Vitamin B12-intrinsic factor relationships have been studied with the isolated perfused rat liver technique. Approximately 20 times more vitamin B12 is absorbed by the liver in the presence of intrinsic factor than without it. Rapid and significant transfer of vitamin B12 from liver to bile has been observed. The dose-response curve for this system is similar to that reported for the absorption of vitamin B12 through the intestinal tract of pernicious anemia patients. The order and time of addition of vitamin B12 and intrinsic factor to the perfusing blood is critical for absorption of vitamin B12 by the liver. If vitamin B12 is added first, binding substances in the blood rapidly make vitamin B12 unavailable to intrinsic factor added subsequently. If intrinsic factor is added first, gradual inactivation or removal of intrinsic factor by the liver leads to a much slower decrease in efficiency of absorption of subsequently added vitamin B12. These effects are similar to those reported on absorption of vitamin B12 through the human intestinal tract.

It has been reported that three binders of "7Co BIZ are separated by DEAE-cellulose column chromatography following addition of labeled vitamin to normal serum (1, 2). These binders are known as transcobalamin I1 (TCII), transcobalamin I (TCI) and main protein peak binder (MPPB) . TCII and MPPB have an electrophoretic mobility of ,%globulin, while TCI has the mobility of a-globulin ( 2 ) . The molecular size of MPPB is approximately 120,000, similar to that of TCI, and larger than TCII (36,OOO) ( 2 ) .Further observations reveal that intravenously administered TCII 57C0 BIZ and MPPB 57C0 B12 are cleared rapidly from plasma while T C I 57C0 B12 shows a much slower pattern (2-4). Finkler and Hall (5) reported that HeLa cells take up TCII but not TCI bound TO BIZ. Retief et al. (6) observed that TCII delivers TO BIZ to erythrocytes at a faster rate than TCI. Haught et al. ( 7 ) recently reported that rat T C I I has no function in B12 uptake by perfused rat liver.In this study uptake by isolated perfused rat liver of VO B12 bound to the three binders was investigated. Naterials and Methods. Preparation of L 7 C~ B1! binders. The binder used in each perfusion experiment was prepared from 30 ml of serum, as follows. Serum from normal subjects or pernicious anemia (PA) patients was kept a t -20" before use. 300 pg of TO BIZ (sp act around 200 mCi/mg; purchased from Philips-Duphar, Holland) were adtded/ ml to 30 rnl of thawed serum. The solution was allowed to stand at 37" for at least 15 min, then was dialyzed in the cold room against 0.0175 M sodium phosphate buffer pH 6.3, for 24 hr.DEAE-cellulose, Schleicher and Schuell, No. 70, of ion exchange capacity between 0.90 to 0.95 mEq/g, was packed after proper preparation, into 3 X 6-cm columns ( 2 ) .The following buffers were used for elution a t 4 O and flow rate of 30 ml/hr: 0.0175 AL? sodium phosphate buffer, pH 6.3 (600 ml) ; 0.04 M sodium phosphate buffer, p H 5.9 (1000 m l ) ; 0.1 M sodium phosphate buffer, pH 5.8 (500 ml); and 0.4 M sodium phosphate buffer, p H 5.2 (700 ml). Buffer sohtions were made in distilled water containing 0.09% methylparaben and 0.01 % propylparaben (Tenneco Chemicals). TCII was eluted with the 0.04 M buffer, MPPB with the 0.1 M buffer and TCI with the 0.4 M buffer (2). Eluates containing binders were dialyzed and freeze dried, No further attempt a t purification was made.Liver perfusion. Rat liver perfusions were carried out as 2 hr experiments according to the procedure described by Miller et al.

No abstract

No abstract

No abstract