Measuring Oral Vitamin B12 Bioavailability Using [13C]-cyanocobalamin in Humans

Abstract

Objectives

To biosynthesize a stable-isotope labeled [ ¹³C]-cyanocobalamin and to measure its bioavailability in humans.

Methods

Salmonella enterica was precultured in Luria-Bertani medium and single-cell colonies were grown in No carbon E medium, supplemented with [ ¹³C]-ethanolamine as a sole-carbon source along with other precursors (dicyanocobinamide and dimethylbenzimidazole) and incubated for 48 h at 30°C and centrifuged. The pellets were resuspended in methanol/sodium cyanide and incubated overnight and extracts were purified by HPLC. The peaks corresponding to standard cyanocobalamin were collected and characterized by MALDI-MS.

After animal testing, healthy young subjects were orally administered with [ ¹³C]-cyanocobalamin in a protocol that included hourly blood sampling for 12 h post-dose, and daily sampling for the next 5 days, to characterize its kinetics at a higher dose than the daily requirement (∼10x, n = 4, 2 males and females) and its appearance was modeled using a two-compartment model with early and late absorption phases. The same model was applied for lower dose at daily requirement (∼2.5 μg, n = 11 males), with sampling for 12 h post-dose. In addition, the effect of co-ingested food (n = 1, male) and the effect of parenteral replenishment of existing body vitamin B ₁₂ stores by intramuscular injection of 1 mg hydroxocobalamin, (n = 3, males) on bioavailability was also studied. The appearance of tracer in plasma was quantified by Q-Exactive-Orbitrap-MS.

Results

Biosynthesized [ ¹³C]-cyanocobalamin was labeled with up to 7– ¹³[C] atoms and at a daily requirement dose (∼2.5 μg), its mean bioavailability was 50.4 ± 8.2%, but this was also dependent on the existing body store of vitamin B ₁₂. Two-weeks after replenishing the body stores, bioavailability increased by 1.5-fold (from 45.6 ± 0.02% to 67.8 ± 0.06%). At a higher dose (∼20 μg), its bioavailability was 6.0 ± 1.2% but the absolute amount absorbed was similar to that with the low dose.

Conclusions

A novel stable isotope-based vitamin B ₁₂ bioavailability assessment is described at low and high doses. This has implications toward rationalizing dosages used in clinical supplementation and public health fortification programs.

Funding Sources

Department of Biotechnology, India (to SD and AVK) and by the Wellcome Trust/DBT India Alliance Margdarshi Fellowshi p [No. IA/M/14/1/501,681] awarded to AVK.