Vitamin B12-binding Protein Research Articles

Combined congenital deficiencies of intrinsic factor and R binder

Coexisting deficiencies of both intrinsic factor (IF) and R binder were identified in an Algerian boy who presented with severe megaloblastic anemia, growth retardation, and neurologic dysfunction with typical features of subacute combined degeneration of the spinal cord. The anemia responded completely to cyanocobalamin and folic acid. IF was absent from gastric juice, but acid secretion and gastric mucosa were normal. R binders were absent from gastric juices as well as from serum, saliva, and polymorphonuclear leukocytes. The patient's father exhibited absence of R binder in his serum with a low serum vitamin B12 level and was asymptomatic. This unique case of simultaneous IF and R binder deficiencies suggests a genetic association between these two functionally and immunologically dissimilar, but structurally close vitamin B12-binding proteins.

Amniotic fluid folate, vitamin B12 and transcobalamins in neural tube defects

Levels of folate, vitamin B12, the vitamin B12 binding proteins, apotranscobalamin I, II and III (TC I, II and III) and the unsaturated vitamin B12 binding capacity (UBBC) were measured in mid-trimester amniotic fluids from normal pregnancies, and from those where the fetus had open spina bifida, anencephaly or omphalocoele, and where the fetus was normal but the mother had had a previous neural tube defect pregnancy. At 15-19 weeks' gestation, vitamin B12 levels were low in the fluids of all the types of abnormal fetuses, and also of normal fetuses where there had been a previous NTD sib. In contradistinction, TC I, II and III and UBBC levels were generally abnormally high in all these groups. Low vitamin B12 levels in the face of high carrier protein levels suggest deranged vitamin B12 production or transport. Since these abnormalities are present in fluids from normal sibs of NTD individuals as well as from those with midline lesions, an inherited defect is implied. We propose that at least part of the genetic predisposition to NTD, and possibly other midline defects, could reside in an abnormality connected with vitamin B12 production, transport or metabolism, and a mechanism is suggested.

Bacterial Lipopolysaccharide Enhances Chemoattractant-Induced Elastase Secretion by Human Neutrophils

Bacterial lipopolysaccharide (LPS) has previously been shown to enhance a number of chemoattractant-induced responses by human neutrophils. The possible role of elastase, a neutral protease with broad substrate specificity, in neutrophil-mediated vascular injury of a variety of diseases prompted us to examine a) whether or not LPS enhances the direct chemoattractant-induced secretion of elastase, b) the quantitative requirements of LPS and chemotactic factors, and c) some structural requirements of LPS for this effect. Our results show that LPS at 10 ng/ml and above, enhanced formyl-methionyl-leucyl-phenylalanine-induced neutrophil secretion of elastase, as well as secretion of myeloperoxidase and vitamin B12-binding protein. This effect was independent of cytochalasins or surface stimulation, and thus may occur during chemotactic factor stimulation in vivo. LPS also enhanced neutrophil secretory responses to the complement fragments C5a, C5a des arg, and, to a lesser degree, to leukotriene B4 and platelet-activating factor. This enhancement effect appeared to require the presence of the lipid A moiety and/or parts of the core polysaccharide but not the O-antigen portion of the LPS molecule. Our findings identify a possible LPS-dependent mechanism of neutrophil elastase-mediated tissue injury in Gram-negative infections.

Type 1 fimbriate Escherichia coli stimulates a unique pattern of degranulation by human polymorphonuclear leukocytes.

Uropathogenic strains of Escherichia coli bearing mannose-sensitive (type 1) fimbriae promote a unique pattern of degranulation from human polymorphonuclear leukocytes (PMN). Significant quantities of the primary (1 degree) and tertiary (3 degree) granule markers, neutral protease-myeloperoxidase and N-acetyl-beta-D-glucosaminidase, respectively, were released by PMN in a dose- and time-dependent manner when stimulated by these defined bacterial strains. Organisms bearing mannose-resistant (P) fimbriae promoted release of only the secondary (2 degree) granule marker, vitamin B12-binding protein. When this pattern of degranulation was compared to that produced by PMN in response to a variety of soluble and particulate stimuli, only the calcium ionophore A23187 similarly triggered 1 degree and 3 degree granule marker release. All the other stimuli tested--zymosan, serum-treated and unopsonized; n-formylmethionyl-leucyl-phenylalanine; and phorbol myristate acetate--promoted release of only the 2 degree granule marker. These results demonstrate selectivity of PMN degranulation in response to a number of transmembrane signals. In addition, the capacity of E. coli to promote PMN degranulation is dependent on its phenotypic fimbrial expression, a surface characteristic which correlates significantly with its relative surface hydrophobicity as measured by binding to octyl Sepharose. Those bacteria demonstrating the greatest hydrophobicity were capable of triggering discharge of all three granule marker proteins. Thus, the mannose-sensitive fimbriae of uropathogenic E. coli may contribute significantly to their potential pathophysiologic role in renal scarring.

