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A low total plasma vitamin B12 supports a clinical suspicion of B12 deficiency, while the interpretation of an unexpectedly normal/high level is marred by controversies. Here, we critically review current knowledge on B12 in blood plasma, including the presence of the so-called "macro-B12". The latter form is most often defined as the fraction of B12 that can be removed by precipitation with polyethylene glycol (PEG), a nonspecific procedure that also removes protein polymers and antibody-bound analytes. Plasma B12 includes B12 attached to transcobalamin and haptocorrin, and an increased concentration of one or both proteins almost always causes an elevation of B12. The total plasma B12 is measured by automated competitive binding assays, often incorrectly referred to as immunoassays, since the binding protein is intrinsic factor and not an antibody. An unexpectedly high level of B12 may be further explored using immunological measurements of haptocorrin and transcobalamin (optionally combined with e.g., size-exclusion chromatography). Nonspecific methods, such as PEG precipitation, are likely to give misleading results and cannot be recommended. Currently, the need for evaluation of a high B12 of unknown etiology is limited since other tests (such as measurements of methylmalonic acid) may better guide the diagnosis of B12 deficiency. [ABSTRACT FROM AUTHOR]

A low total plasma vitamin B12 supports a clinical suspicion of B12 deficiency, while the interpretation of an unexpectedly normal/high level is marred by controversies. Here, we critically review current knowledge on B12 in blood plasma, including the presence of the so-called “macro-B12”. The latter form is most often defined as the fraction of B12 that can be removed by precipitation with polyethylene glycol (PEG), a nonspecific procedure that also removes protein polymers and antibody-bound analytes. Plasma B12 includes B12 attached to transcobalamin and haptocorrin, and an increased concentration of one or both proteins almost always causes an elevation of B12. The total plasma B12 is measured by automated competitive binding assays, often incorrectly referred to as immunoassays, since the binding protein is intrinsic factor and not an antibody. An unexpectedly high level of B12 may be further explored using immunological measurements of haptocorrin and transcobalamin (optionally combined with e.g., size-exclusion chromatography). Nonspecific methods, such as PEG precipitation, are likely to give misleading results and cannot be recommended. Currently, the need for evaluation of a high B12 of unknown etiology is limited since other tests (such as measurements of methylmalonic acid) may better guide the diagnosis of B12 deficiency.

Abstract Objective Measurement of total vitamin B12 (vit B12) concentration raised concerns over early detection of vit B12 deficiency due to its clinical unreliability. In this present article we aimed to assess the efficacy of holo-transcobolamin (active vit B12) for true evaluation of vit B12 deficiency. Methods This retrospective study included 100 participants referred for vit B12 assay. Serum total vit B12, active vit B12 and homocysteine were estimated. Results Our study showed 59% of the total participants with vit B12 deficiency (185 ± 64.62 pg/ml) and 18% with hyper-cobalaminemia (1666.9 ± 367.13 pg/ml) based on their total vit B12 concentrations. A comparative study on total vit B12 and active vit B12 was done which reflected a striking disparity in results. Active vit B12 reported 28.8% patients with vit B12 deficiency (19.8 ± 17.48 pg/ml) and only 16.6% patients with hyper-cobalaminemia (224.14 ± 10 pg/ml). Active vit B12 appeared to be more sensitive (82.35% vs 65%) and specific (46.6% vs. 43.8%) diagnostic marker compared to total vit B12. Pearson Correlation study indicated a strong positive correlation (r = 0.695 at p

This report on vitamin B-12 (B12) is part of the Biomarkers of Nutrition for Development (BOND) Project, which provides state-of-the art information and advice on the selection, use, and interpretation of biomarkers of nutrient exposure, status, and function. As with the other 5 reports in this series, which focused on iodine, folate, zinc, iron, and vitamin A, this B12 report was developed with the assistance of an expert panel (BOND B12 EP) and other experts who provided information during a consultation. The experts reviewed the existing literature in depth in order to consolidate existing relevant information on the biology of B12, including known and possible effects of insufficiency, and available and potential biomarkers of status. Unlike the situation for the other 5 nutrients reviewed during the BOND project, there has been relatively little previous attention paid to B12 status and its biomarkers, so this report is a landmark in terms of the consolidation and interpretation of the available information on B12 nutrition. Historically, most focus has been on diagnosis and treatment of clinical symptoms of B12 deficiency, which result primarily from pernicious anemia or strict vegetarianism. More recently, we have become aware of the high prevalence of B12 insufficiency in populations consuming low amounts of animal-source foods, which can be detected with ≥1 serum biomarker but presents the new challenge of identifying functional consequences that may require public health interventions.

Objective: Measurement of total vitamin B12 (vit B12) concentration raised concerns over early detection of vit B12 deficiency due to its clinical unreliability. In this present article we aimed to assess the efficacy of holo-transcobolamin (active vit B12) for true evaluation of vit B12 deficiency. Methods: This retrospective study included 100 participants referred for vit B12 assay. Serum total vit B12, active vit B12 and homocysteine were estimated. Results: Our study showed 59% of the total participants with vit B12 deficiency (185 ± 64.62 pg/ml) and 18% with hyper-cobalaminemia (1666.9 ± 367.13 pg/ml) based on their total vit B12 concentrations. A comparative study on total vit B12 and active vit B12 was done which reflected a striking disparity in results. Active vit B12 reported 28.8% patients with vit B12 deficiency (19.8 ± 17.48 pg/ml) and only 16.6% patients with hyper-cobalaminemia (224.14 ± 10 pg/ml). Active vit B12 appeared to be more sensitive (82.35% vs 65%) and specific (46.6% vs. 43.8%) diagnostic marker compared to total vit B12. Pearson Correlation study indicated a strong positive correlation (r = 0.695 at p < 0.01) hence justified use of the two methods. Conclusion: We claim that active vit B12 is a much more reliable biomarker than total vit B12 for early diagnosis of vit B12 deficiency. [ABSTRACT FROM AUTHOR]

