Identification and quantitation of cobalamin and cobalamin analogues in human feces

Abstract

Background: Cobalamin (vitamin B-12) and cobalamin analogues are present in human feces, but a complete identification has not been established, and the amounts present have not been determined.

Objectives: We aimed to develop a liquid chromatography-mass spectrometry method for cobalamin and cobalamin analogues and to identify and quantitiate the amounts present in human feces.

Design: Fecal samples were obtained from 20 human subjects in good general health. The samples were analyzed for the presence and amounts of cobalamin and 12 cobalamin analogues that were synthesized with and without the incorporation of stable isotopes.

Results: Cobalamin and 7 cobalamin analogues were identified and quantitated in human feces. The mean for the total amount present in 18 subjects whose daily intake was < or = 25 microg cobalamin from vitamin supplements was 1309 ng cobalamin equivalents/g wet wt of feces. Cobalamin (1.4%) and cobinamide (1.8%) (an incomplete corrinoid) represented a small portion of the total amount. Six cobalamin analogues that contain a base other than the 5,6-dimethylbenzimidizidole in cobalamin were present. The bases and their mean amounts (in %) are 2-methyladenine (60.6%), p-cresol (16.3%), adenine (12.5%), 2-(methylthio)adenine (15.5%), 5-hydroxybenzimidazole (1.8%), and phenol (0.1%). One subject ingested 1 mg cobalamin/d and another ingested 2 mg cobalamin/d, and they appeared to convert most of the cobalamin to cobinamide and the 4 analogues that contain the bases-2-methyladenine, p-cresol, adenine, and 2-(methylthio)adenine.

Conclusions: Cobalamin analogues are present in human feces and account for > 98% of the total of cobalamin plus cobalamin analogues. A major portion of large amounts of ingested cobalamin appears to be converted to cobalamin analogues.

Figure 1

Structure of cyanocobalamin (CN-Cbl) (upper section) and the structures (lower section) of the base portion of CN-Cbl [10 (numbers in circles)] and 11 CN-Cbl analogues (–9) and (–13) that differ from CN-Cbl only in the base portion. * CN-Cbi (cyanocobalamin) (1) lacks the base, ribose and phosphate moieties of CN-Cbl.

Figure 2

Liquid chromatography-mass spectrometry chromatogram of cyanocobalamin (CN-Cbl) and 7 CN-Cbl analogues that were present in a fecal sample from control subject AN01. Cba, cobamide. Each of the 8 panels represents a plot of arbitrary units in 1000’s versus a 2-min time period in which the endogenous unlabeled form was present in the upper half of each panel and the added labeled form was present in the lower half. The number in a circle (top left of each panel) refers to the structure of the base in Figure 1. M⁺ represents the ion that was monitored. “Gain” refers to the extent to which the electron multiplier voltage was amplified; it was always the same for the upper and lower portions of each panel. The time (min) to the right of each peak is the peak retention time. AMT is the calculated amount (in ng) of endogenous unlabeled CN-Cbl or CN-Cbl analogue, expressed as CN-Cbl equivalents, per g of wet wt of the fecal sample that was analyzed.