B12-binding Protein Research Articles

Abstract 2785: Beyond the role of transcobalamin 1 (TCN1) as a vitamin B12-binding protein in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is a common form of primary liver cancer and is associated with poor prognosis. Although sorafenib and lenvatinib have shown promising results in HCC treatment, their clinical benefits for patients have been limited owing to drug resistance and tumor relapse. This may be associated with the presence of tumor-initiating cells (T-ICs), which require further investigation to identify the key pathways or molecules involved in their regulation. Through in vitro enrichment of liver T-IC populations via a series of sphere passages under treatment with chemotherapeutic drugs, subsequent bulk RNA sequencing analysis identified a vitamin B-12 binding protein, transcobalamin 1 (TCN1), as the most upregulated molecule in such enriched T-IC populations. This result was consistent with the enrichment profiles of the stem cell signature in TCN1High HCC samples in the clinical dataset. TCN1 is a secretory protein expressed at high levels in salivary glands and glandular cells; however, its function beyond its native role as a vitamin B12-binding protein in the regulation of T-ICs is unclear. Using lentiviral shRNA overexpression and knockdown approaches, we identified a critical role for TCN1 in regulating the properties of T-ICs, including self-renewal, invasiveness, tumorigenicity, and drug resistance, through in vitro and in vivo assays. Clinically, we also observed a substantial increase in TCN1 mRNA signatures in HCC samples, which was associated with poor prognosis of HCC patients. By analyzing the TCGA-LIHC dataset with confirmation by immunoprecipitation, we report, for the first time, that TCN1 directly interacts with integrin subunit alpha 3 (ITGA3), thereby regulating the Notch signaling pathway. Furthermore, ELISA results revealed the presence of the secretory form of TCN1 in HCC cells, which exerts a T-IC-enhancing effect on HCC cells via autocrine regulation. In conclusion, our study provides novel insights into how TCN1 functions in HCC via a non-canonical mechanism, which goes beyond its conventional role as a vitamin B12-binding protein and uncovers a potential novel therapeutic target for HCC. Citation Format: Gregory Kenneth Muliawan, Carmen Oi Ning Leung, Terence Kin Wah Lee. Beyond the role of transcobalamin 1 (TCN1) as a vitamin B12-binding protein in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2785.

220 Increased transcobalamin 1 gene expression in keratinocytes of lesional psoriatic epidermis

The TCN1 gene encodes transcobalamin I, a vitamin B12 binding protein, with unknown function in the skin. While transcriptomics data indicate an enhanced mRNA expression of TCN1 in psoriasis vulgaris, protein expression, function, and mechanisms of induction have not yet been investigated. Therefore, we examined the mRNA and protein expression of TCN1 specifically in the epidermis of healthy and psoriatic skin using qPCR and immunofluorescence staining, respectively. As compared to non-lesional and healthy epidermis, TCN1 mRNA and protein expression was significantly increased in lesional psoriatic epidermis (p<0.001, lesional: [n=13], non-lesional: [n=3], healthy: [n=5]).

Nutrient smuggling: Commensal gut bacteria‐derived extracellular vesicles scavenge vitamin B12 and related cobamides for microbe and host acquisition

AbstractThe processes by which bacteria proactively scavenge essential nutrients in crowded environments such as the gastrointestinal tract are not fully understood. In this context, we observed that bacterial extracellular vesicles (BEVs) produced by the human commensal gut microbe Bacteroides thetaiotaomicron contain multiple high‐affinity vitamin B12 binding proteins suggesting that the vesicles play a role in micronutrient scavenging. Vitamin B12 belongs to the cobamide family of cofactors that regulate microbial communities through their limited bioavailability. We show that B. thetaiotaomicron derived BEVs bind a variety of cobamides and not only deliver them back to the parental bacterium but also sequester the micronutrient from competing bacteria. Additionally, Caco‐2 cells, representing a model intestinal epithelial barrier, acquire cobamide‐bound vesicles and traffic them to lysosomes, thereby mimicking the physiological cobalamin‐specific intrinsic factor‐mediated uptake process. Our findings identify a novel cobamide binding activity associated with BEVs with far‐reaching implications for microbiota and host health.

