Unsaturated Vitamin B12 Binding Capacity Research Articles

Transcobalamin II deficiency with methylmalonic aciduria in three sisters

Transcobalamin II (TC II) is a plasma protein that binds vitamin B12 (cobalamin, Cbl) and facilitates cellular Cbl uptake by receptor-mediated endocytosis. In autosomal recessive TC II deficiency, intracellular Cbl deficiency results in an early onset of megaloblastic anaemia that may be accompanied by neurological abnormalities. Inadequate treatment may lead to neurological abnormalities. We describe three sisters, the daughters of first cousins of Moroccan origin, with TC II deficiency requiring continuous and long-term vitamin B12 treatment. The diagnosis was suspected from the finding of low unsaturated vitamin B12 binding capacity and confirmed by absence of detectable TC II by radioimmunoassay and by inability of cultured fibroblasts to synthesize TC II.

Potential of measurements of unsaturated vitamin B12-binding capacity in serial colonoscopic biopsy specimens for global and regional assessments of disease severity in inflammatory bowel disease.

We investigated whether measurements of unsaturated vitamin B12-binding capacity (UBBC), in homogenates of serial colonoscopic biopsy specimens, could be used as objective measures of disease severity in ulcerative colitis (UC) and Crohn's disease (CD). On a regional basis UBBC activity correlated with and showed good agreement with endoscopic and histologic activity scores (r = 0.8 and 0.6, respectively, for UC, and r = 0.7 and 0.7, respectively, for CD). For global assessment aggregate UBBC, endoscopic and histologic scores were compared with standard clinical activity scores. In UC, correlations with the van Hees index were r = 0.7, 0.8, and 0.7, respectively, and UBBC assays accurately reflected both regional and global disease activity. In CD, correlations with the CDAI were -0.1, 0.7, and 0.6, respectively. Thus, aggregate UBBC scores failed to reflect disease activity in CD, in which focal deep ulcers may produce high symptom scores but in which adjacent specimens may show no acute inflammation.

Serum Unsaturated Vitamin B<sub>12</sub> Proteins in Adult Patients with Sickle Cell Anaemia (Hb SS)

The unsaturated vitamin B12 binding capacity (UBBC) of serum, the three transcobalamins (TC I, TC II, TC III) and serum cobalamin have been studied in adult sickle cell patients with Hb SS. An increase in the level of the binding capacities of TC I and TC III resulting in an increase in UBBC was observed in these patients. There was no difference in the level of the binding capacity of TC II, while there was a significant reduction in the level of serum cobalamin. These changes in the pattern of these binding proteins are discussed in relation to the changes of these binding proteins in paediatric sickle cell patients with Hb SS. The results indicate that the pattern of these binding proteins changes with age among sickle cell patients resulting in an increased level of the binding capacities of TC I and TC III. These increases lend support to the view that these two similar binding proteins are involved in the defence mechanism of the body.

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.

Immunohistochemical localisation of vitamin B12 R-binder in the human digestive tract.

The distribution of vitamin B12 R-binder in the human digestive tract was studied using an indirect immunoperoxidase technique. Positive staining for R-binder was found in the mucous cells and ductal epithelial cells of the salivary glands and the oesophageal glands. In normal gastric mucosa, no positive staining for R-binder was found, but in the area with intestinal metaplasia, the columnar epithelial cells and goblet cells showed positive staining. Epithelial cells of the gallbladder, intrahepatic bile ducts and pancreatic ducts were also positive for R-binder. In the small intestine and colon, R-binder was found in the columnar epithelial cells and goblet cells. The measurement of unsaturated vitamin B12 binding capacity and cobalamin content in the extracts from intestinal mucosa also indicated the presence of R-binder in the intestinal mucosa.

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.

Elevated serum vitamin B12 in cystic fibrosis.

In 62 patients with cystic fibrosis the serum vitamin B12 concentration ranged from 160-2600 pmol/l with a mean of 1 105 pmol/l. Both vitamin B12-binding proteins in the serum, transcobalamin II and R-binders, carried increased amounts of vitamin B12, but showed relatively normal levels of unsaturated vitamin B12-binding capacity. This combination is rather typical for hepatic dysfunction, although the recurrent pulmonary infections might exert an upward effect on plasma R-binder concentration through increased turnover of myeloid cells. A significant positive correlation between transcobalamin II-vitamin B12 and serum alkaline phosphatase suggests that transcobalamin II-bound vitamin B12 might be an early indicator of focal biliary cirrhosis, which is known to occur in these patients.

