Vitamin B12 Status Upon Short-Term Intervention with a Vegan Diet-A Randomized Controlled Trial in Healthy Participants

Abstract

Vegans are at an increased risk for certain micronutrient deficiencies, foremost of vitamin B₁₂. Little is known about the short-term effects of dietary change to plant-based nutrition on vitamin B₁₂ metabolism. Systemic biomarkers of vitamin B₁₂ status, namely, serum vitamin B₁₂ and holotranscobalamin, may respond quickly to a reduced intake of vitamin B₁₂. To test this hypothesis, 53 healthy omnivore subjects were randomized to a controlled unsupplemented vegan diet (VD, n = 26) or meat-rich diet (MD, n = 27) for 4 weeks. Vitamin B₁₂ status was examined by measurement of serum vitamin B₁₂, holotranscobalamin (holo-TC), methylmalonic acid (MMA) and total plasma homocysteine (tHcy). Holo-TC decreased significantly in the VD compared to the MD group after four weeks of intervention, whereas metabolites MMA and tHcy were unaffected. Body weight remained stable in both groups. VD intervention led to a significant reduction of cholesterol intake, and adequate profiles of nutrient and micronutrient status. Lower intake of vitamin B₁₂ was observed in VD, which was mirrored by a lower concentration of serum vitamin B₁₂ and reduced holo-TC after 4 weeks. Plasma holo-TC may be a fast-responding biomarker to monitor adequate supply of vitamin B₁₂ in plant-based individuals.

Keywords: fatty acids; holotranscobalamin; micronutrient; vegan nutrition; vitamin B12.

Figure 1

Vitamin B₁₂ metabolism. Dietary vitamin B₁₂ is transported through the digestive system and absorbed in the intestine where it binds to apo-transcobalamin (apo-TC) to form holotranscobalamin (holo-TC). Holo-TC is the bioactive form of vitamin B₁₂. Holo-TC reaches circulation and it is then taken up by all cells in the body via receptor-mediated endocytosis (the transcobalamin (TC) receptor is also known as CD320). Once in the cell, vitamin B₁₂ is freed from TC in the lysosome and exported into the cytosol. Downstream cytosolic processing and trafficking events ensure that vitamin B₁₂ ultimately reaches cytosolic methionine synthase and mitochondrial methylmalonyl-CoA mutase. Nutritional deficiency of vitamin B₁₂ blocks the reactions catalyzed by methionine synthase and methylmalonyl-CoA, resulting in the accumulation of their respective substrates, homocysteine (Hcy) and methylmalonyl-CoA (MMA-CoA). Hcy and MMA are toxic to cell metabolism; therefore, cells export these metabolites into circulation under conditions of B₁₂ deficiency. This manifests clinically as elevated concentration of Hcy and MMA in plasma or serum. A vascular endothelial cell is used as a generic model. This figure was modified from reference [19].

Figure 2

Flow chart depicting randomization scheme of participants into vegan (VD) and meat-rich diets (MD).

Figure 3

Holotranscobalamin (Panel A), methylmalonic acid (Panel B), vitamin B₁₂ (Panel C), and 25-OH-Vitamin D₂/D₃ (Panel D) in serum before and after 4-week dietary intervention in VD (blue line) and MD (red line). Serum vitamin B₁₂ (ng/L) and holotranscobalamin concentrations (pmol/L) showed a significantly decrease in vegans (vitamin B₁₂: 362.8 ± 110.9 to 296.1 ± 94.1 ng/L, p < 0.001; holotranscobalamin: 67.3 ± 23.5 to 43.6 ± 20.0 pmol/L, p < 0.001) compared to meat-rich subjects (vitamin B₁₂: 391.2 ± 159.2 to 391.8 ± 143.0 ng/L, p = 0.919; holotranscobalamin: 69.7 ± 29.7 to 64.4 ± 28.7 pmol/L, p = 0.041). Holotranscobalamin decreased by more than 30% upon dietary intervention, whereas serum vitamin B₁₂ decreased by 18%_. Error bars show ± 1 standard error. Baseline and end values as well as statistical comparisons are shown in Table 2. Significant differences between groups are marked with an asterisk (*: p < 0.05).

Figure 4

Relationship of serum holotranscobalamin and vitamin B₁₂ in the two randomized groups after a 4-week dietary intervention. Vitamin B₁₂ (ng/L) correlates positively with holotranscobalamin concentration (pmol/L) both after intervention in VD (Panel A, r = 0.695, p < 0.001) and in MD (Panel B, r = 0.484, p = 0.010). Dispersion lines show the 95% confidence interval of the linear regression fit.

Figure 5

Cystathionine (Panel A), cysteine (Panel B), glutathione (Panel C), homocysteine (Panel D), methionine (Panel E), and methionine sulfoxide (Panel F) in plasma before and after 4-week dietary intervention in VD (blue line) and MD (red line). Error bars show ± 1 standard error. Baseline and end values as well as statistical comparisons are shown in Table 3.

Figure 6

Relationship of serum vitamin B₁₂ with cellular markers of vitamin B₁₂, tHcy, and MMA, and with other marker metabolites of B-vitamin status. Data points of participants in the vegan and meat-rich diets are shown as blue and red dots, respectively. Correlations are shown for serum vitamin B₁₂ (ng/L) and total homocysteine (µmol/L; r = 0.280, p = 0.042) (Panel A), serum vitamin B₁₂ and methylmalonic acid (µmol/L; r = 0.280, p = 0.042) (Panel B), serum vitamin B₁₂ and methionine (µmol/L; r = −0.154, p = 0.272) (Panel C), serum vitamin B₁₂ and serum cysteine (µmol/L; r = 0.147, p = 0.293) (Panel D), serum vitamin B₁₂ and cystathionine (µmol/L; r = −0.126, p = 0.367) (Panel E), and serum vitamin B₁₂ and glutathione (µmol/L; r = 0.110, p = 0.434) (Panel F). Dispersion lines show the 95% confidence interval of the linear regression fit.

Figure 7

Associations of total cysteine and total homocysteine and influence of diet on B-vitamin status of VD (blue) and MD (red). (Panel A) Correlation of log of total cysteine and log of total homocysteine before trial. (Panel B) Correlation of log of total cysteine and log of total homocysteine after trial. (Panel C) Assessment of B-vitamin status using the tHcy:tCys ratio proposed by Ulvik et al. [17] at baseline and after 4-week intervention, sorted by assigned diet. Ratio shows non-significantly decrease in vegan group and non-significantly increase in meat-rich group, sorted by time (green baseline, orange end); error bars show ± 1 standard error.

Figure 8

Arachidonic acid (Panel A), docosahexaenoic acid (Panel B), eicosenoic acid (Panel C), linoleic acid (Panel D), linolenic acid (Panel E), and oleic acid (Panel F) in serum before and after 4-week dietary intervention in VD (blue line) and MD (red line). Baseline value of arachidonic acid of MD group was 3.7 ± 1.8 µM and increased significantly to 6.4 ± 8.0 µM (p = 0.021), arachidonic acid of VD group remained stable (p = 0.424). Error bars show ± 1 standard error. Baseline and end values as well as statistical comparisons are shown in Table 3. Significant differences between groups are marked with an asterisk (*: p < 0.05).