Determination of plasma vitamin B12 (B12) is a frequently requested laboratory analysis, mainly employed to establish B12 deficiency. However, an increased level of B12 is a common unexpected finding that may be related to an increased concentration of one of the B12 binding proteins, haptocorrin or transcobalamin. This paper describes the extensive laboratory evaluation of a patient with an elevated level of plasma B12 with various well-established assays. Initial studies suggested the presence of a macromolecule consisting of haptocorrin bound B12. Specific determinations of the B12-binding proteins revealed normal amounts of haptocorrin but a markedly increase in both total and B12 saturated transcobalamin (holo-TC). The results are in accord with the presence of macro-transcobalamin. These experiments reveal that determination of the nature of the B12-macromolecules is troublesome due to differences in assays applied to measure these proteins. In addition, this publication creates awareness of macro-holo-TC as a cause of an unexplained increased B12 level. [ABSTRACT FROM AUTHOR]

Cellular uptake of vitamin B12 in humans is mediated by the endocytosis of the B12 carrier protein transcobalamin (TC) via its cognate cell surface receptor TCblR, encoded by the CD320 gene. Because CD320 expression is associated with the cell cycle and upregulated in highly proliferating cells including cancer cells, this uptake route is a potential target for cancer therapy. We developed and characterized four camelid nanobodies that bind holo‐TC (TC in complex with B12) or the interface of the human holo‐TC:TCblR complex with nanomolar affinities. We determined X‐ray crystal structures of these nanobodies bound to holo‐TC:TCblR, which enabled us to map their binding epitopes. When conjugated to the model toxin saporin, three of our nanobodies caused growth inhibition of HEK293T cells and therefore have the potential to inhibit the growth of human cancer cells. We visualized the cellular binding and endocytic uptake of the most potent nanobody (TC‐Nb4) using fluorescent light microscopy. The co‐crystal structure of holo‐TC:TCblR with another nanobody (TC‐Nb34) revealed novel features of the interface of TC and the LDLR‐A1 domain of TCblR, rationalizing the decrease in the affinity of TC‐B12 binding caused by the Δ88 mutation in CD320. [ABSTRACT FROM AUTHOR]

Abstract Background Transcobalamin deficiency is a rare autosomal recessive inborn error of cobalamin transport (prevalence:

Abstract Background Transcobalamin (TC) transports vitamin B12 from blood into cells. TC II deficiency is a rare autosomal recessive disorder. It is characterized by failure to thrive, diarrhoea, pallor, anaemia, pancytopenia or agammaglobulinemia. It is usually confirmed by molecular analysis of the TCN2 gene. We report a 2-month-old girl with two novel mutations, which were first reported in humans. Case presentation We present a 2-month-old Chinese girl with pancytopenia, severe combined immunodeficiency disease, and megaloblastic anaemia. Targeted next-generation sequencing (NGS) was performed, which detected compound heterozygous variants in exon 7 of the TCN2 gene (Mutation 1: c.1033 C > T; Mutation 2: c.1017-1031delinsGTAACAGAGATGGTT). These mutations result in stop codons in TCN2. The c.1033C > T mutation causes a stop at codon 345 (p.Gln345Ter), and the c.1017-1031delinsGTAACAGAGATGGTT mutation causes a stop at codon 340 (p.Leu340Ter). After being diagnosed, she was treated with intramuscular 1 mg hydroxycobalamin (OH-Cbl) every day for 2 months. The CBC value returned to normal after half a month. The peripheral blood lymphocyte subsets and immunoglobulin recovered after 2 months. Then, the dosage of OH-Cbl was gradually reduced. Conclusions TC II deficiency is a serious complication that requires lifelong treatment. Its diagnosis is difficult due to the lack of clearly identifiable symptoms. Genetic testing should be performed as early as possible if this disease is suspected. The specific observations of this case report make a considerable contribution to the literature and provide a reference for the diagnosis and treatment of future cases.

Vitamin B12 and folate deficiency have been linked to the susceptibility of developing depression in the postpartum period. Transcobalamin-2 (TCN-2) and reduced folate carrier (RFC-1) are essential for the cellular uptake of vitamin B12 and folate respectively. Therefore, functional genetic polymorphism of both may lead to the intracellular deficiency of vitamin B12 & folate. We observed the association of TCN-2 C776G and RFC-1 G80A polymorphism with postpartum depression, and its effect on the circulating markers of vitamin B12 and folate deficiency. Women were screened at 6 weeks postpartum for the probability of depression by EPDS score, and a score of >10 was taken as a cut-off (n = 434; 217 in each group). Plasma was used for the estimation of homocysteine, methylmalonic acid (MMA), 5-methyl tetrahydrofolate, and holotranscobalamin by commercially available ELISA kits, and whole blood was used for extraction of DNA, which was further genotyped by real-time PCR using Taqman probes. No difference was observed between the distribution of genotypes and alleles of TCN-2 C776G and RFC-1 G80A between women with and without probable depression. On comparing the effect of various genotypes on circulating levels of metabolites, we observed TCN2 C776G CG genotype to be associated with lower 5-methyl THF levels whereas RFC-1 G80A GA genotype was associated with lower vitamin B12 levels. These results suggest the metabolic effect of transporter genotype in folate-supplemented postpartum women.

