Overall, our data suggest that it is important to take this polymorphism into account when studying associations between total TC and clinical outcome. Background: We developed microbiological assays (MBAs) to identify determinants and to establish reference values for cobalamin bound to transcobalamin [holotranscobalamin (holoTC)] and total TC in plasma. This association remained significant after adjustment for age and creatinine concentration, but became nonsignificant after inclusion of tHcy in the model. and you may need to create a new Wiley Online Library account. Helga Refsum, Carole Johnston, Anne Berit Guttormsen, Ebba Nexo, Holotranscobalamin and Total Transcobalamin in Human Plasma: Determination, Determinants, and Reference Values in Healthy Adults, Clinical Chemistry, Volume 52, Issue 1, 1 January 2006, Pages 129–137, https://doi.org/10.1373/clinchem.2005.054619. Hvas AM, Nexo E. Holotranscobalamin—a first choice assay for diagnosing early vitamin B deficiency?. Collapse Section. Chanarin I, Muir M. Demonstration of vitamin B12 analogues in human sera not detected by microbiological assay. Redox status and protein binding of plasma homocysteine and other aminothiols in patients with hyperhomocysteinemia due to cobalamin deficiency. We are indebted to Halvard Bergesen, Elfrid Blomdal, Beate Olsen, Ove Netland, Otto Baarholm (Bergen), Anna-Lisa Christensen (Aarhus), Cynthia Prendergast, and Donald Warden (Oxford) for their expert technical assistance. Miller JW, Ramos MI, Garrod MG, Flynn MA, Green R. Transcobalamin II 775G>C polymorphism and indices of vitamin B12 status in healthy older adults. The online version of this article (doi:10.1007/s10545‐013‐9664‐5) contains supplementary material, which is available to authorized users. Axis-Shield donated the magnetic beads, calibrators, and control samples for holoTC. In the total TC assay, incubation of the sample in the presence of a cyanocobalamin concentration ∼10 times the mean total TC concentration ensured maximum saturation without demanding repeated washing steps to remove excess cyanocobalamin. The genotype effect was more pronounced when total TC concentrations were measured by ELISA (geometric means, 957 vs 863 vs 764 pmol/L; P <0.001, adjusted for age and sex). We compared the variation between duplicate measurements of total TC, holoTC, and TC saturation with 20 samples, all measured on the same day with the same calibration curve. Furthermore, measurement of total TC concentrations may be useful for diagnosis of inherited TC deficiency. BjorkeMonsen AL, Ueland PM, Schneede J. Low vitamin B-12 status in confirmed Alzheimer’s disease as revealed by serum holotranscobalamin. The commercial RIA for holoTC (13), however, requires 400–800 μL of plasma, an amount that makes this assay unsuitable for studies with limited available sample volume. Further analyses revealed that the age effect was limited to women, and the sex differences were confined to those ≤45 years (see Fig. Here, a brief review will be given of this problem and of the … The concentration of holoTC in the stock solution was determined (a) from the absorbance at 362 nm, based on a molar absorptivity of 30 000 cm−1 (18), and (b) by determination in a vitamin B12 assay. Summary. Quantitative methods for measurement of transcobalamin II. Plasma cobalamin was measured by MBA using a colistin sulfate–resistant strain of L. leichmannii (16)(17). It took up B 12 when either form of B 12 or liver B 12 was given by mouth. Because of limited data collection, a shortcoming of our study, we do not have data on use of oral contraceptives or hormone replacement therapy, nor do we have any measurements of iron status, which is of central importance for evaluating associations to hematologic factors. Incontrast, the two subjects lacking seminal vesicles dem-onstrated sharply decreased transcobalamin … Holotranscobalamin (holoTC) is the portion of cobalamin that is bound to the transport protein transcobalamin (TC), which facilitates cellular uptake of cobalamin . For comparison between groups, the Student t-test for independent samples, ANOVA, or the χ2 test was used. In most cases presented, at least weekly injections (1 mg IM) were necessary to ensure optimal treatment. In these healthy blood donors, serum creatinine was not associated with total TC in either sex, and it was not associated (women) or was weakly associated (men) with holoTC. Pseudovitamin B(12) is the predominant cobamide of an algal health food, spirulina tablets. In regression analyses including