This chapter discusses the determination of cobalamins in biological material, and mainly concerns with “true cobalamins.” In animal tissues, five different cobalamins have been identified in cells and extracellular fluids: MeCbl, AdoCbl, OH-Cbl, CN-Cbl, and HSO3-Cbl. In addition, several cobalamin-like substances (i.e., other corrinoids and cobalamin analogs) are found in animal and bacterial materials under various conditions. The major aims of a cobalamin determination are (1) the accurate estimation of total cobalamin content, and (2) estimation of the relative amount of each cobalamin form. The determination of cobalamins from certain compartments and speculatively extrapolate these principles to other compartments are discussed in the chapter. The assay depends on a preliminary extraction of cobalamins from tissue or fluid, further isolation and/or purification of the cobalamins before resolution of the different forms, and quantification of each form. Two methods are presented in the chapter. One, using thin-layer chromatography (TLC), is a refinement of earlier methods. The other employs high performance liquid chromatography (HPLC). The recovery of each cobalamin form from each compartment should be complete in the ideal assay. Since unknown biological materials may contain cobalamin-binding substances of varying stability in the extraction procedure, some compartments may be overestimated at the expense of other compartments. For example, cobalamins bound to transcobalamin are liberated easily by acidification, while their liberation from haptocorrin requires boiling in addition. Thus recovery studies are essential when examining biological materials that might contain uncharacterized cobalamin-binding proteins. Series: Methods in Enzymology (Book 123) Hardcover: 512 pages Publisher: Academic Press; 1 edition (April 11, 1986) Language: English ISBN-10: 0121820238 ISBN-13: 978-0121820237 Product Dimensions: 6 x 1.1 x 9 inches Link Download http://nitroflare.com/view/C37C271F97C172Ehttps://drive.google.com/drive/folders/1yLBzZ1rSQoNjmWeJTZ3WGQHg04L1
