Association between vitamin B₁₂-containing supplement consumption and prevalence of biochemically defined B₁₂ deficiency in adults in NHANES III (Third National Health and Nutrition Examination Survey)

Abstract

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 2·4 μg may be insufficient for those aged >50 years.

The Institute of Medicine (IOM) extensively reviewed the available data on vitamin B₁₂ (B₁₂) deficiency and requirements, and has identified biochemical B₁₂ deficiency and effective methods to reduce risk of B₁₂ deficiency as high priorities for research(1). The Recommended Daily Allowance (RDA) was set at 120% of the daily requirement and is expected to protect 97–98% of healthy persons from deficiency. Based on the lower bioavailability of food-bound B₁₂ in older individuals, the IOM recommended adults over the age of 50 years meet the RDA for B₁₂ of 2·4 μg by consuming synthetic vitamin B₁₂ in B₁₂-fortified foods or B₁₂ supplements(1). Previously, three population-based studies examined biochemical indicators of B₁₂ deficiency (serum B₁₂ and/or homocysteine and methylmalonic acid (MMA) concentrations) in relation to vitamin supplement status and concluded that B₁₂-containing supplements may protect against B₁₂ deficiency(2-4). However, all three studies used data from elderly, racially homogeneous cohorts with relatively small sample sizes, limiting the generalizability of their findings to the US adult population. Therefore, we used data from NHANES III (Third National Health and Nutrition Examination Survey) to further explore the relationship between B₁₂ supplement use and biochemically defined B₁₂ deficiency in a large, nationally representative population sample of the US adult population.

Methods

Subjects

NHANES III was conducted with a complex, multistage probability design(5). Persons older than 60 years as well as African-American and Mexican-Americans were over-sampled to more precisely assess US health and nutritional measures. Importantly, NHANES III was the first NHANES survey without an upper age limit, thus improving the estimates of health and nutrition in older segments of the population. The survey design was approved by the National Center for Health Statistics institutional review board (IRB) and participants gave written informed consent prior to undergoing survey procedures(5); the present analysis was thus deemed exempt from review by the Emory University IRB. The survey was divided into two phases (Phase 1, 1988–91 and Phase 2, 1991–4), designed to allow individual or combined phase analysis. Because homocysteine concentrations were measured only in Phase 2, the current analysis focuses on data from non-pregnant adults (age 18 years and older) in Phase 2 (September 1991–October 1994) for whom supplement use information and laboratory analyses of serum B₁₂ and homocysteine are available (n 8394). Laboratory measurements, including serum B₁₂ and total serum homocysteine concentrations, were collected and measured in accordance with NHANES III procedures(5-7).

Per NHANES protocol, persons who were institutionalized, had haemophilia or had undergone chemotherapy for cancer within the previous four weeks were excluded from phlebotomy and thus from the present analysis. Serum B₁₂ concentrations were analysed with a commercially available radioprotein binding assay kit (Quantaphase II from Bio-Rad Laboratories, Hercules, CA, USA; during NHANES III, Phase 2). EDTA-treated plasma was not available from NHANES III and serum homocysteine concentration was measured by HPLC with fluorometric detection. Further survey procedure details, including how demographic, medical history and medication and supplement intakes were collected, may be found in the NHANES III plan and operation(5) and in the data documentation on the NHANES website (http://www.cdc.gov/nchs/nhanes.htm). In addition, pregnant females (n 167) and persons who reported taking antiretroviral prescription medication for HIV (n 7) were excluded because B₁₂ nutriture is altered in these populations(8,9).

Analysis

There is no gold standard test or definition for B₁₂ deficiency. We used a conservative serum B₁₂ cut-off point of <148 pmol/l (200 pg/ml)(10) to define low B₁₂ concentration. Subjects with serum B₁₂<148 pmol/l and concomitant serum homocysteine >10·0 μmol/l (1·35 μg/l) were defined as biochemically defined B₁₂-deficient.

The primary exposure variable of interest, B₁₂-containing supplement use, was ascertained by calculating the total (single dose) supplemental B₁₂ intake among supplement users from the NHANES III PUVITMIN and SUPLCONC data sets, which define supplement use in this cohort. Supplement users were defined as having total oral B₁₂ supplement intake >0 μg. Supplement dose categories were defined as: 0 μg (non-users), >0 to 6 μg (89% of whom ingested 6 μg), >6 to 25 μg (88% of whom ingested 9–25 μg) and >25 μg. The categories were chosen such that the lowest category of use included the dose found in many over-the-counter multivitamin supplements (6 μg). The second category of supplement users includes the 25 μg amount found in many senior daily multivitamin supplements, and the highest category included the higher doses (100, 500, 1000 μg) found in high-dose B-vitamin supplements.

