Absorption and retention of free and milk protein-bound cyano- and hydroxocobalamins. An experimental study in rats.

INTRODUCTION: Cobalamin/Vitamin B12 (Cbl) is an essential vitamin, supplied mainly as hydroxocobalamin (OHCbl) by animal products, including cows' milk. Cyanocobalamin (CNCbl) is the usual form in vitamin pills. The aim was to explore absorption and tissue accumulation of two Cbl forms, administered alone or bound to milk protein. MATERIALS AND METHODS: We synthesized labeled OH[(57)Co]Cbl from commercially available CN[(57)Co]Cbl. Recombinant bovine transcobalamin (rbTC) was produced in yeast and skimmed milk obtained off the shelf. Male Wistar rats (250-300 g) received labeled Cbl by gastric gavage. First, we administered CN[(57)Co]Cbl, free or rbTC-bound (n = 15 in each group). Rats were sacrificed after two, 24, and 48 h. In the following studies, rats were sacrificed after 24 h. We compared absorption of free or rbTC-bound CN[(57)Co]Cbl added to cows' milk and analogous absorption of OH[(57)Co]Cbl, free or rbTC-bound, to absorption of free CN[(57)Co]Cbl, (n = 10 in each group). Blood, tissues, 24-h urine and feces were collected. Labeled Cbl was measured using a gamma counter. Results are expressed as percentage of administered dose. RESULTS: Absorptions of CNCbl and OHCbl were neither influenced by rbTC-binding nor administration in milk. Absorption increased in the first 24 h with no further tissue accumulation during the subsequent 24 h. Accumulation of free CNCbl and (OHCbl) was 1.4, (4.1) (liver); 20.2, (16.4) (kidney); and 0.05, (0.02) (plasma)% 24 h after administration. Total organ accumulations were 21.6, (20.5)%. While total accumulations of CNCbl and OHCbl were equal, distributions between liver, kidney, and plasma showed significant differences (p < 0.0001; p = 0.01; p < 0.0001). CONCLUSIONS: Cbl added to milk (spiked with rbTC) has high bioavailability matching that of free Cbl. OHCbl and CNCbl are absorbed equally well, but much more OHCbl accumulated in the liver. Benefits of oral supplementation with OHCbl compared to CNCbl should be investigated.

Transcobalamin derived from bovine milk stimulates apical uptake of vitamin B12 into human intestinal epithelial cells.

Intestinal uptake of vitamin B12 (hereafter B12) is impaired in a significant proportion of the human population. This impairment is due to inherited or acquired defects in the expression or function of proteins involved in the binding of diet-derived B12 and its uptake into intestinal cells. Bovine milk is an abundant source of bioavailable B12 wherein it is complexed with transcobalamin. In humans, transcobalamin functions primarily as a circulatory protein, which binds B12 following its absorption and delivers it to peripheral tissues via its cognate receptor, CD320. In the current study, the transcobalamin-B12 complex was purified from cows' milk and its ability to stimulate uptake of B12 into cultured bovine, mouse and human cell lines was assessed. Bovine milk-derived transcobalamin-B12 complex was absorbed by all cell types tested, suggesting that the uptake mechanism is conserved across species. Furthermore, the complex stimulated the uptake of B12 via the apical surface of differentiated Caco-2 human intestinal epithelial cells. These findings suggest the presence of an alternative transcobalamin-mediated uptake pathway for B12 in the human intestine other than that mediated by the gastric glycoprotein, intrinsic factor. Our findings highlight the potential for transcobalamin-B12 complex derived from bovine milk to be used as a natural bioavailable alternative to orally administered free B12 to overcome B12 malabsorption.

Potential host-defense role of a human milk vitamin B-12-binding protein, haptocorrin, in the gastrointestinal tract of breastfed infants, as assessed with porcine haptocorrin in vitro.

