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BACKGROUND: Vitamin B12 is primarily transported from plasma to cells by Transcobalamin. Deficiency of Transcobalamin is a rare autosomal recessive disorder that results in unavailability of cobalamin in cells and accumulation of homocysteine and methylmalonic acid. CASE REPORT: We report a case of a 2-year-old male child with persistent pancytopenia, recurrent infections, and megaloblastic anemia. Next-generation sequencing identified a novel variant in exon 8 of TCN2 gene. Substantial improvement has been observed following administration of high doses of parenteral methylcobalamin. CONCLUSION: In patients with unresolved pancytopenia and megaloblastic anemia, Transcobalamin deficiency should be investigated and treated promptly to prevent any irreversible and harmful outcome.

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BACKGROUND & AIMS: Holo-Transcobalamin (holo-TC) is the biologically active form of vitamin B12, a vitamin essential in human metabolism. The association between vitamin B12 (total cobalamin) and mortality risk has been controversially reported, whereas the relation between holo-TC and survival is unknown. In a population-based sample (n = 862, female share 42.8%, median age 62.3 years), we related serum holo-TC to the risk of all-cause mortality. METHODS: We measured serum holo-TC by electro-chemiluminescence. Multivariable-adjusted Cox proportional hazards regression models were used to quantify the association between serum holo-TC and all-cause mortality. RESULTS: Over a median follow-up time of 10.9 years, n = 99 individuals died. We did not find significant associations between serum holo-TC and the risk of all-cause mortality (HR: 1.00 [95% CI 0.97-1.03] per 5-point increment in holo-TC), neither in the overall sample, nor in subgroups stratified by sex, diabetes, or hypertension. CONCLUSION: The biologically active form of vitamin B12, holo-TC, is not related to the risk of all-cause mortality in a moderate-sized sample from the general population.

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Background: SLE is a complex autoimmune disease with deleterious effects on various organs. Accumulating evidence has shown abnormal vitamin B12 and one-carbon flux contribute to immune dysfunction. Transcobalamin II (TCN2) belongs to the vitamin B12-binding protein family responsible for the cellular uptake of vitamin B12. The role of TCN2 in SLE is still unclear. Methods: We collected clinical information and blood from 51 patients with SLE and 28 healthy controls. RNA sequencing analysis, qPCR, and western blot confirmed the alteration of TCN2 in disease monocytes. The correlation between TCN2 expression and clinical features and serological abnormalities was analyzed. TCN2 heterozygous knockout THP1 cells were used to explore the effects of TCN2 dysfunction on monocytes. CCK-8 assay and EdU staining were used to detect cell proliferation. ELISA was conducted to assess vitamin B12, glutathione, and cytokines changes. UHPLC-MRM-MS/MS was used to detect changes in the intermediates of the one-carbon cycle. Flow cytometry is used to detect cell cycle, ROS, mitoROS, and CD14 changes. Results: Elevated TCN2 in monocytes was correlated positively with disease progression and specific tissue injuries. Using CD14+ monocytes and TCN2 genetically modified THP1 cell lines, we found that the TCN2 was induced by LPS in serum from SLE patients. TCN2 heterozygous knockout inhibited cellular vitamin B12 uptake and one-carbon metabolism, leading to cell proliferation arrest and decreased Toll-like receptor 4 (TLR4)-mediated CCL2 release. Methionine cycle metabolites, s-adenosylmethionine and homocysteine, rescued these effects, whereas folate treatment proved to be ineffective. Folate deficiency also failed to replicate the impact of TCN2 downregulation on THP1 inflammatory response. Conclusion: Our study elucidated the unique involvement of TCN2-driven one-carbon flux on SLE-associated monocyte behavior. Increased TCN2 may promote disease progression and tissue damage by enhancing one-carbon flux, fostering monocyte proliferation, and exacerbating TLR4 mediated inflammatory responses. The inhibition of TCN2 may be a promising therapeutic approach to ameliorate SLE.