Predictors of B₁₂ and/or homocysteine concentrations which could potentially confound the analysis include age, race, impaired renal function, smoking(11), heavy alcohol intake(12,13),hypothyroidism(14) and folate deficiency. Whites tend to have lower serum B₁₂ concentrations than blacks or Mexican-Americans(15) and Mexican-American Hispanics tend to have lower homocysteine concentrations than non-Hispanic whites or non-Hispanic blacks(16). Except where noted, potential confounding exposure variables were dichotomized for the univariate analysis and multivariate logistic regression modelling.

After examining the distribution of cases within quartiles of age and ensuring adequate sample size, age was classified as a dichotomous variable to match the age recommendations discussed in the IOM report (18–50 years and >50 years)(1), and included as a continuous variable in the logistic regression models. Those participants who reported being current smokers of cigarettes, pipes or cigars were classified as current smokers. Study participants' average daily alcohol consumption was estimated from the NHANES survey alcohol consumption questions (number of drinks on drinking days×number of days per year drinking/365). If the number of days per year drinking was missing, the daily average was calculated by multiplying the number of drinks on drinking days by the number days per year with 9+ drinks, and then dividing by 365 days per year. Based on the US Department of Agriculture's dietary guidelines for alcohol (no more than one to two drinks of alcohol daily)(17,18) and the definition of heavy drinking given on the Centers for Disease Control and Prevention (CDC) alcohol fact sheet, participants whose calculated average daily alcohol consumption was three or more drinks were classified as ‘heavy drinkers’(17,18).

Due to possible alteration in B₁₂ and/or homocysteine status in renal impairment, serum creatinine was included as a continuous variable in the multivariate analysis. Participants with thyroid-stimulating hormone concentration greater than 5 μIU/l were considered ‘hypothyroid’ for the present analysis(19). Race was classified into three groups, white, black and other, based on NHANES analysis guidelines and available crude sample size(20). Folate is a major determinant of homocysteine concentrations(7,21,22), and folate deficiency was therefore defined as red blood cell (RBC) folate <232 nmol/l (102·6 ng/ml; in home-examined participants, for whom RBC folate analysis was not done, serum folate <13·3 nmol/l (5·89 ng/ml))(6,23).

In all analyses, α = 0·05 was considered significant and 95% confidence intervals are reported. To account for the complex survey design, SURVEY procedures in the SAS statistical software package version 9·1 (SAS Institute Inc., Cary, NC, USA) were used to estimate the population prevalence and prevalence ratio estimates. To calculate crude prevalence ratios, PROCSURVEY FREQ outcome prevalence estimates from the exposed (supplemented participants) are divided by the prevalence estimates in the unexposed (non-supplemented participants).

Unconditional logistic regression techniques were used to evaluate the univariate frequencies of each potential exposure variable and measures of association were computed, adjusting for supplement intake. Those exposures variables which were significantly related to the outcome variable were then included in a multivariate logistic regression model. Exposure variables were then removed from the model by backwards elimination and those exposures which altered the prevalence odds ratio by more than 10% were kept in the model. The prevalence of biochemical B₁₂ deficiency in the NHANES sample meets the rare disease assumption, and adjusted prevalence ratios are approximated from the multivariate logistic regression odds ratios using the method described by Zhang and Yu(24).

Results

Prevalence of biochemical B₁₂ deficiency

Applying the above criteria yielded a crude sample size of 8394 for the analysis of low B₁₂ and 7404 for the analysis of biochemical B₁₂ deficiency (Table 1). The overall estimated US adult population prevalence of biochemically defined B₁₂ deficiency was 1·6%. The prevalence was 1·2% for those aged 18–50 years and 2·5% for those aged >50 years. The prevalence of low serum B₁₂ concentrations was 3·2% for all adults, 2·6% for adults aged 18–50 years and 4·4% for those aged >50 years (Tables 1, 2 and 3).

Table 1.

