Effect of physiological doses of oral vitamin B₁₂ on plasma homocysteine – A randomized, placebo-controlled, double-blind trial in India

Abstract

Background:

Vitamin B₁₂ (B₁₂) deficiency is common in Indians and a major contributor to hyperhomocysteinemia, which may influence fetal growth, risk of type 2 diabetes and cardiovascular disease.

Objective:

To study the effect of physiological doses of B₁₂ and folic acid on plasma total homocysteine (tHcy).

Design:

A cluster randomized, placebo-controlled, double-blind, 2x3 factorial trial, using the family as the randomization unit. Vitamin B₁₂ was given as 2 or 10 μg capsules, with or without 200 μg folic acid, forming six groups (B₀F₀, B₂F₀, B₁₀F₀, B₀F₂₀₀, B₂F₂₀₀, B₁₀F₂₀₀). Plasma tHcy was measured before and after 4 and 12 mo of supplementation.

Results:

Three hundred individuals from 119 families in the Pune Maternal Nutrition Study were randomised. There was no interaction between B₁₂ and folic acid (P=0.14) in relation to tHcy change and their effects were analyzed separately: B₀ vs. B₂ vs. B₁₀; and F₀ vs. F₂₀₀. At 12 mo, tHcy fell by a mean 5.9 (95% CI: −7.8, −4.1) μmol/L in B₂, and by 7.1 (95% CI: −8.9, −5.4) μmol/L in B₁₀, compared to non-significant rise of 1.2 (95% CI: −0.5, 2.9) μmol/L in B₀. B₂ and B₁₀ did not differ significantly. In F₂₀₀, tHcy fell by 4.8 (95% CI: −6.3, −3.3) μmol/L compared to 2.8 (95% CI: −4.3, −1.2) μmol/L in F₀.

Conclusion:

Daily oral supplementation with physiological doses of B₁₂ is an effective community intervention to reduce tHcy. Folic acid (200 μg/d) showed no additional benefit, neither had any unfavourable effects.

INTRODUCTION

Hyperhomocysteinemia is a risk factor for cardiovascular disease (CVD) (Wald et al., 2002), psychiatric disorders (dementia and Alzheimer's disease) (Smith, 2008), and in pregnancy for adverse outcomes including early pregnancy loss, birth defects and low birth weight (LBW) (Vollset et al., 2000; Selhub, 2008). Low vitamin B₁₂(B₁₂) status and hyperhomocysteinemia are common among Indians living in India (Refsum et al., 2001; Yajnik et al., 2006), and those migrated abroad (Chambers et al., 2000; Chandalia et al., 2003). This is largely due to B₁₂ deficiency, even with normal folate status, reflecting vegetarian food habits. In recent years, this has been particularly well documented from Pune, India (Refsum et al., 2001; Yajnik et al., 2006 and 2008). In the Pune Maternal Nutrition Study (PMNS) maternal hyperhomocysteinemia predicted LBW (Yajnik et al., 2005), and neurocognitive impairment in children (Bhate et al., 2008), and low maternal B₁₂ with high erythrocyte folate predicted higher adiposity and higher insulin resistance (IR) in the offspring (Yajnik et al., 2008). Based on these results we propose that B₁₂ supplementation in women of child bearing age may be a simple and effective mass measure to lower the incidence of LBW, adiposity and insulin resistance and thus of T2D and CVD, and also improve neurocognitive function of the children.

In an earlier ‘proof of concept’ trial (Yajnik et al., 2007), we demonstrated that high dose oral B₁₂ supplementation (500 μg alternate d, for 6 wk) reduced circulating tHcy concentrations. We report results of a randomized, placebo-controlled trial of B₁₂ supplementation on plasma homocysteine, using physiological doses over 12 mo.

METHODS

Participants

The participants were families from an ‘extended’ cohort of the PMNS. The PMNS methodology has been reported in detail by Rao in 2001 (Rao et al., 2001). In brief, 2675 married women of childbearing age, living in 6 rural villages near Pune city were recruited, and those who became pregnant were followed up. After the main study, we enrolled an additional 153 pregnant women from the same recruited sample, in order to study the early fetal growth. They did not contribute to the main study, and nutritional and blood measurements were not available during pregnancy. Of these, 119 families remain in follow-up, and the child and parents (349 individuals) were invited to take part in the current study.