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Vitamin B12 (cobalamin or Cbl) functions as a cofactor in two important enzymatic processes in human cells, and life is not sustainable without it. B12 is obtained from food and travels from the stomach, through the intestine, and into the bloodstream by three B12-transporting proteins: salivary haptocorrin (HC), gastric intrinsic factor, and transcobalamin (TC), which all bind B12 with high affinity and require proteolytic degradation to liberate Cbl. After intracellular delivery of dietary B12, Cbl in the aquo/hydroxocobalamin form can coordinate various nucleophiles, for example, GSH, giving rise to glutathionylcobalamin (GSCbl), a naturally occurring form of vitamin B12. 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 B12 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.

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OBJECTIVES: Transcobalamin II (TC) promotes the cellular uptake of cobalamin (Cbl) through receptor-mediated endocytosis of the TC-cbl complex in peripheral tissues. TC deficiency is a rare disorder that causes intracellular Cbl depletion. It presents in early infancy with a failure to thrive, diarrhea, anemia, agammaglobulinemia, and pancytopenia. Data from five TC-deficient patients including clinical, biochemical, and molecular findings, as well as long-term outcomes, were collected. CASE PRESENTATION: Mutation analysis revealed one unreported pathogenic variant in the TCN2 gene. One patient had exocrine pancreatic insufficiency. We conducted a retrospective analysis of C3 and C3/C2 from dried blood samples, as this is implemented for newborn screening (NBS). We detected a marked increase in the C3/C2 ratio in two samples. Treatment was based on parenteral Cbl. Three patients treated before six months of age had an initial favorable outcome, whereas the two treated later or inadequately had neurological impairment. CONCLUSIONS: This is the first report of Argentinean patients with TC deficiency that detected a new variant in TCN2. NBS may be a tool for the early detection of TC deficiency. This data emphasizes that TC deficiency is a severe disorder that requires early detection and long-term, aggressive therapy. Accurate diagnosis is imperative, because early detection and treatment can be life-saving.

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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.

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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.

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BACKGROUND: Transcobalamin II (TCN2) defect is a rare metabolic disorder associated with a range of neurological manifestations, including mild developmental delay, severe intellectual disability, ataxia, and, in some cases, seizures. Cobalamin, an essential nutrient, plays a crucial role in central nervous system myelination. CLINICAL PRESENTATION: We present a family with an index patient who exhibited progressive neurodevelopmental regression starting at 9 months of age, accompanied by myoclonic seizures, ataxia, and tremor. No significant hematological abnormalities were observed. Exome sequencing analysis identified a novel homozygous mutation, c.3G>A - P(Met1I), affecting the acceptor site of intron 4 of the TCN2 gene (chromosome 22: 31003321, NM_000355.4), leading to likely pathogenic variant potentially affecting translation. Following treatment with hydroxocobalamin, the patient demonstrated partial clinical improvement. He has a sibling with overt hematological abnormalities and subtle neurological abnormalities who is homozygous to the same mutation. Both parents are heterozygous for the same mutation. CONCLUSIONS: In infants presenting with unexplained non-specific neurological symptoms, irrespective of classical signs of vitamin B12 deficiency, evaluation for TCN2 defect should be considered. Early diagnosis and appropriate management can lead to favorable outcomes.

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Background: The changes of plasma transcobalamin-II (TCII) and Zinc (Zn) Levels in epileptic patients are not clearly understood. The aim of the current study was to evaluate the plasma contents of TCII and Zn levels in newly-diagnosed epileptic seizure patients, long-standing grand mal epileptic patients following treatment with sodium valproate and healthy control group. Methods: Thirty patients aged 36.76±12.91 years with newly-diagnosed and thirty long-standing grand mal epileptic patients aged 35.56 ±12.77 years were diagnosed based on the clinical symptoms. The control subjects were picked out from healthy individuals and matched to the patients, aged 36.30 ±12.80 years. Plasma Zn and TCN-2 was evaluated via spectrophotometry at 546 nm and 450 nm, respectively, using chimerical kits. Results: Plasma level of TCII in the newly-diagnosed epileptic seizures patients and long-standing grand mal epileptic patients were significantly increased, compared to the healthy controls [14.89 ±3.24 and 21.84± 2.73 vs. 9.55±1.24, (n=30)], respectively. Plasma level of Zn was decreased in the newly-diagnosed epileptic seizure patients, while it was increased in long-standing grand mal epileptic patients compared to the control group [69.28± 6.41 and 80.56 ±6.12 and vs.75.80±1.59, (n=30)], respectively. Conclusion: This study suggests that sodium valproate may disrupt the homeostatic balance of TCII and Zn, and cause abnormality of their serum level in newly-diagnosed epileptic seizure patients and long-standing grand mal epileptic patients. Further research is recommended to identify the underpinning for these changes.