Abnormalities of serum transcobalamins in sickle cell disease (HbSS) in Black Africa.

The unsaturated vitamin B12 binding capacity (UBBC) of serum, the three transcobalamins (TC I, TC II, TC III), and the total leucocyte and neutrophil counts have been studied in paediatric patients with sickle cell disease (SCD). Increase in the level of the binding capacities of TC I and TC II with concomitant increase of UBBC was observed in these children who also had increased total white blood and neutrophil counts. There was a significant reduction in the level of endogenous B12. These abnormalities are discussed in relation to the deficiency of the splenic reticuloendothelial function, immunologic defect, hepatic degenerative changes and aplastic crisis observed in SCD. These results lend support to the view that transcobalamins are involved in the defence mechanism of the body. The significant reduction in serum cobalamin in SCD suggests a higher demand on this vitamin for metabolic functions, especially for the sparing of folate.

The effects of surgery on the activity of neutrophil granule proteins.

Activities of the neutrophil granule-associated proteins beta-glucuronidase, lysozyme and vitamin B12 binding protein were measured, serially, in the cells and serum of 10 patients undergoing total abdominal hysterectomy. The neutrophil leucocytosis which followed total abdominal hysterectomy was accompanied by a fall in the intraneutrophilic activities of all three granule-associated proteins. Intraneutrophilic lysozyme activity and intraneutrophilic vitamin B12 binding capacity were maximally reduced within 4 h of surgery and fell to 62 +/- 13% (mean +/- SEM) and 63 +/- 9% of their preoperative levels, respectively. This contrasted with the activity of intraneutrophilic beta-glucuronidase which was not maximally reduced until 24 h post-surgery when a fall to 80 +/- 6% of the preoperative level was observed. By the fifth postoperative day activities of the three intraneutrophilic granule proteins were increasing and approaching those observed preoperatively. Serum lysozyme and plasma unsaturated vitamin B12 binding capacity (UBBC) rose steadily following surgery and were significantly elevated by the fifth postoperative day. It is suggested that activation and in vivo degranulation of circulating neutrophils may be responsible for these changes in activity of neutrophil granule proteins following surgery.

High serum vitamin B12 binding capacity as a marker of the fibrolamellar variant of hepatocellular carcinoma.

Ten (9.3%) of 107 patients with hepatocellular carcinoma had considerably increased serum unsaturated vitamin B12 binding capacity. All 10 were young (mean 12 years), had no serum alpha-fetoprotein, and no underlying cirrhosis; all had a longer survival compared with patients without increased serum unsaturated vitamin B12 binding capacity in the study. Seven of the 10 patients had fibrolamellar hepatocellular carcinoma, a recently recognised histological variant, which was found in only one young patient without increased serum unsaturated vitamin B12 binding capacity and no alpha-fetoprotein among the remaining 97. This high degree of correlation between increased serum unsaturated vitamin B12 binding capacity and fibrolamellar hepatocellular carcinoma has not been reported before. Increased serum unsaturated vitamin B12 binding capacity may be of considerable help in diagnosis, prognosis, and monitoring treatment of this well-defined group of patients with hepatocellular carcinoma but no alpha-fetoprotein.

Mucosal Enzymes in Human Inflammatory Bowel Disease with Reference to Neutrophil Granulocytes as Mediators of Tissue Injury

1. Biopsies of rectal mucosa were obtained for histology and enzyme analysis from 32 patients with inflammatory and functional bowel disorders, and the biopsies were classified morphologically as active colitis, quiescent colitis or normal. 2. Supernatant fractions of biopsy homogenates were assayed for their content of the proteolytic enzymes alpha-chymotrypsin, elastase and cathepsin D, and of protein, unsaturated vitamin B12-binding capacity, lysozyme, myeloperoxidase and N-acetyl-beta-glucosaminidase. 3. Mean unsaturated vitamin B12-binding capacity was significantly raised above normal in the active colitic mucosa, and mean lysozyme activity was raised above normal in both active and quiescent mucosae. 4. In active colitic mucosa there was no rise above normal in mean activities of any of the proteolytic enzymes, though a significant fall below normal occurred in mean N-acetyl-beta-glucosaminidase activity in the active colitic group.

