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PubMed1987

Purification and some properties of cytosolic cobalamin-binding protein in Euglena gracilis.

In Euglena gracilis SM-ZK, a bleached mutant of E. gracilis z, the cobalamin- (Cbl-)binding activity was distributed in cytosol (49.2%), mitochondria (20.3%) and microsomal fraction (20.4%). The cytosolic Cbl-binding activity gave two major peaks in isoelectric focusing. The Cbl-binding protein with pI 3.2 was purified 6500-fold in a yield of 19.9%, and that with pI 4.7 5800-fold in a yield of 11.9%. The monomeric Mr values of both Cbl-binding proteins were about 66,000. The Cbl-binding activity of both proteins showed a very low pH-dependency, and thiol groups and metal ions were not concerned with the Cbl-binding activities. The Ks values of the Cbl-binding proteins with pI 3.8 and 4.7 for CN-Cbl were 1.0 and 2.0 nM respectively. The Cbl-binding protein with pI 3.8 was shown to be immunologically identical with the protein with pI 4.7 by double-immunodiffusion experiments against antibody to the protein with pI 3.8. The two cytosolic Cbl-binding proteins did not show the activities of Cbl-dependent enzymes in E. gracilis, N5-methyltetrahydrofolate:homocysteine methyltransferase, methylmalonyl-CoA mutase and ribonucleotide reductase, suggesting that the two cytosolic Cbl-binding proteins play a physiological role as intracellular Cbl carriers.

Animals↗
PubMed1992

Thrombospondin receptor expression in human neutrophils coincides with the release of a subpopulation of specific granules.

The extracellular matrix (ECM) protein thrombospondin (TSP) binds specifically to polymorphonuclear leucocyte (PMN) surface receptors and promotes cell adhesion and motility. TSP receptor expression increases 30-fold after activation with the synthetic chemotactic peptide, N-formylmethionyl-leucylphenylalanine (FMLP) or the Ca2+ ionophore A23187, in combination with cytochalasin B. The expression of TSP receptors was correlated with the exocytosis of both specific and azurophil granules. Newly expressed TSP receptors are not derived from easily mobilized specific granules since agents that trigger some specific granule release [phorbol myristate acetate (PMA), FMLP or ionophore A23187 alone] do not increase TSP receptor expression. In this study we used the anion-channel blocker, 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS) to investigate the source of these newly expressed receptors. When PMNs were exposed to cytochalasin B and FMLP or to cytochalasin B and ionophore A23187 in the presence of 30-100 microM-DIDS, TSP receptor expression increased coincidently with vitamin B12-binding protein release from specific granules. Under these same conditions, the release of the azurophil granule component, myeloperoxidase, was significantly inhibited. Using agonists that cause release of specific granules, or both specific granules and azurophil granules, we determined that DIDS blocked the release of PMA-mobilized specific granules and cytochalasin B plus FMLP- or cytochalasin B plus ionophore A23187-mobilized myeloperoxidase-containing azurophil granules but not specific granules mobilized by cytochalasin B plus FMLP or cytochalasin B plus ionophore A23187. These results suggested that PMNs contain at least two subpopulations of specific granules: one that is easily mobilized, lacks TSP receptors and is inhibitable by DIDS, and one that is difficult to mobilize, contains a large pool of TSP receptors and the release of which is enhanced in the presence of DIDS.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1984

The regulatory roles of liver and kidney in cobalamin (vitamin B12) metabolism in the rat: the uptake and intracellular binding of cobalamin and the activity of the cobalamin-dependent enzymes in response to varying cobalamin supply.

To examine possible regulatory roles of liver and kidney in cobalamin metabolism, specific activities of the two cobalamin-dependent enzymes, uptake in vivo of cyano [57Co]cobalamin [( 57Co]CNCbl) and the binding of [57Co]Cbl to intracellular proteins were measured in normal, cobalamin-loaded and cobalamin-deficient rats. Cobalamin deficiency and cobalamin loading produced greater changes in cobalamin concentration in the kidney than in the liver. Although cobalamin deficiency resulted in a decrease in total methylmalonyl-coenzyme A mutase (methylmalonyl-CoA mutase) in both organs, cobalamin loading had no effect. Neither deficiency nor loading altered total methyltransferase activity. The holoenzyme activities of both enzymes correlated with changes in tissue cobalamin levels. Uptake of [57Co]Cbl indicated that the kidney, in contrast to the liver, increased its uptake during loading and reduced it during deficiency, suggesting a possible regulatory role for this organ. In the normal rat, 24 h after injection of [57Co]CNCbl, 0.3% of the administered [57Co]Cbl was present in the liver as free cobalamin. By contrast, in the kidney, over 13% of the [57Co]Cbl was present in the free form. During deficiency free renal [57Co]Cbl was reduced to 0.6% of the administered [57Co]Cbl whereas in cobalamin-loaded rats it was increased to more than 27%. It is concluded that alterations in tissue cobalamin levels resulting from differences in cobalamin supply are due to changes in the large pool of free cobalamin present in the kidney and not to changes in the intracellular binding.

