Total Transcobalamin Research Articles

Vitamin B12 and its binding proteins in patients with non-small cell lung cancer referred to fast-track diagnostic work-up for lung cancer

High cobalamin levels have previously been associated with short-term cancer risk, including lung cancer. We explored whether levels of cobalamin and/or its binding proteins are useful as a diagnostic tool in patients suspected of non-small cell lung cancer. We included 889 patients referred for fast-track diagnosis of lung cancer to Aarhus University Hospital, Denmark. We analyzed plasma concentrations of cobalamin, transcobalamin, holotranscobalamin and haptocorrin. Information on lung cancer diagnosis was retrieved from a national database. The study cohort showed levels above reference intervals for cobalamin 12%, holotranscobalamin 25%, transcobalamin 9% and haptocorrin 36% (all p-values <.05). We observed no difference in cobalamin or holotranscobalamin levels when comparing patients diagnosed with non-small cell lung cancer (n = 161, 18%) to patients without lung cancer (n = 742, 80%), while transcobalamin showed minor differences. Haptocorrin was significantly higher in those with cancer, mainly among patients with adenocarcinoma (n = 94). A comparison of patients with the highest vs. lowest quartile levels of haptocorrin yielded an adjusted odds ratio for adenocarcinoma of 2.39 (95% confidence interval: 1.26–4.55). However, ROC curve analyzes showed haptocorrin (AUC = 0.55) and total transcobalamin (AUC = 0.56) to be poor diagnostic markers for lung cancer. A high proportion of patients suspected for non-small cell lung cancer showed increased levels of cobalamin-binding proteins. We thereby confirm the association between non-small cell lung cancer and high cobalamin levels and found that haptocorrin was the major underlying factor causing high cobalamin levels. However, none of these biomarkers were of diagnostic use among patients referred for suspected lung cancer.

Measurement of total Transcobalamin employing a commercially available assay for Active B12

IntroductionVitamin B12 deficiency is mostly caused by insufficient gastro-intestinal absorption and in rare conditions by Transcobalamin (TC) deficiency. Unsaturated Transcobalamin (apoTC) can be measured by a binding assay using radiolabeled cobalamin. The Active B12 test analyzes saturated Transcobalamin (holoTC) and we hypothesize that this test can be used to measure total TC by additional in vitro saturation with cobalamin. MethodsSerum was saturated in vitro (16 times dilution) with a cyanocobalamin solution and total TC was selectively measured with the Abbott Active B12 test. ApoTC was calculated by subtracting endogenous holoTC from total TC after correction for dilution. Linearity was determined with a pool serum dilution series. Precision was investigated according to the CLSI EP15 protocol. Method comparison was performed against a binding assay using radiolabeled cobalamin. Reference values were determined in 100 healthy controls. ResultsThe method was linear in the range of 240 to 1933pmol/L (R2=0.997, lack of fit F=1.61). Precision of low- and high-pool total TC in serum were; 5.2% and 4.3% respectively. Method comparison against a radiolabeled cobalamin binding assay showed a proportional bias of 30% (y=0.70x+126). Total TC reference values were determined at 500–1276pmol/L. ConclusionWe describe a rapid method to quantify total TC, which can be implemented on routine platforms using commercial Active B12 tests. In addition, apoTC can be assessed by subtracting endogenous holoTC concentration which can be measured in the same run, securing the same calibration level for all three parameters (holoTC, apoTC and total TC). This method is applicable in clinical diagnostics and in larger epidemiological studies.

First trimester serum levels of the soluble transcobalamin receptor, holo-transcobalamin, and total transcobalamin in relation to preeclampsia risk