Heteronuclear NMR studies of cobalamins. 31P NMR observations of cobalamins bound to a haptocorrin from chicken serum.

A vitamin B12-binding protein (haptocorrin) from chicken serum has been purified to homogeneity by photodissociative affinity chromatography and characterized by gel electrophoresis and UV-visible spectrophotometry of its aquocobalamin, hydroxocobalamin, and cyanocobalamin complexes. The haptocorrin is a glycoprotein with a molecular mass of about 70 kDa and a protein moiety of about 40 kDa. 31P NMR resonances of the haptocorrin-cobalamin complexes are relatively broad singlets (with or without proton decoupling) shifted downfield by 0.7-1.0 ppm from the position of the free cobalamin resonances. From the line width data, the relaxation of the phosphorus nucleus is found to be dominated by chemical shift anisotropy with a very minor (13%) component from dipolar interaction with the two nearest neighbor protons. The rotational correlation time of the haptocorrin at 25 degrees C is estimated to be 85 ns and the activation energy for rotational correlation 3.9 +/- 0.3 kcal mol-1. The downfield shift of the 31P resonances of cobalamins upon binding to the haptocorrin cannot be due to hydrogen bonding phosphodiester moiety or displacement of the axial base by a group on the protein. Calculations also show that the downfield shift is very unlikely to be due to dipolar deshielding of the phosphorus nucleus by the ring current of an aromatic residue of the protein. It is concluded that the downfield shift of the 31P resonance must be due to sterically induced changes in phosphodiester conformation which may, or may not, involve steric compression of the axial Co-N bond.

Effects of storage and radiation on human neutrophil function in vitro

To better understand the process of time-related functional deterioration which occurs in human polymorphonuclear leukocytes (PMNs), we examined the effects of in vitro storage on multiple functional parameters of human PMNs. Single-donor, phlebotomy-collected PMNs were stored at both room temperature and 37 degrees C for 24 and 48 h, then compared to fresh cells from the same donor. Similar numbers of cells were recovered from each storage condition. Cell viability decreased after 37 degrees C storage for 48 h. Cells stored at room temperature for 24 h showed significant depression of multiple functions (bactericidal activity, chemotaxis, aggregation, superoxide production, and oxygen consumption) compared to fresh cells. They contained less vitamin B12 binding protein activity than fresh cells, and by fluorescence-activated cell-sorter analysis, their forward light scatter and membrane depolarization responses were abnormal. For all parameters examined, cells stored at 37 degrees C were more abnormal than cells stored at room temperature. Stored cells from a patient with myeloperoxidase deficiency lost bactericidal and chemotactic activity after storage at 37 degrees C for 24 h, but cells from a patient with chronic granulomatous disease retained their original bactericidal and chemotactic activity after 37 degrees C storage for 24 h. Radiation, in doses used to prevent graft vs. host disease in leukocyte-transfusion recipients (2500-5000 rads) caused a significant decrease in the mean percentage of continuous flow centrifugation leukapheresis (CFCL) collected PMNs capable of reducing nitroblue tetrazolium. Human PMNs show deterioration of multiple in vitro functions when they are stored and are susceptible to damage by radiation when they are collected by CFCL.