Associations between potential causal factors and colorectal cancer risk: A systematic review and meta-analysis of Mendelian randomization studies.

To summarize the associations between potential causal factors and colorectal cancer (CRC) risk based on existing Mendelian randomization studies. This systematic review and meta-analysis involved a literature search in Embase and Medline. All published articles using Mendelian randomization to explore potential causal factors of CRC were included. Studies that reported Mendelian randomization estimates of standard deviation changes in exposures were included in the meta-analysis. Subgroup analyses based on sex and anatomical sites were performed. One hundred and ninety studies presented in 51 articles were included in systematic review, and 114 studies conducted in 32 articles were included in the meta-analysis. Adult body mass index, waist circumference, waist hip ratio, body height, body fat percentage, arm fat ratio, childhood obesity, lifetime cigarette consumption, short sleep, coffee consumption, and blood levels of vitamin B12 , arachidonic acid, stearic acid, and insulin-like growth factor binding protein 3 were positively associated with CRC risk. Conversely, acceleration-vector-magnitude physical activity, milk consumption, and blood levels of adiponectin, linoleic acid, α-linolenic acid, oleic acid, palmitoleic acid, interleukin-6 receptor subunit-α, and tumor necrosis factor were inversely associated with CRC risk. Most obesity-related anthropometric characteristics, several unhealthy lifestyles, and blood levels of some micronutrients, fatty acids, and diabetes-related biomarkers were positively associated with CRC risk. In contrast, some lifestyles and blood levels of some fatty acids and inflammatory biomarkers were inversely associated with CRC risk. Future studies with more valid genetic variants are needed for factors with discrepancies between Mendelian randomization and epidemiological studies.

TCN1 is a potential prognostic biomarker and correlates with immune infiltrates in lung adenocarcinoma

BackgroundAround the world, lung cancer is the leading cause of cancer-related death. Lung adenocarcinomas are among the most common diagnosed forms of lung cancer, whose overall survival has not improved significantly, which makes finding an effective therapeutic target vital. Transcobalamin (TCN1) is a vitamin B12-binding protein which regulates cobalamin homeostasis. In tumor tissues, TCN1 is expressed highly, and its expression is correlated with cancer aggressiveness and poor prognosis according to recent studies and bioinformatic analyses. However, its effect on lung adenocarcinoma (LUAD) is unknown.MethodsWe evaluated whether TCN1 shows diagnostic and prognostic value in LUAD using bioinformatic analysis. In particular, various databases and analysis tools were used to determine TCN1’s relationship with LUAD, including TCGA, GTEx, GEO, STRING, and TISIDB.ResultsAs compared to normal lung tissue, the level of TCN1 expression in LUAD tissues was significantly higher (P < 0.001). TCN1 also had a good ability to distinguish lung adenocarcinoma from non-lung adenocarcinoma samples [area under the curve (AUC) = 0.788]. According to univariate Cox statistics, high expression levels of TCN1 correlate with poor overall survival (OS) in LUAD (P < 0.001). Moreover, based on a multivariate Cox analysis, TCN1 expression was independently correlated with OS (P = 0.011). GO/KEGG and GSEA indicated enrichment in epidermal cell differentiation (P < 0.0005), keratinocyte differentiation (P < 0.0005), neuroactive ligand–receptor interaction (P < 0.0005), epithelial–mesenchymal transition (P = 0.029, FDR = 0.023) and TNFA signaling via NFKB (P = 0.029, FDR = 0.023). Furthermore, TCN1 is associated with immune infiltration based on an analysis of immune cell infiltration.ConclusionsIn summary, TCN1 could be used as a prognostic and diagnostic biomarker and provide deeper perspectives for the development of therapies and prognostic markers in LUAD.

An unusual light-sensing function for coenzyme B12 in bacterial transcription regulator CarH.