Background: Human placenta expresses CD320, a receptor that ensures the uptake of holo-transcobalamin (holoTC). Soluble CD320 (sCD320) is present in the circulation and its concentration increases during pregnancy.Aims: To investigate a possible association of sCD320, holoTC and total transcobalamin (TC) with the risk of subsequent preeclampsia using serum samples from asymptomatic first trimester pregnant women. Moreover, we aimed to establish reference intervals of the aforementioned biomarkers for first trimester pregnant women who remained healthy throughout pregnancy.Study design: This study was a retrospective case-control study that we performed on biobank serum samples. Cases (n = 50) and controls (n = 198) (matched for gestational age and date of sample collection) were asymptomatic women in early pregnancy [median (range) gestational age = 10 (8–12) weeks]. Cases developed preeclampsia while the controls remained normotensive throughout pregnancy. We measured the serum concentration of sCD320, holoTC, and total TC by using in-house ELISA methods.Results: First trimester median concentrations of sCD320, holoTC and total TC were not significantly different between cases and controls. The odd ratio for developing preeclampsia based on exposure to low or high levels of sCD320, holoTC or total TC at first trimester was not significant. The reference intervals (2.5–97.5% percentiles (median)) derived from the controls were 50–170 (90) pmol\\L for sCD320, 20–140 (70) pmol\\L for holoTC and 560–1300 (810) pmol\\L for total TC.Conclusions: The risk of preeclampsia is not predicted by first trimester serum concentrations of sCD320, holoTC or total TC. The first trimester reference intervals for the three parameters is reported.

Association of a transcobalamin II genetic variant with falsely low results for the holotranscobalamin immunoassay.

The clinical use of holotranscobalamin (holoTC) testing to evaluate vitamin B12 status has increased in recent years. We present two patients (African Caribbean and Indian heritage), in which the holoTC assay indicated severe B12 deficiency (< 5 pmol/L). Additional clinical tests revealed that these patients had normal levels of total vitamin B12 in blood and unremarkable levels of two other markers of vitamin B12 status, homocysteine and methylmalonic acid. We hypothesized that these patients carry a variant in the transcobalamin gene (TCN2) that influences the most widely commercially available holoTC test - Active-B12 (Axis-Shield Diagnostics Ltd). Exon sequencing of the TCN2 gene was carried out. Protein characterization included total transcobalamin (TCN2) detection by Western blot, and holoTC by (57) Co-labelled B12 binding followed by size fractionation. Exon sequencing of TCN2 revealed both patients were homozygous for the minor allele of rs35838082 (p.R215W). Western blot and chromatographic analyses revealed that the serum of these patients contains intact transcobalamin and that this variant-containing protein binds vitamin B12 . The variant is rare in Caucasians (minor allele frequency (MAF) < 0·01) but more common in South Asians (MAF ~ 0·02) and those of African origin (MAF ~ 0·25). The impeded ability to detect normal levels of holoTC in these two patients may be due to this variant interfering with the detection of holoTC by one or both of the monoclonal antibodies currently employed in the Active-B12 test. Laboratories should be aware of this variant and use confirmatory tests when applicable.

Vitamin B12 and its binding proteins in hepatocellular carcinoma and chronic liver diseases

Background. The vitamin B12 (B12)-binding protein haptocorrin (HC) has proven to be a potentially useful biomarker in patients with fibrolamellar hepatocellular carcinoma (HCC). Little is known concerning the level of HC and other B12-related proteins in patients with HCC as compared to patients with other chronic liver diseases (CLDs) and healthy controls. We hypothesized that HC could be a biomarker of HCC. Aims. To investigate levels of HC and B12-related proteins in HCC compared to CLDs and healthy controls. Methods. We investigated two patient populations: A cross-sectional cohort of HCC patients (n = 130), CLD patients (n = 102) and healthy controls (n = 46) and a cohort of 38 HCC patients studied at baseline and 1, 4, and 12 weeks following ablative treatment. Patients were evaluated by standard biochemistry, Child–Pugh-score and Barcelona Clinic Liver Cancer (BCLC) classification. We analyzed total B12 by routine methods and HC, transcobalamin (TC), B12 saturated TC (holoTC), and the soluble cell surface receptor for holoTC (sCD320) by in-house enzyme-linked immunosorbent assay. Results. HC showed higher median (range) levels for both HCC (590 [290–5860]) and CLD patients (620 [310–4010]) compared to controls (460 [250–2020]) (p < 0.01). Total B12, TC, holoTC, and sCD320 showed elevated levels in both HCC and CLD compared to controls. Only holoTC changed following treatment, without a concurrent change in TC. Conclusion. B12 and B12-related proteins (total B12, HC, TC, holoTC, and sCD320) show elevations in both HCC and CLD patients compared to controls, suggesting a relation to CLD in general rather than to primary liver cancer. Thus, HC is not useful as a biomarker for HCC.