The study was approved by the KEM Hospital Ethics Committee. Exclusion criteria were: unwillingness to participate, pregnancy, anemia (hemoglobin (Hb) <9 g/dL), already taking supplements containing iron, folic acid and/or B₁₂ for 10 or more days, or on treatment with drugs known to impair the absorption or utilization of folic acid or B₁₂ (e.g. phenytoin, antacids). We obtained informed written consent from the parents and informed written assent from the children (mean age 9 y).

For blood collection (June to November 2006) the families were brought to the Research Centre, the evening before the study. A standard vegetarian dinner was provided, after which they rested. A fasting blood sample was collected in the morning.

Study design and intervention

The trial was double blinded. We planned to test three levels of B₁₂ supplementation (none, 2 and 10 μg) and each of these at two levels of folic acid supplementation (none and 200 μg), forming 6 groups (A = B₀F₀, B = B₂F₀, C = B₁₀F₀, D = B₀F₂₀₀, E = B₂F₂₀₀, F = B₁₀F₂₀₀) (Figure 1). Randomization was computer-based. The unit of randomization was the family, making it a cluster randomized trial. We stratified the families by the children's baseline plasma B₁₂ concentrations; those below and above the median value were equally distributed in six groups. Within each group, the statistician randomly allocated codes (A to F) to the participant families. The contents of the capsules were known only to the pharmacist until the end of the trial. The codes were revealed only after data analysis. The study capsules were manufactured in six different colors. The supplements were dispensed monthly in containers labeled with the participant's name and capsule group (A through F). All members of the family received the same colored capsules. The participants were advised to take one capsule orally, daily before breakfast. The number of dispensed capsules and those returned at each monthly home visit were counted to calculate the compliance. At each monthly home visit we recorded adverse events and treatment of intercurrent illnesses, if any. Participants who took medicine containing folic acid and/or B₁₂ for more than 10 days were omitted from data analysis. The duration of supplementation was 12 mo, and took place between April 2007 and March 2008. Laboratory analysis of the study medication at the beginning and end of the study period revealed similar potency of the capsules.

FIGURE 1.

Participant flow and follow up

Measurements

Blood samples were collected at baseline and 4 and 12 mo after supplementation and were measured in separate batches. The samples were collected in EDTA tubes, kept on ice and spun within one h (2500 g × 30 min) and plasma aliquots were stored (−70°C) until further analysis. Hemoglobin was measured within one hour of blood collection on a Beckman Coulter analyser (A^C.T diff ™, Miami, Florida). Plasma creatinine was measured on an Alcyon 300 automated analyser (Abbott Laboratories, Abbott Park, IL, USA) using Jaffe's method. Plasma B₁₂and folate were measured by microbiological assay using a colistin sulfate-resistant strain of L. leichmanii (Kelleher et al., 1987, ​1991) and a chloramphenicol-resistant strain of L. casei (Horne and Patterson, 1988; Tamura et al., 1990), with inter-batch CV <8% and <7% respectively. Plasma tHcy was measured by fluorescence polarization immunoassay (Abbott, IL, USA; CV <8%) (Shipchandler and Moore, 1995).

Height was measured to the nearest 0.1 cm using a wall-mounted stadiometer (CMS Instruments, London, UK), and body weight to the nearest 0.005 kg (Conveigh, Electronic Instruments Ltd., Mumbai, India). Dietary intake of B₁₂ and folate rich foods was recorded by food frequency questionnaire in children at the beginning and end of 12 mo.

Definitions

Compliance was defined as taking ≥ 80% of the dispensed capsules, hyperhomocysteinemia as plasma tHcy concentrations >15 μmol/L (in adults), >10 μmol/L (in children) (Refsum et al., 2004), and B₁₂ and folate deficiency as concentrations <150 pmol/L (Refsum et al., 2001) and <7 nmol/L (Clarke et al., 2004) respectively.

Statistical methods

The data are presented as mean and standard deviation (SD). Though B₁₂ and tHcy concentrations were not normally distributed, we used parametric tests for differences between group means, which are normally distributed as per the central limit theorem. We used ANOVA to test the differences between supplementation groups, adjusting for the cluster design. There was no difference between clustered and un-clustered analysis (statistically insignificant intraclass correlation). The change in the prevalence of hyperhomocysteinemia from baseline was tested by McNemar's test.

