The present study is to determine the beneficial effect of regular consumption of adequate milk on vitamin B-12 status in predominantly young vitamin B-12 deficient vegetarian Indians who had no folate deficiency. Milk was only found to be a significant contributor to vitamin B-12 intake in these participants.
Fifty seven percent of the participants had vitamin B-12 deficiency and 65% had hyperhomocysteinemia, 84% had low plasma holo-TC (<35 pmol/L) concentrations. However, none had any clinical (neurological/anemia) symptoms of vitamin B-12 deficiency. They could be termed as subjects with Sub Clinical Cobalamin deficiency. One half of the participants with normal vitamin B-12 status (plasma vitamin B-12 >148 pmol/L and tHcy <15 μ mol/L) had lower plasma holo-TC concentrations (median 27.7 pmol/L) than normal white Caucasians [5, 6, 29, 30]. The participants were consuming less than 1.4 μ g vitamin B-12 in daily diet. There was a significant association between dietary intake of vitamin B-12 and plasma vitamin B-12 and holo-TC concentrations which is in agreement with other studies [5, 7, 29].
Holo-TC has a short half-life [31, 32] and is therefore proposed to reflect recent vitamin B-12 absorption. Once in circulation, holo-TC is taken up into cells within minutes [33, 34]. Until the cells are saturated with holo-TC, most of it is taken up so quickly that no large changes are observed initially in blood. Significant changes in the plasma holo-TC concentrations can be measured when the intake of vitamin B-12 is sufficient to saturate the cells with vitamin B-12. A dose dependent Holo-TC rise during 24 hrs following vitamin B-12 administration suggested that the levels are influenced by recent absorption . Plasma holo-TC concentration significantly increased (0-120%, p = 0.0001) in vitamin B-12 deficient participants 24 hrs. following the milk load (Figure 2). Bhat et al  found a significant increase in plasma holo-TC concentration (0-150%) 24 hrs after loading with 6 μg of cyanocobalamin in vitamin B-12 deficient subjects while Bor et al  found an increase (0-108%) in normal subjects after 27 μg load. Bor et al  and Castel-Roberts et al  reported that measuring the rise in plasma holo-TC concentration 24 hrs after vitamin B-12 load is better to assess recent intestinal absorption than measuring plasma vitamin B-12 alone. One fourth of the participants showed <12.5% increase in holo-TC concentration after milk load. They may have mal-absorption or the ingested vitamin B-12 may have been used to saturate the cells. However, their plasma tHcy was reduced in vitamin B-12 deficient participants. These subjects need further evaluation for their gastrointestinal absorption status. Whereas, the participants with normal plasma B-12 concentration who did not show any increase in plasma holo-TC concentrations had basal level of >45 pmol/L with <12.5 μ mol/L of plasma tHcy which implies that plasma holo-TC concentration reaches a plateau when basal plasma holo-TC concentration exceeds 45 pmol/L in vitamin B-12 deficients, similar to the observation by Bhat et al  who reported that plasma Holo-TC concentration increased from 10.6 pmol/L to 24.9 in adult Indian males and 30.5 pmol/L in females after ingesting 6 μ g of cyanocobalamin and 7.7 to 45.7 in males and 9.7 to 47.5 pmol/L in females after 30 μ g of cyanocobalamin ingestion.
Regular consumption of milk for 14 days increased the circulating concentration of vitamin B-12, holo-TC and decreased tHcy concentration in vitamin B-12 deficient participants indicating efficient metabolic effects (Table 4).
Carmel  categorized available biomarkers as those that directly measured plasma vitamin B-12 and those that measured metabolites that accumulated with inadequate amounts of vitamin B-12. Serum holo-TC and vitamin B-12 measured circulating vitamin B-12 concentrations. These two therefore reflected the broad vitamin B-12 status from high risk of severe deficiency to adequacy. Miller et al  stated that holo-TC and total vitamin B-12 have equal diagnostic accuracy in screening for metabolic vitamin B-12 deficiency. Measurement of both holo-TC and total vitamin B-12 provided a better screen for vitamin B-12 deficiency than either assay alone.