Prevalence and unadjusted and adjusted prevalence ratio of low serum vitamin B₁₂ concentration and biochemically defined vitamin B₁₂ deficiency by B₁₂ supplement dose for all persons aged 18 years and older in NHANES III (Third National Health and Nutrition Examination Survey; Phase 2, 1991–4)

	Low serum B12*					Biochemically defined B12 deficiency†
B12-containing supplement use	Prevalence (%)	95% CI	Unadj. PR	Adj. PR‡	Adj. 95% CI	Prevalence (%)	95% CI	Unadj. PR	Adj. PR§	95% CI
All adults (n 8394∥)	3·2	2·2, 4·2	–	–	–	1·6	1·1, 2·1	–	–	–
Supplement non-users (n 6192)	3·9	2·8, 5·0	(referent)	–	–	2·2	1·6, 2·7	(referent)	–	–
Supplement users
Any amount (n 2202)	1·7¶	0·7, 2·8	0·4	0·6	0·3, 1·0	0·4**	0·0, 0·9	0·2	0·3	0·1, 0·8
>0 to 6 μg (n 1377)	1·8	0·6, 3·0	0·5	0·6	0·3, 1·1	0·5	0·0, 1·2	0·2	0·3	0·1, 1·3
>6 to 25 μg (n 515)	2·0	0·0, 4·7	0·5	0·6	0·2, 2·0	0·5	0·0, 1·5	0·2	0·3	0·1, 1·6
>25 μg (n 310)	1·1	0·0, 3·0	0·3	0·4	0·1, 2·0	0·2	0·0, 0·6	0·1	0·0††	0·0, 1·2

Unadj., unadjusted; Adj., adjusted; PR, prevalence ratio.

The PROCSURVEY FREQ procedure in SAS version 9·1 (SAS Institute Inc., Cary, NC, USA) and the NHANES III, Phase 2 data sets were used to obtain these US population prevalence proportion estimates in adults aged 18 years and older.

^*Low serum B₁₂: serum B₁₂ <148 pmol/l.

^†Biochemically defined B₁₂ deficiency: serum B₁₂ <148 pmol/l with serum homocysteine >10 μmol/l.

^‡Adj. PR=prevalence ratio adjusted for age (continuous), gender, race (black race or other), folate deficiency status and education (<8 years v. >8 years).

^§Adj. PR=prevalence ratio adjusted for age (continuous), gender, race (black race or other), folate deficiency status, serum creatinine and education (<8 years v. >8 years).

^∥Nine hundred and seventy-two participants had missing homocysteine measurements, so estimates are based on 7404 survey participants for the bio-chemical B₁₂ deficiency analysis and on 8394 survey participants for the low B₁₂ analysis.

^¶Rao–Scott P value=0·001 compared with supplement non-users.

^**Rao–Scott P value=0·0002 compared with supplement non-users.

^††PR=0·02, rounded to 0·0.

Table 2.

Prevalence and unadjusted and adjusted prevalence ratio of low serum vitamin B₁₂ concentration and biochemically defined vitamin B₁₂ deficiency by B₁₂ supplement dose for adults aged 18–50 years in NHANES III (Third National Health and Nutrition Examination Survey; Phase 2, 1991–4)

	Low serum B12*					Biochemically defined B12 deficiency†
B12-containing supplement use	Prevalence (%)	95% CI	Unadj. PR	Adj. PR‡	Adj. 95% CI	Prevalence (%)	95% CI	Unadj. PR	Adj. PR§	95% CI
Adults aged 18–50 years (n 4944∥)	2·6	1·4, 3·8	–	–	–	1·2	0·5, 1·8	–	–	–
Supplement non-users (n 3768)	2·9	1·6, 4·3	(referent)	–	–	1·5	0·7, 2·3	(referent)	–	–
Supplement users
Any amount (n 1176)	1·9¶	0·5, 3·3	0·7	0·8	0·4, 1·7	0·4**	0·0, 1·1	0·3	0·5	0·1, 2·2
>0 to 6 μg (n 754)	1·8 (10 cases)	0·3, 3·2	0·6	0·8	0·4, 1·5	0·6 (2 cases)	0·0, 1·7	0·4	0·6	0·1, 2·9
>6 to 25 μg (n 261)	2·5 (3 cases)	0·0, 6·0	0·9	1·0	0·3, 3·3	0 (no cases)	n/a	n/a	<0·001	n/a
>25 μg (n 161)	1·6 (2 cases)	0·0, 4·3	0·6	0·7	0·1, 4·8	0·3 (1 case)	0·0, 0·9	0·2	0·3	0·0, 3·5

Unadj., unadjusted; Adj., adjusted; PR, prevalence ratio; n/a, not applicable.