Peroxidase-containing microgranules in human neutrophils: physical, morphological, cytochemical, and secretory properties

A microgranule fraction, isolated from human neutrophils by using a novel high-resolution Percoll density gradient system contained granules with the lowest density and diameter when compared with 12 other isopycnic granule fractions. Ultrastructurally, from 34% to 50% of the microgranules showed homogeneous diaminobenzidine (DAB) staining under conditions for localizing peroxidase reactivity. The presence of myeloperoxidase (MPO) was further confirmed by biochemical and spectral analysis and immunodiffusion methods. Periodate-thiocarbohydrazide- silver proteinate (PA-TCH-SP) intensely stained vicinal glycols in the matrix of greater than 97% microgranules in contrast to the weak or absent staining seen in larger primary granules. Directly sampled segmented neutrophils contained small DAB- and PA-TCH-SP-positive granules, which often appeared in clusters. These DAB-positive microgranules selectively remained within the cells after stimulation of exocytosis with the calcium ionophore A23187. The enriched DAB- positive microgranule fraction recovered from A23187-treated cells also contained lysozyme and beta-glucuronidase but lacked vitamin B12 binding protein activity. A similar small, DAB- and PA-TCH-SP-positive granule type was also identified in normal promyelocytes and was the predominant or only granule type observed in leukemic or preleukemic myeloid cells from four patients. This study demonstrates a unique subpopulation of MPO-containing microgranules in normal and leukemic human myeloid cells that are distinguished from (other) primary granules by their extremely low density, small size, content of complex carbohydrates, and resistance to secretion.

Peroxidase-Containing Microgranules in Human Neutrophils: Physical, Morphological, Cytochemical, and Secretory Properties

A microgranule fraction, isolated from human neutrophils by using a novel high-resolution Percoll density gradient system contained granules with the lowest density and diameter when compared with 12 other isopycnic granule fractions. Ultrastructurally, from 34% to 50% of the microgranules showed homogeneous diaminobenzidine (DAB) staining under conditions for localizing peroxidase reactivity. The presence of myeloperoxidase (MPO) was further confirmed by biochemical and spectral analysis and immunodiffusion methods. Periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) intensely stained vicinal glycols in the matrix of greater than 97% microgranules in contrast to the weak or absent staining seen in larger primary granules. Directly sampled segmented neutrophils contained small DAB- and PA-TCH-SP-positive granules, which often appeared in clusters. These DAB-positive microgranules selectively remained within the cells after stimulation of exocytosis with the calcium ionophore A23187. The enriched DAB-positive microgranule fraction recovered from A23187-treated cells also contained lysozyme and beta-glucuronidase but lacked vitamin B12 binding protein activity. A similar small, DAB- and PA-TCH-SP-positive granule type was also identified in normal promyelocytes and was the predominant or only granule type observed in leukemic or preleukemic myeloid cells from four patients. This study demonstrates a unique subpopulation of MPO-containing microgranules in normal and leukemic human myeloid cells that are distinguished from (other) primary granules by their extremely low density, small size, content of complex carbohydrates, and resistance to secretion.

Development of the phagocytic and cidal capacity during maturation of myeloid cells: studies on cells from patients with chronic myelogenous leukaemia.

Myeloid cells from peripheral blood of patients with chronic myelogenous leukaemia were isolated and fractionated by density gradient centrifugation using Lymphoprep gradient followed by discontinuous Percoll gradients. Six fractions were obtained, each enriched in one of the morphologically identifiable types of myeloid cells from myeloblasts to polymorphonuclear neutrophils. Each of these cell types were functionally and biochemically characterized. The development of the capacities for phagocytosis and killing of yeast cells and the ability to generate a respiratory burst of phagocytosis correlated closely with the content of cytochrome b and vitamin B12-binding protein, a marker of specific granules. These results support the notion that the specific granules provide the developing neutrophil with components which are essential for its microbicidal activity.

The 1986 Herman award lecture. Nutrition science as a continually unfolding story: the folate and vitamin B-12 paradigm

An award address presents a comprehensive technical review discusses the current state of knowledge concerning the development of nerve damage in vitamin B12 deficiency and its alleviation by folic acid. Specific attention is given to: the folate and vitamin B12 paradigm: the iron mineral model and the importance of moderation in supplementation; folate and vitamin B12 models; observations that laboratory test results become abnormal for the individual before it exceeds the range of normal for the laboratory; vitamin B12 transport; the confounding variable of iron deficiency; the absorption of vitamin B12 and its analogues; radioassays for cobalamins and noncobalamin corrinoids; the argument that vitamin B12 in food and that synthesized is not the same; the role of liver receptors for beta-galactosyl end groups of glycoproteins; vitamin B12-binding protein research involving the Australia antigen; and the sequence of events in folate deficiency etiology. Controversies in developing minimal and maximal daily requirements for folate and vitamin B12 also are discussed.(wz)

Oxidative inactivation of myeloperoxidase released from human neutrophils.

