Vitamin B12 (B12, cobalamin) is one of the greatest nutritional concerns for vegetarians as vitamin B12 is primarily present in foods of animal origin [1
]. B12 deficiency is identified by a decrease in total plasma B12, as well as by B12 bound to its transport protein transcobalamin (holotranscobalamin, holoTC) and increase in the metabolites–plasma homocysteine (Hcy) and methylmalonic acid (MMA) [2
]. Increased concentrations of MMA and Hcy are due to decreased activity of methylmalonyl-CoA mutase and methoionine synthase, respectively [1
]. While severe deficiency can cause megaloblastic anemia and permanent neurological damage, early physiological manifestations are generally subtle [1
Two main etiologic factors play a role in developing vitamin B12 deficiency: Inadequate dietary intake and/or vitamin B12 malabsorption. Asymptomatic Indian lactovegetarians, who make up more than half of the Indian population, have distinctly lower vitamin B12 concentrations than non-vegetarians across geographic regions of India [7
]. These lactovegetarians were also found to have increased plasma levels of Hcy and MMA, as well as lowered holoTC, all indicating vitamin B12 deficiency [7
]. The options of increasing the overall vitamin B12 status of a deficient population include vitamin supplementations, fortification or targeted dietary recommendations.
Epidemiological and observational studies show that B12 status correlates with the consumption of animal products [5
]. Among those, endogenous B12 in dairy products and especially milk appears to be highly bioavailable [11
]. In accordance, intervention studies have revealed that the B12 status of vegetarians is positively associated with their intake of milk [7
] and that both cow and buffalo milk [12
] match supplements with B12 capsules in improving biomarkers of B12 deficiency [8
]. The native form of B12 in milk is mainly hydroxo-B12 (HO-B12) [8
], whereas the synthetic variant cyano-B12 (CN-B12) is widely used in supplements and fortified foods. Employing an animal model, we have previously demonstrated that, though the two forms of B12 are absorbed alike, they distribute differently in the body [14
]. CN-B12 accumulated mainly in the kidneys, whereas HO-B12 predominantly targeted the liver. In both human and animal studies, CN-B12 supplementation caused the highest increase in plasma B12, yet more biologically active B12 coenzymes were retrieved from the tissues of the HO-B12 supplemented animals [16
]. These findings question whether the two vitamin forms are of equal value for the improvement of B12 status.
A recent eight weeks intervention study in elderly Australians with subclinical deficiency of B12 revealed that cow’s milk whey powder, unlike devoid of B12 soy protein isolate, increased plasma holoTC and attenuated the buildup of MMA and Hcy [17
]. Studies, carried out so far, leave us with a question, how to normalize and maintain B12 status in lactovegetarians with a borderline B12 deficiency. The purpose of this investigation was to directly compare the efficacy of treatment with whey powder and CN-B12 in capsules (both equivalent to 5.6 µg of B12).
We studied the biomarkers, related to B12 status, in a lactovegetarian Indian population supplemented with a daily dose of 5.6 µg B12, administered as a divided portion with 10–12 h intervals (either two capsules of CN-B12 or two servings of whey powder with endogenous HO-B12). Blood samples were collected at baseline (week 0) and every second week during eight weeks of supplementation, plus two weeks post-intervention.
The present study supports and expands our previous data [8
] on supplementations with CN-B12 vs. HO-B12 (the latter being either in capsules or present in cow and buffalo milk). Here, we confirm the equal value of the two vitamin forms in relation to improving metabolic biomarkers (Hcy and MMA) and underscore that measurements of just plasma B12 would give a false impression of CN-B12 superiority. For the first time, we demonstrate that the difference in plasma B12 following supplementation with CN-B12 vs. HO-B12 may well be driven by retention of unmetabolized CN-B12 on haptocorrin (and to a lesser degree on transcobalamin). Importantly, we establish that eight weeks of treatment with a high physiological dose of B12 is insufficient to normalize biomarkers of B12 status—even when dividing the dose into two daily servings in order to prevent overload of the B12 uptake system, characterized by a limited capacity for each separate B12 intake.
Our study has some limitations. The conclusions would be strengthened had the study groups been increased and the intervention period (as well as the follow-up period) prolonged. The capsule group had a rather uneven representation of men (n
= 2) and women (n
= 15). However, our unpublished comparison of men vs. women in previously studied cohorts [8
] did not reveal any significant difference in the responses of women as compared to men. Concerning the analysis of B12 forms on transcobalamin and haptocorrin, we were not able to examine the individual samples, but only pooled samples.