Vitamin B 12 (cobalamin or Cbl) functions as a cofactor in two important enzymatic processes in human cells, and life is not sustainable without it. B 12 is obtained from food and travels from the stomach, through the intestine, and into the bloodstream by three B 12 -transporting proteins: salivary haptocorrin (HC), gastric intrinsic factor, and transcobalamin (TC), which all bind B 12 with high affinity and require proteolytic degradation to liberate Cbl. After intracellular delivery of dietary B 12 , Cbl in the aquo/hydroxocobalamin form can coordinate various nucleophiles, for example, GSH, giving rise to glutathionylcobalamin (GSCbl), a naturally occurring form of vitamin B 12 . Currently, there is no data showing whether GSCbl is recognized and transported in the human body. Our crystallographic data shows for the first time the complex between a vitamin B 12 transporter and GSCbl, which compared to aquo/hydroxocobalamin, binds TC equally well. Furthermore, sequence analysis and structural comparisons show that TC recognizes and transports GSCbl and that the residues involved are conserved among TCs from different organisms. Interestingly, haptocorrin and intrinsic factor are not structurally tailored to bind GSCbl. This study provides new insights into the interactions between TC and Cbl., Competing Interests: Conflict of interests The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: In humans, vitamin B-12 (cobalamin) transport involves 3 paralogous proteins: transcobalamin, haptocorrin, and intrinsic factor. Zebrafish (Danio rerio) express 3 genes that encode proteins homologous to known B-12 carrier proteins: tcn2 (a transcobalamin ortholog) and 2 atypical β-domain-only homologs, tcnba and tcnbb.Objectives: Given the orthologous relation between zebrafish Tcn2 and human transcobalamin, we hypothesized that zebrafish carrying null mutations of tcn2 would exhibit phenotypes consistent with vitamin B-12 deficiency.Methods: First-generation and second-generation tcn2-/- zebrafish were characterized using phenotypic assessments, metabolic analyses, viability studies, and transcriptomics.Results: Homozygous tcn2-/- fish produced from a heterozygous cross are viable and fertile but exhibit reduced growth, which persists into adulthood. When first-generation female tcn2-/- fish are bred, their offspring exhibit gross developmental and metabolic defects. These phenotypes are observed in all offspring from a tcn2-/- female regardless of the genotype of the male mating partner, suggesting a maternal effect, and can be rescued with vitamin B-12 supplementation. Transcriptome analyses indicate that offspring from a tcn2-/- female exhibit expression profiles distinct from those of offspring from a tcn2+/+ female, which demonstrate dysregulation of visual perception, fatty acid metabolism, and neurotransmitter signaling pathways.Conclusions: Our findings suggest that the deposition of vitamin B-12 in the yolk by tcn2-/- females may be insufficient to support the early development of their offspring. These data present a compelling model to study the effects of vitamin B-12 deficiency on early development, with a particular emphasis on transgenerational effects and gene-environment interactions. [ABSTRACT FROM AUTHOR]

Background: Transcobalamin deficiency is a rare autosomal recessive inborn error of cobalamin transport (prevalence: < 1/1000000) which clinically manifests in early infancy. Case presentation: We describe the case of a 31 years old woman who at the age of 30 days presented with the classical clinical and laboratory signs of an inborn error of vitamin B12 metabolism. Family history revealed a sister who died at the age of 3 months with a similar clinical syndrome and with pancytopenia. She was started on empirical intramuscular (IM) cobalamin supplements (injections of hydroxocobalamin 1 mg/day for 1 week and then 1 mg twice a week) and several transfusions of washed and concentrated red blood cells. With these treatments a clear improvement in symptoms was observed, with the disappearance of vomiting, diarrhea and normalization of the full blood count. At 8 years of age injections were stopped for about two and a half months causing the appearance of pancytopenia. IM hydroxocobalamin was then restarted sine die. The definitive diagnosis could only be established at 29 years of age when a genetic evaluation revealed the homozygous c.1115_1116delCA mutation of TCN2 gene (p.Q373GfsX38). Currently she is healthy and she is taking 1 mg of IM hydroxocobalamin once a week. Conclusions: Our case report highlights that early detection of TC deficiency and early initiation of aggressive IM treatment is likely associated with disease control and an overall favorable outcome. [ABSTRACT FROM AUTHOR]

Cobalamin, also known as vitamin B12, is a water-soluble vitamin. Cobalamin deficiency can be frequently seen in people all around the world. It can have non-specific symptoms, and in patients who are in a very critical state, it can lead to neurological or hematological abnormalities. While pernicious anemia used to be the main cause, it now accounts for a smaller number of cases, with food-bound cobalamin malabsorption being more common. Early diagnosis and appropriate management are crucial to avoid severe complications like spinal cord degeneration and pancytopenia. The primary method of treatment has been injections of vitamin B12 which are given through the intramuscular route but now the oral replacement therapy has also been very effective in treating the patients. There is increasing evidence linking increased levels of vitamin B12 to hematological and hepatic disorders, particularly cancers. This review has primarily highlighted the metabolism, clinical manifestations, diagnosis, and treatment of cobalamin deficiency in the past decade., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2024, Jajoo et al.)