The change in plasma tHcy concentrations was adjusted for the baseline tHcy concentrations, age and gender. There was no significant interaction between the effects of B₁₂ and folic acid supplementation on change in plasma tHcy concentration (P=0.14; Figure 1 in supplementary information). Therefore, the effects of 2 μg and 10 μg B₁₂ supplementation were tested against no B₁₂, combining the folic acid supplementation groups (B₀= B₀F₀, B₀F_200; B₂= B₂F₀, B₂F₂₀₀; B₁₀= B₁₀F_0, B₁₀F₂₀₀) and the effect of 200 μg folic acid was tested against no folic acid by combining the B₁₂ supplementation groups (F₀= B₀F₀, B₂F₀, B₁₀F₀; F₂₀₀= B₀F₂₀₀, B₂F_200, B₁₀F₂₀₀). The relative benefit for hyperhomocysteinemia was calculated by taking ratios of absolute benefit in different supplementation groups against non-supplemented group. The number needed to treat (NNT) was calculated as the reciprocal of the absolute risk reduction (ARR) for hyperhomocysteinemia at the end of 12 mo. All analyses were done using STATA, version 7.0 (STATA Inc. College Station, Texas, USA).

RESULTS

Recruitment and Participant flow (Figure 1)

Of the 119 families (349 individuals) in the extended PMNS cohort, 307 individuals were willing to participate (88% response), seven (1 pregnant, 6 anemic) were excluded and 300 (106 children, 93 fathers and 101 mothers) were randomized. During the intervention, 1 woman became pregnant and 5 participants received B₁₂ containing medication (from family physician) and were excluded from the analysis. Three participants withdrew and one was lost to follow up after collection of the 4 mo sample; they were analysed using the Last Observation Carried Forward (LOCF) method. Thus the final analysis includes 294 participants (106 children, 92 fathers and 96 mothers).

Baseline characteristics

Table 1 shows the basic characteristics of the 300 participants. Seventy two percent fathers, 48% mothers and 27% children were B₁₂ deficient, and 75% fathers, 35% mothers and 47% children were hyperhomocysteinemic. In contrast, only 14% fathers, 8% mothers and 2% children had folate deficiency. Baseline B₁₂, folate and tHcy concentrations were similar in the different supplementation groups.

TABLE 1.

Baseline parameters in children and parents

Physical and biochemical parameters	Children n= 106	Fathers n= 93	Mothers n= 101
Age (y)	9.0 (0.2)	36.8 (3.7)	30.4 (3.1)
Weight (kg)	21.9 (2.9)	59.2 (10.0)	47.9 (8.3)
Height (cm)	126.4 (5.4)	165.6 (7.0)	155.3 (5.4)
BMI (kg/m2)	13.7 (1.4)	21.6 (3.3)	20.4 (3.5)
< 18.5 kg/m2 (%)	Boys 42.61	21.5	37.6
> 25 kg/m2 (%)	Girls 55.81	17.2	13.9
Hemoglobin (g/dL)	12.5 (0.9)	14.3 (1.2)	12.2 (1.4)
Plasma creatinine (mg/dL)	0.6 (0.1)	0.9 (0.1)	0.8 (0.1)
Plasma B12 concentration (pmol/L)	203 (83)	130 (65)	161 (77)
Plasma vitamin B12 < 150 pmol/L (%)	26.7	72.2	48.4
Plasma folate concentration (nmol/L)	18.9 (6.3)	16.3 (5.5)	16.8 (6.8)
Plasma folate < 7 nmol/L (%)	1.9	14.4	8.3
Plasma tHcy concentration (μmol/L)	10.7 (3.8)	31.4 (22.6)	14.6 (7.8)
Plasma tHcy > 15 μmol/L (adults) and > 10 μmol/L (children) (%)	47	75.3	34.7

All values are mean (SD) unless specified.

¹% of children < −2 SD of age and gender specific BMI (WHO Reference population).

Compliance

Seventy one percent (n=210) participants returned <20% of the dispensed capsules over 12 mo and were defined as ‘compliers’. Fourteen percent participants consumed 70-80%, 6% consumed 60-70%, another 6% consumed 50-60% and remaining 3% consumed <50% of the dispensed dose. The mean plasma tHcy concentration, fall in plasma tHcy concentration and prevalence of hyperhomocysteinemia at 4 and 12 mo were similar in the compliers (n=210) and non-compliers (n=84) (Table 1 in supplementary information). Overall compliance rates were similar at 4 and 12 mo.

The frequency of consumption of folate and B₁₂ rich foods in children was similar at baseline and after 12 mo.

Plasma vitamin B₁₂ and folate concentrations (Table 2)

TABLE 2.