BACKGROUND: Limited information exists on the biological role of a vitamin B-12-binding protein, haptocorrin, in human milk. The expression of haptocorrin by human mammary epithelial cells and its presence in human milk suggest a potential physiologic function in breastfed infants. OBJECTIVE: We investigated the extent to which haptocorrin could withstand proteolytic degradation and exert antimicrobial activity under in vitro conditions designed to simulate the gastrointestinal tract of breastfed infants. DESIGN: An in vitro model that simulates infant gastric and intestinal digestion was developed. The structural stability of porcine haptocorrin after exposure to digestive enzymes (pepsin and pancreatin) was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot analysis, column chromatography, and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The antimicrobial activity of haptocorrin was determined by incubating haptocorrin with enteropathogenic Escherichia coli O127 strain 2348/69 and monitoring bacterial growth. RESULTS: The structural analysis of haptocorrin exposed to enzymes did not show a decrease in molecular weight, which indicated that haptocorrin can survive proteolytic degradation. Both haptocorrin exposed to digestive enzymes and undigested haptocorrin inhibited the growth of enteropathogenic E. coli and did so to a similar extent. Thus, haptocorrin in vitro not only retains its structure after exposure to proteases but also exhibits antimicrobial activity. CONCLUSION: These results suggest that haptocorrin may exert a host-defense function against pathogens in the gastrointestinal tracts of breastfed infants.

Synthesis and secretion of transcobalamin II by cultured astrocytes derived from human brain tissue.

Astrocytes derived from human brain tissue secreted a single cobalamin (vitamin B12, Cbl) binding protein over a 4 day period in culture. Cycloheximide reversibly inhibited the release, and the binding protein was identified as transcobalamin II (TCII) based on molecular size, reaction with anti-human TCII antiserum, precipitation with 2.0 M ammonium sulfate and its ability to bind radioactive cyanocobalamin. It also enhanced the cellular incorporation of the vitamin. Our data show that cultured cells from human brain synthesize and secrete TCII and suggests that at least some of the TCII known to be present in cerebrospinal fluid may originate from within the central nervous system.

Effects of cobalamin, cobalamin analogues and cobalamin binding proteins on P388D1 mouse leukemic cells in culture.

Cobalamin-deficient P388D1 mouse leukemic cells were created by propagation in a cyanocobalamin-free medium in which the original fetal bovine serum was replaced by bovine serum albumin. These cobalamin-deficient cells gradually ceased to multiply when the medium contained 5-methyltetrahydrofolate. The growth of cells that had been cultured with this coenzyme was recovered following the addition of cyanocobalamin (CNCbl), at concentrations above 37 pM. In contrast to the effect of CNCbl, cobinamide, and cobalamin analogues prepared from hydroxy cobalamin by reaction with ascorbic acid, did not have a growth-inducing effect on these cells, nor did these analogues inhibit CNCbl-dependent growth. Transcobalamin II-cobalamin complex had a remarkably stimulating effect on cell growth. The growth inducing effect became apparent with a cobalamin concentration of only 0.37 pM. This was about 1/100th the level of free cobalamin required for cell growth. However, no growth-inducing effect was seen at an R protein-bound cobalamin concentration of 37 pM, indicating that once cobalamin has been bound to R protein, it loses its growth-promoting effect on these cells in culture.

Expression of transcobalamin II receptors by human leukemia K562 and HL-60 cells.