According to Green , low vitamin B-12 status was indicated by being below the lower the reference range (for vitamin B-12 < 148 pmol/L; for holo TC, <35 pmol/L), whereas for indirect measures of metabolites (methylmalonic acid or homocysteine), low vitamin B-12 status measures would be indicated by a level above the upper limit of the reference range (for methylmalonic acid, >260 nmol/L; for homocysteine, >12 μmol/L). The markers he mentioned were homocysteine for detection of either vitamin B-12 or folate deficiency and methylmalonic acid for vitamin B-12 deficiency only.
However, Valente et al  suggested a diagnostic strategy using holo-TC as the front-line test. The cutoffs for deficiency were defined as 20 pmol/L for holo-TC and 123 pmol/L for serum vitamin B-12 after studying employees and medical students of a local hospital at Dundee, UK. We defined the participants as vitamin B-12 deficient who showed <148 pmol/L of plasma vitamin B-12 concentration.
Yajnik et al  observed that the habitual intake of green leafy vegetables or other vegetables did not affect plasma vitamin B-12 or tHcy concentrations. They  have reported that the oral vitamin B-12 supplementation reduced plasma tHcy concentration in first two weeks but did not achieve normal plasma tHcy concentrations even after 6 wks. Deshmukh et al  studied the effects of physiological doses of oral vitamin B-12 on plasma tHcy and observed that the reduction was 5.5 and 6.8 μ mol/L after 4 and 12 months of 2 μ g supplementation per day respectively, while supplementation of 10 ug per day for 6 and 12 months reduced plasma tHcy concentration by 5.6 and 6.9 μ mol/L respectively. There was no advantage in supplementing with higher dose. It has been observed in healthy adults that about 50% of a 1-μg dose of vitamin B-12 is absorbed from food, while only 20% of a 5 μ g dose, and 5% of a 25 μ g dose is absorbed . Therefore, intervention with at least 1.0 to 1.54 μ g of additional vitamin B-12 available from milk every day producing beneficial metabolic effect, increasing plasma vitamin B-12 and reducing tHcy, is advocated.
The novelty of the present study is that it used milk supplementation to measure both absorption capacity and efficiency to reduce plasma tHcy levels. Bor et al , von Castel-Roberts et al  and Bhat et al , used free cyanocobalamin to assess intestinal vitamin B-12 absorption while Deshmukh et al  used free cyanocobalamin for lowering plasma homocysteine levels. Although cyanocobalamin increases circulating levels of cobalamin, its ability to increase tissue levels of the active form of vitamin B-12 can be limited in a large number of subclinical and clinical conditions. The activation of cyanocobalamin to either adenosyl cobalamin or methyl cobalamin does not occur instantly, possibly occurring over 1-2 months  and requires decyanation, interaction of glutathione and in the case of methyl cobalamin, S-adenosyl methionine and the active form of folic acid. Qubeck study conducted on pigs showed that the intestinal absorption of vitamin B-12 from milk was higher than that of the synthetic form of vitamin B-12 by comparing the net fluxes of vitamin B-12 across the portal drained viscera after ingestion of milk or synthetic vitamin B-12 . Vitamin B-12 in milk is in the form of coenzyme (adenosyl cobalamin), a major form in cellular tissues, where it is retained in the mitochondria, facilitating faster metabolic actions .
Even though the increase in plasma vitamin B-12 concentrations was modest (29.6 pmol/L) following milk intake by the participants, such an increase would be beneficial in vitamin B-12 deficient subjects. The increase in plasma holo-TC concentration after regular milk intake helped in lowering plasma tHcy concentrations (by 7 μmol/L).
The limitation of the present study is that we have not measured plasma methylmalonic acid concentration which is a specific biomarker for vitamin B-12 deficiency status. The other marker, tHcy gets accumulated in both vitamin B-12 and folate deficiency. However, none of our participants had folate deficiency. The participants with >148 pmol/L of plasma vitamin B-12 concentrations were not studied after 14 days consuming 400 ml milk every day.
The recommended dietary allowance (1 μg/day) for Indians seems to be inadequate. Even though there was significant reduction in plasma tHcy concentration 14 days after providing regular dietary vitamin B-12 (2.5 to 3 μg) in the form dairy products, the levels did not reach to normal levels. Effect of long term daily intake of milk on vitamin B-12 status should form a part of future study.