Within 1 min of stimulation of human neutrophils by the chemotactic peptide (N-formyl-L-methionyl-L-leucyl-L-phenylalanine) plus cytochalasin B, myeloperoxidase (together with other granule enzymes) was secreted and detected extracellularly. In contrast with the other granule constituents assayed (vitamin B12-binding protein and beta-glucuronidase), the activity of released myeloperoxidase rapidly decreased, so that, by 10 min after stimulation, only about 5% of the total cellular activity was detected. This inactivation was shown to be dependent on oxidant generation during the respiratory burst, since inactivation was not observed (a) after stimulation of anaerobic suspensions or (b) after release from neutrophils from a patient with chronic granulomatous disease; purified myeloperoxidase was rapidly inactivated after incubation with H2O2, presumably owing to the formation of an inactive enzyme-H2O2 complex. These results show that experiments designed to assess the role of myeloperoxidase in neutrophil functions which utilize assays based on peroxidase activity will grossly underestimate this enzyme if oxidant generation during the respiratory burst has also been activated.

Human polymorphonuclear neutrophil activation with arachidonic acid.

The capacity of arachidonic acid (AA) to stimulate granule exocytosis from human polymorphonuclear neutrophils (PMNs) was investigated. AA induced the selected extracellular release of azurophil (myeloperoxidase, lysozyme) and specific (lysozyme, vitamin B12 binding protein) granule constituents from human PMNs in a time- and concentration-dependent manner. Cytochalasin B (CB) enhanced but was not required for PMN activation with AA. Although extracellular calcium had no effect on granule exocytosis, AA did stimulate the mobilization of intracellular sequestered Ca2+ which resulted in an increase in cytosolic-free Ca2+ ([Ca2+]i) as reflected by increased fluorescence of Fura-2-treated cells. AA stimulated Ca2+/phospholipid-dependent protein kinase C (PK-C) activity in PMNs. 4,4'-Diisothiocyano-2,2'-disulphonic acid stilbene (DIDS), an anion channel blocker, caused a concentration-dependent inhibition of granule enzyme release. Activation of PMNs with AA was unaffected by the lipoxygenase/cycle-oxygenase inhibitors, 5,8,11, 14-eicosatetraynoic acid (ETYA) and benoxaprofen, a lipoxygenase inhibitor, 6, 9, deepoxy-6,9-(phenylimino) delta 6,8-prostaglandin 1(1) (piriprost potassium) or a pure cyclo-oxygenase inhibitor, flurbiprofen. These data define the properties of AA as a secretory stimulus for human PMNs.

Human neutrophil plasminogen activator is localized in specific granules and is translocated to the cell surface by exocytosis.

The subcellular localization of plasminogen activator (PA) in human neutrophils was studied. The cells were disrupted by nitrogen cavitation and fractionated on Percoll density gradients into three major components containing the plasma membranes, the specific granules, and the azurophilic granules. The biochemical markers we used to identify these organelles were alkaline phosphatase, vitamin B12-binding protein, and beta-glucuronidase, respectively. Using the radioactive fibrin plate method, PA activity and plasminogen-independent fibrinolytic activity were measured. In resting neutrophils, PA was associated mainly with the membranes of the specific granules. In five individual experiments the activity of this fraction varied from 79 to 100% of the total; the remaining activity was found to be associated with the plasma membrane, and no activity was present in the azurophilic granules. In neutrophils that were activated by exposure to PMA (20 ng/ml for 15 min at 37 degrees C), the total recoverable PA activity remained unchanged; however, the main peak of activity (85% of total) shifted from the specific granules to the plasma membranes. The magnitude of the reduction of the enzyme in the specific granules paralleled that of vitamin B12-binding protein. PMA-activated, intact neutrophils had approximately 12-fold more surface-bound PA activity than resting cells. Recovery of PA activity from neutrophils was critically dependent on pretreatment of the intact cells with DFP before cavitation; 100-fold more PA activity was detected in DFP-pretreated cells. At the same time, this pretreatment reduced the plasminogen-independent fibrinolytic activity by approximately sevenfold. We determined that PA present in the neutrophils is of the urokinase (UK) type and that the enzyme is produced and stored as a pro-UK, a form insensitive to DFP inhibition. The reduction in the level of proteases (measured as fibrinolytic activity) and the resistance of pro-UK to DFP are most likely the two major reasons for the greatly improved recovery of PA from the DFP-pretreated cells. These findings show that in resting neutrophils PA is stored in the specific granules, and that during activation, it translocates to the outer surface of the plasma membranes, thus equipping the cell with an ecto-proteolytic potential.

