Anes J M, Beck R A, Brink J J, Goldberg R J
Framingham Institute for Food Science, Technology and Nutritional Biochemistry, Framingham State College, MA 01701.
J Chromatogr B Biomed Appl. 1994 Oct 3;660(1):180-5. doi: 10.1016/0378-4347(94)00260-6.
The previously reported ability of SP-Sephadex C25 column chromatography for partitioning biologically important cobalamins has been modified to include analytical separation of nitritocobalamin (NO2-Cbl) and nitrosocobalamin (NO-Cbl). Gel column dimensions (1.5 x 11.0 cm), a low eluent flow-rate (125 microliters/min), collection of small eluate fractions (160 microliters) plus maintenance of He saturated mobile and gel phases all combined to eliminate ordinarily confusing proximal elution of NO2-Cbl and NO-Cb1 with sulfitocobalamin (SO3-Cbl) and cyanocobalamin. Cobalamin elution profiles from the gel column were monitored by direct radiometric analysis of 57Co-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-Cb1 normally coexisting in prepared NO2-Cbl standards.
先前报道的SP-葡聚糖凝胶C25柱色谱法分离具有生物学重要性的钴胺素的能力已得到改进，以包括对亚硝酸钴胺（NO2-Cbl）和亚硝基钴胺（NO-Cbl）的分析分离。凝胶柱尺寸（1.5×11.0厘米）、低洗脱液流速（125微升/分钟）、收集小份洗脱液（160微升）以及保持氦饱和的流动相和凝胶相，所有这些结合起来消除了通常令人困惑的NO2-Cbl和NO-Cbl与亚硫酸钴胺（SO3-Cbl）和氰钴胺的近端洗脱。通过对57Co标记的钴胺素标准品进行直接放射性分析或对样品量高达10.0纳克的钴胺素进行竞争性内因子放射分析，监测凝胶柱上钴胺素的洗脱曲线。未能采用此处详述的色谱条件，完全掩盖了暴露于膳食亚硫酸盐的雏鸡脑组织中与SO3-Cbl共存的NO2-Cbl的分析，并导致在制备的NO2-Cbl标准品中通常共存的NO-Cbl被忽视。