Cobalamin and haptocorrin in human milk and cobalamin-related variables in mother and child: a 9-mo longitudinal study

Measurement of milk cobalamin is hampered by the high content of the cobalamin-binding protein haptocorrin, and limited data are available relating trustworthy measures of milk cobalamin to cobalamin status in healthy mothers and their children. The objectives were to explore the concentration of cobalamin and haptocorrin in foremilk and hindmilk during the first 9 mo of lactation and to relate these results to biomarkers of an impaired cobalamin status of mother and child. Milk samples from 25 mothers were collected at 2 wk, 4 mo, and 9 mo postpartum for the measurement of cobalamin and haptocorrin. Plasma samples from a larger cohort of lactating mothers (n = 107) and their infants (n = 108) were collected at the same time points for the measurement of cobalamin, holotranscobalamin, total transcobalamin, total haptocorrin, and methylmalonic acid. Median (range) concentrations of cobalamin in hindmilk were 760 (210-1880), 290 (140-690), and 440 (160-1940) pmol/L at 2 wk, 4 mo, and 9 mo, respectively; the respective haptocorrin concentrations were 25 (9-102), 22 (4-100), and 180 (30-460) nmol/L. We found slightly lower values in foremilk. A decrease in milk cobalamin at 4 mo was associated with decreases in plasma cobalamin (P , 0.0001) and holotranscobalamin (P , 0.0001) in the infants. Strong positive associations in paired maternal-infant cobalamin concentrations were found at all time points. Foremilk and hindmilk contained comparable amounts of cobalamin and haptocorrin, but marked changes were observed during 9 mo of lactation. At 4 mo, low concentrations of milk cobalamin mirrored biochemical changes in infants, which suggests an impaired cobalamin status and indicates that nutrition from only mother's milk may not be sufficient for the supply of cobalamin from this age. This trial was registered by the Danish Data Protection Agency at www.datatilsynet.dk/english as 2008-41-2185.

Three family members with elevated plasma cobalamin, transcobalamin and soluble transcobalamin receptor (sCD320)

Plasma cobalamin is requested in order to diagnose cobalamin deficiency and low levels confirm a deficient state. Here, we present three family members with unexpected high levels of cobalamin. We included a patient referred for cobalamin measurement due to neurological symptoms, her son and her daughter. Mother and son both suffered from myotonic dystrophy type II, while the daughter tested negative for this disease. Blood samples were analyzed for cobalamin, haptocorrin, transcobalamin, holoTC, and sCD320. We employed gel filtration and antibody precipitation for further characterization. The protein coding region of the TCN2 gene, encoding transcobalamin, was sequenced. The patient, her {son} and [daughter] all had cobalamin levels above the measurement range of the routine method employed (>1476 pmol/L). Total transcobalamin and (holoTC) were 5980 (1500), {5260 (2410)} and [5630 (1340)] pmol/L, which is well above the upper reference limits of 1500 (160) pmol/L. The sCD320 concentration was also well above the upper reference limit of 97 arb.u.: 1340, {1510} and [1090] arb.u. Haptocorrin levels were within the reference range and no signs of cobalamin deficiency were found. DNA sequencing of the TCN2 gene revealed several known polymorphisms not associated with highly elevated transcobalamin levels. Upon gel filtration, sCD320 eluted as a larger molecule than previously reported. By incubation with anti-transcobalamin antibodies, we precipitated both transcobalamin and part of sCD320. The high cobalamin levels were mainly explained by high levels of holoTC, possibly caused by complex formation with its soluble receptor, sCD320. The family occurrence points to a genetic explanation.

Cobalamin Related Parameters and Disease Patterns in Patients with Increased Serum Cobalamin Levels

