Vitamin B 12 (cobalamin, Cbl) is absorbed in the distal ileum with the help of a specific binding protein intrinsic factor (IF) and appears in the circulation bound to another carrier transcobalamin (TC) [1]. Tissue uptake of the TCAECbl complex (holo-TC) is mediated by specific receptors on the surface of the plasma membrane [2]. Holo-TC represents Cbl available for cellular uptake and a decrease in its level would indicate reduced absorption of the vitamin as well as systemic Cbl deficiency. Two new methods have recently been described for the measurement of holo-TC in plasma samples [3,4]. Both methods employ TC-specific antibodies to capture the protein from plasma but lack the specificity needed for direct measurement of holo-TC in serum. The antigenic determinants and the functional domains of TC have not been identified.Cloning [5][6][7] and recent expression of several kindred Cbl-binding proteins [8][9][10][11] Recombinant human transcobalamin (TC) was probed with 17 monoclonal antibodies (mAbs), using surface plasmon resonance measurements. These experiments identified five distinct epitope clusters on the surface of holo-TC. Western blot analysis of the CNBr cleavage fragments of TC allowed us to distribute the epitopes between two regions, which spanned either the second quarter of the TC sequence GQLA…TAAM or the C-terminal peptide LEPA…LVSW(316-427). Proteolytic fragments of TC and the synthetic peptides were used to further specify the epitope map and define the functional domains of TC. Only one antibody showed some interference with cobalamin (Cbl) binding to TC, and the corresponding epitope was situated at the C-terminal stretch TQAS…QLLR(372-399). We explored the receptor-blocking effect of several mAbs and heparin to identify TC domains essential for the interaction between holo-TC and the receptor. The receptor-related epitopes were located within the TC sequence GQLA…HHSV(103-159). The putative heparin-binding site corresponded to a positively charged segment , which also seemed to be necessary for receptor binding. We conclude that conformational changes in TC upon Cbl binding are accompanied by the convergence of multiple domains, and only the assembled conformation of the protein (i.e. holo-TC) has high affinity for the receptor. Co]cyano-Cbl; IF, intrinsic factor; RU, resonance unit; SPR, surface plasmon resonance; TC, transcobalamin; TC p , recombinant human transcobalamin produced in a plant system; TC p11 , TC y31 ,…, the proteolytic fragments of TC p and TC y with the indicated molecular mass; TC y , recombinant human transcobalamin produced in yeast.
We present a new filter assay for the determination of glycohemoglobin as a unique application of the boronic acid affinity principle. With the use of a water-soluble blue-colored boronic acid derivative and a specific precipitation method for hemoglobin, total hemoglobin including bound boronic acid is precipitated and collected on a filter strip before quantification. Hemoglobin and boronic acid are quantified by a dual-wavelength reflectometric measurement, and the result is reported directly as percent glycohemoglobin. The test is simple, quick, and designed as a doctors’ office test for the monitoring and management of diabetes. The imprecision of the assay is <4% over the range 3–18% Hb A1c, and the method is linear up to at least 20% Hb A1c. Comparisons with four well-established glycohemoglobin methods yielded correlation coefficients ranging from 0.94 to 0.99, with slopes from 0.94 to 1.01.
Carbohydrate-deficient transferrin (CDT) may now be the most valuable biological marker for diagnosis of alcohol abuse. We compared the diagnostic performance of two new CDT tests, Axis %CDT turbidimetric immunoassay (TIA) and Axis %CDT HPLC, against Specialty Laboratories’ isoelectric focusing/immunoblotting/laser densitometry (IEF/IB/LD). Both Axis tests include one-half the concentration of trisialotransferrin isoforms in their CDT quantitation schemes. Considering an alcohol abuse prevalence of 7%, Axis %CDT TIA shows a sensitivity of 87% at 98% specificity and a positive predictive value (PPV) of 0.75; %CDT HPLC shows a sensitivity of 87% at 100% specificity for a PPV of 1, and the IEF/IB/LD shows 81% sensitivity at 94% specificity for a PPV of 0.5. All three CDT tests show the same negative predictive value (0.98). Both Axis procedures perform better than IEF/IB/LD in the diagnosis of alcohol abuse; %CDT TIA is available in several semiautomated, cost-effective formats.
CDT (carbohydrate-deficient transferrin) has been identified as a specific marker for chronically elevated alcohol consumption. We investigated the sensitivity and accuracy of using relative concentrations of different isotransferrins in serum for diagnosis of chronically elevated alcohol consumption. The different transferrin variants (isoforms) were quantified by HPLC. Including the trisialo-transferrin fraction into the definition of %CDT resulted in an increased accuracy in the detection of chronically elevated alcohol intake in a study among 17 heavy drinkers, 25 healthy individuals with moderate alcohol consumption and nine total abstainers. The results also suggest that desialylation of transferrin is a gradually continuing process, rather than one leading to a single end-result separating asialo-, mono- and disialo-transferrins from trisialo-, tetrasialo-, pentasialo- and higher sialo-transferrins.
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