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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.

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Introduction: Increasing evidence implicates proteostatic dysfunction as an early event in the development of frontotemporal dementia (FTD). This study aimed to explore potential cerebrospinal fluid (CSF) biomarkers associated with the proteolytic systems in genetic FTD caused by CHMP2B mutation. Methods: Combining solid-phase extraction and parallel reaction monitoring mass spectrometry, a panel of 47 peptides derived from 20 proteins was analyzed in CSF from 31 members of the Danish CHMP2B-FTD family. Results: Compared with family controls, mutation carriers had significantly higher levels of complement C9, lysozyme and transcobalamin II, and lower levels of ubiquitin, cathepsin B, and amyloid precursor protein. Discussion: Lower CSF ubiquitin concentrations in CHMP2B mutation carriers indicate that ubiquitin levels relate to the specific disease pathology, rather than all-cause neurodegeneration. Increased lysozyme and complement proteins may indicate innate immune activation. Altered levels of amyloid precursor protein and cathepsins have previously been associated with impaired lysosomal proteolysis in FTD. Highlights: CSF markers of proteostasis were explored in CHMP2B-mediated frontotemporal dementia (FTD).31 members of the Danish CHMP2B-FTD family were included.We used solid-phase extraction and parallel reaction monitoring mass spectrometry.Six protein levels were significantly altered in CHMP2B-FTD compared with controls.Lower CSF ubiquitin levels in patients suggest association with disease mechanisms.

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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.

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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.

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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.

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Transcobalamin (TC) deficiency is a rare autosomal recessive disease characterized by megaloblastic anemia. It is caused by cellular vitamin B12 depletion, which subsequently results in elevated levels of homocysteine and methylmalonic acid. This disease is usually diagnosed by genetic analysis of the TCN2 gene. Here, we described a 2.2-month-old Chinese girl with TC deficiency presenting with diarrhea, fever and poor feeding. Whole-exome sequencing detected a pair of compound-heterozygous mutations in TCN2 gene, c.754-12C>G and c.1031_1032delGA (p.R344Tfs*20). To our knowledge, it is the first time that they were identified and reported in TC deficiency. This study contributes to a better understanding of the TC deficiency, expanding the spectrum of TCN2 mutations in this disorder and also supporting the early diagnosis and proper treatment of similar cases in the future.

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BACKGROUND: Transcobalamin deficiency is a rare inborn metabolic disorder, characterized by pancytopenia, megaloblastic anemia, failure to thrive, diarrhea, and psychomotor retardation. CASE PRESENTATION: We describe a patient who first presented at 3 months of age, with pancytopenia, hepatosplenomegaly, recurrent infection, metabolic acidosis, and acute hemolytic crisis. Extensive hematologic and immunologic investigations did not identify inherited bone marrow failure syndrome, acute leukemia or its related disorders. Whole exome sequencing identified a novel homozygous TCN2 mutation, c.428-2A > G and mRNA study confirmed an aberrant transcription of exon 4 skipping. The mutant protein is predicted to have an in-fame 51 amino acids deletion (NP_000346:p.Gly143_Val193del). The patient exhibited marked clinical improvement following hydroxocobalamin treatment. CONCLUSIONS: Transcobalamin deficiency should be investigated in infants with unexplained pancytopenia and acute hemolytic crisis with or without typical evidence of vitamin B12 deficiency.

