Tore Forsingdal Hardlei1, Ebba Nexo. 1. Department of Clinical Biochemistry, AS, Aarhus University Hospital, Aarhus, Denmark. tore.forsingdal.hardlei@ki.au.dk
Abstract
BACKGROUND: 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 <or=10% and <or=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.
BACKGROUND: 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 <or=10% and <or=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.
Authors: Tito Borner; Jayme L Workinger; Ian C Tinsley; Samantha M Fortin; Lauren M Stein; Oleg G Chepurny; George G Holz; Aleksandra J Wierzba; Dorota Gryko; Ebba Nexø; Evan D Shaulson; Ankur Bamezai; Valentina A Rodriguez Da Silva; Bart C De Jonghe; Matthew R Hayes; Robert P Doyle Journal: Cell Rep Date: 2020-06-16 Impact factor: 9.423
Authors: Michael J Dunphy; Annette M Sysel; Joseph A Lupica; Kristie Griffith; Taylor Sherrod; Joseph A Bauer Journal: Chromatographia Date: 2014-04-01 Impact factor: 2.044
Authors: Katrine Hygum; Dorte L Lildballe; Eva H Greibe; Anne L Morkbak; Steen S Poulsen; Boe S Sorensen; Torben E Petersen; Ebba Nexo Journal: PLoS One Date: 2011-05-31 Impact factor: 3.240