Plasma membrane receptors for the serum cobalamin-binding protein transcobalamin II (TCII) were identified on human leukemia K562 and HL-60 cells using immunoaffinity-purified human TCII labeled with [57Co]cyanocobalamin. The Bmax values for TCII receptors on proliferating K562 and HL-60 cells were 4,500 and 2,700 per cell, respectively. Corresponding dissociation constants (kd) were 8.0 x 10(-11) mol/L and 9.0 x 10(-11) mol/L. Rabbit TCII also bound to K562 and HL-60 cells but with slightly reduced affinities. Calcium was required for the binding of transcobalamin II to K562 cells. Brief treatment of these cells with trypsin resulted in almost total loss of surface binding activity. After removal of trypsin, surface receptors for TCII slowly reappeared, reaching pretrypsin treatment densities only after 24 hours. Reappearance of receptors was blocked by cycloheximide. TCII receptor densities on K562 and HL-60 cells correlated inversely with the concentration of cobalamin in the culture medium. This suggests that intracellular stores of cobalamin may affect the expression of transcobalamin receptors. Nonproliferating stationary-phase K562 cells had low TCII receptor densities (less than 1,200 receptors/cell). However, the density of TCII receptors increased substantially when cells were subcultured in fresh medium. Up-regulation of receptor expression coincided with increased 3H-thymidine incorporation, which preceded the resumption of cellular proliferation as measured by cell density. In the presence of cytosine arabinoside, which induces erythroid differentiation, K562 cells down-regulated expression of TCII receptors. When HL-60 cells were subcultured in fresh medium containing dimethysulfoxide to induce granulocytic differentiation, the up-regulation of TCII receptors was suppressed. This event occurred well before a diminution of 3H-thymidine incorporation and cessation of proliferation. Thus, changes in the regulation of expression of TCII receptors correlate with both the proliferative and differentiation status of cells.

The release of granule components from human polymorphonuclear leukocytes in response to both phagocytic and chemical stimuli.

The differential effects of phagocytic and chemical stimuli on neutrophil enzyme and specific protein release were compared. Phorbol myristate acetate (PMA) stimulated release of the specific granule matrix marker, vitamin B-12-binding protein in a dose-dependent manner. Subcellular fractionation by sucrose density gradient centrifugation indicated that the residual vitamin B-12-binding protein is associated with the specific granule fraction. In contrast, neutral alpha-glucosidase and adenosine diphosphatase, associated with specific granule membranes, were not released by PMA. Subcellular fractionation studies suggest that fusion of the specific granule membrane and plasma membrane occurs, thus translocating the adenosine diphosphatase to the cell surface. The relevance of this finding to the possible role of nucleoside phosphatases in limiting platelet aggregation is discussed. Serum-treated zymosan particles also caused a selective release of vitamin B-12-binding protein from the specific granule without release of alpha-glucosidase and adenosine diphosphatase. Neither PMA nor opsonized zymosan caused significant release of azurophil, tertiary granule or cytosol marker enzymes.

Mechanism of uptake of cobalamin into enterocyte mitochondria.

To study the mechanism which cobalamin is taken up by the mitochondria, 57Co-cyanocobalamin-binder complex and freshly prepared mitochondria were prepared from the enterocytes of rats. Subsequently, the binder complex was incubated together with mitochondria in a calcium-containing medium in vitro. Uptake of cobalamin was determined by measuring the radioactivities bound to the mitochondria. Consequently, lysosomal and microsomal binders enhanced cobalamin uptake into the mitochondria, but intrinsic factor did not. It was found that the uptake into the mitochondria was inhibited by previous treatment with calcium-chelating agents. The uptake was completely restored after addition of calcium ions to the mitochondria.

The effect of digestive enzymes on the binding and bacteriostatic properties of lactoferrin and vitamin B12 binder in human milk.

Human milk contains unsaturated lactoferrin and vitamin B12 binding protein. It has been suggested that these proteins may exert antibacterial effects in the intestine of the breast fed infant, but the effect of the intestinal environment on the antibacterial effect of these proteins has not been described. In this study human milk was treated with pepsin and trypsin and the influence of digestion on iron and vitamin B12 binding capacity, bacterial uptake of iron and vitamin B12 from milk and bacteriostatic effect was studied. Pepsin digestion had no effect on vitamin B12 binding capacity, or the ability of bacteria to take up vitamin B12, or the growth inhibitory effect on a vitamin B12 dependent strain. In contrast, trypsin digestion did not affect iron binding or bacteriostatic effects attributable to lactoferrin. The. findings support an in vivo bacteriostatic role for lactoferrin in the breast fed neonate's intestine but do not support a similar role for the vitamin B12 binding protein.