The PROCSURVEY FREQ procedure in SAS version 9·1 (SAS Institute Inc., Cary, NC, USA) and the NHANES III, Phase 2 data sets were used to obtain these US population prevalence proportion estimates in adults aged 18–50 years.

^*Low serum B₁₂: serum B₁₂ <148 pmol/l.

^†Biochemically defined B₁₂ deficiency: serum B₁₂ <148 pmol/l with serum homocysteine >10 μmol/l.

^‡Adj. PR=prevalence ratio adjusted for age (continuous), gender, race (black race or other), folate deficiency status and education (<8 years v. >8 years).

^§Adj. PR=prevalence ratio adjusted for age (continuous), gender, race (black race or other), folate deficiency status, serum creatinine and education (<8 years v. >8 years).

^∥Estimates are based on 7404 survey participants for the biochemical B₁₂ deficiency analysis and on 8376 survey participants for the low B₁₂ analysis.

^¶Rao–Scott P value=0·2 compared with supplement non-users.

^**Rao–Scott P value=0·06 compared with supplement non-users.

Table 3.

Prevalence and unadjusted and adjusted prevalence ratio of low serum vitamin B₁₂ concentration and biochemically defined vitamin B₁₂ deficiency by B₁₂ supplement dose for adults aged >50 years in NHANES III (Third National Health and Nutrition Examination Survey; Phase 2, 1991–4)

	Low serum B12*					Biochemically defined B12 deficiency†
B12-containing supplement use	Prevalence (%)	95% CI	Unadj. PR	Adj. PR‡	Adj. 95% CI	Prevalence (%)	95% CI	Unadj. PR	Adj. PR‡	95% CI
Adults aged >50 years (n 3450)	4·4	3·1, 5·7	–	–	–	2·5	1·7, 3·4	–	–	–
Supplement non-users (n 2424)	5·9	4·1, 7·7	(referent)	–	–	3·7	2·5, 4·8	(referent)	–	–
Supplement users
Any amount (n 1026)	1·5§	0·0, 3·2	0·3	0·3	0·1, 1·7	0·4¶	0, 1·1	0·3	0·4	0·1, 1·7
>0 to 6 μg (n 623)	1·9 (5 cases)	0·0, 4·7	0·3	0·4	0·1, 1·6	0·1 (2 cases)	0·0, 0·3	0·2	0·5	0·1, 3·0
>6 to 25 μg (n 254)	1·2 (3 cases)	0·0, 3·4	0·2	0·3	0·0, 1·3	1·3 (2 cases)	0·0, 3·9	0·2	0·4	0·1, 2·4
>25 μg (n 149)	0·2 (1 case)	0·0, 0·5	0·0∥	0·0∥	0·0, 0·3	0·0** (no cases)	–	0·0** (no cases)	<0·001	–

Unadj., unadjusted; Adj., adjusted; PR, prevalence ratio.

The PROCSURVEY FREQ procedure in SAS version 9·1 (SAS Institute Inc., Cary, NC, USA) and the NHANES III, Phase 2 data sets were used to obtain these US population prevalence proportion estimates in adults aged >50 years.

^*Low serum B₁₂: serum B₁₂ <148 pmol/l.

^†Biochemically defined B₁₂ deficiency: serum B₁₂ <148 pmol/l with serum homocysteine >10 μmol/l.

^‡Adj. PR=prevalence ratio adjusted for age (continuous) gender, race (black race or other) and folate deficiency status.

^§Rao–Scott P value=0·001 compared with supplement non-users.

^∥Rao–Scott P value=0·0002 compared with supplement non-users.

^¶Point estimate for PR was 0·03, rounded to 0·0.

^**There were no cases of biochemical B₁₂ deficiency in the 149 survey participants over age 50 who were taking >25 μg of supplemental B₁₂.

B₁₂-containing supplement consumption and prevalence ratios

Supplement consumption was associated with lower prevalence of both low serum B₁₂ concentration and biochemically determined B₁₂ deficiency in the total population and in both age categories (18–50 and >50 years, Tables 1-3). Among the over-50 s taking >0–6 μg or >6–25 μg, supplements reduced the occurrence of both outcomes by 60–70%. The prevalence was even lower among those consuming >25 μg.