age, sex, tHcy, cysteine, holoTC, and the TCN2 genotype, the genotype was by far the strongest determinant of total TC (partial R for MBA = −0.27, for ELISA = −0.43). In relation to cysteine, one possible explanation could be that low holoTC is associated with high tHcy, which may displace cysteine from plasma (31). Total TC was measured as holoTC after the binding sites had been saturated with cyanocobalamin. Microbiological assay for vitamin B12 performed in 96-well microtitre plates. There were no differences in holoTC and total TC concentrations in fasting vs nonfasting individuals, and we found no correlation between time since last meal and holoTC or total TC concentrations. Armstrong RA, Eperjesi F, Gilmartin B. b Volumes in microliters. E-mail. Quantification of holo-transcobalamin, a marker of vitamin B12 deficiency. Associations between holoTC and total TC and other variables were assessed by Spearman correlation and multiple linear regression analysis. Using all samples from the reference population, we found that holoTC by MBA strongly correlated (r = 0.95, Spearman correlation) with holoTC by RIA (13), but the values were consistently ∼20% higher by MBA than by RIA, as confirmed by a Bland–Altman plot (see Fig. 11 ), these calibrator samples corresponded to plasma concentrations of 0, 52, 77, 103, 129, 157, 206, 310, 413, 516, 723, and 930 pmol/L. There was no significant sex difference in total TC concentrations, but total TC increased with age in women but not in men (see Fig. In the total TC and holoTC assays, however, we measured cobalamin bound to TC. Diagnosis of TC deficiency is suspected based on megaloblastic anemia, elevation of total plasma homocysteine, and blood or urine methylmalonic acid. 2. In contrast, TC saturation becomes lower. Conclusions: Our MBAs for TC and holoTC required low plasma volume and performed acceptably compared with other methods. However, our data show that these proportions change according to cobalamin status: at low concentrations, a higher proportion of cobalamin exists as holoTC, suggesting that holoTC is better maintained than haptocorrins. However, entering tHcy and possible confounders in the regression model strengthened the association. Address correspondence to this author at: Oxford Centre for Gene Function, University Laboratory of Physiology, University of Oxford, Parks Rd, Oxford OX1 3P, United Kingdom. The solution was then diluted to 100 μg/L in water, divided into aliquots, and stored at −80 °C. Reference values were 670–1270 pmol/L for TC and 42–157 pmol/L for holoTC, but they differed according to age and sex. Further analyses showed that the positive association between creatinine and holoTC was confined to men >45 years of age (rs = 0.25). We examined blood samples from 500 healthy blood donors for physiologic and biochemical variables associated with holoTC and total TC. Matrix effects were tested by paired-sample t-test. Further investigations to elaborate accurate age- and sex-specific reference values are warranted. A 50 mg/L solution of cyanocobalamin of was first prepared and the concentration checked by the absorbance at 362 nm, based on a molar absorptivity of 30 000 cm−1. The characteristics of the total population and for men and women separately are listed in Table S2 in the online Data Supplement. 1. Cobalamin (B 12) has three carrier proteins namely haptocorrin (formerly transcobalamin I) which binds majority of B 12, transcobalamin (TC) (formerly transcobalamin II)—which is responsible for the … RIA for Serum Holo-Transcobalamin: Method Evaluation in the Clinical Laboratory and Reference Interval Saila Loikas,1* Minna Lo¨ppo¨nen,2 Pauli Suominen,1 Jan Møller,3 Kerttu Irjala,1 Raimo Isoaho,2,4 Sirkka-Liisa Kivela¨,4,5 Pertti Koskinen,1 and Tarja-Terttu Pelliniemi1 Background: Decreased serum holo-transcobalamin … The correlation coefficient was relatively low, however, possibly as a consequence of low between-person variability in total TC, because correlation between measurements is dependent on the variability between samples (29). Stupperich E, Nexo E. Effect of the cobalt-N coordination on the cobamide recognition by the human vitamin B12 binding proteins intrinsic factor, transcobalamin and haptocorrin. Transcobalamin II (TC II), a plasma vitamin B 12 binding protein, took up cyanocobalamin and hydroxocobalamin when added to plasma in amounts of 1.0 & 25 pg/ml of plasma. Spearman rank correlations. Transcobalamin deficiency is a disorder that impairs the transport of cobalamin (also known as vitamin B12) within the body. PMID: 12413492 