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PubMed1982

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.

Blood Proteins↗
PubMed1982

Localization of leucine aminopeptidase and vitamin B-12 binding protein in rabbit peripheral blood polymorphonuclear leukocytes.

Polymorphonuclear leukocytes were isolated from the peripheral blood of rabbits by Ficoll-Hypaque centrifugation followed by dextran sedimentation. The granulocytes were homogenized in isotonic sucrose and subjected to analytical subcellular fractionation by sucrose density gradient centrifugation. Leucine aminopeptidase, when assayed with L-leucine-7-amido-4-methyl-coumarin as substrate, showed a similar distribution to N-acetyl-beta-glucosaminidase and thus is localized to the tertiary granules. There was no leucine aminopeptidase associated with the plasma membrane of these cells. Further experiments with purified plasma membranes and inhibitor studies using diazotized sulphanilic acid further confirmed that leucine aminopeptidase had a purely intracellular localization. Vitamin B-12 binding protein showed a similar localization to alkaline phosphatase indicating that, as in human polymorphonuclear leukocytes, vitamin B-12 binding protein is located to the specific granules.

Animals↗
PubMed1985

Characterization and immunohistochemical localization of rat salivary cobalamin-binding protein and comparison with human salivary haptocorrin.

Rat saliva contains a cobalamin-binding protein that binds cobalamin as well as cobinamide. The protein binds cobalamin with an affinity constant of 8 X 10(10) l X mol-1, and it binds cobalamin over a more narrow pH range (pH 7.5-10) than does human haptocorrin. It has a Stokes radius of 2.45 nm as compared to the Stokes radius of 4.50 nm for human haptocorrin. Upon isoelectricfocusing it dissociates into four strong bands with pI between 7 and 8, while human haptocorrin dissociates into acid isoproteins. Since human haptocorrin binds to concanavalin A while rat haptocorrin does not, we suggest that rat haptocorrin lacks carbohydrate. The substance concentration of rat saliva haptocorrin is 0.04-12.9 nmol X l-1 (median 7.5 nmol X l-1, n = 9) for control animals. After stimulation with isoproterenol, a beta-adrenergic agent, the substance concentration is 46.4-96.6 nmol X l-1 (median 69.7 nmol X l-1, n = 8). Immunohistochemical studies show haptocorrin in the secretory acini of the submandibular and parotid glands of the rat. In the human submandibular gland, the protein is detected both in the mucous secretory acini and in the intercalated ducts.

Animals↗
PubMed1977

Cobalamin binding proteins in stomach and serum from various animal species data for B12 binding capacities and molecular sizes of the binding proteins.

1. The unsaturated cobalamin-binding capacity of stomach mucosa and serum from 37 animals and the size of the binders have been measured. 2. The binding capacity in stomach mucosa was from 1 to 600 nmole kg-1 wet wt, the highest values occurring in pig, guinea pig, porpoise and earthworm. In serum it varied from 0.4 to 270 nmole 1(-1), the highest values occurring in duck, grass snake, toad and spiny dogfish. 3. The size in terms of Stokes radius of the cobalamin binders in stomach mucosa varied from 1 nm in hagfish (Mr approximately 14,000) to 7 nm in lamprey (Mr approximately 210,000). In serum it was from 1.4 nm (Mr approximately 17,000) to 7.4 nm (Mr approximately 240,000) both in frog.

Animals↗
PubMed1992

Enzyme-linked immunoassay of haptocorrin: analysis of milk and granulocytes.

An enzyme-linked, sandwich-type immunoassay method was described for quantitive assay of haptocorrin. The ranges of haptocorrin concentrations measurable by the method were comparable with those by radioimmunoassay. Using the method, the mean +/- SD of haptocorrin was 4.83 +/- 1.23 micrograms/ml in the colostrum (4th and 5th days after parturition), 3.17 +/- 1.77 micrograms/ml in the mature milk, and 21 ng/10(4) granulocytes (634 ng/mg protein). Haptocorrin from milk showed a homogeneous band at 66 kDa on SDS polyacrylamide electrophoresis followed by immunoblotting, while that from the granulocytes exhibited several closely aligned bands at 80 kDa and one or two bands at around 20 kDa.

Colostrum↗
PubMed1989

Protein kinase C is not involved in secretion by permeabilized human neutrophils.