Animals↗
PubMed1985

A comparison of cobalamin binding by liver and kidney in rat and man.

Binding of cobalamin (Cbl) was compared in liver and kidney plasma membranes prepared from rat and human tissues. Single, high-affinity, saturable (200 pmol/l), binding sites for TC II-Cbl were found in all tissues; by contrast no receptors were present for free cobalamin, for which only non-specific adsorption occurred. Binding constants for TC II-CNCbl determined for liver and kidney plasma membranes were of a similar magnitude. Mean values for Ka (litre/nmol) were 16.7 (rat liver), 18.8 (rat kidney), 8.0 (human liver) and 7.5 (human kidney). Results for binding TC II-OHCbl instead of TC II-CNCbl showed no difference in Ka and Bmax. values, although the non-specific adsorption was decreased to a third. Competitive inhibition results showed that the receptors are specific for the TC II molecule and that binding is unaffected either by the cobalamin moiety or by the presence of free cobalamin. Degradation of the receptor protein molecules by trypsin (10 micrograms/ml) resulted in 90% inhibition of binding. It is concluded that differences between liver and kidney in cobalamin uptake and accumulation cannot be attributed to differences in their TC II receptors.

Animals↗
PubMed2003

Genetic determinants of the homocysteine level.

Elevated total homocysteine (tHcy) plasma concentrations indicate folate and/or vitamin B12 deficiency and are associated with cardiovascular disease and neural tube defects. Evidence has accumulated that folate-, vitamin B12-, and Hcy-metabolism are under genetic control. Because Hcy metabolism is impaired in renal failure, MTHFR 677 C>T, GCP2 1561C>T, RFC1 80G>A, and TCN2 776G>C may further aggravate hyperhomocysteinemia in these patients. The most consistent effect on tHcy plasma concentrations is observed for 677C>T of MTHFR, whereas GCP2, RFC1, and TCN2 polymorphisms show no major effect on tHcy concentrations. Much is yet to be learned about the impact of genetic variants on tHcy levels, human diseases, the genetic-nutrient interactions, as well as the pharmacogenetic consequences in Hcy and vitamin metabolism.

Antigens, Surface↗
PubMed1999

Human haptocorrin in hepatocellular carcinoma.

This study aimed to determine whether haptocorrin (HC), a vitamin B12 binder, is stored in hepatic cells and whether this storage is modified by hepatic carcinogenesis. It was carried out using immunohistochemistry on different liver tissues (normal liver and steatosis, N = 22; cirrhosis, N = 13; and hepatocellular carcinoma, N = 31). No significant immunostaining of HC was detected in noncancerous biopsies with the exception of in one case of cirrhosis. Hepatocellular carcinoma (HCC) sections showed a weak to moderate cytoplasmic staining of cancerous cells (93% of cases) and of noncancerous hepatocytes surrounding the tumor (95%) of cases. Sections with pseudoglandular structures showed a moderate to strong staining of their secretion products. These results and previous studies would seem to confirm the hypothesis that the raised HC serum level observed in HCC is due both to the increased hepatic synthesis of HC and to a decreased uptake by the liver of the particular isoform of this glycoprotein present in the serum of HCC patients.

Biomarkers↗
PubMed1978

Cobalamin analogues are present in human plasma and can mask cobalamin deficiency because current radioisotope dilution assays are not specific for true cobalamin.

Since R protein binds cobalamin (vitamin B12) and cobalamin analogues, whereas intrinsic factor is highly specific for true cobalamin, we compared the serum cobalamin values obtained with these proteins in radioisotope dilution assays. With R protein, eight of 21 patients with cobalamin deficiency had serum cobalamin levels (mean, 204, range, 85 to 355 pg per milliliter) that overlapped with values for 74 normal subjects (mean, 576, range, 220 to 1230). With intrinsic factor, no patient values (mean, 36, range, less than 10 to 78 pg per milliliter) overlapped with the normal values (mean, 322, range, 130 to 785). Paper chromatography showed that these differences were due to the presence of cobalamin analogues. R protein constituted 51 to 85 per cent of the cobalamin-binding protein in 10 commercial serum cobalamin assay kits, which were said to contain "intrinsic factor". Human plasma contains cobalamin analogues that can mask cobalamin deficiency with current radioisotope dilution assays.

