NITRITOCOBALAMIN AND NITROSOCOBALAMIN MAY BE CONFUSED WITH SULFITOCOBALAMIN USING CATION-EXCHANGE CHROMATOGRAPHY

The previously reported ability of SP-Sephadex C-25 column chromatography for partitioning biologically important cobalamins has been modified to include analytical separation of nitritocobalamin (NO2-Cbl) and nitrosocabalamin (NO2-Cbl). Gel column dimensions (1.5 x 11.0 cm), a low eluent flow-rate (125 mu l/min), collection of small eluate fractions (160 mu l) plus maintenance of He saturated mobile and gel phases all combined to eliminate ordinarily confusing proximal elution of NO2-Cbl and NO-Cbl with sulfitocobalamin (SO3-Cbl) and cyanocobalamin. Cobalamin elution profiles from the gel column were monitored by direct radiometric analysis of Co-57-labelled cobalamin standards or competitive intrinsic factor radioassays for cobalamin sample sizes up to 10.0 ng. Failure to implement the chromatographic conditions detailed here totally obscured analysis of NO2-Cbl coexisting with SO3-Cbl in brain tissues for chicks exposed to dietary sulfites and caused oversight of NO-Cbl normally coexisting in prepared NO2-Cbl standards.