The generally accepted sequence of intracellular signal transduction involves: (1) cell surface receptor-ligand interactions; (2) activation of G-proteins; (3) activation of phospholipase C, leading to inositol phosphate (IP3), and diacylglycerol production; (4) parallel mobilization of intracellular Ca2+ by IP3, and; (5) activation of protein kinase C (PKC) by diacylglycerol and Ca2+, leading to; (6) cellular responses. Human neutrophils appear to utilize this cascade, at least in general, and some, but not all, elements of the intracellular signal cascade known to be operating in intact cells also function in permeabilized cell systems. We have previously shown that permeabilized neutrophils can be induced to secrete lysosomal enzymes in response to elevated levels of Ca2+ alone and this secretion can be synergistically enhanced by the presence of guanine nucleotides. We now show that Ca2+, in the presence and absence of guanine nucleotides, can stimulate the production of soluble inositol phosphates. Furthermore, neomycin, a putative inhibitor of phospholipase C, can block Ca2(+)-induced secretion. These data thus suggest a role for phospholipase C activity or its products in the transduction process. The next enzymatic activity 'downstream' is PKC. Consequently, we looked at the role Mg-ATP, one of the substrates of PKC, plays in degranulation by permeabilized neutrophils, We found no obligatory role for this nucleotide in the secretory process. We then looked at the activity of oleoyl-acetyl-glycerol (OAG), a synthetic diacylglycerol and PKC agonist, on degranulation. We found that OAG was largely additive with Ca2+. Another PKC agonist, phorbol myristate acetate (PMA), also did not display notable synergy. Finally, inhibitors of PKC activity were not capable of blocking secretion, either in the presence or absence of guanine nucleotides. Thus, while circumstantial evidence seems to point towards a requirement for phospholipase C activation and diacylglycerol production in secretion, we were unable to demonstrate the next putative step in signal transduction, namely activation of PKC.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Release of azurophilic granule contents in fMLP-stimulated neutrophils requires two activation signals, one of which is a rise in cytosolic free Ca2+.

We have used a continuous spectrofluorimetric method to analyse the role of cytosolic free Ca2+ ([Ca2+]i) in the lysosomal enzyme release from the azurophilic granules in human neutrophils stimulated with f-Met-Leu-Phe (fMLP) in the presence of cytochalasin B. Measurements were performed with the beta-glucuronidase substrate 4-methylumbelliferyl-beta-D-glucuronide. We found that the transient rise in [Ca2+]i induced by fMLP is a necessary signal to obtain maximal degranulation. When this Ca2+ transient is prevented by the Ca2+ chelator BAPTA, degranulation can still be induced by a stimulated Ca2+ influx, albeit to a lower extent. We also studied the degranulation process in the neutrophils of a patient with a generalized chemotactic defect. Release of beta-glucuronidase from the patient's neutrophils could not be induced despite the occurrence of a normal Ca2+ response and normal degranulation of specific granules. We conclude that, besides an increase in [Ca2+]i, an additional signal is required for the fusion of azurophilic granules with the plasma membrane in human neutrophils.

Calcium↗
PubMed2006

Plasma holotranscobalamin compared with plasma cobalamins for assessment of vitamin B12 absorption; optimisation of a non-radioactive vitamin B12 absorption test (CobaSorb).

BACKGROUND: A recently developed non-radioactive vitamin B(12) absorption test (CobaSorb) was further explored to identify the best marker for reflection of vitamin B(12) absorption and to determine the duration of the test. METHODS: Seventy-eight healthy individuals (age 21-81 years) were given three oral doses of 9 microg vitamin B(12) per day for 5 successive days. Non-fasting blood samples were collected on days 1 to 5 before administration of vitamin B(12) and on day 8. Cobalamins and holotranscobalamin were measured. RESULTS: Performance of the vitamin B(12) absorption test was evaluated in individuals with holotranscobalamin or cobalamins below the 75% percentiles. We used a change greater than 2xCV(day-to-day) in holotranscobalamin (22%) and cobalamins (12%) to indicate a change caused by absorption of vitamin B(12). Among individuals with a baseline holotranscobalamin below the 75% percentile (<75 pmol/L, n=57), 98% had an increase in holotranscobalamin >22% from day 1 to day 3. In contrast, only 72% of the individuals with baseline cobalamins below the 75% percentile (<335 pmol/L, n=57) had an increase in cobalamins >12%. CONCLUSIONS: In healthy individuals with baseline holotranscobalamin <75 pmol/L, vitamin B(12) absorption is well reflected by an increase in holotranscobalamin after 2 days administration of oral vitamin B(12).

Adult↗
PubMed2005

Glycosylation independent measurement of the cobalamin binding protein haptocorrin.

