Helicobacter pylori Prevalence and Circulating Micronutrient Levels in a Low-Income United States Population

Infection with Helicobacter pylori (H. pylori), a gram-negative spiral bacterium that induces chronic gastritis in half of the world's population, is the leading cause of gastric cancer (1), the second most deadly cancer worldwide (2). Within both high- and low-risk countries, gastric cancer incidence, like H. pylori prevalence, is also strongly associated with low socioeconomic status (3). Recently, a study of trends in incidence of noncardia gastric cancer (the type most significantly associated with H. pylori) found that incidence was generally decreasing in the United States as expected, although age-specific analyses suggest that the decline may be beginning to end (4). In the United States, where gastric cancer is uncommon, there is still a large racial disparity, as African Americans have almost twice the likelihood of being diagnosed with gastric cancer as whites (5). Similarly, the overall H. pylori prevalence in the United States is estimated to be relatively low at around 30% (although 50%–60% for African Americans; ref. 6), but a surprising 80% of a biracial sample of Southern Community Cohort Study (SCCS) participants, a primarily low-income study population from the southeastern United States, were recently found to be seropositive for H. pylori (7).

Because of the generally consistent findings from observational studies that implicate diet in the etiology of gastric cancer, in 2007, an expert panel of the World Cancer Research Fund judged that there is probably an evidence that fruit intake protects against gastric cancer (8). However, although a number of randomized trials of vitamin supplementation (including nutrients such as β-carotene, ascorbic acid, folic acid, α-tocopherol, selenium, and zinc) have been conducted, only 2 of 7 studies have reported benefits in the prevention of gastric premalignancy and 2 of 9 studies have found significant protective effects for gastric cancer (9).

Another hypothesis for the association of nutrient levels and gastric cancer risk relates to the role of H. pylori. Instead of high nutrient levels protecting against H. pylori infection, it has been suggested that infection with H. pylori itself reduces the absorption of nutrients (10, 11). This hypothesis is based on the fact that chronic H. pylori infection is generally begun in childhood, and thus nutrient levels measured among adolescents and adults are more likely to reflect H. pylori status, rather than the other way around. Furthermore, while nutrients are not absorbed in the stomach, H. pylori infection affects acid secretion that is vital for nutrient absorption. Several studies have found that the concentration of certain micronutrients found in plasma, gastric juice, and gastric mucosa is lower in H. pylori-infected subjects (12).

In this study, we examined the association between H. pylori prevalence and circulating levels of micronutrients in the SCCS, representing a low-income U.S. population with high prevalence of H. pylori infection.

From 2002 to 2009, the SCCS recruited approximately 86,000 men and women, ages 40 to 79, from 12 southeastern states (13). Those who enrolled in person at community health centers (approximately 86%) completed a comprehensive computer-assisted in-person interview, providing information on demographics, anthropometrics, medical history, and cancer risk factors (questionnaire available at: http://www.southerncommunitystudy.org). A food frequency questionnaire (FFQ), validated in this population, was used to collect information on usual diet (14, 15). Those recruited by mail (approximately 14%) completed the same baseline survey, by paper. Race was self-reported with participants instructed to choose all applicable racial/ethnic categories. Venous blood samples (20 mL) were collected from participants at the time of their baseline interview at the community health center, then refrigerated and shipped overnight to Vanderbilt University, Nashville, TN, to be centrifuged the next day and stored at −80°C. By using a 2×2×3×3 factorial design with 22 individuals selected within each of the 36 strata defined by self-reported race (African American/white), sex, smoking status (current/former/never), and body mass index (BMI, 18–24.9/25–29.9/30–45 kg/m²), 792 of the 12,162 participants who enrolled in the study from March 2002 to October 2004 and donated a blood sample at baseline were randomly selected. While this design meant that some populations from within the entire SCCS were oversampled (including whites, nonsmokers, and nonobese participants), it provided a balanced distribution across these factors in consideration of other blood biomarkers being measured in addition to H. pylori.