Objectives: Proper metabolism of the folate is crucial for maintaining DNA integrity, chromosome structure, methylation, as well as gene expression, and thus, folate is speculated to contribute to the etiology of different disorders. Since the etiology of autism spectrum disorder (ASD) is believed to be influenced by both genetic and environmental factors, we hypothesized that functional single nucleotide polymorphisms (SNPs) affecting folate metabolic pathway may have a causal role in the etiology of ASD. Methods: We analyzed three SNPs, rs2071010, rs2298444 and rs1801198 (in the folate receptor 1, folate receptor 2 and transcobalamin 2, respectively), in 867 ethnically matched subjects including 206 ASD probands and 286 controls. Plasma vitamin B6 and folate were measured in age-matched probands and controls. Results: ASD probands showed a higher frequency of rs2298444 'A' allele (P = 0.01) and genotypes with 'A' allele (P = 0.03) when compared with the controls. rs1801198 'C' allele and 'CG' genotype also showed higher occurrence in the probands (P = 0.009 and 0.005, respectively). Gender-based stratified analysis revealed a significant higher frequency of rs2298444 'A' allele (P = 0.003), genotypes with rs2298444 'A' allele (P = 0.003) and rs1801198 CG (P = 0.001) in the male probands. Studied variants also showed statistically significant associations with ASD-associated traits measured by the Childhood Autism Rating Scale. ASD subjects exhibited gross deficiency in vitamin B6 level when compared with age-matched controls (P < 0.001), which correlated with risk genetic variants. Discussion: We infer from this pioneering study on eastern Indian subjects that vitamin B6 deficiency, along with risk gene variants, may affect ASD-associated symptoms, warranting further investigation in large cohorts. [ABSTRACT FROM AUTHOR]

Background/objectivesA common polymorphism, C776G, in the plasma B12 transport protein transcobalamin (TC), encodes for either proline or arginine at codon 259. This polymorphism may affect the affinity of TC for B12 and subsequent delivery of B12 to tissues.Subjects/methodsTC genotype and its associations with indicators of B12 status, including total B12, holotranscobalamin (holoTC), methylmalonic acid and homocysteine, were evaluated in a cohort of elderly Latinos (N=554, age 60-93 years) from the Sacramento Area Latino Study on Aging (SALSA).ResultsThe distribution of TC genotypes was 41.3% homozygous reference (776CC) and 11.6% homozygous variant (776GG). No differences between the homozygous genotypes were observed in total B12, holoTC, methylmalonic acid or homocysteine. The holoTC/total B12 ratio was lower in the 776GG group compared with the 776CC group (P=0.04). Significant interactions of TC genotype with total B12 (P=0.04) and with holoTC (P< or =0.03) were observed such that mean homocysteine concentrations and the odds ratios for hyperhomocysteinemia (>13 micromol/l) were higher in the 776CC subjects compared with all carriers of the G allele (776CG and 776GG combined) when total B12 (

Background: Gaucher disease (GD) is a lysosomal disorder caused by biallelic pathogenic mutations in the GBA1 gene that encodes beta-glucosidase (GCase), and more rarely, by a deficiency in the GCase activator, saposin C. Clinically, GD manifests with heterogeneous multiorgan involvement mainly affecting hematological, hepatic and neurological axes. This disorder is divided into three types, based on the absence (type I) or presence and severity (types II and III) of involvement of the central nervous system. At the cellular level, deficiency of GBA1 disturbs lysosomal storage with buildup of glucocerebroside. The consequences of disturbed lysosomal metabolism on biochemical pathways that require lysosomal processing are unknown. Abnormal systemic markers of cobalamin (Cbl, B12) metabolism have been reported in patients with GD, suggesting impairments in lysosomal handling of Cbl or in its downstream utilization events. Methods: Cultured skin fibroblasts from control humans (n = 3), from patients with GD types I (n = 1), II (n = 1) and III (n = 1) and an asymptomatic carrier of GD were examined for their GCase enzymatic activity and lysosomal compartment intactness. Control human and GD fibroblasts were cultured in growth medium with and without 500 nM hydroxocobalamin supplementation. Cellular cobalamin status was examined via determination of metabolomic markers in cell lysate (intracellular) and conditioned culture medium (extracellular). The presence of transcobalamin (TC) in whole cell lysates was examined by Western blot. Results: Cultured skin fibroblasts from GD patients exhibited reduced GCase activity compared to healthy individuals and an asymptomatic carrier of GD, demonstrating a preserved disease phenotype in this cell type. The concentrations of total homocysteine (tHcy), methylmalonic acid (MMA), cysteine (Cys) and methionine (Met) in GD cells were comparable to control levels, except in one patient with GD III. The response of these metabolomic markers to supplementation with hydroxocobalamin (HOCbl) yielded variable results. The content of transcobalamin in whole cell lysates was comparable in control human and GD patients. Conclusions: Our results indicate that cobalamin transport and cellular processing pathways are overall protected from lysosomal storage damage in GD fibroblasts. Extending these studies to hepatocytes, macrophages and plasma will shed light on cell- and compartment-specific vitamin B12 metabolism in Gaucher disease. [ABSTRACT FROM AUTHOR]

Background: Vitamin B12 deficiency is a common disorder. In circulation, vitamin B12 is bound to transcobalamin (holotranscobalamin), which is considered the active form of cobalamin. The objective of this study was to evaluate the analytical performance of the Roche Elecsys Active B12 immunoassay. Methods: Limit of quantification and linearity were assessed according to CLSI EP17-A2 and EP-6A guidelines. Precision and bias of Roche Active B12 test against Architect ci8200 (Abbott) were performed according to CLSI EP-5 A3 guideline at three European laboratories. Precision requirements were set at <4% for within-run precision and <15% for intermediate precision. Requirements for method comparison were set at a slope of 1.00 ± 20%. In addition, 95% reference interval was conducted in European adults according to CLSI-EP28 guideline. Results: The assay was shown to be linear and precise and met the requirements. Lot-to-lot variation and lab-to-lab variation both were ≤4%. Method comparison with Abbott Architect showed a significant bias of 9 pmol/L, which increases to up to 23 pmol/L around the current medical decision point. The 95% reference interval of the Roche Elecsys Active B12 test was determined at 37–188 pmol/L. Conclusion: Although Roche Elecsys Active B12 immunoassay met the requirements, this test has a positive bias of 9 pmol/L, which led to an increase in lower limit of reference interval compared with Abbott test. This test first needs to be clinically validated before it can be applied as a first-line screening test in the diagnosis of vitamin B12 deficiency. [ABSTRACT FROM AUTHOR]