Quantitative analysis of the cytosolic free calcium dependency of exocytosis from three subcellular compartments in intact human neutrophils.

Cytosolic free calcium concentration, [Ca2+]i, and exocytosis of azurophil granules (beta-glucuronidase), specific granules (vitamin B12-binding protein), and secretory vesicles (gelatinase) were measured concomitantly in intact human neutrophils under steady state [Ca2+]i. The cells were loaded with the fluorescent calcium indicator quin2 in the presence or absence of extracellular Ca2+, and steady state [Ca2+]i levels ranging from 20 to greater than 2,000 nM were obtained by adding the Ca2+ ionophore ionomycin at various concentrations of extracellular calcium. The extent of exocytosis from the three granule populations was found to be a function of [Ca2+]i. The minimal [Ca2+]i that caused significant release (threshold [Ca2+]i) was approximately 200-300 nM and was similar for all three compartments. Marked differences, however, were found when the [Ca2+]i for half-maximal exocytosis (EC50) was determined. In the absence of cytochalasin B the EC50 was 1,100 +/- 220 nM and 1,600 +/- 510 nM for specific granules and secretory vesicles, respectively, and approximately 6,000 nM for azurophil granules. Cytochalasin B did not affect the threshold [Ca2+]i but decreased the EC50 and enhanced the rate of exocytosis. In the presence of cytochalasin B the EC50 was approximately 600 nM both for secretory vesicles and specific granules, and approximately 2,600 nM for azurophil granules. The addition of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine dramatically changed the [Ca2+]i dependency of granule secretion: It decreased the threshold [Ca2+]i to less than 20 and less than 50 nM, and the EC50 to 50 and 200 nM for specific and azurophil granules, respectively, and it significantly increased the rate of exocytosis. Thus, the additional signal(s) provided by receptor activation markedly lower(s) the Ca2+ requirement of the exocytotic process. Furthermore, these results indicate that the secretion from three different granule populations within the same cell type are differently modulated by [Ca2+]i.

Metabolic comparison between basophils and other leukocytes from human blood.

Basophilic granulocytes were purified from the blood of normal individuals by successive isopyknic centrifugation and elutriation centrifugation. Starting with the leukocyte-rich fraction of 500 ml of blood, we recovered 31 to 80% (mean 51%, n = 20) of the basophils in 45 to 87% purity (mean 69%, n = 23). The contaminating cells were mainly lymphocytes. The basophils were greater than 98% vital (exclusion of ethidium bromide and hydrolysis of fluorescein diacetate). The histamine content of the basophils was 1.1 to 2 pg/cell (mean 1.6 pg/cell, n = 22). With anti-IgE, 30 to 50% of the histamine was released; with phorbol myristic acetate (PMA) or the calcium ionophore A23187, 70 to 100% of the histamine was released. Serum-opsonized zymosan (STZ) did not induce histamine release. Reactions with monoclonal antibodies revealed that the basophils expressed the C3bi receptor (CR3) and the leukocyte function-associated antigen 1 (LFA1), but not the gp 150,95 antigen, the C3b receptor (CR1), or the low avidity Fc gamma receptor. Basophils carry class I but not class II HLA antigens. During incubation of the basophils with serum-opsonized Staphylococcus aureus or Escherichia coli, these bacteria were neither phagocytized nor killed. STZ, PMA, A23187, or anti-IgE did not initiate an "oxidative burst" in the basophils. This was tested with oxygen consumption, cytochrome c reduction, NBT reduction, chemiluminescence, and release of hydrogen peroxide. Moreover, we did not detect cytochrome b558, superoxide dismutase, catalase, or peroxidase in the basophils. Of the typical granule-associated enzymes lysozyme, Vitamin B12-binding protein, and beta-glucuronidase, only beta-glucuronidase was present in the basophils in detectable amounts. This enzyme was released, together with histamine, on incubation of the cells with PMA, A23187, or anti-IgE, but not with STZ. We conclude that basophils from normal human blood are not phagocytes and are probably not involved in the oxidative defense of the host against foreign antigens.