BackgroundMeasurement of serum cobalamin levels is routinely used to diagnose cobalamin deficiency. Surprisingly, approximately 15% of patients have high cobalamin levels and no consensus exists regarding the clinical implications.MethodsHospital-treated patients above 18 years of age referred for serum cobalamin measurement were included in groups of patients [percentage cobalamin supplemented] with low (<200 pmol/L, n = 200 [6%]), normal (200–600, n = 202 [6%]) high (601–1000, n = 217 [27%]) and very high (>1000, n = 199 [53%]) cobalamin levels. Total and cobalamin-saturated (holo) transcobalamin, total haptocorrin, soluble TC receptor, sCD320, and methylmalonic acid were analyzed. Data on diagnoses and medical prescriptions was obtained through medical files and the Aarhus University Prescription Database.ResultsAmong patients not cobalamin supplemented median total haptocorrin and holo transcobalamin levels were markedly higher in the groups with high/very high cobalamin levels compared to groups with low/normal cobalamin levels. Median total transcobalamin and sCD320 levels were similar across the groups. A number of diagnoses were significantly associated to very high Cbl levels (odds ratio (95% confidence interval)): alcoholism (5.74 (2.76–11.96)), liver disease (8.53 (3.59–20.23)), and cancer (5.48 (2.85–10.55)). Elevated haptocorrin levels were seen in patients with alcoholism, cancer, liver-, renal-, autoimmune-, and bronchopulmonary disease. No clinical associations to sCD320 and total and holo transcobalamin levels were found.ConclusionIn non-supplemented patients, high cobalamin levels were associated to high haptocorrin levels, and several diagnoses, including alcoholism, liver disease and cancer. Our study emphasizes that clinicians should take high serum cobalamin levels into consideration in the diagnostic process.

Bioinformatic and Genetic Association Analysis of MicroRNA Target Sites in One-Carbon Metabolism Genes

One-carbon metabolism (OCM) is linked to DNA synthesis and methylation, amino acid metabolism and cell proliferation. OCM dysfunction has been associated with increased risk for various diseases, including cancer and neural tube defects. MicroRNAs (miRNAs) are ∼22 nt RNA regulators that have been implicated in a wide array of basic cellular processes, such as differentiation and metabolism. Accordingly, mis-regulation of miRNA expression and/or activity can underlie complex disease etiology. We examined the possibility of OCM regulation by miRNAs. Using computational miRNA target prediction methods and Monte-Carlo based statistical analyses, we identified two candidate miRNA “master regulators” (miR-22 and miR-125) and one candidate pair of “master co-regulators” (miR-344-5p/484 and miR-488) that may influence the expression of a significant number of genes involved in OCM. Interestingly, miR-22 and miR-125 are significantly up-regulated in cells grown under low-folate conditions. In a complementary analysis, we identified 15 single nucleotide polymorphisms (SNPs) that are located within predicted miRNA target sites in OCM genes. We genotyped these 15 SNPs in a population of healthy individuals (age 18–28, n = 2,506) that was previously phenotyped for various serum metabolites related to OCM. Prior to correction for multiple testing, we detected significant associations between TCblR rs9426 and methylmalonic acid (p = 0.045), total homocysteine levels (tHcy) (p = 0.033), serum B12 (p < 0.0001), holo transcobalamin (p < 0.0001) and total transcobalamin (p < 0.0001); and between MTHFR rs1537514 and red blood cell folate (p < 0.0001). However, upon further genetic analysis, we determined that in each case, a linked missense SNP is the more likely causative variant. Nonetheless, our Monte-Carlo based in silico simulations suggest that miRNAs could play an important role in the regulation of OCM.

Associations of Folate, Vitamin B12, Homocysteine, and Folate-Pathway Polymorphisms with Prostate-Specific Antigen Velocity in Men with Localized Prostate Cancer

Vitamin B(12), holo-haptocorrin, and the folate-pathway single-nucleotide polymorphisms MTR 2756A>G and SHMT1 1420C>T have been associated with an increased risk of prostate cancer. We investigated whether these and other elements of folate metabolism were associated with prostate-specific antigen (PSA) velocity (PSAV) as a proxy measure of prostate cancer progression in men with localized prostate cancer. We measured plasma folate, B(12), holo-haptocorrin, holo-transcobalamin, total transcobalamin, and total homocysteine at diagnosis in 424 men (ages 45-70 years) with localized prostate cancer in a U.K.-wide population-based cohort. Thirteen folate-pathway single-nucleotide polymorphisms were genotyped for 311 of these men. Postdiagnosis PSAV (continuous measure and with a threshold set a priori at 2 ng/mL/y) was estimated from repeat PSA measurements. Median follow-up time was 2.5 (range, 0.8-5.6) years. Vitamin B(12), holo-haptocorrin, holo-transcobalamin, total transcobalamin, and total homocysteine were not associated with postdiagnosis PSAV. Folate was associated with an increased risk of PSAV >2 ng/mL/y [odds ratio (OR) per unit increase in log(e) concentration, 1.57; 95% confidence interval (95% CI), 0.98-2.51; P = 0.06]. MTRR 66A>G (rs1801394) was associated with a reduced risk (recessive model OR, 0.33; 95% CI, 0.11-0.97; P = 0.04), and SHMT1 1420C>T (rs1979277) with an increased risk (per-allele OR, 1.49; 95% CI, 0.93-2.37; P = 0.09) of PSAV >2 ng/mL/y. We found weak evidence that higher folate levels may be associated with faster progression of localized prostate cancer. Long-term follow-up is needed to test associations with metastases and mortality, and the observed genetic effects require replication.