The liver mass constitutes hepatocytes expressing receptors for vitamin B12 (B12)-bound transporters in circulation. However, intrahepatic and circulating B12 interrelationship levels remain unclear. We assessed the intracellular B12 levels at various circulating B12 concentrations in human HepG2 cell-line and liver tissue levels of B12 in the C57BL/6 mouse model. In HepG2 cells treated with a range of B12 concentrations, the intracellular and circulatory B12 levels, transcript and protein levels of B12 receptor (CD320) and transporter (TCN2) were determined using immunoassays, qRT-PCR and Western blot, respectively. Similar assessments were done in plasma and liver tissue of C57BL/6 mice, previously fed a diet of either a high or low B12 (30.82 µg B12/kg and 7.49 µg B12/kg, respectively) for 8–10 weeks. The physiological B12 status (0.15–1 nM) resulted in increased levels of intracellular B12 in HepG2 cells compared to supraphysiological levels of B12 (>1 nM). Gene and protein expression of CD320 and TCN2 were also higher at physiological levels of B12. Progressively increasing extracellular B12 to supraphysiological levels led to relative decreased levels of intracellular B12, lower expression of gene and protein levels of CD320 and TCN2. Similar results were observed in liver tissue from mice fed on a low B12 diet verses high B12 diet. These findings suggest that unlike supraphysiological B12, physiological levels of B12 in the extracellular media or circulation accelerates active transport of B12, and expression of CD320 and TCN2, resulting in higher relative uptake of B12 in hepatocytes.

Far from being devoid of life, Antarctic waters are home to Cryonotothenioidea, which represent one of the fascinating cases of evolutionary adaptation to extreme environmental conditions in vertebrates. Thanks to a series of unique morphological and physiological peculiarities, which include the paradigmatic case of loss of hemoglobin in the family Channichthyidae, these fish survive and thrive at sub-zero temperatures. While some of the distinctive features of such adaptations have been known for decades, our knowledge of their genetic and molecular bases is still limited. We generated a reference de novo assembly of the icefish Chionodraco hamatus transcriptome and used this resource for a large-scale comparative analysis among five red-blooded Cryonotothenioidea, the sub-Antarctic notothenioid Eleginops maclovinus and seven temperate teleost species. Our investigation targeted the gills, a tissue of primary importance for gaseous exchange, osmoregulation, ammonia excretion, and its role in fish immunity. One hundred and twenty genes were identified as significantly up-regulated in Antarctic species and surprisingly shared by red- and white-blooded notothenioids, unveiling several previously unreported molecular players that might have contributed to the evolutionary success of Cryonotothenioidea in Antarctica. In particular, we detected cobalamin deficiency signatures and discussed the possible biological implications of this condition concerning hematological alterations and the heavy parasitic loads typically observed in all Cryonotothenioidea.

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This review gives an overview of clinical characteristics, treatment and outcome of nutritional and acquired cobalamin (Cbl; synonym: vitamin B12) deficiencies, inborn errors of Cbl absorption and intracellular trafficking, as well as methylenetetrahydrofolate dehydrogenase (MTHFD1) and methylene tetrahydrofolate reductase (MTHFR) deficiencies, which impair Cbl‐dependent remethylation. Acquired and inborn Cbl‐related disorders and MTHFR deficiency cause multisystem, often severe disease. Failure to thrive, neurocognitive or psychiatric symptoms, eye disease, bone marrow alterations, microangiopathy and thromboembolic events are characteristic. The recently identified MTHFD1 defect additionally presents with severe immune deficiency. Deficient Cbl‐dependent enzymes cause reduced methylation capacity and metabolite toxicity. Further net‐effects of perturbed Cbl function or reduced Cbl supply causing oxidative stress, altered cytokine regulation or immune functions are discussed. [ABSTRACT FROM AUTHOR]

This is the fifth in the series of reviews developed as part of the Biomarkers of Nutrition for Development (BOND) program. The BOND Iron Expert Panel (I-EP) reviewed the extant knowledge regarding iron biology, public health implications, and the relative usefulness of currently available biomarkers of iron status from deficiency to overload. Approaches to assessing intake, including bioavailability, are also covered. The report also covers technical and laboratory considerations for the use of available biomarkers of iron status, and concludes with a description of research priorities along with a brief discussion of new biomarkers with potential for use across the spectrum of activities related to the study of iron in human health. The I-EP concluded that current iron biomarkers are reliable for accurately assessing many aspects of iron nutrition. However, a clear distinction is made between the relative strengths of biomarkers to assess hematological consequences of iron deficiency versus other putative functional outcomes, particularly the relationship between maternal and fetal iron status during pregnancy, birth outcomes, and infant cognitive, motor and emotional development. The I-EP also highlighted the importance of considering the confounding effects of inflammation and infection on the interpretation of iron biomarker results, as well as the impact of life stage. Finally, alternative approaches to the evaluation of the risk for nutritional iron overload at the population level are presented, because the currently designated upper limits for the biomarker generally employed (serum ferritin) may not differentiate between true iron overload and the effects of subclinical inflammation. [ABSTRACT FROM AUTHOR]