Adult↗
PubMed1985

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.

Absorption↗
PubMed2004

Determination of vitamin B12 using the enzyme glycerol dehydrase.

Glycerol dehydrase is an enzyme that catalyzes dehydration of glycerol into beta-propionaldehyde. It requires 5'-deoxyadenosylcobalamin, one of the forms of vitamin B12, as a coenzyme. The enzyme is inactivated in vitro by all forms of vitamin B12 stoichiometrically. The objective of this study was to determine vitamin B12 content by utilizing the inactivation of the enzyme by vitamin B12. After various examinations, an excellent standard curve was obtained up to 1 pmol vitamin B12 using 14 mU of the enzyme per tube. Glycerol dehydrase does not respond to vitamin B12 if it is bound to haptocorrin, a vitamin B12-binding protein. This necessitates a procedure for extraction of vitamin B12 from samples before assay. The enzyme was less inactivated by 5'-deoxyadenosylcobalamin than any other form of vitamin B12. However, this did not matter because all forms of vitamin B12 were converted into cyanocobalamin during the extraction procedure cited above, which was performed in a buffer containing potassium cyanide.

Aldehydes↗
PubMed1990

Epidermal growth factor and haptocorrin in nasal secretion.

We report that nasal secretion contains the growth-promoting peptide epidermal growth factor (EGF) and a cobalamin-binding protein. Based on its amino-terminal sequence the cobalamin binding protein is classified as haptocorrin (HC). We have investigated the concentration of these components, the volume secreted, and the concentration of total protein after nasal challenge with methacholine, methacholine + ephedrine, histamine and serotonin. The EGF and HC concentrations do not differ for the four challenge agents employed. Range and median for all values obtained are 0.1-2.5 (0.7) nmol/l (n = 49) for EGF and 50-405 (190) nmol/l (n = 63) for HC. After challenge with methacholine the amount of secretion (80-630 (200) mg (n = 14) and the protein concentration (1.5-7.8 (3.5) g/l (n = 14] are lower than for any other challenge. After challenge with serotonin or histamine the amount secreted is 60-1125 (480) mg (n = 39). The concentration of protein is significantly higher (p less than 0.01) after challenge with serotonin (1.4-56.0 (17.0) g/l (n = 18] than after challenge with histamine (2.0-25.0 (5.0) g/l (n = 20].

Adult↗
PubMed1997

Atherogenesis and the homocysteine-folate-cobalamin triad: do we need standardized analyses?

BACKGROUND: Bioscientists, physicians and nutritionists are newly interested in the homocysteine-folate-cobalamin triad, in part because homocysteine may be important both in atherogenesis and thrombogenesis. Homocysteine imbalance may be an early marker for cobalamin disorders because cobalamin is a cofactor in remethylation of homocysteine to methionine. METHODS: In 139 men and 32 women of similar mean age of 65 years, we measured markers which have been cited as risk for atherosclerosis: serum homocysteine, folate, total cobalamin, holotranscobalamin I and II, (TCI and TCII), total serum cholesterol (SCHOL), high density lipoprotein cholesterol (HDLC), triglycerides (STG) as well as red blood cell (RBC) folate, food records and body composition by whole body counting of potassium-forty (40K). RESULTS: Statistical relationships among the data showed healthy women had lower mean serum homocysteine and their mean RBC folate and TCI and TCII were higher than men. Eighty-three subjects had TCII much lower than 60 pg/ml (subnormal), yet only 11 of these men and two women had total cobalamin < 200 pg/ml (abnormal). Fifty-two subjects with serum homocysteine greater than 17.5 nmol/ml had TCII less than 60 pg/ml, suggesting serum homocysteine may be a marker for early cobalamin negative balance. None of the subjects in the study had serum folate below abnormal values, i.e., less than 1.6 mg/ml. All subjects had RBC folate within normal range. Serum homocysteine showed inverse relationship with RBC folate and serum total cobalamin, TCI and TCII. CONCLUSIONS: 1) importance of using serum holotranscobalamin TCI and TCII as markers of cobalamin deficiency, 2) necessity to use documented quantitative components of dietary intake if strong comparisons are to be made among quantitative values of serum or plasma homocysteine, folate, cobalamin, and nutrients in food intake.

Adult↗