Blocking and binding type antibodies against all major vitamin B12-binders in a pernicious anaemia serum.

The simultaneous occurrence of blocking and binding antibodies to intrinsic factor (IF), transcobalamin (TC- II, TC I) and other R type vitamin B12 proteins in the serum of a patient with treated pernicious anaemia (PA) is reported here for the first time. The dialysed and purified immunoglobulin-G (IgG), but not the immunoglobulin-M (IgM), from a PA patient neutralized the total unsaturated vitamin B12 binding capacity (UB12BC) of human gastric juice, saliva and serum and also of rabbit serum, suggesting that the PA IgG contained blocking antibodies against, IF, TC II, TC I and other R-binders. In addition, the PA IgG and IgM preparations contained binding antibodies since they could form macromolecular complexes with 57Co-B12 bound to IF, TC I or TC II so that each one of the latter was totally excluded from Sephadex G-200. The presence of the binding antibodies was further confirmed by the formation of radioactive precipitation lines on agarose with each one of the vitamin B12-binders bound to 58Co-B12. The PA serum did not exhibit any measurable UB12BC after dialysis against 7.5 M guanidine-HCl followed by renaturation with phosphate buffer (pH 7.4). But it did form TC I and TC II complexes with 57Co-B12 when the latter was added during the renaturation step indicating that the serum contained circulating immunoglobulin-TC complexes. The blocking antibodies should be distinguished from the previously described binding antibodies. The blocking of the binding of vitamin B12 to TCs resulted in relatively lower serum vitamin B12 levels in the present case in contrast to the presence of binding antibodies where high serum vitamin B12 levels have been reported. In addition, the binding antibodies form immunocomplexes with TCs which can easily be detected because they can bind radioactive vitamin B12 while the corresponding immunocomplexes of blocking antibodies are hidden because they prevent the binding of the vitamin to TCs.

Serum vitamin B12 levels and vitamin B12-binding proteins of serum and saliva of healthy Nigerians and Europeans

Serum vitamin B12 and unsaturated vitamin B12-binding capacity (UBBC) are known to be higher among people living in the tropics and subtropics than among Europeans. However, the cause of this phenomenon is not known nor have the individual transcobalamins (TC) been adequately examined. Therefore, we studied the serum vitamin B12, UBBC, and individual TC levels and salivary UBBC in 30 Nigerians and 10 Europeans living in northern Nigeria. Results have also been compared to those from white Americans living in the United States. The serum vitamin B12 tended to be higher in elite Nigerians than in the Europeans living in Nigeria, but there was considerable overlap. However, since all three groups in Nigeria had significantly higher levels than Americans an acquired cause associated with living in Africa is strongly suspected. No definite correlation with liver disease was apparent, but such an etiology could not be ruled out. The UBBC was clearly elevated in Nigerians compared to the Europeans, but UBBC and TC levels were indistinguishable between Europeans and Americans. TC I was higher in the Nigerians but the most striking difference was in TC II levels, which deserves further study. UBBC and TC values did not correlate with leukocyte counts or liver test results. It was not clear whether TC differences between Europeans and Nigerians were genetic or acquired. However, elevation of serum vitamin B12 and TC levels were independent of each other and probably had different causes.

