A nested case-control study of methemoglobinemia risk factors in children of Transylvania, Romania.

In this nested case-control study, we investigated the risk factors for methemoglobinemia (MHG) in 71 children in the Transylvania region of Romania. This study was unique in that the exposures for cases and controls were calculated as continuous values and were reported in milligrams per kilogram per day of nitrate/nitrite based on careful dietary reconstruction and environmental sampling. This procedure allowed us to compare point estimates of nitrate/nitrite exposure with other continuous, categoric, and ranked risk factors such as the presence or absence of diarrheal disease, reported severity of diarrheal disease, the use of vitamin supplements, the presence, absence, and/or duration of breast-feeding, and whether or not first-generation relatives experienced MHG. Analysis of these factors and exposure levels using both univariate and multivariate whole-model tests was performed to understand the relative significance of risk factors at varying levels of exposure to the development of MHG. Univariate and multifactorial analysis of risk factors for MHG underscored that, for this population, MHG is most strongly associated with nitrate/nitrite exposure through the dietary route (p = 0.0318), via feeding of formula and tea made with water containing high levels of nitrates, and that breast-feeding protects infants younger than 6 months of age (p = 0.0244). Our findings also raise questions about the role of diarrheal disease in the development of MHG, as likelihood ratios (likelihood 4.323, p = 0.0376) and multifactorial analysis indicated a significant role for diarrheal disease for some individuals.

Exposure methodology and findings for dietary nitrate exposures in children of Transylvania, Romania.

AIMS: The primary focus of this exposure assessment work involved developing an exposure model and determining a numerical point estimate of the amount of biologically relevant nitrate/nitrite exposure that occurred for each child in the study. This assessment was done in support of two epidemiological studies. The first study was an epidemiological cohort study (three cohorts based on nitrate/nitrite exposure) that explored the relationship between high nitrate/nitrite exposure and neuropsychological development. The second study was a nested case/control study (cases of methemoglobinemia versus disease-free controls) that sought to explore the relationship between MHG and various risk factors for the disease. METHODS: This work uses both dietary survey and environmental sampling and modeling in order to develop two point estimates of nitrate exposure in milligrams per kilogram per day of nitrite (the biologically active form of the hemoglobin-oxidizing agent) for the first 6 months of the child's life (2-months-of-age and 6-months-of-age point estimates). Methodologies included proxy interviews of primary caregivers, review of existing medical and environmental sampling and analysis. RESULTS: Exposure to nitrate--nitrogen (with final calculations converted to the biologically active form of the toxin, nitrite) was categorized as high, medium, and low as determined from the distribution of the data derived from final exposure calculations at both the 2-months-of-age point estimate and at the 6-months-of-age point estimate. These tertiles correspond to greater-than-or-equal1.5 mg/kg/day nitrite-nitrogen for high-exposure individuals, <1.5-> or = 0.1 mg/kg/day for medium-exposure individuals, and <0.1 mg/kg/day for low-exposure individuals. Analyses illustrate that over 90% of the nitrate exposure occurred through the consumption of liquids (water) at the 2-months-of-age point estimate while at the 6-months-of-age point estimate, a 10-fold change in the amount of solid consumables occurred. CONCLUSIONS: Final exposure calculations were well differentiated into three tertiles based on a point estimate of average daily intake of nitrite in milligrams per kilogram body weight per day at roughly 2 and 6 months of age. These categories of exposure, based on the exposure model point estimate, correspond well with the exposure estimates as estimated only on the basis of cohort status and their corresponding nitrate/nitrite well water levels. Comparisons of these two sets of data illustrate that following the MHG incident, Cohort II shifted places with Cohort I to become the high-exposure cohort. Further, the predictive ability of the exposure assessment in regard to the outcome of MHG was estimated using a Likelihood Ratio and Pearson's Crosstab analysis. This was performed on the 2-month-of-age point estimate. Likelihood Ratio and Pearson's chi-square were 39.40 and 33.74, respectively, with a probability of achieving these fits by chance alone of <0.0001. This indicates clearly that the children who experienced MHG were also the children at the 2-month-of-age point estimate who had received the highest exposure to nitrate/nitrite through their diet.