Circulating Folate, Vitamin B12, Homocysteine, Vitamin B12 Transport Proteins, and Risk of Prostate Cancer: a Case-Control Study, Systematic Review, and Meta-analysis

Disturbed folate metabolism is associated with an increased risk of some cancers. Our objective was to determine whether blood levels of folate, vitamin B(12), and related metabolites were associated with prostate cancer risk. Matched case-control study nested within the U.K. population-based Prostate testing for cancer and Treatment (ProtecT) study of prostate-specific antigen-detected prostate cancer in men ages 50 to 69 years. Plasma concentrations of folate, B(12) (cobalamin), holo-haptocorrin, holo-transcobalamin total transcobalamin, and total homocysteine (tHcy) were measured in 1,461 cases and 1,507 controls. ProtecT study estimates for associations of folate, B(12), and tHcy with prostate cancer risk were included in a meta-analysis, based on a systematic review. In the ProtecT study, increased B(12) and holo-haptocorrin concentrations showed positive associations with prostate cancer risk [highest versus lowest quartile of B(12) odds ratio (OR) = 1.17 (95% confidence interval, 0.95-1.43); P(trend) = 0.06; highest versus lowest quartile of holo-haptocorrin OR = 1.27 (1.04-1.56); P(trend) = 0.01]; folate, holo-transcobalamin, and tHcy were not associated with prostate cancer risk. In the meta-analysis, circulating B(12) levels were associated with an increased prostate cancer risk [pooled OR = 1.10 (1.01-1.19) per 100 pmol/L increase in B(12); P = 0.002]; the pooled OR for the association of folate with prostate cancer was positive [OR = 1.11 (0.96-1.28) per 10 nmol/L; P = 0.2) and conventionally statistically significant if ProtecT (the only case-control study) was excluded [OR = 1.18 (1.00-1.40) per 10 nmol/L; P = 0.02]. Vitamin B(12) and (in cohort studies) folate were associated with increased prostate cancer risk. Given current controversies over mandatory fortification, further research is needed to determine whether these are causal associations.

Daily intake of 4 to 7 μg dietary vitamin B-12 is associated with steady concentrations of vitamin B-12–related biomarkers in a healthy young population

Studies have questioned whether the current Recommended Dietary Allowance (RDA) of 2.4 microg vitamin B-12/d is adequate. We examined the association between dietary vitamin B-12 intake and biomarkers of vitamin B-12 status. Dietary vitamin B-12 intake was estimated, and biomarkers of vitamin B-12 status were measured, in healthy men and women (n = 299; age range: 18-50 y) who were recruited from a Florida community. The National Cancer Institute Diet History Questionnaire was used. Plasma cobalamin, total transcobalamin, holo-transcobalamin, methylmalonic acid (MMA), total homocysteine (tHcy), and autoantibodies against intrinsic factor (IF) and Helicobacter pylori were analyzed in blood samples. Antibodies to H. pylori were detected in 12% of subjects (35/299), and negative results for IF antibodies were obtained for all subjects. The intake of vitamin B-12 correlated significantly with cobalamin, holo-transcobalamin, MMA, and tHcy. Subjects were divided into quintiles on the basis of their dietary vitamin B-12 intake (range: 0.42-22.7 microg/d), and biomarkers of vitamin B-12 status were plotted against estimated dietary vitamin B-12 intake. All biomarkers appeared to level off at a daily dietary vitamin B-12 intake between 4.2 and 7.0 microg. In persons with normal absorption, our data indicate that an intake of 4-7 microg vitamin B-12/d is associated with an adequate vitamin B-12 status, which suggests that the current RDA of 2.4 microg vitamin B-12/d might be inadequate for optimal biomarker status even in a healthy population between 18 and 50 y of age.

