods
This cross-sectional study was conducted between November 2003 and November 2004 The inclusion criteria was limited to patients diagnosed with gastritis by endoscopy and not taking any vitamin B 12 supplements.Patients were excluded from the study if they had any underlying medical condition which may have caused vitamin B 12 deficiency, if they had previous administration of H. pylori eradication therapy, or if they had renal failure or liver disease.Pregnant women were also excluded.
For each patient enrolled, information about age, sex, smoking and symptoms suffered was recorded.Blood was collected aseptically in EDTA and plain vacutainer tubes for laboratory investigations.Complete Blood Count (CBC) was performed using Sysmex Analyzer (Sysmex, Japan) to determine the MCV.Vitamin B 12 levels was determined by a competitive chemiluminescent immunoassay using an IMMULITE automated analyzer (DPC, CA, USA).
Gastric biopsies from the antrum and duodenal biopsies were obtained during endoscopy.The rapid urease which is a qualitative test for the detection of the urease enzyme produced by H. pylori was conducted using the CLO-test (Biomer'ieux, France).H. pylori hydrolyzes the urea rapidly and the color change of the indicator within 20 minutes to one hour was indicative of a positive test.If negative after one hour, the test was read again after 24 hours.
Poly Stat Helicobacter pylori test kit (Polymedco Inc.USA) was used for the qualitative detection of anti-Helicobacter pylori IgG in the human serum.Histopathology was performed on formalin fixed paraffin embedded biopsies.Sections were cut at 5 microns thick and stained with eosin and methylene blue.The sections were then evaluated for the presence of H. pylori by a pathologist.
Statistical analysis was conducted using SPSS version 12 (SPSS inc., Chicago).The presence of association between the different parameters was assessed by by Chi-Square and correlation coefficients was also determined (p<0.05 was taken as statistically siginificant).

Results
Sixty patients (29 females and 31 males) who fulfilled the inclusion and exclusion criteria were enrolled in the study.H. pylori was detected in 97.7% (42/43) of the patients diagnosed with gastritis.Histopathology was used to demonstrate the presence of the characteristic curved or S-shaped H. pylori and to provide information about the nature of the patient's gastritis.CLO test and serology revealed an infection rate of H. pylori in 71.7% (43/60) of the study population.Among those positive for H. pylori, the rate of infection was higher in males, 55.8% (24/43) versus 44.2% (19/43) in females.The age range of the patients was between 18 and 82 years (mean 43.4 +17.3 years).With respect to age, the population was divided into three groups: group I (18 -39 years), group II (40-59 years) and group III (> 60 years).Our results indicated that H. pylori was present in 56.7% of group I and 21.7% in groups II and III (Table 1).With respect to smoking, 30.2% (11/43) of the H. pylori positive patients were smokers.The lower limit of normal serum vitamin B 12 level is set at 200 pg/ml [13] and the reference level used at the Mayo Clinic is 200 to 650 pg/ml [14].The mean vitamin B 12 level (+ SEM) was 264.5 + 22.9 pg/ml in the total patient population (n=56).Four values were excluded from the mean because the levels were extremely elevated (1088, 1103, 1185, 1200).The cutoff level to determine vitamin B 12 deficiency was taken as <200 pg/ml, and based on this measure, 67.4% (29/43) of the H. pylori infected patients had Vitamin B 12 deficiency.The mean vitamin B 12 level (+ SEM) for the H. pylori infected patients was 207.7+21.9 as compared to 419.7+39.8 for the H. pylori negative patients.There was significant difference by the independent t-test (p= 0.00) between the means of vitamin B 12 levels of H. pylori negative and H. pylori positive patients at 0.05 level of significance.MCV was elevated (>90 fl) in 67.4% of the H pylori infected patients (29/43) (Table 2).There was significant association (p<0.05) between presence of H. pylori infection and vitamin B 12 deficiency.Pernicious anemia is a type of megalobastic anemia due to Vitamin B 12 deficiency.In order to establish an association between H. pylori infection and vitamin B 12 deficiency, we have chosen patients symptomatic for H. pylori infection.Our results showed that 71.7 % of the patients with gastritis were infected with H. pylori, and 67.4% of these patients had pernicious anemia as seen in the elevated MCV levels.In this study, selection of patients was restricted to those who had no inherent problems related to vitamin B 12 absorption or malnutrition.H. pylori and related gastric pathology emerges as a major factor responsible for vitamin B 12 deficiency.Several studies on the relationship between H. pylori, gastritis and vitamin B 12 deficiency have been conducted [9,11].Our results agree with the results obtained by these studies.The main variables in this study were H. pylori infection and vitamin B 12 deficiency.Our results indicated a significant association between the two (P<0.05).In conclusion, the presence of H.pylori in the gastric mucosa can be considered as an important factor in causing vitamin B 12 deficiency.In developing countries, where there is a high prevalence of H. pylori infections, one expects to also find vitamin B 12 deficiency in certain members of the population.Therefore, diagnostic evaluation of vitamin B 12 deficiency must include tests for H. pylori.

Table 1 .
General characteristics of the parameters monitored.

Table 2 .
Association of vitamin B 12 and MCV in H. pylori infected cases.