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Objective: To explore the association between vitamin B12 (B12)-containing supplement use, low B12 concentrations and biochemically defined B12 deficiency in US adults. Design: A cross-sectional study with adjustment for survey design. Prevalence ratios for two age groups (18-50 and >50 years) were estimated using unconditional logistic models. Outcome measures included prevalence of low serum B12 concentration (<148 pmol/l) and biochemical B12 deficiency (serum B12<148 pmol/l with concomitant homocysteine<10 μmol/l). Setting: A population survey of health and nutritional measures. Subjects: Subjects were non-institutionalized adults, aged 18 years and older, who participated in Phase 2 of NHANES III (Third National Health and Nutrition Examination Survey). Results: Low B12 concentrations were less prevalent among persons consuming B12-containing supplements (P=0·001) with an adjusted prevalence ratio of 0·6 (95% CI 0·3, 1·0). Biochemical B12 deficiency showed a similar trend (P=0·0002), with an adjusted prevalence ratio of 0·3 (95% CI 0·1, 0·8). Prevalence ratios were similar in adults <50 years of age, although the prevalence of low B12 and biochemical deficiency was proportionally higher. Conclusions: Consumption of B12-containing supplements was associated with at least 50% lower prevalence of both low serum B12 and biochemical B12 deficiency in a nationally representative sample of US adults, suggesting increased consumption of B12 from supplements or from fortified foods may reduce the prevalence of B12 deficiency. Additionally, the current Recommended Daily Allowance for B12 of 2·4μg may be insufficient for those aged .50 years. [ABSTRACT FROM AUTHOR]