Exudation primes human and guinea pig neutrophils for subsequent responsiveness to the chemotactic peptide N-formylmethionylleucylphenylalanine and increases complement component C3bi receptor expression.

After circulating in the vascular system a short time, polymorphonuclear leukocytes (PMN) migrate to extravascular sites in response to chemotactic stimuli. Prestimulation of PMN in vitro by secretagogues has been shown to increase their number of N-formylmethionylleucylphenylalanine (fmet-leu-phe) and complement component C3bi (CR3) receptors. We investigated whether the same phenomenon occurred in vivo, comparing characteristics of human skin chamber and guinea pig peritoneal exudate and blood PMN. Exudate PMN of both species contained approximately 28% less of the specific granule marker vitamin B12-binding protein (P less than 0.01) but a similar amount of the azurophil granule marker beta-glucuronidase. The total number of fmet-leu-phe receptors was 5.9 times higher in guinea pig exudate than in blood PMN (P less than 0.01) and 2.9 times higher in human exudate than in blood PMN (P less than 0.02). All exudate PMN and most blood PMN preparations showed a high affinity receptor (Kd approximately 2.3 X 10(-8) M) and a low affinity receptor (approximately 1.5 X 10(-7) M). The upregulation of fmet-leu-phe receptors in exudate PMN correlated with an improved responsiveness to fmet-leu-phe induced membrane depolarization, oxidative metabolism, and chemotaxis. In addition, the concentration of fmet-leu-phe that produced a half-maximal response of chemotaxis, superoxide production, and membrane potential depolarization was 10-fold lower in exudate PMN than in blood PMN. Human exudate PMN had a twofold increased C3bi receptor expression compared with blood PMN. Thus, a preferential loss of specific granules is associated with increased number of high and low affinity fmet-leu-phe receptors and increased C3bi receptor expression not only in vitro, but also in vivo. The data indicate that exudation primes PMN for their subsequent responsiveness to fmet-leu-phe, a modification that may be crucial for efficient antimicrobial host defense.

Is cytochrome b558 translocated into the plasma membrane from granules during the activation of neutrophils?

Two laboratories (Borregaard et al. (1984) J. Biol. Chem. 259, 47; Ohno et al. (1985) J. Biol. Chem. 260, 2409) have reported that a b-type cytochrome (b558) was translocated into plasma membranes from specific granules in activated neutrophils. In an attempt to examine the cytochrome b translocation, porcine neutrophils were activated by treatment with surface-active agents such as myristate (MA) and phorbol myristate acetate (PMA), and then the postnuclear supernatants of both activated and unactivated cells were fractionated by Percoll density gradient centrifugation with a Zonal rotor. In activated neutrophils, high O2- generating activity was found in the plasma membrane fraction, which showed a peak of Na, K-ATPase activity as a marker enzyme. Cytochrome b558 was recovered 74 to 78% in the plasma membrane fraction and 14 to 16% in granules in either activated or unactivated cells. No change in specific content of cytochrome b558 was observed in plasma membranes before and after activation of cells. Furthermore, in both activated and unactivated cells, vitamin B12-binding protein, a specific granule marker, was mainly found in the bottom fractions and scarcely at all in plasma membranes. These results suggest that no translocation of cytochrome b558 occurs during activation of neutrophils.