Coenzyme B12 is one of the most complex cofactors found in nature and synthesized de novo by certain groups of bacteria. Although its use in various enzymatic reactions is well characterized, only recently an unusual light-sensing function has been ascribed to coenzyme B12. It has been reported that the coenzyme B12 binding protein CarH, found in the carotenoid biosynthesis pathway of several thermostable bacteria, binds to the promoter region of DNA and suppresses transcription. To overcome the harmful effects of light-induced damage in the cells, CarH releases DNA in the presence of light and promotes transcription and synthesis of carotenoids, thereby working as a photoreceptor. CarH is able to achieve this by exploiting the photosensitive nature of the CoC bond between the adenosyl moiety and the cobalt atom in the coenzyme B12 molecule. Extensive structural and spectroscopy studies provided a mechanistic understanding of the molecular basis of this unique light-sensitive reaction. Most studies on CarH have used the ortholog from the thermostable bacterium Thermus thermophilus, due to the ease with which it can be expressed and purified in high quantities. In this chapter we give an overview of this intriguing class of photoreceptors and report a step-by-step protocol for expression, purification and spectroscopy experiments (both static and time-resolved techniques) employed in our laboratory to study CarH from T. thermophilus. We hope the contents of this chapter will be of interest to the wider coenzyme B12 community and apprise them of the potential and possibilities of using coenzyme B12 as a light-sensing probe in a protein scaffold.

Vitamin B12 and transcobalamin in bovine milk: Genetic variation and genome-wide association with loci along the genome

In human nutrition, bovine milk is an essential source of bioavailable vitamin B12 and B12-binding proteins, including transcobalamin. In this study, we estimated genetic parameters for milk content of vitamin B12 and transcobalamin using milk samples from 341 and 663 Danish Holstein cows, respectively. Additionally, we conducted whole-genome association analysis to identify SNP and genes associated with vitamin B12 and transcobalamin. Our results indicated moderate to high heritability for vitamin B12 (0.37 ± 0.18) and transcobalamin (0.61 ± 0.13) content in the Danish Holstein. With a significance threshold of –log10P-value > 5.87, significant associations were detected between SNP in Bos taurus autosome (BTA)17 and the log-transformed transcobalamin content of milk; no significant association was detected for vitamin B12. The significant region in BTA17 was imputed to full sequence for further fine mapping, and the SNP with the most significant associations to transcobalamin were assigned to the transcobalamin 2 (TCN2) gene.

Four families of folate-independent methionine synthases.

Although most organisms synthesize methionine from homocysteine and methyl folates, some have "core" methionine synthases that lack folate-binding domains and use other methyl donors. In vitro, the characterized core synthases use methylcobalamin as a methyl donor, but in vivo, they probably rely on corrinoid (vitamin B12-binding) proteins. We identified four families of core methionine synthases that are distantly related to each other (under 30% pairwise amino acid identity). From the characterized enzymes, we identified the families MesA, which is found in methanogens, and MesB, which is found in anaerobic bacteria and archaea with the Wood-Ljungdahl pathway. A third uncharacterized family, MesC, is found in anaerobic archaea that have the Wood-Ljungdahl pathway and lack known forms of methionine synthase. We predict that most members of the MesB and MesC families accept methyl groups from the iron-sulfur corrinoid protein of that pathway. The fourth family, MesD, is found only in aerobic bacteria. Using transposon mutants and complementation, we show that MesD does not require 5-methyltetrahydrofolate or cobalamin. Instead, MesD requires an uncharacterized protein family (DUF1852) and oxygen for activity.

Expression of Specific Granule Markers on the Cell Surface of Neutrophil Cytoplasts

The widespread assumption that cytoplasts generated from human polymorphonuclear leukocytes (PMNs) are vesicles consisting solely of cytoplasm surrounded by plasma membrane and devoid of granule activity remains to be tested. PMN cytoplasts were prepared by centrifugation of intact cells on a Ficoll step gradient in the presence of cytochalasin B. Two granule membrane markers, Mol, a fluorometrically detectable antigen, and cytochrome b, both of which have been shown to translocate to the plasma membrane during granule release, were compared for their activity in cytoplasts and intact PMNs. We found that the amount of Mol detected on the plasma membrane of intact PMNs, as compared with other membrane markers (such as antigens LFA-1 and beta 2m), increased 1.6-fold upon exposure of PMNs to Ficoll plus cytochalasin B prior to centrifugation. Another twofold increase in Mol expression occurred upon cytoplast preparation. Release of the granule enzymes, vitamin B12-binding protein, and lysozyme were also followed and correlated well (r = .78 and .92) with the amount of Mol antigen present on the cell surface. Cytochrome b was also found to be higher (1.4-fold) on plasma membranes isolated from cytoplasts than on plasma membranes isolated from intact control cells. These results indicate that some fusion of granule membranes and plasma membranes occurred during treatment of PMNs with Ficoll plus cytochalasin b and during cytoplast preparation.