Cobalamin [Cbl (or B12)] deficiency causes megaloblastic anemia and a variety of neuropathies. However, homeostatic mechanisms of cyanocobalamin (CNCbl) and other Cbls by vascular endothelial cells are poorly understood. Herein, we describe our investigation into whether cultured bovine aortic endothelial cells (BAECs) perform transcytosis of B12, namely, the complex formed between serum transcobalamin and B12, designated as holo-transcobalamin (holo-TC). We show that cultured BAECs endocytose [57Co]-CNCbl-TC (source material) via the CD320 receptor. The bound Cbl is transported across the cell both via exocytosis in its free form, [57Co]-CNCbl, and via transcytosis as [57Co]-CNCbl-TC. Transcellular mobilization of Cbl occurred in a bidirectional manner. A portion of the endocytosed [57Co]-CNCbl was enzymatically processed by methylmalonic aciduria combined with homocystinuria type C (cblC) with subsequent formation of hydroxocobalamin, methylcobalamin, and adenosylcobalamin, which were also transported across the cell in a bidirectional manner. This demonstrates that transport mechanisms for Cbl in vascular endothelial cells do not discriminate between various β-axial ligands of the vitamin. Competition studies with apoprotein- and holo-TC and holo-intrinsic factor showed that only holo-TC was effective at inhibiting transcellular transport of Cbl. Incubation of BAECs with a blocking antibody against the extracellular domain of the CD320 receptor inhibited uptake and transcytosis by ~40%. This study reveals that endothelial cells recycle uncommitted intracellular Cbl for downstream usage by other cell types and suggests that the endothelium is self-sufficient for the specific acquisition and subsequent distribution of circulating B12via the CD320 receptor. We posit that the endothelial lining of the vasculature is an essential component for the maintenance of serum-tissue homeostasis of B12. [ABSTRACT FROM AUTHOR]

Background: Vitamin B-12 (cobalamin) deficiency may produce severe neurologic and hematologic manifestations. Approximately 20-25% of circulating cobalamin binds to transcobalamin 2 (TCN2), which is referred to as active vitamin B-12. The G allele of the TCN2 c.776G>C (rs1801198) polymorphism has been associated with a lower plasma concentration of holotranscobalamin. However, genotype association studies on rs1801198 have led to conflicting results regarding its influence on one-carbon metabolism (OCM) markers or its association with pathologic conditions. Objective: We assessed the association of rs1801198 genotypes with OCM marker concentrations and primary risks of congenital abnormalities, cancer, and Alzheimer disease. Design: We conducted a systematic review of the literature that was published from January 1966 to February 2017 and included all studies that assessed the association between rs1801198 and OCM markers or a pathologic condition. Results: Thirty-four studies met the inclusion criteria. Subjects with the rs1801198 GG genotype had significantly lower concentrations of holotranscobalamin [standardized mean difference (SMD): -0.445 (95% CI: -0.673, --0.217; P < 0.001); I² = 48.16% (95% CI: 0.00%, 78.10%; P = 0.07)] and higher concentrations of homocysteine (European descent only) [SMD: 0.070 (95% CI: 0.020, 0.120; P = 0.01); I² = 0.00% (95% CI: 0.00%, 49.59%; P = 0.73)] than did subjects with the rs1801198 CC genotype. The meta-analysis on the association between rs1801198 and methylmalonic acid (MMA) lacked statistical power. No significant difference was observed regarding cobalamin, folate, and red blood cell folate. No significant association was observed between rs1801198 and primary risks of congenital abnormalities, cancer, or Alzheimer disease. Conclusions: Meta-analysis results indicate an influence of rs1801198 on holotranscobalamin and homocysteine concentrations in European-descent subjects. In addition, well-designed and -powered studies should be conducted for assessing the association between rs1801198 and MMA and clinical manifestations that are linked to a decreased availability of cobalamin. This review was registered at www.crd.york.ac.uk/prospero as CRD42017058504. [ABSTRACT FROM AUTHOR]

Objectives: The study aimed to investigate the association of Crohn's disease (CD) with transcobalamin II (TCN2) polymorphisms and serum homocysteine, folate, and vitamin B 12 levels. Methods: TCN2 (rs1801198, rs9606756) were genotyped by iMLDR in 389 CD patients and 746 controls. Furthermore, 102 CD patients and 153 controls were randomly selected for examination of serum homocysteine, folate, and vitamin B 12 levels by enzymatic cycling assay and chemiluminescence immunoassay, respectively. Results: Mutant allele (G) and genotype (AG + GG) of (rs9606756) were higher in CD patients than in controls (both p < 0.05). So were they in ileocolonic CD patients and stricturing CD patients compared to controls (all p < 0.05). Mutant allele (G) and genotype (CG + GG) of (rs1801198) were more prevalent in stricturing CD patients than in controls (both p < 0.05). Compared to controls, average homocysteine level was enhanced in CD patients (p = 0.003), whereas average folate and vitamin B 12 levels were reduced in CD patients (both p < 0.001). The prevalence of hyperhomocysteinemia, folate deficiency, and vitamin B 12 deficiency was higher in CD patients than in controls (all p < 0.01). Both folate deficiency and vitamin B 12 deficiency were independently related to risk of CD (both p < 0.01). Conclusion: TCN2 (rs1801198, rs9606756) polymorphisms as well as folate deficiency and vitamin B 12 deficiency are correlated with CD. [ABSTRACT FROM AUTHOR]