Mutations of TCN1 Cause Transcobalamin I Deficiency with Low Serum Cobalamin Levels That Are Indistinguishable From Cobalamin Deficiency.

Abstract 1989Poster Board I-1011Low serum cobalamin levels have diverse origins. Indeed, in 21-22% of cases low cobalamin levels do not even reflect cobalamin deficiency, as judged by metabolic criteria, and in most of the rest they reflect subclinical, purely biochemical cobalamin insufficiency. These complicate the interpretation of low cobalamin levels and have engendered controversies. Transcobalamin (TC) I deficiency causes spuriously low cobalamin levels because this binding protein normally carries >70% of the cobalamin in the blood, yet cellular cobalamin status remains normal despite the low plasma cobalamin levels. The entity is often misdiagnosed as cobalamin deficiency and incorrectly treated with cobalamin, and it is thought to be rare. Diagnosis is especially elusive if TC I deficiency is mild. We recently described two TCN1 nonsense mutations, 270delG and 315C>T, in 8 affected members of 3 non-Caucasian families, that caused TC I deficiency that was severe (absence of TC I and very low cobalamin) when both alleles are affected or mild (low plasma TC I and low or low-normal cobalamin) when only one allele was affected (Carmel et al, Br J Haematol, in press). We now describe the new association between TC I deficiency and a TCN1 polymorphism known to be more common in Caucasians. Patients and Methods: Plasma total TC I was assayed by radioimmunassay. TCN1 was sequenced as previously described, using 9 primer sets to sequence each of the 9 exons, in 4 unrelated patients with unexplained low cobalamin levels, in all of whom cobalamin deficiency and malabsorption were ruled out by metabolic and absorption studies. Available relatives were similarly tested. Results: All 4 unrelated propositi with mild TC I deficiency (plasma TC I 87-136 pmol/l, normal 165-454; serum cobalamin 94-155 pmol/l, normal 162-664) were found to be heterozygotes for a 999G>T missense mutation that causes substitution of tyrosine, a large hydrophobic amino acid, for the smaller, negatively charged aspartic acid at position 301. These 4 families of Caucasian ancestry also included 3 relatives with equally mild TC I deficiency (TC I 117-185; cobalamin 111-271), all of whom had the same mutation. In contrast, all 4 relatives with normal TC I levels (TC I 205-367; cobalamin 210-345) had no mutations. Conclusions: The association of heterozygosity for 999G>T with mild TC I deficiency in 7 affected members of 4 Caucasian families and its absence in all 4 unaffected members of these families suggest that this common mutation is responsible for TC I deficiency. It also adheres to the association of heterozygosity for our previously identified TCN1 mutations (270delG and 315C>T) with mild TC I-deficiency. The former occurred in non-Caucasian families, particularly of African ancestry, and were often linked in unexplained, possibly indirect ways with Hemoglobin S. The findings also suggest TCN1 mutational diversity among different ethnic groups. The known preponderance of the 999G>T polymorphism in European subjects is compatible with our ethnic findings. It may also explain why whites have significantly lower cobalamin and TC I levels than do blacks. Finally, the relatively high frequency of 999G>T polymorphism (11.6% + 0.02% heterozygosity frequency; NCBI website) resembles the 15% frequency of mild TC I deficiency reported in a TC I survey that suggested that TC I deficiency is a surprisingly common cause of low serum cobalamin levels and may often be hereditary. These TCN1 mutations should expand the ability to diagnose TC I deficiency, which can be a difficult diagnosis to make because TC I immunoassay is rarely available. They should also allow more reliable study of hereditary TC I deficiency and its frequency as a cause of low cobalamin levels. A better understanding of the entity may help clarify the currently mysterious role of TC I. Disclosures:Carmel:Emisphere Biotechnology: Consultancy; New York Methodist Hopital: Patent application pending on work presented here. Kelman:New York Methodist Hospital: Patent application on work presented here.