Mean concentrations of plasma vitamin B₁₂, folate and tHcy in the B₀, B₂, B₁₀, F₀ and F₂₀₀ groups at baseline, 4 and 12 mo

	B0 n=102	B2 n=94	B10 n=98	F0 n=143	F200 n=151
Mean compliance at 12 months (%)	82	84	87	86	82
Plasma B12 pmol/L
• Baseline	171 (76)	168 (85)	159 (83)	163 (84)	169 (79)
• 4 mo	181 (141)	267 (158)***	326 (158)***	267 (191)***	248 (131)***
• 12 mo	201 (69)***	242 (73)***	307 (119)***	252 (114)***	247 (84)***
Plasma folate nmol/L
• Baseline	13.9 (5.7)	12.6 (4.3)	13.5 (3.8)	13.2 (5.6)	13.5 (5.6)
• 4 mo	24.9 (15.6)***	24.6 (15.7)***	24.2 (14.3)***	15.5 (6.4)***	33.1 (16.1)***
• 12 mo	23.7 (15.2)***	20.2 (11.4)**	19.7 (11.5)	14.6 (6.3)***	27.8 (14.4)***
Plasma tHcy μmol/L
• Baseline	17.6 (15.3)	19.7 (19.0)	18.5 (14.3)	19.8 (17.3)	17.5 (15.1)
• 4 mo	18.5 (17.1)	14.2 (10.6)***	12.9 (9.4)***	17.2 (14.5)*	13.4 (11.3)***
• 12 mo	19.3 (16.8)	12.9 (7.9)***	11.6 (7.4)***	16.3 (13.7)**	13.1 (10.3)***

Values in mean (SD);

^*P < 0.05,

^**P < 0.01,

^***P < 0.001 different from baseline concentration

At baseline 48% participants were B₁₂ deficient. Plasma B₁₂ concentrations increased significantly in those who received B₁₂ supplements. At 12 mo the rise was 64% in those who received 2 μg (B₂) and 119% in those who received 10 μg (B₁₀). In both groups this was similar to the rise achieved by 4 mo. Plasma B₁₂ concentrations were higher in the B₁₀ compared to the B₂ group. After 12 mo of supplementation 6% of the B₂ and 2% of the B₁₀ group remained B₁₂ deficient. Participants who did not receive B₁₂ (B₀) also showed a rise in plasma B₁₂ concentration (33% above baseline) after 12 mo.

Plasma folate concentrations increased by 112% in those who received folic acid (F₂₀₀) and by 18.8% in the group who did not (F₀). At baseline 8% participants were folate deficient; after 12 mo this reduced to 0% in the supplemented and to 6% in the non-supplemented group.

Plasma total homocysteine (tHcy) concentration

B₁₂ supplementation (Figure 2, Table 2)

FIGURE 2.

Effect of 12 mo of supplementation with vitamin B₁₂ (B₀, B₂, B₁₀) on plasma tHcy

2a Change in tHcy (mean and 95% CI) over 12 mo in 3 groups (0 line indicates baseline tHcy concentration); 2b Proportion with hyperhomocysteinemia at baseline and at 12 mo in 3 groups

Plasma tHcy concentrations fell in the B₂ and B₁₀ groups, and showed little change in the B₀ group. The fall was greater in those with higher baseline concentrations (r = −0.6, P=0.000). We therefore adjusted the change in plasma tHcy concentrations for baseline concentrations. The change in plasma tHcy concentrations was not related to the baseline plasma B₁₂ and folate concentrations. The baseline-adjusted fall was 5.9 (95% CI: −7.8, −4.1) μmol/L in the B₂ group and 7.1 (95% CI: −8.9, −5.4) μmol/L in the B₁₀ group (not significantly different). The B₀ group showed a non-significant rise of 1.2 (95% CI: −0.5, 2.8) μmol/L. Eighty-two percent of the fall was achieved by 4 mo. After 12 mo, in the B₂ group the proportion of hyperhomocysteinemic participants fell from 52% to 39% (P=0.02), in the B₁₀ group from 56% to 21% (P<0.000), and in the B₀ group it increased from 44% to 56% (P=0.02).

Folic acid supplementation (Figure 3, Table 2)

FIGURE 3.

