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

Abstract

AbstractBackground: Transcobalamin (TC) and haptocorrin (HC) are serum corrinoid–binding proteins. We developed new methods for measurement of the corrinoids bound to HC and TC.Methods: TC (n = 10) or HC (n = 138) was immunoprecipitated, and corrinoids were released by enzymatic degradation [subtilisin Carlsberg (EC 3.4.21.62)] of the binding proteins. Binding of the released corrinoids to added unsaturated TC (apoTC) or HC (apoHC) created holoTC (as measure of cobalamins) and holoHC (as measure of corrinoids). holoTC and holoHC were measured by use of ELISA. The amounts of analogs were calculated as the difference between corrinoids and cobalamins. Corrinoids extracted from HC were separated with HPLC after addition of potassium cyanide (n = 3).Results: The corrinoid- and cobalamin-specific assays had a positive linear relation between analyte concentration and assay signal, detection limits of 8 and 4 pmol/L, and imprecision values (CV) of ≤10% and ≤13% for concentrations between 45–200 and 12–115 pmol/L, respectively. No analogs were bound to serum TC, whereas the mean (95% reference range) for analogs present on HC was 245 (100–380) pmol/L. On HPLC a substantial amount of the analogs showed elution patterns similar to those of dicyanocobinamide.Conclusions: Our methods for measurement of unmodified corrinoids in serum demonstrate that HC carries cobalamin analogs not recognized by TC, and that on HPLC a substantial part of these analogs elute similarly to cobinamide.