intrinsic-factor and cobinamide

We found in four of five pernicious anemia gastric juices a partly degraded R binder which was cobalamin specific and has an apparent molecular weight of 60-70,000 daltons. Twenty-nine to 74% (4.8-27.0 ng/ml) of the corrinoid binding capacity could not be blocked by cobinamide (a noncobalamin corrinoid). The fifth pernicious anemia gastric juice and five nonpernicious anemia gastric juices had minimal amounts of this binder (2.5 and 2.2 +/- 1.4 ng/ml). Scatchard analysis revealed that cobalamin-specific R binder has 1000-fold lower affinity for cobinamide than cobalamin. Increasing quantities of trypsin and/or chymotrypsin digested increasing amounts of saliva R binder and an increasing percentage of the remaining digest-resistant R binder acquired cobalamin specificity. Partly degraded R binder in pernicious anemia gastric juice was resistant to further proteolysis. Cobalamin-specific R binder, perhaps produced in vivo by the action of refluxed pancreatic enzymes on swallowed R would preferentially bind ingested and/or biliary cobalamin rather than analogue and thereby could play a role in hastening the development of cobalamin deficiency in pernicious anemia.

Topics: Anemia; Anemia, Pernicious; Chromatography, Gel; Cobamides; Gastric Juice; Humans; Intrinsic Factor; Molecular Weight; Pancreas; Receptors, Cell Surface; Saliva; Vitamin B 12

In a study from four laboratories using two commercial vitamin B12 radioassays (impure hog intrinsic factor concentrate containing both intrinsic factor and R binders (IF + R) to measure total corrinoids and the same concentrate presaturated with cobinamide (Cbi) to block B12 binding sites on R binder (IF + R + Cbi) to measure only cobalamins), the rank order of results was generally the same. The concordance between the two tests for classifying sera as normal or deficient was 91% in 311 serum samples. Three percent of sera below the "true B12" (B12 binding to IF + R + Cbi) normal cut-off point were not below the cut-off point for normal "total B12" (B12 binding to IF + R); 6% of sera below the total B12 normal cut-off point were not below true B12 cut-off point. The correlations between Euglena gracilis and the radioassays were 0.80 and 0.83 in the 50 serum samples that also had E. gracilis serum vitamin B12 levels. Lactobacillus leichmannii serum vitamin B12 levels were determined in 49 of the 311 serum samples and results were comparable with results obtained by four radioassay binder systems: IF + R, IF + R + Cbi, highly purified hog IF, and saliva R binder. The closest correlate with L. leichmannii was radioassay using IF + R as binder (r = 0.93), then IF + R + Cbi (r = 0.92), pure IF (r = 0.80), and pure R (r = 0.73). The key to reliable results appears not to reside in a particular assay but rather in determining for each assay its own range of results in participants determined clinically and morphologically normal vs. participants with deficient vitamin B12 (with B12 deficiency defined independently of a serum B12 assay). When laboratory assay results differ from clinical judgment, further evaluation is the appropriate course. There is no "gold standard" for human serum vitamin B12 assay.

Topics: Biological Assay; Cobamides; Diagnostic Errors; Humans; Intrinsic Factor; Radioisotope Dilution Technique; Reagent Kits, Diagnostic; Reference Standards; Vitamin B 12

Topics: Anemia, Pernicious; Cobamides; Humans; Intrinsic Factor

Recent evidence (Kolhouse et al., N. Engl. J. Med. 299: 785-792, 1978) demonstrates that commercial cobalamin (Vitamin B12) radioassay kits contain nonspecific R-protein binding agents that can give falsely normal results in patients who are actually cobalamin deficient. We tested three kits: with "purified" intrinsic factor as the binder, with intrinsic factor and the nonspecific R-protein sites blocked with "cobinamide," and non-purified intrinsic factor-R-protein binder. Results with use of the first two compared well with those by a microbiological assay (Lactobacillus leichmannii) and are in harmony with clinical impressions.

Topics: Biological Assay; Carrier Proteins; Cobamides; Humans; Intrinsic Factor; Lactobacillus; Radioimmunoassay; Reagent Kits, Diagnostic; Transcobalamins

In vitro studies indicate that [(57)Co]cobalamin (Cbl) is preferentially bound to salivary R protein as opposed to intrinsic factor (IF) and that [(57)Co]Cbl bound to R protein is not transferred to IF at either pH 2 or pH 8. Incubation of R protein-[(57)Co]Cbl with pancreatic proteases causes a partial degradation of the R protein moiety and a rapid transfer of [(57)Co]Cbl to IF. We have postulated that the etiology of Cbl malabsorption in pancreatic insufficiency is an inability to partially degrade R protein because of a lack of pancreatic proteases. We have tested this hypothesis by determining the ability of a nonradioactive Cbl analogue, bound with high affinity by R protein but not by IF, to correct the malabsorption of [(57)Co]Cbl in patients with pancreatic insufficiency.R protein bound the Cbl analogue known as cobinamide with affinities that were the same and only 14-fold lower than those for Cbl at pH 8 and pH 2, respectively. Cobinamide was bound by IF with affinities that were 600,000- and 10,000-fold lower than those for Cbl at pH 8 and 2, respectively. The addition of 125 pmol of nonradioactive cobinamide to 0.5 pmol of [(57)Co]Cbl before being added to 1 pmol of R protein and 1 pmol of IF, markedly inhibited the ability of R protein to compete with IF for binding the [(57)Co]Cbl. Similar results were obtained with freshly aspirated gastric juice. This change was essentially indistinguishable from that observed previously when R protein or R protein-[(57)Co]Cbl was incubated in vitro with trypsin. The oral administration of 100 nmol of nonradioactive cobinamide in Schilling tests was equivalent to trypsin in its ability to completely correct the malabsorption of 0.4 nmol of [(57)Co]Cbl in three patients with pancreatic insufficiency. The fact that both trypsin and nonradioactive cobinamide inhibit the ability of R protein to compete with IF for [(57)Co]Cbl binding in vitro, and correct the mal-absorption of [(57)Co]Cbl in patients with pancreatic insufficiency in vivo, supports our hypothesis that the primary defect in Cbl absorption in this disease is an inability to partially degrade R protein because of a lack of pancreatic proteases.

Topics: Adolescent; Adult; Binding, Competitive; Carrier Proteins; Cobamides; Female; Humans; Intestinal Absorption; Intrinsic Factor; Male; Middle Aged; Pancreatic Diseases; Schilling Test; Transcobalamins; Trypsin; Vitamin B 12