Association Between Gelatinase Release and Increased Plasma Membrane Expression of the Mo1 Glycoprotein

Mo1, a glycoprotein heterodimer (gp 155,95) that functions as an adhesion promoting molecule and as the C3bi receptor of human myeloid cells, is expressed in increased amounts in the plasma membrane after exposure of polymorphonuclear leukocytes (PMNs) to various stimuli. Previous studies have suggested that secondary granules represent an intracellular pool of Mo1 that, upon degranulation, fuse with the plasma membrane resulting in a tenfold increase in surface expression of Mo1. To determine the intracellular location of Mo1, we monitored Mo1 expression by immunofluorescence and compared it to the release of myeloperoxidase (MPO, a marker for the primary granules), vitamin B12 binding protein (B12BP, secondary granules), and gelatinase (gelatinase-containing organelles) following exposure to various stimuli. Human neutrophils stimulated with 20 mmol/L fluoride for 16 minutes exhibited a twofold increase in Mo1 expression and gelatinase release but no enhanced release of primary or secondary granular contents. In a similar fashion, incubation of cells at 37 degrees C for five minutes with 7.5 X 10(-9) to 10(-6) mol/L N-formyl-methionyl-leucyl-phenylalanine (FMLP) resulted in significant increases in both surface Mo1 expression (three- to fivefold) and gelatinase release (five- to eightfold) without significant release of either MPO or B12BP. In addition, both the fluoride and FMLP experiments demonstrated that Mo1 up-modulation alone is not sufficient to activate superoxide (O2-) production. These data indicate that at least one intracellular storage pool of Mo1 is the gelatinase-containing organelles and that their fusion with the plasma membrane results in increased expression of Mo1 on the cell surface.

Lysosomal activity regulates Caenorhabditis elegans mitochondrial dynamics through vitamin B12 metabolism

Mitochondrial fission and fusion are highly regulated by energy demand and physiological conditions to control the production, activity, and movement of these organelles. Mitochondria are arrayed in a periodic pattern in Caenorhabditis elegans muscle, but this pattern is disrupted by mutations in the mitochondrial fission component dynamin DRP-1. Here we show that the dramatically disorganized mitochondria caused by a mitochondrial fission-defective dynamin mutation is strongly suppressed to a more periodic pattern by a second mutation in lysosomal biogenesis or acidification. Vitamin B12 is normally imported from the bacterial diet via lysosomal degradation of B12-binding proteins and transport of vitamin B12 to the mitochondrion and cytoplasm. We show that the lysosomal dysfunction induced by gene inactivations of lysosomal biogenesis or acidification factors causes vitamin B12 deficiency. Growth of the C. elegans dynamin mutant on an Escherichia coli strain with low vitamin B12 also strongly suppressed the mitochondrial fission defect. Of the two C. elegans enzymes that require B12, gene inactivation of methionine synthase suppressed the mitochondrial fission defect of a dynamin mutation. We show that lysosomal dysfunction induced mitochondrial biogenesis, which is mediated by vitamin B12 deficiency and methionine restriction. S-adenosylmethionine, the methyl donor of many methylation reactions, including histones, is synthesized from methionine by S-adenosylmethionine synthase; inactivation of the sams-1 S-adenosylmethionine synthase also suppresses the drp-1 fission defect, suggesting that vitamin B12 regulates mitochondrial biogenesis and then affects mitochondrial fission via chromatin pathways.

A Rapid Quantitative Chemiluminescence Immunoassay for Vitamin B12 in Human Serum.