Cutaneous arteritis (CA) is a single-organ vasculitis that exclusively affects the small to medium-sized arteries of the skin. Diagnosis depends on a histological investigation with skin biopsy, which could be burdensome for both patients and clinicians. Moreover, the pathogenesis of CA remains unstudied, and treatment has not yet been established. Herein, we applied our proteome-wide autoantibody screening method to explore autoantibodies in the serum of CA patients. As a result, anti-transcobalamin receptor (TCblR) antibodies (Abs) were specifically detected in 24% of CA patients. Patients with positive anti-TCblR Abs were spared from peripheral neuropathy compared to those with negative anti-TCblR Abs, showing characteristics as CA confined to the skin. In addition, we revealed that anti-TCblR Abs trigger the autocrine loop of interleukin-6 mediated by tripartite motif-containing protein 21 in human endothelial cells and induce periarterial inflammation in murine skin. Furthermore, we demonstrated that methylcobalamin, a ligand of TCblR, ameliorates inflammation caused by anti-TCblR Abs both in vitro and in vivo. Collectively, our investigation unveils the pathologic significance of anti-TCblR Abs in CA and their potential as a diagnostic marker and a pathophysiology-oriented therapeutic target. • Cutaneous arteritis (CA) is a single-organ vasculitis of unknown origin. • Autoantibody screening discovered anti-transcobalamin receptor antibodies in CA. • In vitro and in vivo studies indicated they trigger periarterial inflammation. • Methylcobalamin might be a pathophysiology-oriented therapeutic agent. [ABSTRACT FROM AUTHOR]

Background & Aims: Cellular uptake of the essential nutrient vitamin B12 (cobalamin) occurs via the transcobalamin receptor (TCblR/CD320), a ubiquitous membrane receptor. Polymorphisms in the receptor exist, though the effect of such variants across patient populations is unknown., Methods: We determined CD320 genotype in 377 randomly selected elderly individuals., Results: Three polymorphisms and a codon deletion were identified in the exon 2 region. Haplotype variants had significantly higher holotranscobalamin (holo-TC) values and a higher holo-TC/total cobalamin ratio. TCblR haplotype explained 46% of the variability in holo-TC values., Conclusions: This has significant implications for the clinical utility of the 'combined indicator' of B12 status since it is based on a standard rate of intracellular flux via the TC-Cbl receptor. Modification of the model may be required to account for CD320 haplotype., Competing Interests: Declaration of competing interest No author had any conflict of interest to declare., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Many missense mutations/SNPs of the TCN2 gene (which yield Transcobalamin (TC)) were reported in the literature but no study is available about their effect on binding to vitamin B12(B12) at the structural level experimentally nor computationally. Predict the effect of TC missense mutations/SNPs on binding affinity to B12 and characterize their contacts to B12 at the structural level. TC-B12 binding energy difference from the wildtype (ΔΔGmut) was calculated for 378 alanine scanning mutations and 76 ClinVar missense mutations, repeated on two distinct X-ray structures of holoTC namely 2BB5 and 4ZRP. Destabilizing mutations then went through 100 ns molecular dynamics simulation to study their effect on TC-B12 binding at the structural level employing 2BB5 structure. Out of the studied 454 mutations (378 alanine mutations + 76 ClinVar mutations), 19 were destabilizing representing 17 amino acid locations. Mutation energy results show a neutral effect on B12 binding of several missense SNPs reported in the literature including I23V, G94S, R215W, P259R, S348F, L376S, and R399Q. Compared to the wildtype, all the destabilizing mutations have higher average RMSD-Ligand in the last 25% of the MD simulation trajectories and lower average hydrogen bond count while the other parameters vary. Previously reported TCN2 SNPs with an unknown effect on TC-B12 binding were found to have a neutral effect in the current study based on mutation energy calculations. Also, we reported 17 possible amino acids that destabilize TC-B12 binding upon mutation (four listed in ClinVar) and studied their structural effect computationally.

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. [ABSTRACT FROM AUTHOR]

Background: The 776C→G polymorphism of the vitamin B-12 transport protein transcobalamin gene (TCN2) (rs1801198; Pro259Arg) is associated with a lower holotranscobalamin concentration in plasma. This effect may reduce the availability of vitamin B-12 to tissues even when vitamin B-12 intake is adequate. Clinical outcomes associated with vitamin B-12 insufficiency could potentially be worsened by high folate intake. Objective: We determined the association of the TCN2 776C→G polymorphism and folate intake with peripheral neuropathy in elders with normal plasma concentrations of vitamin B-12. Design: Participants in this cross-sectional study (n = 171) were from a cohort of community-based, home-bound elderly individuals aged ≥60 y who underwent an evaluation by physicians including an assessment for peripheral neuropathy. Participants were administered food-frequency and general health status questionnaires, anthropometric measurements were taken, and a fasting blood sample from each subject was collected. Results: Odds of neuropathy were 3-fold higher for GG genotypes than for CC genotypes (OR: 3.33; 95% CI: 1.15, 9.64). When folate intake was >2 times the Recommended Dietary Allowance (800 μg), GG genotypes had 6.9-fold higher odds of neuropathy than CC genotypes (OR: 6.9; 95% CI: 1.31, 36.36). There was no difference between the genotypes in the odds of peripheral neuropathy when folate intake was ≤800 μg (OR: 1.5; 95% CI: 0.18, 12.33). Conclusion: The TCN2 776C→G polymorphism is associated with increased odds of peripheral neuropathy in the elderly, even with a normal vitamin B-12 status, especially if their folate intake is >2 times the Recommended Dietary Allowance. [ABSTRACT FROM AUTHOR]

Background: Measurement of holotranscobalamin (holoTC) is increasingly used as a screening test for cobalamin (Cbl) deficiency. A level well below the reference interval strongly supports a deficient state. We examined a 21-year-old woman diagnosed as Cbl deficient because of an extremely low holoTC level as measured by the Abbott Architect Assay. Methods: The patient was evaluated for Cbl deficiency employing an in-house holoTC method as well as other routine markers of Cbl status. Further analyses included exploration of the Cbl binding proteins employing gel filtration of a serum sample saturated with 57 Co-labeled Cbl and Sanger sequencing of exons 1-9 and the intron-exon boundaries of the TCN2 gene, the gene coding for transcobalamin (TC). Results: The patient had normal hematological variables throughout. Despite initial treatment with Cbl, holoTC as measured by the Abbott assay remained low, while holoTC measured with the in-house assay was normal, and behaved as TC upon gel-filtration. By Sanger sequencing, we detected a homozygous single point mutation c.855T > A in exon 6 of TCN2, corresponding to a asparagine (Asn) to lysine (Lys) substitution in position 267 of the mature protein. Conclusions: We describe a novel point mutation of the TCN2 gene. The mutation does not seem to interfere with the function of TC, but the mutation may well explain the low level of holoTC detected by the Abbott assay. Our results underscores that mutations of TCN2 have to be considered when implausible holoTC results are obtained. [ABSTRACT FROM AUTHOR]

