Assessment of vitamin B(12) absorption based on the accumulation of orally administered cyanocobalamin on transcobalamin

Abstract

Background: Vitamin B(12), or cobalamin (Cbl), is absorbed in the intestine and transported to the cells bound to transcobalamin (TC). We hypothesize that cyanocobalamin (CNCbl) is absorbed unchanged, thereby allowing measurement of the complex of CNCbl bound to TC (TC-CNCbl) to be used for studying the absorption of the vitamin.

Methods: TC was immunoprecipitated from serum samples obtained from healthy donors at baseline and at 24 h after oral administration of three 9-microg CNCbl doses over 1 day. Cbl was released by treatment with subtilisin Carlsberg. The different forms of Cbl were isolated by HPLC and subsequently quantified with an ELISA-based Cbl assay.

Results: At baseline, the median TC-CNCbl concentration was 1 pmol/L (range, 0-10 pmol/L); the intraindividual variation (SD) was 1.6 pmol/L (n = 31). After CNCbl administration, the TC-CNCbl concentration increased significantly (P = 0.0003, paired t-test), whereas no major changes were observed in any of the other Cbl forms bound to TC (n = 10). Only a moderate additional increase in TC-CNCbl was observed with prolonged (5 days) CNCbl administration (n = 10). We designed an absorption test based on measuring TC-CNCbl at baseline and 24 h after CNCbl intake and established a reference interval for the increase in TC-CNCbl (n = 78). The median absolute increase was 23 pmol/L (range, 6-64 pmol/L), and the relative increase was >3-fold.

Conclusions: Our data demonstrate that CNCbl is absorbed unchanged and accumulates on circulating TC. We suggest that measuring TC-CNCbl will improve the assessment of vitamin B(12) absorption.

Figure 1.

Forms of Cbl on TC before and 24 h after CNCbl intake. Ten healthy donors were administered 3 oral doses of 9 μg CNCbl on a single day. Light-protected blood samples were withdrawn at baseline and 24 h after the first dose. Cbl on serum TC was isolated and separated by HPLC. (A), Chromatograms from 1 representative donor showing the baseline Cbl forms on TC (solid line) and the forms on TC 24 h after supplementation (dotted line). (B), The changes in OHCbl, CNCbl, AdoCbl, and MeCbl found on TC for all 10 donors at 24 h after supplementation. Data are presented as the mean and SD.

Figure 2.

Accumulation of CNCbl on TC after intake of CNCbl. Healthy donors (n = 10) were supplemented daily with 3 oral doses of 9 μg CNCbl for 5 days. TC–CNCbl was measured in serum from blood samples withdrawn at baseline (day 0) and on days 1, 2, 3, 4, and 7 after the first dose.

Figure 3.

Absorption of vitamin B₁₂(CNCbl), as judged by holoTC measurement (CobaSorb) or TC–CNCbl measurement (C-CobaSorb). Donors (n = 78) were administered 3 oral doses of 9 μg CNCbl per day for 5 days. Blood samples were taken at baseline and at timed intervals after the start of supplementation and analyzed. Results for individuals judged able to absorb vitamin B₁₂ (n = 68) with both methods are not indicated. Results for individuals that could not be classified with CobaSorb (n = 8) are indicated with a solid line. A dashed line indicates a donor judged by the CobaSorb assay to be unable to absorb vitamin B₁₂; the dotted line indicates a donor that could not be classified with C-CobaSorb. (A), The CobaSorb test. Measurement of holoTC at baseline and 48 h after first test dose of vitamin B₁₂. Data on the measurement of holoTC is obtained from Hvas et al. (10). (B), The C-CobaSorb test. Measurement of TC–CNCbl at baseline and 24 h after first test dose of vitamin B₁₂.