BACKGROUND: Haptocorrin carries the major part of the circulating vitamin B12. The protein is heavily glycosylated and this may have implications for its measurement. METHODS: We used two different ELISA assays. In one assay, we employed antibodies against native HC and no pre-treatment of samples or calibrators. In the other assay, we used antibodies raised against deglycosylated HC, and deglycosylated the samples and calibrators by treatment with neuraminidase and PNGase prior to analysis. Plasma samples from healthy donors were analysed. RESULTS: The ELISA against native HC showed a high detection limit (71 pmol/l) and a poor linearity for serial dilutions of samples. The ELISA against deglycosylated HC showed a detection limit of 1.6 pmol/l, an excellent linearity between 1.6 and 100 pmol/l (r(2) = 0.99) and an inaccuracy of 5% for concentrations ranging from 250 to 840 pmol/l. The 95% reference interval was 240-680 pmol/l (n = 148). The concentration of HC showed a strong association to plasma cobalamins (p < 0.0001). CONCLUSIONS: An ELISA against native HC does not ensure an equimolar measurement of HC, while this is the case when a glycosylation independent assay is employed. Using this assay, a very strong correlation between total plasma HC and cobalamins in healthy donors is obtained.

Cross Reactions↗
PubMed2004

Biochemical signs of impaired cobalamin status during and after radiotherapy for rectal cancer.

PURPOSE: The aim of the study was to investigate whether pelvic radiotherapy for rectal cancer had a negative impact on cobalamin status. METHODS AND MATERIALS: Consecutive patients receiving pelvic radiotherapy (50 Gy) for rectal cancer were evaluated prospectively (n = 54). Serum cobalamin, holotranscobalamin (holoTC), methylmalonic acid (MMA), and total homocysteine (tHcy) were measured at start and end of radiotherapy, at follow-up 4-6 weeks and 1 year (n = 23) after radiotherapy. RESULTS: Mean serum cobalamin decreased from 306 pmol/L before treatment to 267 pmol/L at the end of radiotherapy (p < 0.001), 247 pmol/L 4-6 weeks after radiotherapy (p < 0.001), and 249 pmol/L 1 year after radiotherapy (p = 0.02). Mean serum MMA was 0.16 micromol/L pretreatment, 0.17 micromol/L at the end of radiotherapy (n.s.), and increased to 0.19 micromol/L after 4-6 weeks (p = 0.007), and to 0.21 micromol/L after 1 year (p < 0.001). There was no change in serum tHcy. Mean serum holoTC was reduced from 111 pmol/L pretreatment to 93 pmol/L 4-6 weeks after radiotherapy (p = 0.002). CONCLUSIONS: The data suggest rapid and persistent decrease in cobalamin status after radiotherapy for rectal cancer, as reflected by reduced serum cobalamin combined with increased serum MMA. This observation, though modest, may motivate routine monitoring of cobalamin status at follow-up after radiotherapy.

Adult↗
PubMed2006

Mitochondrial vitamin B12-binding proteins in patients with inborn errors of cobalamin metabolism.

Inborn errors of vitamin B12 (cobalamin, Cbl) metabolism are autosomal recessive disorders and have been classified into nine distinct complementation classes (cblA-cblH and mut). Disorders affecting methylcobalamin metabolism cause megaloblastic anemia, which may be accompanied by leukopenia and thrombocytopenia, and a variety of neurological problems. Disorders affecting adenosylcobalamin cause methylmalonic acidemia and metabolic acidosis. Previous studies have shown that cobalamin binds to two enzymes in humans: methylmalonyl-CoA mutase in mitochondria and methionine synthase in the cytosol. In this study, cobalamin binding patterns were analyzed in crude mitochondrial fractions obtained from both control and patient fibroblasts that had been incubated with [57Co]cyanocobalamin. Crude mitochondrial fractions from control fibroblasts confirmed that the majority of [57Co]Cbl eluted with methylmalonyl-CoA mutase. However, in six of the nine disorders, at least one previously unidentified mitochondrial cobalamin binding protein was observed to bind [57Co]Cbl. The proportion of [57Co]Cbl that binds, is increased compared to controls when a deficiency in either adenosylcobalamin synthesis or utilization prevents binding to methylmalonyl-CoA mutase. Furthermore, unique cobalamin binding profiles emerged demonstrating how known mutations in these patients affect cobalamin binding to as yet unidentified proteins.

Case-Control Studies↗
PubMed1988

Vitamin B12 R-binder localization in the human uterus: an immunohistochemical study.