Circulating Antibody to Transcobalamin II Causing Retention of Vitamin B12 in the Blood

Abstract A patient with recurrent pulmonary abscess, weight loss, and alcoholism was found to have extremely high serum vitamin B12 and unsaturated vitamin B12-binding capacity (UBBC) levels. While transcobalamin (TC) II was also increased, most of his UBBC was due to an abnormal binding protein which carried greater than 80% of the endogenous vitamin B12 and was not found in his saliva, granulocytes, or urine. This protein was shown to be a complex of TC II and a circulating immunoglobulin (IgGkappa and IgGlambda). Each IgG molecule appeared to bind two TC II molecules. The reacting site did not interfere with the ability of TC II to bind vitamin B12, but did interfere with its ability to transfer the vitamin to cells in vitro. The site was not identical to that reacting with anti-human TC II antibody produced in rabbits. Because of this abnormal complex, 57Co-vitamin B12 injected intravenously was cleared slowly by the patient. However, no metabolic evidence for vitamin B12 deficiency was demonstrable, although the patient initially had megaloblastic anemia apparently due to folate deficiency. The course of the vitamin B12-binding abnormalities was followed over 4 yr and appeared to fluctuate with the status of the patient's illness. The IgG-TC II complex resembled one induced in some patients with pernicious anemia by intensive treatment with long-acting vitamin B12 preparations. The mechanism of induction of the antibody formation in our patient is unknown.

Circulating antibody to transcobalamin II causing retention of vitamin B12 in the blood

A patient with recurrent pulmonary abscess, weight loss, and alcoholism was found to have extremely high serum vitamin B12 and unsaturated vitamin B12-binding capacity (UBBC) levels. While transcobalamin (TC) II was also increased, most of his UBBC was due to an abnormal binding protein which carried greater than 80% of the endogenous vitamin B12 and was not found in his saliva, granulocytes, or urine. This protein was shown to be a complex of TC II and a circulating immunoglobulin (IgGkappa and IgGlambda). Each IgG molecule appeared to bind two TC II molecules. The reacting site did not interfere with the ability of TC II to bind vitamin B12, but did interfere with its ability to transfer the vitamin to cells in vitro. The site was not identical to that reacting with anti-human TC II antibody produced in rabbits. Because of this abnormal complex, 57Co-vitamin B12 injected intravenously was cleared slowly by the patient. However, no metabolic evidence for vitamin B12 deficiency was demonstrable, although the patient initially had megaloblastic anemia apparently due to folate deficiency. The course of the vitamin B12-binding abnormalities was followed over 4 yr and appeared to fluctuate with the status of the patient's illness. The IgG-TC II complex resembled one induced in some patients with pernicious anemia by intensive treatment with long-acting vitamin B12 preparations. The mechanism of induction of the antibody formation in our patient is unknown.

A Perfluorochemical Oxygen Carrier (Fluosol‐43) in a Synthetic Medium Used for Perfusion of Isolated Rat Liver

An emulsion of perfluorotributylamine (Fluosol-43) was used as substitute for the gas-carrying function of erythrocytes in a synthetic medium for perfusion of isolated rat liver. The efficiency and effect of this synthetic gas-carrier were evaluated from measurements of the concentrations of rat albumin, the unsaturated vitamin B12-binding capacity of small-molecular-size vitamin B12-binding proteins (UBBC of SBP), urea nitrogen, glucose, sodium, potassium, alanine amino transferase (ALAT) in the medium, and the incorporation of 14C-lysine into the circulating proteins. Secretion of bile, portal pressure, PO2, PCO2 and pH in the affluent and the effluent mediums were also measured. The results demonstrate that the oxygen-carrying capacity of the medium and the metabolic functions of the liver were higher, and the liver damage less, when the medium included Fluosol-43 than without it. The albumin synthesis and the secretion of bile were as high as has been reported for perfusions with erythrocytes. This indicates that the oxygen carried by the Fluosol-43 was utilised by the liver, and that the metabolic functions were not adversely affected during 4 h of perfusion by the medium containing Fluosol-43.

Marked cyclic leukocytosis-leukopenia in chronic myelogenous leukemia

A spontaneous oscillation of the white blood cell count was observed in a 58 year old man with chronic myelogenous leukemia (CML). Similar cyclic variations were noted in the platelet and reticulocyte counts with no apparent alterations in marrow cellularity to account for such changes. Since direct correlation was noted between white blood cells, platelets, and reticulocyte counts versus spleen size, it suggests that splenic hemopoiesis may be responsible for these cyclic changes. A possible inverse relationship between colony-stimulating factor (CSF) activity and the white blood cell count was noted, suggesting that CSF may be the humoral agent controlling granulocyte production. A direct correlation between the white blood cell count and serum unsaturated vitamin B 12 binding capacity (UBBC) and lysozyme was also noted and further supports the concept that the latter two are measures of the granulocyte pool and metabolism. An inverse relationship between CSF activity and the UBBC suggests that these may be two different entities. Finally a modified form of standard chemotherapy may be effective in inducing remission in cases of CML with marked cyclic leukocytosis-leukopenia.