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Vitamin B12 is assimilated and transported by complex mechanisms that involve three transport proteins, intrinsic factor (IF), haptocorrin (HC) and transcobalamin (TC) and their respective membrane receptors. Vitamin deficiency is mainly due to inadequate dietary intake in vegans, and B12 malabsorption is related to digestive diseases. This review explores the physiology of vitamin B12 absorption and the mechanisms and diseases that produce malabsorption. In the stomach, B12 is released from food carrier proteins and binds to HC. The degradation of HC by pancreatic proteases and the pH change trigger the transfer of B12 to IF in the duodenum. Cubilin and amnionless are the two components of the receptor that mediates the uptake of B12 in the distal ileum. Part of liver B12 is excreted in bile, and undergoes an enterohepatic circulation. The main causes of B12 malabsorption include inherited disorders (Intrinsic factor deficiency, Imerslund-Gräsbeck disease, Addison's pernicious anemia, obesity, bariatric surgery and gastrectomies. Other causes include pancreatic insufficiency, obstructive Jaundice, tropical sprue and celiac disease, bacterial overgrowth, parasitic infestations, Zollinger-Ellison syndrome, inflammatory bowel diseases, chronic radiation enteritis of the distal ileum and short bowel. The assessment of B12 deficit is recommended in the follow-up of subjects with bariatric surgery. The genetic causes of B12 malabsorption are probably underestimated in adult cases with B12 deficit. Despite its high prevalence in the general population and in the elderly, B12 malabsorption cannot be anymore assessed by the Schilling test, pointing out the urgent need for an equivalent reliable test.

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Of the water-soluble vitamins, vitamin B12 (B12) has the lowest daily requirement. It also has several unique properties including a complex pathway for its absorption and assimilation requiring intact gastric and terminal small intestinal function, an enterohepatic pathway, and several dedicated binding proteins and chaperons. The many causes of B12 deficiency include malabsorption and defects in cellular delivery and uptake, as well as limited dietary intake. B12 is required as a cofactor for only two reactions in humans, the cytosolic methionine synthase reaction and the mitochondrial methymalonyl CoA mutase reaction. Disruption of either of these reactions gives rise to B12 deficiency. Although more common with advancing age, because of the higher prevalence of malabsorptive disorders in the elderly, B12 deficiency is widely distributed across all age groups particularly where food insecurity occurs. The consequences and severity of B12 deficiency are variable depending on the degree of deficiency and its duration. Major organ systems affected include the blood, bone marrow and nervous system. Megaloblastic anemia results from a defect in thymidine and therefore DNA synthesis in rapidly dividing cells. Nervous system involvement is varied, some of which results from defective myelin synthesis and repair. Cognitive impairment and psychosis may also occur. Diagnosis of B12 deficiency rests on clinical suspicion followed by laboratory testing, which consists of a panel of tests, that together provide clinically reliable predictive indices. B12 metabolism and deficiency is closely intertwined with folate, another B-vitamin. This chapter explores the various aspects of a unique and fascinating micronutrient.

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Hyperhomocysteinemia is an independent risk factor for atherosclerosis, even in early childhood. A mutation in genes that code homocysteine metabolism enzymes or deficiency of specific vitamin cofactors may cause hyperhomocysteinemia. Vitamin B complex has been correlated with serum homocysteine levels. Any abnormality in its metabolism or nutritional deficiency may lead to hyperhomocysteinemia. Both vitamin B complex and homocysteine levels are partly genetically determined. Specifically, the most studied polymorphism is 677T-C in exon 5 of the 5,10- methylenetetrahydrofolate reductase (MTHFR) gene, which plays an important role in folate's metabolism. This polymorphism has been shown to be correlated with hypertension and cardiovascular disease. Polymorphisms in methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1-like (MTHFD1L) gene have also been correlated with increased risk for coronary artery disease. Other common serious polymorphisms regard the area with high linkage disequilibrium, including the neuroblastoma breakpoint family, NBPF3 gene, and ~ 12-50 kb upstream of the tissue nonspecific alkaline phosphatase gene. Finally, the polymorphisms which have been mostly associated with vitamin B12 concentration are the rs11254363 polymorphism at intron 52 of the intrinsic factor vitamin B12 receptor of the CUBN and the rs526934 polymorphism at intron 8 of transcobalamin I. To sum up, several polymorphisms have already been associated with vitamin B complexes and therefore homocysteine level, highlighting the complex nature of vitamin B genetics.

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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.

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