The localization of vitamin B12 R-binder in the uterus was studied by use of an immunoperoxidase technique. Positive staining by anti-R-binder antiserum was observed in the columnar epithelium of the endocervix (18/18 cases) and in the surface epithelium of the endometrium (8/21 cases). Staining was usually seen in the apical portion of the epithelium; cytoplasmic staining in the endocervical columnar epithelium was intense. The secretory products in the endocervical glands showed positive staining. The endometrial glandular epithelium did not stain (0/24 cases). Metaplastic squamous epithelium of the endocervix showed positive staining (3/18 cases). The native squamous epithelium as well as the stromal components of the cervix, endometrium, and myometrium were negative for R-binder. This study shows that R-binder is localized in the uterus, especially in the endocervical glands. The R-binder in the endocervix may have antimicrobial activity in the uterus as in other organs, such as the intestines and mammary glands.

Adult↗
PubMed2002

Vegetarian lifestyle and monitoring of vitamin B-12 status.

Vegetarians are at risk to develop deficiencies of some essential nutrients, especially vitamin B-12 (cobalamin). Cobalamin occurs in substantial amounts only in foods derived from animals and is essential for one-carbon metabolism and cell division. Low nutritional intake of vitamin B-12 may lead to negative balance and, finally, to functional deficiency when tissue stores of vitamin B-12 are depleted. Early diagnosis of vitamin B-12 deficiency seems to be useful because irreversible neurological damages may be prevented by cobalamin substitution. The search for a specific and sensitive test to diagnose vitamin B-12 deficiency is ongoing. Serum vitamin B-12 measurement is a widely applied standard method. However, the test has poor predictive value. Optimal monitoring of cobalamin status in vegetarians should include the measurement of homocysteine (HCY), methylmalonic acid (MMA), and holotranscobalamin II. Vitamin B-12 deficiency can be divided into four stages. In stages I and II, indicated by a low plasma level of holotranscobalamin II, the plasma and cell stores become depleted. Stage III is characterized by increased levels of HCY and MMA in addition to lowered holotranscobalamin II. In stage IV, clinical signs become recognizable like macroovalocytosis, elevated MCV of erythrocytes or lowered haemoglobin. In our investigations, we have found stage III of vitamin B-12 deficiency in over 60% of vegetarians, thus underlining the importance of cobalamin monitoring in this dietary group.

Analysis of Variance↗
PubMed1984

Biochemistry and clinical chemistry of vitamin B12 transport and the related diseases.

This review focusses on research performed by the author and coworkers. The absorption, turnover and excretion of cobalamin and the pathogenesis of cobalamin deficiency states are described and the laboratory tests used to diagnose these states are discussed. Topics dealt with in detail include: overall turnover, daily need, enterohepatic circulation and excretion of cobalamin and other corrins . The soluble proteins mediating cobalamin transport and their cellular receptors are described and their nomenclature, isolation, structure and mode of action, the role of calcium in the membrane transport, the evolution of these systems and the analogies with transport systems for other substrates are discussed together with deficiency states, especially fish tapeworm anemia and familial selective vitamin B12 malabsorption with proteinuria. Folate deficiency is a relatively rare cause of megaloblastic anemia in Scandinavia but common in North America and explanations for this difference are suggested. The methods of assaying cobalamin in serum and plasma and the performance of radiovitamin B12 absorption tests are critically evaluated. The measurement of intrinsic factor in gastric juice, serum, amniotic fluid and urine is described.

Anemia, Megaloblastic↗
PubMed1977

Primary deficiencies in humoral immunity.

Patients with primary disorders of B lymphocytes and immunoglobulins usually display increased susceptibility to bacterial infections but atopic, autoimmune, and malignant disorders are also more common in these patients. The spectrum of these disorders ranges from a virtual absence of B cells and immunoglobulins to selective deficiencies of immunoglobulin subclasses. The diagnosis is dependent upon the demonstration of the immunologic deficits by specialized laboratory procedures which include the quantitation of immunoglobulins, the formation of antibodies in vivo and in vitro and the demonstration of B cells in the tissues or the peripheral blood. There are five major points in the management of these patients: (1) the delineation of the immunologic defects by laboratory testing, (2) the use of parenterally injected human immunoglobulins, (3) the rapid identification of infecting organisms and the prompt institution of appropriate antibiotic therapy, (4) screening the family for immunodeficiency in those cases which are of genetic origin, and (5) genetic counseling.

Adult↗