For each study subject, 50 μL of serum samples were aliquoted for H. pylori assays. H. pylori multiplex serology, as performed in this study, has been described recently (16). In summary, it is a new antibody detection technology on the basis of fluorescent polystyrene beads (Luminex) and recombinant glutathione S-transferase fusion protein capture (17, 18). For all 15 antigens—the H. pylori proteins UreA, catalase, GroEL, NapA, CagA, CagM, Cagδ, HP0231, VacA, HpaA, Cad, HyuA, Omp, HcpC, and HP0305—previously determined antigen-specific cut-point values were applied by using a bridging panel of 78 previously characterized sera (which included 38 H. pylori negative sera and 40 H. pylori positive sera). All sera were analyzed once within a single assay day. Individuals with seropositivity to more than 3 proteins were considered to be H. pylori seropositive as this definition had shown good agreement with commercial serological assay classification.

To study nutritional biomarkers, plasma or serum for exactly half (396) of the individuals selected by the sampling design mentioned before (11 from each of the 36 strata) was assayed for micronutrient levels. Details on the measurement of plasma α-carotene, β-carotene, β-cryptoxanthin, lutein plus zeaxanthin, lycopene, and α-tocopherol levels using assays based on high-performance liquid chromatography, and the analysis of serum folate levels using a microbiologic assay, have been recently published (15). Retinol was measured by the use of a second channel at a wavelength of 325 nm. Quality control procedures included routine analysis of plasma and serum control pools containing high and low concentrations of each analyte. The coefficients of variation were less than 10% for all analytes and control pools.

Of the 396 participants selected for the nutritional biomarker sample, 310 (78.3%) were included in this study. The available serum for H. pylori multiplex serology was depleted from other assays performed on this group for 77 (19.4%), samples for 3 (0.8%) individuals could not be assayed for H. pylori because of serum handling issues, 2 (0.6%) individuals tested seropositive for CagA but seronegative for H. pylori (as they were each seropositive to less than 4 H. pylori antigens total), and 4 (1.0%) individuals were missing data on the potential confounders of total daily energy intake and/or vitamin supplement use.

To assess differences in demographic and lifestyle characteristics between H. pylori + and H. pylori − individuals, crude linear regression was used for the continuous variables of age and daily energy intake and the Mantel–Haenszel chi-square test was used for the remaining categorical variables. To determine the associations between the prevalence of H. pylori infection and blood nutrient levels, multivariate linear regression was used to calculate least-squares mean micronutrient levels within groups defined by H. pylori status in 2 ways: as a dichotomous variable (H. pylori − versus H. pylori +) and as dummy variables, comparing H. pylori seronegative individuals to those seropositive to H. pylori but not to CagA (H. pylori +, CagA−), and those seropositive to both H. pylori and CagA (H. pylori +, CagA+), and comparing mean micronutrient levels between CagA seropositive and CagA seronegative individuals among those who are H. pylori seropositive. Multivariate linear regression models were also carried out to explore the association of micronutrient levels with seropositivity to each of the 15 individual H. pylori antigens assessed with multiplex serology. For the outcome of circulating levels of each micronutrient, blood nutrient levels were log-transformed as they were not normally distributed. A category of total carotenoids was created by summing the log-transformed values for α-carotene, β-carotene, lycopene, lutein and zeaxanthin, and β-cryptoxanthin. Statistical adjustment was made for age at enrollment (continuous), race (African American/white), sex, BMI (continuous), education (less than high school education/high school or GED/greater than high school education), smoking status (never/former/current of less than or 10 cigarettes per day/current of more than 10 cigarettes per day), regular (i.e., ≥once a month) use of vitamin supplements (primarily multivitamins) in the past year (yes/no), and total energy intake (continuous). Additional adjustment for income, fruit and vegetable intake, dietary fat intake, alcoholic drink consumption, health insurance status, family history of gastric cancer, and state where recruited was considered, but these variables were not associated with both the exposure and outcome in the data, and thus were not included in the final models. To evaluate potential effect modification, the association of H. pylori infection and serum nutrient levels was explored in separate models stratified by race, sex, and smoking status, and a likelihood ratio test was used to compare models with and without the respective interaction terms.

