In the experiments presented in –, we did not detect the presence of the Cbl analogues numbered 5–7, 9 and 11 in and . Recent experiments, however, indicate that small amounts, 9–56 ng/g wet wt, of item 9-[5-MeO,6-MeBZA]CN-Cba-were present in 3 fecal samples from subject AN01 when a vitamin supplement containing 2 mg CH3-Cbl had been ingested on a daily basis for several months. In addition, small amounts (9–63 ng/g wet wt) of item 6, [5-MeOBZA]CN-Cba, have been observed in 6 samples from a single subject involved in another study.
The review by Renz (1
) lists an additional 4 naturally occurring Cbl analogues that we have not synthesized and are not included in . These 4 Cbl analogues contain guanine, hypoxanthine, 2-methylsulfinyl-adenine and 2-methylsulfonyl-adenine in place of the 5,6-diMeBZA contained in Cbl. We are in the process of synthesizing labeled and unlabeled forms of these Cbl analogues (24
), although we have not encountered peaks with the expected ions for any of them in human feces.
The data obtained with control subject AN01 and presented in – show that this subject has an unusual CN-Cbl and CN-Cbl analogue distribution, in that it contains a relatively large amount of [5-OHBZA]CN-Cba and often contains detectable amounts of [Phe]CN-Cba. In addition, the CN-Cbl and CN-Cbl analogue distribution persisted over many months. In future studies, it will be interesting to determine whether this observation holds true for longer periods and for other control subjects and, if so, whether individual distribution patterns are due to environmental or genetic differences, or both. It also will be interesting to determine whether patients with various gastrointestinal diseases have distribution patterns of CN-Cbl and CN-Cbl analogue that are specific to a given disease and, if so, whether these patterns differ from those seen in healthy subjects.
It has been estimated that healthy humans secrete 3–9 ug Cbl/d in their bile and consume 5–15 Cbl/d in their diet (5
). They reabsorb or absorb 3–9 ug/d to maintain their Cbl homeostasis (5
), which leaves 5–15 ug (mean: 10 ug) that could be excreted in the feces each day. On the basis of an average value of 100 g/d for human fecal excretion (26
), one would expect a mean value of CN-Cbl in human feces of 100 ng/g wet wt. Our observed value of 19 ng/g wet wt in 18 control human subjects indicates that an average of about 81% of nonabsorbed Cbl is altered or destroyed in the human gastrointestinal tract. We found an average value for Cbl analogues of 1290 mg/g wet wt, which makes it possible that all 81 ng/g wet wt of the missing Cbl is converted to Cbl analogues.
The data in and indicate that the ingestion by healthy subjects of 1–2 mg of CN-Cbl on a daily or one-time basis causes large increases in the fecal content of CN-Cbi, [Ade]CN-Cba, [2-MeAde]CN-Cba and [p-Cre]CN-Cba, as well as an increase in the fecal content of CN-Cbl. This finding suggests that microorganisms in the human gastrointestinal tract can also convert a large amount of ingested CN-Cbl to these 4 CN-Cbl analogues. Preliminary experiments in which CN-Cbl labeled with 15
in the core, nonbase area was ingested by rats support this possibility, because significant amounts of the 15
label appeared within 24 h in the feces in CN-Cbl and in each of the 4 CN-Cbl analogues just mentioned (RH Allen, SP Stabler, unpublished observations, 2007). Our results and conclusions are also supported by earlier studies by Brandt et al (27
), who described 4 patients with small-bowel overgrowth who had in their intestines bacteria that contained significant amounts of CN-Cbi, [Ade]CN-Cba, [2-MeAde]CN-Cba, and “factor E,” which is a Cbl analogue of unknown structure. More important, they also showed that intestinal bacteria from all 4 patients converted (57
)CN-Cbl into the (57
)CN-forms of all 4 of these CN-Cbl analogues.
On the basis of our observed means for CN-Cbl and the total of CN-Cbl analogues in 18 control subjects of about 19 and 1290 ng/g wet wt, respectively, and an average value of about 100 g wet wt for daily fecal output (25
), we calculate that about 2 ug Cbl is excreted and 130 ug Cbl analogues is formed in and excreted from the human body every 24 h. Thus, about 1 mg Cbl is excreted and 50 mg Cbl analogues is formed and excreted over the course of 1 y. These values are significantly different from estimates for the total human body content of Cbl of only of 2–5 mg (2
). It would be interesting to design studies to determine whether significant amounts of these fecal Cbl analogues traverse the gastrointestinal mucosal barrier in healthy human subjects and in patients with various inflammatory bowel diseases, and whether mechanisms exist to prevent their dissemination to other tissues. These studies are particularly intriguing because another study by Brandt et al (28
), conducted in dogs with surgically constructed, self-filling high intestinal blind loops, showed that Cbl analogues were present there in much greater amounts than Cbl and that they consisted mainly in the form of CN-Cbi, [Ade]CN-Cba and [2-MeAde]CN-Cba. Furthermore, over the course of 1 y, CN-Cbl was gradually replaced in the liver by the accumulation of mainly CN-Cbi and [2-MeAde]CN-Cba and only relatively small amounts of [Ade]CN-Cbl. It is not known whether [Ade]CN-Cba reached the liver in relatively small amounts or whether it was structurally altered or preferentially excreted once it arrived there.
In summary, we have developed a sensitive and specific assay for Cbl and Cbl analogues and have studied the quantities and patterns of these Cbl analogues in human feces. These assays can be used to study the presence of Cbl and Cbl analogues in food and the environment and to explore the fate of ingested Cbl and Cbl analogues in humans.