A more detailed comparison of the current study to our previous works reveals that the daily dose of B12 employed in the current study (5.6 µg for eight weeks) results in a more explicit improvement in the metabolites (Hcy and MMA) than observed in previous supplementation schemes of 1.52 µg for four weeks [8
] or 3 µg for eight weeks [25
], Table 4
. However, none of the three interventions led to complete normalization of the biomarkers, thereby suggesting that the impaired B12 status was not completely reversed by the doses administered.
The responses of metabolites and holoTC (Table 2
and Figure 2
B and Figure 3
A,B) showed some unexpected trends to the respective baselines at the end of our study (weeks 8 and 10). The effect was evident for Hcy and holoTC, but nearly absent for MMA (at least in statistical terms). A backward tendency of MMA was, however, noticed in our previous study [18
]. We have no unequivocal explanation of these observations but can offer a plausible conjecture. Activation of a functioning enzyme indeed provides a drop in its substrate, but this drop is afterward partially reversed, if the enzyme belongs to a metabolic or/and transportation flux. This reversion originates from the fact that the decreased substrate is simultaneously the product of preceding enzymes. Lowering of product inhibition in the preceding enzyme stimulates it to produce more of the compound in question. In a well-mixed in vitro system, these down-up fluctuations are accomplished within seconds, but in a multi-compartment (multi-organ) in vivo system, the return to a new steady-state in blood might take time.
Contrary to the aforementioned markers, total B12 steadily increased and stabilized at a new steady-state level without further fluctuations (Figure 2
A). The levels were, however, different for CN-B12 capsules and whey powder (containing mostly HO-B12). A more pronounced increase in total plasma B12 during supplementation of CN-B12 vs. HO-B12 (especially during the first two weeks) was consistently recorded in our current and preceding studies [8
]. This difference in finally achieved levels depended though on the supplemented dose, showing a low difference at low doses, but a high difference at higher doses (Table 4
Irrespective of a higher build-up in total plasma B12 upon ingestion of CN-B12, this accumulation was not reflected in the metabolic markers, which uniformly indicated that CN-B12 and HO-B12 improved B12 status with equal potency.
The aforementioned accumulation of total plasma B12 upon ingestion of CN-B12 vs. HO-B12 requires a separate discussion. First, we should point out that total plasma B12 is a sum of holoTC (present at a low concentration) and holohaptocorrin (holoHC) (present at a high concentration), reviewed in refs. [1
]. HoloTC is a fast-exchanging carrier (half-life in blood ≈1 h [27
]), which plays an essential role in promoting the cellular uptake of B12 through receptor-mediated endocytosis (involving a specific TC receptor found on all cells). Hitherto, no universal receptor for holoHC has been identified, and the haptocorrin-B12 complex is considered to be of limited importance for B12 tissue delivery. Haptocorrin is believed to accumulate and dispose of the inactive forms of B12, acting as a scavenging protein [27
]. We speculated in our previous paper [8
] that a higher total B12 in blood upon ingestion of CN-B12 might be caused by constrained intracellular processing of this vitamin form, as was the case for the nonconvertible “anti-vitamin” ethylphenyl-Cbl [28
]. The cobalt site of CN-B12 is partially protected by CN-group, which can decelerate transformation of CN-B12 to the active cofactors (at least in comparison to the “unprotected” HO-B12). Such insufficient conversion apparently initiates a cyclic turnover of the unprocessed CN-B12 (cellular uptake → excretion → uptake → excretion …), leading to a gradual accumulation of CN-B12 on the slow-exchanging haptocorrin (with a half-life time in blood of approximately 10 days [27
Confirming our hypothesis about the accumulation of CN-B12 on haptocorrin, we here provide biochemical evidence that the high level of total B12 after administration of CN-B12 indeed originates from CN-B12 bound to haptocorrin. Over-representation of the inactive vitamin on haptocorrin implies that the total plasma B12 (holoHC + holoTC) does not reflect the real quantities of the active B12-cofactors in the tissues (if the ingested form is CN-B12). Therefore, the metabolic markers MMA and Hcy should be aligned rather with transcobalamin-bound B12 (holoTC), because this marker is apparently less sensitive to the supplemented vitamin form.