Release of vitamin B12-binding protein from isolated rat liver perfused with a synthetic medium.

The unsaturated vitamin B12-binding capacity (UBBC) of the small molecular size vitamin B12-binding protein (SBP) release and albumin release into the medium and the fluid pressure in the portal vein were studied with respect to their ability to reflect the state of isolated perfused rat liver. The liver was perfused in vitro with a synthetic medium either with or without a gas carrier. Liver damage was induced by hypoxia or noradrenalin. Variations in experimental conditions resulted in different patterns of parameters reflecting different aspects of the liver state. The fluid pressure in the portal vein reflected sensitively the circulatory state but is not always a criterion of the general condition of the isolated liver. The albumin release is a criterion of functional state but did not always reflect liver damage. However, the amount of UBBC of SBP released into the medium is a parameter reflecting sensitively the functional capacity of the liver to produce protein and also seems to indicate liver damage. The use of this parameter provides supplementary information that cannot be obtained by either of the two more conventional parameters.

Serum vitamin B12 content and UBBC in gravid and fetal rabbits.

It is well known that human serum vitamin B12 decreases progressively during pregnancy (1-3) with lowest levels present at term. Within 4-6 weeks post partum, the maternal serum B12 level returns to normal or higher (4-5). In general, fetal cord blood contains more vitamin B12 than comparable maternal blood (3). The unsaturated vitamin B12 binding capacity (UBBC) of the mother increases during pregnancy and returns to normal within a month of parturition (6, 7). Similar phenomenon have also been found in rats (1). Although pregnant women and gravid experimental rats appear to follow the same pattern with regard to serum vitamin B12, no definitive rationale has as yet been offered to explain these effects. Rabbits are known to contain the highest serum vitamin B12 content and UBBC of all laboratory mammals so far tested (8). Because rabbits are often used as models for fetal development, it was of interest to determine the serum B12 and UBBC during gravidity and in the fetus. Materials. Samples. Sera of gravid New Zealand rabbits, fetuses near term and sera of both sexes during growth and development were obtained commercially.1 Sera from New Zealand strain rabbits that were maintained in the laboratory were used for comparative purposes. Sera of normal pregnant women were obtained a few hours prior to parturition and cord blood was obtained from their normal healthy infants. All sera were maintained frozen at −20° until analyzed. Vitamin B12 assay. Vitamin B12 was determined by a competitive binding assay using porcine intrinsic factor (IF)2 as the binding protein, high specific activity 57Co labelled cyanocobalamin (15 μCi/μg B12-60 μCi/μg B12)3 and equilibrium dialysis briefly described as follows:

The three transcobalamins in myeloproliferative disorders and acute leukaemia.

The unsaturated vitamin B12 binding capacity of whole serum (UBBC) and of the three transcobalamins (TC) has been studied in patients with various haematological diseases including myeloproliferative disorders (MPD) and acute leukaemia. The binding capacity of TC I and TC III was increased in MPD; TC I being particularly high in chronic granulocytic leukaemia (CGL) and TC III especially raised in polycythaemia rubra vera (PRV) and in infectious leucocytosis. The binding capacity of both TC I and TC III correlated with blood neutrophil count and the ratio TC III/TC I was low in CGL and increased in PRV. TC II was increased in acute myelogenous leukaemia, during remission and blast cell crisis of CGL and in refractory anaemia with excess of myeloblasts but not in acute lymphoblastic leukaemia (ALL). TC II correlated inversely with blood neutrophil count. There is an inverse ratio between TC II and TC I at least in myelogenous leukaemia. These abnormalities are discussed in relation to granulocyte kinetics. TC III and TC I reflect probably the total body granulocyte pool and share some biochemical and immunological properties supporting the view that they have a common origin in the more mature stages of the granulocyte cell line while TC II probably originates partly in more primitive granulocytes.