Effect of 12 mo of supplementation with folic acid (F₀, F₂₀₀) on plasma tHcy

3a Change in tHcy (mean and 95% CI) over 12 mo in 2 groups (0 line indicates baseline tHcy concentration), 3b Proportion with hyperhomocysteinemia at baseline and at 12 mo in 2 groups

The F₀ and F₂₀₀ groups showed similar fall in plasma tHcy concentration: F₀ 2.8 (95% CI: −4.3, −1.2) μmol/L and F₂₀₀ 4.8 (95% CI: −6.3, −3.3) μmol/L. In the F₀ group the proportion of hyperhomocysteinemic participants fell from 53% to 44%, P=0.07 and in the F₂₀₀ group from 48% to 34%, P=0.003.

Table 3 shows the number of hyperhomocysteinemic individuals in different supplementation groups who became normohomocysteinemic (‘responded’) or remained hyperhomocysteinemic (‘not responded’) after 12 mo. The relative benefit of supplementation was similar in the two B₁₂ supplemented groups (B₂ and B₁₀), but was higher in the B₁₀ compared to the F₂₀₀ group. The number needed to treat (NNT) was 4 for B₂, 2 for B₁₀ and 10 for F₂₀₀ group.

TABLE 3.

Relative benefit and number needed to treat (NNT) for hyperhomocysteinemia in different supplementation groups after 12 mo

Groups	Responded (n)	Not responded (n)	Relative benefit (95% CI)	Number needed to treat (NNT)
B0	6	39
B2	18	29	2.87 (1.25, 6.58)	4
B10	39	16	5.32 (2.48, 11.4)	2
F0	29	47
F200	34	37	1.25 (0.86, 1.82)	10

(Responded = number of hyperhomocysteinemic participants who became normohomocysteinemic, Not responded = number of hyperhomocysteinemic participants who remained hyperhomocysteinemic at the end of trial).

Side effects

There were 62 responses from 46 participants during the study period. One woman reported an accidental injury requiring hospital admission, which was not attributable to supplementation. Other responses were classified into positive (increased appetite, weight gain, sense of wellbeing; n=40) and negative (abdominal pain and acidity, feeling unwell; n=22). There was no obvious clustering of side effects in any particular intervention group.

DISCUSSION

This is the first community-based randomized trial of B₁₂ supplementation in an Indian population with substantial B₁₂ deficiency due to low dietary intake. We found that both 2 and 10 μg/d of oral B₁₂ (cyanocobalamin) significantly reduced plasma tHcy concentrations in otherwise healthy, free-living, rural participants. Eighty two percent of the effect was achieved by 4 mo. Overall, the two doses of B₁₂ were similarly effective in reducing plasma tHcy concentrations. Folic acid by itself had no additional effect on plasma tHcy reduction, over placebo or in combination with B₁₂ (Table 2 in supplementary information).

The relatively large effect of such small doses of B₁₂ is probably related to the high prevalence of vitamin B₁₂ deficiency and hyperhomocysteinemia in this population (Refsum et al., 2001; Yajnik et al., 2006, 2008). Without B₁₂ supplementation, hyperhomocysteinemia increased by 12% over the 12 mo period. Using the cut-point of 15 μmol/L (adults) and 10 μmol/L (children), we found there was a 13% fall in the hyperhomocysteinemia with 2 μg/d and 35% fall with 10 μg/d of B₁₂ from the baseline. The large effect of supplementation was also evident in the small numbers needed to treat: only 4 hyperhomocysteinemic individuals needed to be treated with 2 μg/d of B₁₂ for 12 mo for 1 to become normohomocysteinemic, and only 2 with 10 μg/d B₁₂. If the relationship of maternal B₁₂ deficiency and hyperhomocysteinemia with fetal outcomes is causal vitamin B₁₂ intervention could translate into a substantial reduction in the incidence of low birth weight, diabetes and CVD in this community as well as improvement in cognitive function based on our previous findings (Yajnik et al., 2005, 2008; Bhate et al., 2008).

Although we knew that B₁₂ deficiency was common in this population (Refsum et al., 2001; Yajnik et al., 2006, 2008), we used a placebo to maintain the scientific rigor and included an arm with only folic acid to test comparative effects, especially in view of proposed food fortification in India. Despite doubling of plasma folate concentrations, folic acid by itself had no effect on circulating tHcy concentrations; neither did it enhance the effect of B₁₂ (Figure 1, supplementary information). This supports our contention that folate deficiency is not common in this population (Refsum et al., 2001; Yajnik et al., 2006, 2008). Although this dose of folic acid was not associated with any adverse effects over 12 mo, the proposal for folic acid fortification in India for prevention of first occurrence neural tube defects (The Flour Fortification Initiative, 2007) needs to be formally investigated, including co-fortification with B₁₂.