To examine the association of H. pylori status and dietary intake of micronutrients, secondary analyses were done by micronutrients levels of intake as derived from the FFQ, also log-transformed due to a skewed distribution. Multivariate linear regression was again used to calculate least-squares mean dietary micronutrient levels within groups defined by H. pylori status, as described before.

In the results presented in this article, the mean levels of micronutrients have been backtransformed so to be scientifically meaningful. The percentage difference in levels as shown in the tables, however, reflects the difference between the log-transformed values, and thus is slightly different from what one would calculate by using the backtransformed values presented.

All statistical analyses were done by SAS 9.2 (SAS Institute).

Of the 310 individuals included in this study, 244 (79%) were seropositive for H. pylori. H. pylori seropositive individuals were more likely to be African American, have less than a high school education, and a first-degree family history of stomach cancer than H. pylori seronegative individuals (Table 1).

For the combined category of total carotenoids and all individual micronutrients, the mean circulating level as derived from serum assays was higher among H. pylori − individuals than H. pylori + individuals. The differences in mean levels for H. pylori + individuals, compared with H. pylori − individuals, were statistically significant for β-carotene, folate, and retinol (decreases of 27.6%, 18.6%, and 9.7%, respectively; Table 2).

When examining the associations between circulating nutrient levels and H. pylori prevalence by CagA status with 1 exception (lutein and zeaxanthin), H. pylori − individuals had the highest mean levels of all micronutrients with lower levels for H. pylori +, CagA− individuals, and the lowest levels among H. pylori +, CagA+ individuals. By comparing H. pylori +, CagA− individuals to H. pylori − individuals, statistical significance for the difference in mean levels was achieved only for folate (an 18.1% difference). For H. pylori +, CagA+ individuals, however, significant differences in mean levels compared with H. pylori − individuals were found with β-carotene, folate, total carotenoids, and retinol (decreases of 38.9%, 19.1%, 17.0%, and 11.7%, respectively). Furthermore, H. pylori +, CagA+ individuals had significantly lower levels of β-carotene, lycopene, and total carotenoids than H. pylori+, CagA− individuals (decreases of 26.4%, 15.6%, and 13.4%, respectively; Table 3). By including H. pylori seroprevalence by CagA status, the r² value of the model for our strongest association, with β-carotene, increased from 0.23 to 0.27 for the model without any information on H. pylori, a small increase despite the strong and significant effect of H. pylori status found in the model.

Of the other H. pylori antigens explored, only seropositivity to VacA also seemed to be associated with micronutrient levels. However, VacA is strongly correlated with CagA (Pearson correlation coefficient = 0.38, P < 0.0001), and after adjusting for CagA seropositivity, the associations with VacA seropositivity were no longer present.

When stratifying by smoking status (current versus never/former), H. pylori + smokers had lower levels of almost all carotenoids than H. pylori + nonsmokers, and there was a consistent trend of a greater percent decrease in blood carotenoid levels among the H. pylori +, CagA+ current smokers than among the H. pylori +, CagA+ noncurrent smokers, when comparing to H. pylori − individuals within the same smoking category, but the differences were not statistically significant (see Appendix Table 1). The associations between H. pylori status and circulating nutrient levels did not vary when examining the relationships separately by sex or race (data not shown).

To further explore the association between H. pylori status and micronutrient levels, we ran the same models using as the outcome micronutrient intake as determined by the FFQ, rather than the blood levels as used previously. While H. pylori + individuals tended to have lower mean intakes than H. pylori − individuals, the differences were neither large nor significant, and there was no trend of decreasing levels as before when moving from the categories of H. pylori − to H. pylori +, CagA− to H. pylori +, CagA+ (Table 4).

In this low-income highly H. pylori-prevalent population in the southeastern United States, H. pylori prevalence, particularly CagA+ H. pylori strains, was associated with lower blood levels of β-carotene, folate, and retinol.

The few previous studies of the association between H. pylori infection and plasma carotenoid and folate levels have generally found null results (19–22). Compared with the present study, these small (ranging from 44 to 86 individuals) clinic-based studies had less power and could not investigate specific H. pylori proteins. Inverse associations have, however, been observed between H. pylori infection and levels of β-carotene in gastric juice (20).