A longitudinal study of serum cobalamins and its binding proteins in lactating women

To examine longitudinal changes in serum cobalamins, transcobalamin (TC) and haptocorrin (HC) during lactation and to investigate the influence of vitamin B12 supplementation on these parameters. A 9-month follow-up study. Lactating mothers (N=89) including 23 supplemented with vitamin B12 (1-18 microg/daily), 41 partly supplemented and 25 not supplemented. Blood samples collected 3 weeks (baseline) and 4 and 9 months post-partum were analysed for cobalamins, TC and HC. Both the total concentration and the cobalamin-saturated form (holo) of TC and HC were analysed. No significant differences were observed in serum cobalamins or its binding proteins related to supplementation with vitamin B12 or the duration of lactation. Serum cobalamins remained unchanged from 3 weeks to 9 months post-partum. Total TC (holoTC) (median+/-s.e. pmol/l) decreased between 3 weeks (710+/-23 (85+/-12)) and 9 months (602+/-21 (76+/-11)) (P<0.0001 (P=0.0002)), whereas total HC (holoHC) increased from (422+/-11 (300+/-9)) at 4 months to (455+/-13 (317+/-10)) to 9 months post-partum (P<0.0001 (P<0.0001)). We report a decrease in TC and an increase in HC during a 9-month period post-partum. No differences were observed between the vitamin B12-supplemented and the unsupplemented groups. Thus, supplementation with vitamin B12 has no impact on the circulating level of serum cobalamins or its binding proteins in a Danish population of lactating mothers.

The Cobalamin-Binding Proteins Transcobalamin and Haptocorrin in Maternal and Cord Blood Sera at Birth

Two proteins carry vitamin B12 in plasma. Transcobalamin (TC) carries approximately 25% of total plasma vitamin B12 and is 6% to 20% saturated with cobalamin. Haptocorrin (HC) binds approximately 80% of total cobalamin and is largely saturated with cobalamin. We investigated the distribution and the relationship between concentrations of cobalamin, total and holo forms of TC, and HC in blood samples from pregnant women just before delivery (n = 92) and in cord blood samples from their newborn babies. We also investigated the relationship between these proteins and concentrations of methylmalonic acid (MMA), the functional marker of vitamin B12 status. Concentrations of total serum cobalamin, total HC, holoHC, and percentage of HC saturation were higher in cord blood than in the maternal blood (mean cobalamin, 268 vs 188 pmol/L; total HC, 648 vs 538 pmol/L; holoHC, 441 vs 237 pmol/L; HC saturation, 70% vs 47%). Moreover, total TC was low in cord blood, whereas both holoTC and TC saturation were higher in cord blood than in the maternal blood (mean total TC, 654 vs 1002 pmol/L; holoTC, 118 vs 53 pmol/L; TC saturation, 19.8% vs 5.4%). Higher maternal serum cobalamin was associated with higher cord blood holoTC and TC saturation (P <0.05). Gestational age was also a significant determinant of baby total TC, TC saturation, total HC, and holoHC. The close correlation between the amounts of holoTC present in cord blood and in maternal serum supports the importance of maternal cobalamin status for ensuring a sufficient supply to the baby.

Holotranscobalamin and Total Transcobalamin in Human Plasma: Determination, Determinants, and Reference Values in Healthy Adults

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. We captured holoTC with magnetic beads with TC antibodies and used a conventional MBA for cobalamin measurements. Total TC was determined as holoTC after TC was saturated with cyanocobalamin. The new assays were compared with published methods. Determinants and reference values were determined in 500 blood donors, ages 18-69 years. Determination of cobalamin, holoTC, and TC by MBA required <150 microL. HoloTC and TC by MBA correlated with holoTC by RIA (r = 0.95) and TC by ELISA (r = 0.79), respectively. Between-day CVs for holoTC and total TC were 4%-9%. Women had lower holoTC than men, but only at age < or = 45 years. In multivariate regression analyses, holoTC was positively associated with age (in women only), creatinine (in men only), and plasma concentrations of total TC, folate, and cysteine, but inversely correlated with homocysteine and methylmalonic acid. 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. Reference values were 670-1270 pmol/L for TC and 42-157 pmol/L for holoTC, but they differed according to age and sex. Our MBAs for TC and holoTC required low plasma volume and performed acceptably compared with other methods. Determinants of holoTC and TC differed between men and women and according to age. Separate reference intervals for holoTC should be considered in younger women.