optimal binding of Cbl by human TC II is supported … The mean CVs were 1.6% (range, 0.1%–7.5%) for total TC, 1.7% (0%–5.8%) for holoTC, and 2.6% (0%–10%) for TC saturation. Imprecision of TC saturation was difficult to assess because we usually measured total TC and holoTC on different days. 1972 Jan; 22 (1):33–42. We lacked data on the use of hormonal replacement therapy or oral contraceptives; however, we observed the same low concentrations in women 35–45 years of age, a group that traditionally does not use oral contraceptives—in 40-year-old Norwegians, only 3.5% of the women used combined hormonal contraceptives (42). About 30% of plasma cobalamin is bound to transcobalamin II while the remaining part is bound to haptocorrin, but only the part of circulating cobalamin attached to transcobalamin II is the biologically active form and transcobalamin II mediates the entry of cobalamin into a variety of cell types other than hepatocytes [3,4,5]. For holoTC, the major change, compared with the published HoloTC RIA [Axis Shield ASA (13)], was that the RIA has been replaced with the MBA developed for cobalamin determination. The precise physiological role of HC within the complex process of Cbl internalization is not yet fully discovered. A randomized placebo-controlled study. Bjorksten KS, Dige N, Nexo E. Long-term memory correlates negatively with plasma transcobalamin in healthy elderly with normal plasma cobalamin levels. Transcobalamin II expression is regulated by transcription factor(s) binding to a hexameric sequence (TGGTCC) in the promoter region. Microtiter plates (flat-bottomed 96-well microtest plates; well volume, 360 μL) were from Sarstedt, and adhesive sealing film for the plates was from Fisher Scientific. … Using samples from the reference population (see below), we compared holoTC results obtained by MBA with those from a commercial holoTC assay (13). Median age was 44 years; 49% of the donors were women, 21.7% were smokers, and 92.5% were nonfasting at the time of blood collection. We assessed bacterial growth by measuring absorbance at 590 nm with a plate reader. It is confirmed by studying the synthesis of TC in cultured fibroblasts, or by molecular analysis of the TCN2 gene. Both holoTC and total TC are increased in renal failure (32)(33), and serum creatinine is an important determinant of holoTC concentrations in elderly persons and in individuals with impaired cobalamin status (9)(30). Bloomfield FJ, Scott JM. Levey AS, Perrone RD, Madias NE. Afman LA, Van Der Put NM, Thomas CM, Trijbels JM, Blom HJ. Bland JM, Altman DG. McCaddon A, Blennow K, Hudson P, Hughes A, Barber J, Gray R, et al. Zetterberg H, Nexo E, Regland B, Minthon L, Boson R, Palmer M, et al. With our MBA, concentrations of holoTC and total TC in EDTA plasma, heparin plasma, and serum were similar, whereas citrate plasma had significantly lower concentrations than other matrixes (81% relative to EDTA for holoTC, 86% for total TC). 1972 Jan; 22 (1):33–42. Nexo E, Engbaek F, Ueland PM, Westby C, O’Gorman P, Johnston C, et al. For total TC, we used 3 pools of human plasma; in pool 1 (P1) the concentration was above the upper reference limit, in P2 within the reference interval, and in P3 in the low end of the reference interval. In the commercial assay, the magnetic beads for capturing TC are the same, but cobalamins are determined by RIA. Cobalamin is obtained from the diet; this vitamin is found in animal products such as meat, eggs, and shellfish. News & … S1 and S5 were then mixed so that samples S2, S3, and S4 contained 50%, 25%, and 12.5% of S1 and 50%, 75%, and 87.5% of S5, respectively. Nexo E, Christensen AL, Petersen TE, Fedosov SN. 4: Has a low potential for abuse relative to those in schedule 3. function as a storage protein and as scavenger of inactive Cbl-analogues. The new assays were compared with published methods. Tubes with anticoagulant were placed on ice, and serum tubes were kept at room temperature. Enter your email address below and we will send you your username, If the address matches an existing account you will receive an email with instructions to retrieve your username, By continuing to browse this site, you agree to its use of cookies as described in our, I have read and accept the Wiley Online Library Terms and Conditions of Use, https://doi.org/10.1007/s10545-013-9664-5. In regression analyses, this association remained significant, and for holoTC, it became stronger after adjustment for age, tHcy, creatinine, or the other variables listed in Table 11 . A stock of Lactobacillus leichmannii (NCIB 12519, ATCC 43787) was provided by Professor John M. Scott, Departments of Biochemistry and Clinical Medicine, Trinity College, Dublin, Ireland. Serum creatinine and renal function. Refsum H, Smith AD, Ueland PM, Nexo E, Clarke R, McPartlin J, et al. Cobalamin (vitamin B12) is essential for 1-carbon metabolism and cell division. Fractionation of vitamin B 12 binders in a normal human serum pool showed a distribution of 92% Transcobalamin II (TCII) and 8% Transcobalamin I (TCI) on DEAE‐cellulose chromatography. Total TC by MBA had a correlation coefficient of 0.79 with total TC by ELISA (14), and the values were ∼6% higher than those obtained by ELISA. « hide 10 20 30 40 50 mrhlgaflfl lgvlgaltem ceipemdshl veklgqhllp wmdrlslehl 60 70 80 90 100 npsiyvglrl sslqagtked lylhslklgy qqcllgsafs eddgdcqgkp 110 120 130 140 150 smgqlalyll alrancefvr ghkgdrlvsq … Markedly skewed variables (cobalamin, holoTC, total TC, TC saturation, tHcy, MMA, folate) were logarithmically transformed before statistical analysis. The “true concentrations” of S2, S3, and S4 were calculated from the fractional composition of S1 and S5 and then compared with the measured results. In addition, we assessed between-day variation for holoTC by analyzing the control samples for holoTC provided by Axis-Shield. Identification of a new vitamin B 12 binder (transcobalamin 3) in normal human serum. Holo-transcobalamin is an early marker of changes in cobalamin homeostasis. In the ELISA, γ-globulins from 2 polyclonal rabbit antibodies against recombinant human TC were used as capture and detection antibodies, and recombinant human TC was used as a calibrator. The plate reader, with a 590 nm multiscan filter, was from Thermo Electron Corporation. This finding was related to a complete shift in cobalamin concentrations to the left, which was not associated with metabolic effects on tHcy or MMA. The relationship of total TC to cobalamin status is not as well understood, but together with holoTC, total TC measurements can be used to determine TC saturation, which may be better than holoTC alone for detecting changes in cobalamin status (4)(6). As expected (5)(9)(30), holoTC concentrations were independently associated with tHcy and MMA, but the associations were relatively weak. This in vivo function is supported by the promotion … Significant correlations are indicated in bold. This observed difference is probably caused by slightly different holoTC concentrations in different calibrator batches. Br J Haematol. S2 and Table S3 in the online Data Supplement). Using the new assays, we searched for determinants and determined reference values for plasma holoTC and total TC in healthy adults. Rothenberg SP, Quadros EV. The between-day CVs were 3.8%–5.7% for the high control samples and 4.9%–7.3% for the low control samples. Get emergency medical help if you have any of these signs of an allergic reaction: hives; chest tightness, difficulty breathing; swelling of your face, lips, … The growth medium with the L. leichmannii was prepared as follows: 6.2 g of Difco B12 assay medium and 150 μL of Tween 80 were mixed with 100 mL of sterile H2O, heated, and boiled for 3 min. Refsum H, Smith AD. Transcobalamin codon 259 polymorphism in HT-29 and Caco-2 cells and in Caucasians: relation to transcobalamin and homocysteine concentration in blood. Riedel B, . Learn more. Br J Haematol. The MBAs for holoTC, total TC, and cobalamin are described in detail in Fig. As expected, the interrelationships between cobalamin, holoTC, and TC saturation were strong (Table 11 ). Here we present a series of 30 patients with TC deficiency, including an update on multiple previously published patients, in order to evaluate the different treatment strategies and provide information about long term outcome. Mansoor AM, Ueland PM, Svardal AM. This relationship between holoTC and folate might indicate that individuals with good folate status also tend to have good cobalamin status. PubMed is a searchable database of medical literature and lists journal articles that discuss Transcobalamin 1 deficiency. We included 2 additional concentrations: 30 pmol/L was prepared with a starting volume of 75 μL of 40 pmol/L calibrator, and 200 pmol/L with 125 μL of 160 pmol/L calibrator. This study was supported by a European Union Demonstration Project on the diagnostic utility of holoTC (QLK3-CT-2002-01775) and The Advanced Research Program of Norway. Carmel R, Vasireddy H, Aurangzeb I, George K. High serum cobalamin levels in the clinical setting—clinical associations and holo-transcobalamin