Presence of cobalamin analogues in animal tissues

Abstract

Cobalamin (Cbl, has been extracted and isolated from a number of animal tissues by using (i) reverse-affinity chromatography on R protein-Sepharose followed by adsorption to and elution from charcoal-coated agarose and (ii) paper chromatography. Radioisotope dilution assays showed that only 75-97% of the Cbl chromatographed in the position of crystalline Cbl. The remaining 3-25% was present in a number of slower and faster moving fractions. This suggested that Cbl analogues are present in animal tissues because appropriate controls ruled out the possibility that this material was artifactually derived from Cbl during the extraction and purification procedures. With a large-scale isolation from rabbit kidney, the material in five such fractions contained cobalt and had absorption spectra that were similar to but different from the spectrum of Cbl, indicating that they were Cbl analogues. Compared to Cbl, these Cbl analogues had decreased but definite affinities for Cbl-binding proteins with the following order of strength of binding: R protein > transcobalamin II > intrinsic factor. Compared to Cbl, they also had decreased but definite growth-promoting activity for two microorganisms, Euglena gracifis and Lactobacillus leichmannii, which require Cbl for growth. These Cbl analogues differed from each other and from 18 synthetic Cbl analogues, including the most common Cbl analogues synthesized by microorganisms, in at least one of the above features. These studies indicate that animal tissues contain a number of Cbl analogues whose origins, structures, and biologic activities remain to be determined.

A New Principle for Measurement of Cobalamin and Corrinoids, Used for Studies of Cobalamin Analogs on Serum Haptocorrin

“…At that time, studies of Cbl analogs in biological samples were based on extraction procedures that involved acidic or alkaline solutions, addition of KCN, and sometimes heating before quantification of the corrinoids with radioisotope dilution assays (20,21 ). There has been discussion as to whether these extraction procedures could alter the molecular form of Cbl, thereby creating the Cbl analogs as artifacts (22,23 ).…”

Nitrous oxide has multiple deleterious effects on cobalamin metabolism and causes decreases in activities of both mammalian cobalamin-dependent enzymes in rats.

“…The possibility that the formation of Cbl analogues might play a role in the neurologic abnormalities seen in chronic N20 exposure is intriguing in view of the previous suggestion (36) that variations in levels of endogenous Cbl analogues in patients with Cbl deficiency might explain why some patients develop only hematologic or neurologic abnormalities while others develop both. Definitive answers to these questions as well as to the question of whether endogenous Cbl analogues present in mammalian tissues (44) are the same as those formed from Cbl by N20, will require further studies.…”

“…Data to support this concept are presented in Table III 10,27, and 4% in liver, kidney, and brain, respectively. Levels of the total of Cbl and Cbl analogues, as measured with the radioisotope dilution assay using R protein,4 showed smaller but significant decreases to 35, 46, and 24% of control values, in liver, kidney, and brain, 4Cbl analogues may not be measured to their full extent with the R protein assay since some Cbl analogues have slight to moderately reduced affinities for R protein, relative to Cbl (44). respectively.…”

Inhibition of cobalamin-dependent enzymes by cobalamin analogues in rats.

“…Previous investigations have shown that there are a variety of mechanisms which appear to prevent the absorption and tissue dissemination of certain of these cbl analogues (16)(17)(18). Previous studies have also shown that there are cbl analogues ofunknown structure in human serum (19), animal tissues (20), and foodstuffs and vitamin supplements (21), though their effects on cbl metabolism are largely unknown.…”

“…The concentration of cbl and cbl analogues was determined spectrophotometrically at 367.5 nm in 0.1 M KCN. A molar extinction coefficient of 30,800 M-' cm-' was used for each analogue except for CN-cbl [13-epi] for which a value of 20,600 MWcm- (16) was used. The final analogue solutions gave a single peak when they were analyzed for purity by HPLC utilizing separation methods previously described (25).…”