Serum vitamin B12 binders in south african blacks with hepatocellular carcinoma

Sera from 242 South African blacks with hepatocellular carcinoma were assayed for unsaturated vitamin B12 binding capacity (UBBC) and vitamin B12 activity. Six patients were younger than 20 years of age, and 24% were younger than 30 years of age. Eighty-four percent of the patients had a slightly raised UBBC and 86% had a slightly elevated vitamin B12 value, but in no patient was an exceptionally high UBBC present. Serum UBBC and vitamin B12 were not higher in younger patients, and raised UBBC values were not related to serum alpha-fetoprotein values. Serum UBBC and vitamin B12 concentrations were not significantly different in patients with and without coexisting cirrhosis. In none of the patients with a UBBC above 3000 pg/ml was the fibrolamellar variant of hepatocellular carcinoma present. The authors conclude that South African blacks with hepatocellular carcinoma do not secrete an abnormal vitamin B12 binding protein.

Vitamin B12 absorption in the neonatal piglet. 2. Resistance of the vitamin B12-binding protein in sows' milk to proteolysis in vivo.

It has been postulated that the vitamin B12-binding protein in the sows' milk may facilitate the intestinal absorption of vitamin B12 in the piglet. This implies that the binder is not rapidly and completely degraded by gut proteases, and the present experiments were devised to test this assumption. Piglets aged 7 and 28 d were given a test feed of sows' milk whey both with and without cyano[3H]cobalamin to saturate the binder, and with 14C-labelled polyethylene glycol (PEG) as a marker. Control piglets were given a milk substitute containing no vitamin B12-binder. At 80 min after the test meal the piglets were killed and the contents of the stomach and small intestine were removed for analysis. Recovery of [14C]PEG, mainly from the middle and distal portions of the small intestine, ranged from 49 to 78%. Apparent loss of vitamin B12-binder from the intestine was calculated from the ratio, PEG:binder. In both 7- and 28-d-old piglets, the content of saturated binder in the middle and lower small intestine was about 20% depleted relative to that in the test fed. With the unsaturated binder the loss was more variable and generally greater, at about 50%. At slaughter the stomach was nearly empty and the high unsaturated binding capacity of the contents was attributed to endogenous binders. However, in the middle and lower small intestine, the binding capacity was derived mainly from sows' milk given with the test meal. No free cyano[3H]cobalamin was found in the intestinal contents of piglets given sows' milk to which enough cyano[3H]cobalamin had been added to saturate the binder. There was no change in the molecular weight of the vitamin B12-binder during its passage down the intestine, as judged by its behaviour on filtration in Sephadex gel G-150. The results indicate that a high proportion of saturated binder, and a smaller proportion of unsaturated binder, survived unchanged in the intestine of piglets at both ages.

Vitamin B12 absorption in the neonatal piglet. 3. Influence of vitamin B12-binding protein from sows' milk on uptake of vitamin B12 by microvillus membrane vesicles prepared from small intestine of the piglet.

The influence of the vitamin B12-binding protein isolated from sows' milk on the uptake of vitamin B12 was investigated using microvillus membrane vesicles prepared from the small intestine of 7- and 28-d-old piglets. Uptake of radioiodinated purified binder was also examined. The binder strongly promoted uptake of vitamin B12 at both ages; in the absence of the binder there was little uptake. The uptake mechanism was specific and operative in vesicles prepared from all regions of the small intestine. Uptake was a rapid process, exhibiting saturation kinetics, with a pH optimum at about 7.0, and dependent on the presence of magnesium or calcium ions for maximum activity. Affinity constants of the binding sites for the milk binder were determined. Competition experiments using complexes of the binder with vitamin B12 and with non-cobalamin analogues (cobinamide and Co-alpha-[2-methyladenyl]cobamide) showed that the bound analogues competed with bound vitamin B12 for uptake but with lower efficiency. Intrinsic factor also promoted vitamin B12 uptake by the vesicles but it did not compete with the milk binder for the same binding sites. It promoted uptake only in microvilli isolated from the lower third of the small intestine, and was more effective with preparations from 28-d-old piglets, whereas the milk binder was more effective with the 7-d-old piglets. Porcine gastric cobalophilin competed with the milk cobalophilin, but with lower efficiency. It was concluded that a specific transport mechanism for absorption of vitamin B12, mediated by the vitamin B12-binder in milk, exists at the intestinal brush border of neonatal piglets and strongly reinforces the developing intrinsic factor-mediated mechanism during the early days or weeks of life. It is suggested that the binder in the milk has a wider physiological significance and acts also as a 'host protective' factor and as a scavenger of adventitious vitamin B12.