Among all B₁₂-supplement users, the distribution of total supplemental intakes was skewed, with a mean of 31·4 μg/d and a median of 6·0 μg/d (range 0·19–2075 μg/d). Of participants who consumed B₁₂-containing supplements, 99·8% consumed at least 2·4 μg B₁₂/d (the RDA suggested by the IOM) and 80·7% consumed 6–25 μg B₁₂/d (the amounts most commonly found in multivitamin supplements). Of supplement users in the second supplement dose category, >0 to 6 μg, most (89%) participants took 6 μg, the dose found in ‘regular’ (non-geriatric) multivitamin supplements. Only 2·1% of supplement users took large doses (250 μg or more) of supplemental B₁₂ and no cases of biochemical B₁₂ deficiency occurred in supplement users taking >75 μg of B₁₂ daily.

Univariate analysis and multivariate logistic regression modelling

Logistic regression analysis of each potentially confounding exposure variable (with adjustment for NHANES design and supplement status) revealed that age <50 years and 8 years or less of education were both significantly associated with biochemical B₁₂ deficiency. Odds ratio point estimates of biochemical B₁₂ deficiency were above unity for male gender, non-white or African-American race, as well as hypothyroid state, but none of these associations was significant. For each demographic exposure category (old and young age categories, gender, white and black races, and all education levels) B₁₂ supplement consumption was associated with reduced odds of biochemical B₁₂ deficiency.

After adjusting for survey design, 17·9% of persons had elevated serum creatinine. However, the prevalence of low serum B₁₂ concentration and biochemically defined B₁₂ deficiency in persons with and without elevated creatinine (>1·1 mg/dl for females, >1·4 mg/dl for males) was not significantly different (P = 0·5 for low B₁₂ and P = 0·09 for biochemically determined B₁₂ deficiency). Similarly, the prevalence of low B₁₂ in participants aged >50 years with normal creatinine (4·1%) was not significantly different from that in older participants with elevated creatinine (5·2%, P = 0·26).

Multivariate logistic regression model analysis revealed that hypothyroidism, heavy alcohol use and smoking status were not significant predictors of low serum B₁₂ or biochemical B₁₂ deficiency. Although lower educational level showed a statistically significant association with biochemical B₁₂ deficiency in the univariate analysis, removing it from the model did not change the prevalence ratio estimates. Similarly, removing serum creatinine concentration from the multivariate model did not affect the point estimates or confidence intervals.

Discussion

Consistent with our a priori hypothesis, we found that intake of supplements providing 6 or 25 μg of vitamin B₁₂ was associated with reductions in the prevalence of biochemical B₁₂ deficiency and low serum B₁₂ concentrations in US adults (aged both 18 years and older and above 50 years). The majority of supplement users met or exceeded the IOM RDA of B₁₂ intake (2·4 μg) because the supplemental B₁₂ quantities commonly found in over-the-counter multivitamin supplements range from 6 to 25 μg. Although we cannot conclude that there is a significant benefit to consuming 6 μg v. 2·4 μg supplement, or 25 μg v. 6 μg supplement, our data suggest that the prevalence of B₁₂ deficiency (defined as low serum B₁₂ or biochemical deficiency) among persons aged >50 years who consume B₁₂ supplements is half to two-thirds the prevalence in persons who do not consume B₁₂ supplements; the difference between prevalence for supplement users v. non-users in persons aged 18–50 years is about a third. The prevalence reduction was even higher in persons taking >25 μg supplemental B₁₂ daily.

Although there was overlap in the confidence intervals, the trend we observed of decreasing prevalence of low serum B₁₂ and biochemical B₁₂ deficiency with increasing B₁₂ supplement dose is consistent with results seen in vitamin supplement intervention trials in the elderly(25-28). In three of these studies, 10–500 μg of supplemental B₁₂ increased serum B₁₂ concentrations by 18·5–166 pmol/l (25–225 pg/ml)(25-27). Along with our findings, these studies indicate that daily doses of supplemental B₁₂ as low as 6 μg can shift the distribution of B₁₂ concentrations upwards in the general population and reduce biochemical B₁₂ deficiency prevalence.

About 2% of people aged >50 years will still have low B₁₂ concentrations despite consuming 6–25 μg of synthetic B₁₂. Daily consumption of more than 75 μg B₁₂ may nearly eliminate low B₁₂ and biochemical B₁₂ deficiency, but more research is needed to determine how much supplement is needed by those individuals for whom 6 or 25 μg/d does not prevent low B₁₂ or biochemical B₁₂ deficiency.