changes. Most centres adjusted the treatment regimen based on monitoring CBC, total plasma homocysteine, plasma and urine methylmalonic acid, as well as, clinical status. To assess variation in the measurement of TC saturation, we used 20 samples from the reference population (see below) that differed in TC saturation, holoTC, and total TC concentration. Interestingly, the difference in total TC concentrations was most pronounced for the TCN2 776GG genotype, a finding that suggests that one or both assays give a systematic error depending on genotype, a matter that needs further elucidation. Finally, continuing IM treatment into adulthood appears to be beneficial. The effect of moderate hypovolaemia on microcirculation in healthy older blood donors. Thus, the typical finding in individuals with low cobalamin concentrations will be a relatively high holoTC/cobalamin ratio, whereas TC saturation will be low. Herrmann W, Obeid R, Schorr H, Geisel J. The purity of the recombinant holoTC was determined by sodium dodecyl sulfate electrophoresis and spectroscopically from the ratio of the protein component and the cobalamin component. Over a 15-month period, 2 technicians performed 75 different runs, with 3 batches of control samples. Using an MBA to detect the cobalamin moiety, we measured cobalamin, holoTC, and total TC with only ∼150 μL of plasma. In addition, we found that younger women had lower reference values for cobalamin, holoTC, and TC saturation than the rest of the population. We observed the same pattern in relation to age and sex when we adjusted for hematocrit or excluded individuals with low hematocrits. Intrinsic factor–based assays, such as holoTC by RIA, have low affinity for analogs (26). This association was also present when we replaced the MBA values with holoTC by RIA and total TC by ELISA (rs = 0.16). The observed effects in young women may be related to hormonal factors, because estrogen intake in men (39) and use of oral contraceptives in premenopausal women (40)(41) have been associated with lower cobalamin and holoTC concentrations. Transcobalamin-bound vitamin B(12), or holotranscobalamin (HoloTC), a biologically active form, is believed to be a sensitive marker of vitamin B(12) deficiency. It can sometimes resemble neonatal leukemia or severe combined immunodeficiency disease. End-stage renal disease increases plasma transcobalamin and neutralizes influence of TCN 776C>G polymorphism. Vikelsoe J, Bechgaard E, Magid E. A procedure for the evaluation of precision and accuracy of analytical methods. The clinical consequences of cobalamin deficiency include megaloblastic anemia and neurologic disease. Transcobalamin II (TC II) transports vitamin B12 to the tissues. Multi- or B-vitamin supplements were used by 15.6%. The reference values in the total population and according to age and sex are listed in Table 22 . c Incubation for longer times, such as overnight, is possible. For all study participants, total TC was associated with holoTC and number of TCN2 766C alleles; in female participants only, total TC was also associated with age, homocysteine, and cysteine. Between-day CVs were 1%–2% lower when we used duplicate rather than singular measurements (see Table S1 in the online Data Supplement). HoloTC and TC by MBA correlated with holoTC by RIA (r = 0.95) and TC by ELISA (r = 0.79), respectively. It thus is unlikely that the presence of analogs explains the systematic 20% difference in holoTC measurements between the MBA and RIA. pared with normal (t = 3.369, P< 0.01) while transcobalamin IIremained normal (t = 1.391). The “true values” of S1 and S5 were estimated as the overall means obtained from the between-day experiments. We assessed the within-day CV by measuring 10 parallels of samples S1 through S5 for cobalamin and for holoTC, and 10 parallels of pools P1 through P3 for total TC. Comparison of methods was performed by Spearman correlations and by Bland–Altman plots (25). Methods: We captured holoTC with magnetic beads with TC antibodies and used a conventional MBA for cobalamin measurements. An inability to transport cobalamin within the body results in cells that lack cobalamin, which they need for many functions including cell growth and division (proliferation) and DNA production. d See Materials and Methods for additional details. The magnetic separator was from Abgene. The holoTC calibrator set was also used for total TC; these were analyzed as the holoTC calibrators, i.e., without saturation with cyanocobalamin (Fig. Search for other works by this author on: Department