Background: Transcobalamin is a key placental protein involved in transport of vitamin B12 to the fetus. However, few data currently exist on the ability of the placenta to modify vitamin B12 transporter expression, particularly in high-risk populations such as pregnant adolescents.Objective: This study was conducted to determine the impact of maternal and neonatal serum vitamin B12 concentrations on placental transcobalamin (TC) expression in a cohort of healthy pregnant adolescents in the United States.Design: Serum vitamin B12 concentrations were measured in maternal blood samples at mid-gestation (26.4 ± 2.8 weeks) and delivery (39.8 ± 1.4 weeks) and infant cord blood samples at birth. Placentas were collected at delivery and TC mRNA expression (ΔΔCt) and TC protein abundance (TC:α-actin) were evaluated. Linear and binomial regression models were used to examine the associations of maternal serum (mid-gestation, delivery) and cord blood vitamin B12 concentrations with placental TC mRNA expression and protein abundance (n = 63).Results: Maternal serum vitamin B12 concentrations at mid-gestation or delivery were not significantly associated with placental TC mRNA expression or TC protein abundance (p > 0.05). Higher placental TC protein abundance was associated with increased cord blood vitamin B12 concentrations (p = 0.003).Conclusions: Higher placental TC protein abundance was associated with higher cord blood vitamin B12 concentrations, suggesting a potential role in vitamin B12 transport to the fetus. [ABSTRACT FROM AUTHOR]

Vitamin B12 (cobalamin, Cbl) cofactors adenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl) are required for the activity of the enzymes methylmalonyl-CoA mutase (MCM) and methionine synthase (MS). Inborn errors of Cbl metabolism are rare Mendelian disorders associated with hematological and neurological manifestations, and elevations of methylmalonic acid and/or homocysteine in the blood and urine. We describe a patient whose fibroblasts had decreased functional activity ofMCM andMS and decreased synthesis of AdoCbl and MeCbl (3.4% and 1.0% of cellular Cbl, respectively). The defect in cultured patient fibroblasts complemented those from all known complementation groups. Patient cells accumulated transcobalamin-bound–Cbl, a complex which usually dissociates in the lysosome to release free Cbl.Whole-exome sequencing identified putative disease-causing variants c.851T>G (p.L284∗) and c.1019C>T (p.T340I) in transcription factor ZNF143. Proximity biotinylation analysis confirmed the interaction between ZNF143 andHCFC1, a protein that regulates expression of the Cbl trafficking enzyme MMACHC. qRT-PCR analysis revealed low MMACHC expression levels both in patient fibroblasts, and in control fibroblasts incubated with ZNF143 siRNA. [ABSTRACT FROM AUTHOR]

Introduction Cobalamin/Vitamin B12 (Cbl) is an essential vitamin, supplied mainly as hydroxocobalamin (OHCbl) by animal products, including cows' milk. Cyanocobalamin (CNCbl) is the usual form in vitamin pills. The aim was to explore absorption and tissue accumulation of two Cbl forms, administered alone or bound to milk protein. Materials and methods We synthesized labeled OH[ 57 Co]Cbl from commercially available CN[ 57 Co]Cbl. Recombinant bovine transcobalamin (rbTC) was produced in yeast and skimmed milk obtained off the shelf. Male Wistar rats (250–300 g) received labeled Cbl by gastric gavage. First, we administered CN[ 57 Co]Cbl, free or rbTC-bound (n = 15 in each group). Rats were sacrificed after two, 24, and 48 h. In the following studies, rats were sacrificed after 24 h. We compared absorption of free or rbTC-bound CN[ 57 Co]Cbl added to cows' milk and analogous absorption of OH[ 57 Co]Cbl, free or rbTC-bound, to absorption of free CN[ 57 Co]Cbl, (n = 10 in each group). Blood, tissues, 24-h urine and feces were collected. Labeled Cbl was measured using a gamma counter. Results are expressed as percentage of administered dose. Results Absorptions of CNCbl and OHCbl were neither influenced by rbTC-binding nor administration in milk. Absorption increased in the first 24 h with no further tissue accumulation during the subsequent 24 h. Accumulation of free CNCbl and (OHCbl) was 1.4, (4.1) (liver); 20.2, (16.4) (kidney); and 0.05, (0.02) (plasma)% 24 h after administration. Total organ accumulations were 21.6, (20.5)%. While total accumulations of CNCbl and OHCbl were equal, distributions between liver, kidney, and plasma showed significant differences (p < 0.0001; p = 0.01; p < 0.0001). Conclusions Cbl added to milk (spiked with rbTC) has high bioavailability matching that of free Cbl. OHCbl and CNCbl are absorbed equally well, but much more OHCbl accumulated in the liver. Benefits of oral supplementation with OHCbl compared to CNCbl should be investigated. [ABSTRACT FROM AUTHOR]

Background 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). Design Exon sequencing of the TCN2 gene was carried out. Protein characterization included total transcobalamin (TCN2) detection by Western blot, and holoTC by 57Co-labelled B12 binding followed by size fractionation. Results 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). Conclusions 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. [ABSTRACT FROM AUTHOR]