Circadian variation of holo-transcobalamin (holo-TC) and related markers

We examined the circadian variation of holo-transcobalamin (holo-TC), total transcobalamin (total TC) and plasma cobalamins, and the association between food intake and fluctuations in the biochemical markers. A total of 17 healthy women (mean age 33 years; range 24-40) participated. The subjects were admitted at 08:00 h after an overnight fast, and blood samples were obtained every 20 min for 24 h from 12:00 h. Holo-TC and total TC were measured by an ELISA, and plasma cobalamins were measured by a routine method (Centaur, Bayer). All subjects, except one, had 12:00 h levels within the reference interval for all variables studied, and all subjects had normal renal function as judged from plasma creatinine. We found a small intra-individual variation (12%, 10%, 10%) and a considerable inter-individual variation (56%, 26%, 43%) in the level of holo-TC, total TC and plasma cobalamins, respectively. During the night, the absolute values of all components decreased, as did plasma albumin. There was no systematic association between intake of food and fluctuation of the biochemical markers. In conclusion, we found no significant circadian variation in serum holo-TC in healthy vitamin-replete subjects on a standard diet. This supports the view that holo-TC is a marker of long-term vitamin B(12) status, and that samples may also be obtained from non-fasting subjects.

Holo-Transcobalamin Is an Early Marker of Changes in Cobalamin Homeostasis. A Randomized Placebo-controlled Study

We examined the effect of oral vitamin B(12) treatment on fluctuations in plasma total cobalamin and its binding proteins transcobalamin (TC) and haptocorrin (HC). Patients (n = 88; age range, 38-80 years) undergoing coronary angiography (part of the homocysteine-lowering Western Norway B-Vitamin Intervention Trial) were allocated to daily oral treatment with (a) vitamin B(12) (0.4 mg), folic acid (0.8 mg), and vitamin B(6) (40 mg); (b) vitamin B(12) and folic acid; (c) vitamin B(6); or (d) placebo. EDTA blood was obtained before treatment and 3, 14, 28, and 84 days thereafter. The intraindividual variation for patients not treated with B(12) was approximately 10% for plasma total cobalamin, total TC, apo-TC, and apo-HC, and <20% for holo-TC and TC saturation. In B(12)-treated patients, the maximum change in concentrations was observed already after 3 days for total TC (-16%), holo-TC (+54%), and TC saturation (+82%). At this time holo-HC (+20%) and plasma total cobalamin (+28%) showed an initial burst, but had increased further at 84 days. All changes were highly significant compared with the control group (P <0.0001). Oral vitamin B(12) treatment produces maximal effects on total TC, holo-TC, and TC saturation within 3 days, whereas maximal increases in holo-HC and plasma total cobalamin occur later. The results support the view that holo-TC is an early marker of changes in cobalamin homeostasis.

Measurement of Transcobalamin by ELISA

Transcobalamin is essential for the cellular internalization of cobalamin. Methods to quantify the unsaturated protein are available, but few attempts have been made to develop methods to quantify the sum of unsaturated and cobalamin saturated transcobalamin. gamma-Globulins from two polyclonal rabbit antibodies against recombinant human transcobalamin were used as capture and detection antibodies, and recombinant human transcobalamin was used as calibrator in an ELISA design. The ELISA is specific for transcobalamin and has a detection limit of <1.6 pmol/L. The imprecision (CV) is 4-6% for mean concentrations of 13-70 pmol/L. The central 95% interval for serum from healthy blood donors (n = 77) was approximately 600-1500 pmol/L and showed limited variation with age and sex. No correlation was observed between the marker of acute phase reaction, C-reactive protein, and transcobalamin in plasma. The ELISA measures total transcobalamin in serum and thus can be used for measurement of transcobalamin in patients treated with cobalamin.

The effect of oral contraceptives on vitamin B12 metabolism

Serum vitamin vitamin B12 levels were determined in 199 women who were on a regnnen of oral contraceptives of either the combination or sequential type and in a control group of 196 women. The group using oral contraceptive agents.(OCA) had significantly lower serum levels of vitamin B12 vitamin as compared to those of the control group. A total of 19 women using OCAs had serum vitamin B12 levels that were lower than normal values. However, the Schilling test and urinary methylmalonate excretion in this group were normal. The OCA group had a significantly lower total serum vitamin B12 binding capacity, a lower total transcobalamin I level, and a higher transcobalamin III level. Our study suggests that the fall of serum vitamin B12 in OCA users is due to the changes in vitamin B12 binders of serum arid does not represent vitamin B12 deficiency. Therefore, aceording to our data, there is no justification for vitamin B12 supplementation in users of oral contraceptives.