of Anaesthesia and Intensive Care, © 2006 The American Association for Clinical Chemistry, This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (, Chemical Characterization and Quantification of Circulating Intact PTH and PTH Fragments by High-Resolution Mass Spectrometry in Chronic Renal Failure, Jagged Ends of Cell-Free DNA: Rebranding Fragmentomics in Modern Liquid Biopsy Diagnostics, Is It Time to Move On? More surprising is our observation that holoTC was independently associated with plasma folate and total cysteine. Results obtained by MBA were used, but with few exceptions (listed below), we found very similar results for holoTC by RIA and total TC by ELISA. Using 30 samples with variable holoTC (range, 28–227 pmol/L) and total TC concentrations (range, 477–2088 pmol/L), we searched for matrix effects by measuring cobalamin, holoTC, and total TC in serum and in EDTA-, heparin-, and citrate plasma. The remaining proportion is bound to haptocorrins (4)(5), the function of which is not known. The full text of this article hosted at iucr.org is unavailable due to technical difficulties. The performance of the MBA was comparable to other assays (5)(13)(14)(15). Neither smoking status nor use of B-vitamin or multivitamin supplements was associated with cobalamin, holoTC, or total TC. The usefulness of holotranscobalamin in predicting vitamin B12 status in different clinical settings. The funding sources had no influence on the study design, experiments, and data analyses or on the decision to submit the manuscript for publication. We acknowledge the staff of the Blood Bank at Haukeland University Hospital for help in collecting blood samples. STABLER2, and M.D. The association between holoTC and tHcy or MMA remained significant in multivariate regression analyses. Haptocorrin (HC), also commonly known as the R-protein, or the R-factor, or previously referred to as transcobalamin … In women ≤45 years of age, there was a complete shift of the holoTC distribution toward lower concentrations (see Fig. Effects of oral contraceptives and hormone replacement therapy on markers of cobalamin status. We changed this assay into a microbiological assay (MBA) that uses less plasma volume and further modified it for use in determining total TC. Because we used one-tenth the plasma volume for total TC compared with holoTC, the results were multiplied by 10. Each aliquot was thawed only once. Furthermore, the 2 methods have a different approach: MBA measures the cobalamin moiety (16), whereas ELISA measures the protein moiety (14). Brattström L, Israelsson B, Olsson A, Andersson A, Hultberg B. Holo-transcobalamin concentration and transcobalamin saturation reflect recent vitamin B12 absorption better than does serum vitamin B12. Results: Determination of cobalamin, holoTC, and TC by MBA required <150 μL. Husek P. Simultaneous profile analysis of plasma amino and organic acids by capillary gas chromatography. There was a weak but significant positive association between holoTC and total TC, which remained significant in linear regression models after adjustment for age, sex, TCN2 genotypes, and the other variables listed in Table 11 . The essential function of haptocorrin is protection of the acid-sensitive vitamin B 12 while it moves through the stomach.. Function. Between-day CVs for holoTC and total TC were 4%–9%. Kelleher BP, O’Broin SD. HoloTC was inversely associated with tHcy and MMA concentrations. We assessed linearity for the holoTC assay with 5 samples with interrelated concentrations (see Table S1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol52/issue1/). We assessed holoTC linearity with samples S1 through S5, as described previously (19)(20). Another potential weakness with this population was that we collected blood at the end of a routine blood donor session. Abstract. NX_P20061 - TCN1 - Transcobalamin-1 - Function. 3. There was no effect of the polymorphism on cobalamin or holoTC concentrations in the total group, but an analysis confined to participants >45 years of age showed a significant difference in holoTC concentrations according to genotype in women (geometric mean, 102 vs 86 vs 80 pmol/L; P = 0.041, adjusted for age) but not in men (P = 0.22). The optimized procedures for cobalamin, holoTC, total TC, and their calibrators are described in Fig. a In addition to a zero sample, there were 7 calibrators for holoTC (and total TC) that covered the range 10–200 pmol/L (corresponding to 100-2000 pmol/L for total TC), and 11 calibrators for cobalamin that covered the range 52–930 pmol/L.