Given the success of a proof of concept study by Winkels et al.(29), our data suggest that a B₁₂ fortification programme that delivered 6 to 25 μg of synthetic vitamin B₁₂ to most of the population may be highly effective in decreasing the prevalence of both low B₁₂ concentrations and biochemically defined B₁₂ deficiency. Were such a programme implemented, there would be fewer patients with low serum B₁₂ and biochemically defined B₁₂ deficiency for clinicians to investigate. Several studies have reported that adults with suboptimal B₁₂ status have poorer cognitive functioning or could improve with supplementation(30-32). However, results from other B-vitamin supplement trials have failed to yield clinically measurable benefit(33,34). Further investigations are required to determine whether improving B₁₂ status with fortification would result in improved functional status or clinical benefit.

The prevalence of low B₁₂ in NHANES III (3·2%) was similar to that previously observed in NHANES III (3%)(7), but was lower than that observed in other cohort studies in the USA (5·3–14·5%)(2,3,35). However, those studies focused on racially homogeneous, elderly cohorts rather than the entire adult population, and they had relatively small sample sizes. Increased age and white race have both been associated with lower B₁₂ concentrations(15) and estimates from predominantly older, white cohorts may therefore overestimate the national prevalence of B₁₂ deficiency. The higher cut-off points for defining low B₁₂ (<221 pmol/l or 300 pg/ml) in three non-institutionalized adult populations(2,3,35) and two outpatient source populations(35,36) may also explain the lower prevalence of B₁₂ deficiency in our study population. Overall, our study supports the results of previous studies, but widens their generalizability for the US population.

We did not examine the limited physical examination data in NHANES III addressing whether persons with biochemically determined B₁₂ deficiency exhibited clinical signs of B₁₂ deficiency. Although 6 to 25 μg of supplemental B₁₂ appears to prevent most preclinical deficiency indicators in those aged 18 years and older, higher doses may be warranted in persons over 50 years old or with clinical signs such as anaemia and/or neuropathy(25). We remind clinicians that patients with clinical symptoms consistent with clinical B₁₂ deficiency disease should be worked up and treated with an appropriate dose of vitamin B₁₂.

The primary strength of our analysis is that it examines a nationally representative population sample of ambulatory persons in the USA. With the largest sample size reported to date (over 8000 participants), we were able to adjust prevalence ratio estimates for the most common potential confounding exposures, including age, race, educational level, renal insufficiency and folate status.

A potential weakness of our study is the absence of MMA data to use as a criterion for biochemical determination of B₁₂ deficiency. An elevated MMA concentration is typically considered a more specific confirmatory indicator of B₁₂ deficiency than elevated homocysteine. Because NHANES III MMA data are not publicly available, we chose a homocysteine cut-off that is within the range expected in B₁₂- and folate-replete individuals(23,37). Another potential weakness of our study is the incomplete exclusion of HIV-positive individuals(38). Given that low B₁₂ concentrations are reportedly less likely in HIV after antiretroviral therapy is started(8), those excluded were least likely to be cases. HIV patients use supplements at a higher rate (over 70%) than the general population and the participants not excluded would likely increase the number of exposed controls, potentially resulting in bias towards the null. However, at the estimated prevalence of HIV of 0·34%(38), unidentified HIV-positive individuals would have virtually no impact on the prevalence of B₁₂ deficiency and prevalence ratio estimates in our data.

In conclusion, consumption of supplements with 6 or 25 μg of vitamin B₁₂ was associated with about a 50% reduction in the prevalence of low B₁₂ concentrations and biochemically defined B₁₂ deficiency. These findings suggest that approximately three million cases of low serum B₁₂ and biochemical B₁₂ deficiency in the US population may be prevented if all adults consumed 6–25 μg B₁₂/d from supplements or fortified foods. We also found low vitamin B₁₂ concentrations and biochemically defined vitamin B₁₂ deficiency in 1–2% of persons aged >50 years who were consuming 6 to 25 μg of B₁₂ from supplements. This relatively high prevalence in older supplement users suggests that the current RDA of 2·4 μg/d may be too low for this age group. As Berry et al. noted, people who have low serum concentrations of B₁₂ may have problems in absorbing free B₁₂(39) and have the preclinical pernicious anaemia that Carmel et al. have identified among those over 50 years of age(10). Clinicians should be vigilant for subtle signs and symptoms of pernicious anaemia and clinical vitamin B₁₂ deficiency, particularly in older adults who have low concentration of serum B₁₂ and/or biochemically defined vitamin B₁₂ deficiency in spite of consuming vitamin B₁₂ supplements, and manage these patients appropriately.