The effect of supplementation on plasma tHcy was unrelated to the compliance (Table 1 in supplementary information), perhaps because the overall compliance was good (72%). Another explanation is that the dose of B₁₂ over 4 and 12 mo was more than necessary to achieve the effect. This observation is reassuring for future public health interventions.

Major strengths of our study are that it was community-based and included apparently healthy children and adults, rather than being targeted at high-risk groups or patients. The participation rate was high and compliance was maintained at high levels throughout the 12 mo. The factorial design allowed us to look at independent effects of B₁₂ and folic acid in comparison to their combination and the placebo. We used physiological rather than pharmacological doses of vitamins, with a view to translate our findings into future public health programs. The difficulty in obtaining specially manufactured capsules led to a five months gap between the baseline data collection and commencement of the intervention. However this was distributed similarly in different groups and therefore should not affect our results.

The striking reduction in plasma tHcy concentrations with small doses of 2 and 10 μg of B₁₂ merits discussion. In a recent study we have demonstrated that 3 doses of 2 μg of B₁₂ at 6 hr intervals not only raised plasma B₁₂ concentrations but also caused a significant (though small) fall in tHcy concentrations within 24 hr of the first dose (Bhat et al., 2009). This is perhaps a reflection of a B₁₂ deficient state and high baseline plasma tHcy concentrations. The almost similar effect of 2 and 10 μg doses is perhaps related to characteristics of intestinal B₁₂ absorption, which is predominantly by an active (intrinsic factor-mediated) mechanism which saturates after a 1.5 to 2 μg dose (Carmel, 2008). Only about 1% of absorption is by passive absorption (by diffusion).

In addition to these considerations the duration of supplementation is also an important determinant of the effect. Small doses over a long time might be equally effective as a large dose over a short time (Carmel, 2008). Our previous study (in vegetarian women) used a large dose of oral B₁₂ (500 μg every alternate day for 6 wk) (Yajnik et al., 2007). In 2 wk (total dose 3 mg B₁₂) plasma tHcy concentrations fell from 18.0 to 13.0 μmol/L, which remained static over the next 4 wk (total dose 9 mg B₁₂). In the present study 0.72 mg of B₁₂ (2 μg/d × 12 mo) achieved a similar effect, 82% of which was achieved by 4 mo with 0.24 mg B₁₂.

The majority of published studies of B vitamin supplementation have been in predominantly non-vegetarian western populations, in whom folate deficiency is the main determinant of hyperhomocysteinemia (Selhub, 2008). After folic acid fortification of foods in these populations, the attention has now shifted towards B₁₂ deficient groups like the elderly, in whom B₁₂ deficiency is thought to be due to ‘atrophic gastritis’, rather than dietary deficiency. This causes food cobalamin malabsorption, which could require large doses of B₁₂ to be effective (Eussen et al., 2005), although recent studies have shown efficacy with smaller doses (Bor et al., 2006; Blacher et al., 2007) as well as foods fortified with folic acid, B₁₂ and/or B₆ in the elderly (Tucker et al., 2004; Dhonukshe-Rutten et al., 2005; van Vliet et al., 2007; Winkles et al., 2008).

Our trial can be considered a public health scale ‘proof of principle’ study, following on from a high-dose, short-term intervention we reported in a small group of volunteers (Yajnik et al., 2007). The two studies have demonstrated an unequivocal role for B₁₂ deficiency as contributing to hyperhomocysteinemia in our population. It is of interest that our interventions have not reduced the plasma tHcy concentrations to those in age-matched Europids, suggesting that other factors also contribute to hyperhomocysteinemia in this population. Such factors may be protein malnutrition (Ingenbleek et al., 2002), low methionine intake (Elshorbagy et al., 2009), or deficiency of riboflavin (Hustad et al., 2000) or pyridoxine (Selhub J, 1999). However, it is rewarding that we were able to shift the distribution of plasma homocysteine to more favorable concentrations and this might contribute to a better risk reduction in the population than concentrating on the relatively smaller number with hyperhomocysteinemia (Rose G, 1985). There is scope for further investigation to find the etiology of the residual hyperhomocysteinemia, including the role of “tropical sprue-like” conditions.

In the meanwhile, public health specialists may build on our results and plan large-scale community based strategies to improve B₁₂ nutrition of Indians at different stages of the life-cycle. Of particular relevance will be to include B₁₂ along with folic acid in the National Nutritional Anemia Control Program or in the proposed food-fortification.