The strong associations between H. pylori status and blood nutrient levels observed in this study are made more interesting by the fact that the dietary intake of these micronutrients as determined by an FFQ was not associated with H. pylori status, lending support for the theory that H. pylori infection impairs the absorption of nutrients. Our data suggest that H. pylori seronegative and seropositive individuals have similar diets with regard to nutritional content, but their circulating levels of certain micronutrients are significantly different, most strongly for β-carotene, whereby H. pylori +, CagA+ individuals have a mean circulating level of only 7.35 μg/dL, compared with 12.03 μg/dL for H. pylori − individuals (P = 0.0004), and 9.99 μg/dL for H. pylori +, CagA− individuals (P = 0.007). In contrast, the mean dietary intake of β-carotene among H. pylori +, CagA+ individuals of 3,181.4 mcg/day is only slightly less than the mean of 3,370.3 mcg/day for H. pylori − individuals (P = 0.59), and, in fact, is greater than the mean of 3,125.5 among H. pylori +, CagA− individuals (P = 0.84). And although FFQ-estimated intakes have been significantly correlated with blood levels of these nutrients in this population, the correlation coefficients for β-carotene and folate were still only at 0.28 and 0.26, respectively, indicating that there remains much room for divergence (Signorello, 2010 #375).

A number of hypotheses have been proposed as to the mechanism by which H. pylori infection reduces nutrient absorption, primarily thorough the bacteria's ability to modify the secretion of hydrochloric acid and the increase in pH (10, 12). Of course, it is also possible that high levels of such nutrients impair the viability of H. pylori (23). A recent study in Korean isolates found that β-carotene inhibited the expression of inducible nitric oxide synthase and cyclooxygenase-2 in H. pylori-infected gastric epithelial cells (24). Furthermore, it has been suggested by in vivo and in vitro experiments that certain carotenoids exhibit antimicrobial activity against H. pylori (23). Previously observed associations between low levels of plasma carotenoids and gastric cancer risk (25–27) in conjunction with our findings of associations between H. pylori seroprevalence and low levels of circulating micronutrients could reflect both H. pylori's direct association with the disease, by inducing chronic gastritis and its indirect association with lower nutrient levels, through H. pylori infection reducing the absorption of antioxidants.

In the present study, the median level of plasma β-carotene among SCCS participants was 9.0 μg/dL (and a significantly lower 7.4 μg/dL among current smokers), similar to that reported in a Shanghai population (9.2 μg/dL; ref. 27), where serum micronutrients were inversely related to gastric cancer risk, and somewhat higher than that seen in Linxian (6.4 μg/dL; ref. 28), where intervention trials showed reduced gastric cancer incidence following nutrient supplementation with a combination of β-carotene, selenium, and vitamin E. SCCS β-carotene levels were significantly lower than those found at baseline in the European Prospective Investigation into Cancer and Nutrition (median, 19.0 μg/dL; ref. 26), and in a Colombian vitamin supplementation trial (baseline presupplementation median, 23.9 μg/dL; ref. 29). Our findings suggest that beyond supplement intervention trials, there are opportunities for both improving nutritional status and reducing gastric cancer incidence in high-risk populations by directly dealing with the potential underlying cause—that of H. pylori infection. Methods to eliminate endemic H. pylori infection could include vaccine development and eradication schemes that are aided by vitamin supplementation.

The low-income population represented by the SCCS thus seems to have low circulating levels of antioxidants, particularly β-carotene, and a high prevalence of H. pylori infection, especially the virulent CagA+ H. pylori strains, and our data suggest that this association may be due to the ability of H. pylori to impair the absorption of nutrients. The presence of this combination of risk factors for gastric cancer also suggests that this population is a high-risk group, with a need for future studies to further explore the role of H. pylori infection on nutrition and gastric cancer risk as the cohort is followed in the coming years.

No potential conflicts of interest were disclosed.

This research was supported by the National Cancer Institute (Grants R01 CA092447 and P30 CA68485) and American Cancer Society—Institutional Research Grant to Vanderbilt University (Grant ACS-IRG-58-009-50).

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