Enzymatic extraction of cobalamin from monoclonal antibody captured haptocorrin and transcobalamin.
OBJECTIVE
Current extraction methods for cobalamins from serum influence the molecular characteristics of the vitamin. Therefore, an extraction procedure that leaves the cobalamins unchanged is needed.


METHODS
Monoclonal antibodies towards transcobalamin (TC) and haptocorrin (HC) (in house) linked to magnetic microspheres were used for precipitation of the proteins. The cobalamins were subsequently released by degradation of TC or HC with subtilisin Carlsberg (EC.3.4.21.14).


RESULTS
Recovery for the extraction of (57)Co-cyanocobalamin ((57)Co-hydroxycobalamin) was higher than 99 (93)% and 92 (93)% from TC and HC respectively (n=10 (3)). No change in retention time and recovery was observed for (57)Co-hydroxycobalamin analyzed with HPLC before and after extraction with subtilisin Carlsberg. Endogenous cobalamin in serum was extracted from TC and HC and measured with routine methods. Enzymatic extracted cobalamin constituted 118-187% of that measured in serum with routine methods (n=16).


CONCLUSIONS
We present a method that allows extraction of cobalamins from their binding proteins without modifying the vitamin. The procedure may be useful especially for extractions aiming at characterizing the cobalamins bound to individual cobalamin binding proteins.
Hardlei
Tore Forsingdal
TF
Department of Clinical Biochemistry, AS, Aarhus University Hospital, Norrebrogade 44, DK 8000 Aarhus, Denmark. thard@as.aaa.dk
Morkbak
Anne Louise
AL
Nexo
Ebba
E
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002918:Chymotrypsin / Q000378:metabolism; D003037:Cobalt Radioisotopes / Q000302:isolation & purification; D019286:Endopeptidase K / Q000378:metabolism; D005260:Female; D051379:Mice; D013381:Subtilisins / Q000378:metabolism*; D013820:Thermolysin / Q000378:metabolism; D014155:Transcobalamins / Q000276:immunology* / Q000378:metabolism*; D014805:Vitamin B 12 / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D003037:Cobalt Radioisotopes; D014155:Transcobalamins; D013381:Subtilisins; D002918:Chymotrypsin; D019286:Endopeptidase K; D013820:Thermolysin; D014805:Vitamin B 12