Vitamin B12 is very important for the human body. Early diagnosis of vitamin B12 deficiency is essential because it is associated with many problems such as fatigue, lethargy, depression, poor memory, breathlessness, headaches, pale skin, mania, psychosis, etc. Most of the vitamin B12 molecules in serum bind to specific proteins as a complex; so, although researchers have developed some sensitive methods to quantify vitamin B12, few can be used for human serum detection of vitamin B12 because of disturbed releasing process. In this study, we are aiming to develop a rapid and accurate chemiluminescence immunoassay (CLIA) for human serum testing. In this study, we studied and optimized labelling of biotin molecules to vitamin B12 binding protein which had been extracted from hog gastric mucus, labelling of vitamin B12 molecules to horse radish peroxidase (HRP), releasing of vitamin B12 from its bound state in serum, concentrations of reagents, and incubation times. The developed method shows good performance and thermo-stability. The limit of detection (LOD) is 41 pg/mL, the recovery rate is 90.7% - 107.4%. The intra-assay CV is 4.4% - 8.0% and the inter-assay CV is 4.5% - 12.1%. Cross reactivity (CR) values are 49.4% for hydroxocobalamin and lower than 0.1% for vitamin B1, B2, B3, B9, and C. The method comparison results show that the correlation coefficient (R2) is 0.91, with a slight negative bias of -4.1% [95% limits of agreement (± 1.96 SD); -39.6% to 31.6%]. The developed method shows high sensitivity and specificity and good correlation using a Beckman Coulter instrument while the whole test needs only 40 minutes. It can fully satisfy the very busy clinical labs.

Cobamides.

Vitamin B12 is the only known essential human micronutrient made exclusively by prokaryotes. Kennedy and Taga introduce us to the world of cobamides-those cobalt-containing compounds, like B12, that appear to be the proprietary domain of our microbial partners.

Functional and phylogenetic characterization of noncanonical vitamin B12–binding proteins in zebrafish suggests involvement in cobalamin transport

In humans, transport of food-derived cobalamin (vitamin B12) from the digestive system into the bloodstream involves three paralogous proteins: transcobalamin (TC), haptocorrin (HC), and intrinsic factor (IF). Each of these proteins contains two domains, an α-domain and a β-domain, which together form a cleft in which cobalamin binds. Zebrafish (Danio rerio) are thought to possess only a single cobalamin transport protein, referred to as Tcn2, which is a transcobalamin homolog. Here, we used CRISPR/Cas9 mutagenesis to create null alleles of tcn2 in zebrafish. Fish homozygous for tcn2-null alleles were viable and exhibited no obvious developmentally or behaviorally abnormal phenotypes. For this reason, we hypothesized that previously unidentified cobalamin-carrier proteins encoded in the zebrafish genome may provide an additional pathway for cobalamin transport. We identified genes predicted to code for two such proteins, Tcn-beta-a (Tcnba) and Tcn-beta-b (Tcnbb), which differ from all previously characterized cobalamin transport proteins as they lack the α-domain. These β-domain-only proteins are representative of an undescribed class of cobalamin-carrier proteins that are highly conserved throughout the ray-finned fishes. We observed that the genes encoding the three cobalamin transport homologs, tcn2, tcnba, and tcnbb, are expressed in unique spatial and temporal patterns in the developing zebrafish. Moreover, exogenously expressed recombinant Tcnba and Tcnbb bound cobalamin with high affinity, comparable with binding by full-length Tcn2. Taken together, our results suggest that this noncanonical protein structure has evolved to fully function as a cobalamin-carrier protein, thereby allowing for a compensatory cobalamin transport mechanism in the tcn2-/- zebrafish.

Human Intrinsic Factor Expression for Bioavailable Vitamin B12 Enrichment in Microalgae.

Dietary supplements and functional foods are becoming increasingly popular complements to regular diets. A recurring ingredient is the essential cofactor vitamin B12 (B12). Microalgae are making their way into the dietary supplement and functional food market but do not produce B12, and their B12 content is very variable. In this study, the suitability of using the human B12-binding protein intrinsic factor (IF) to enrich bioavailable B12 using microalgae was tested. The IF protein was successfully expressed from the nuclear genome of the model microalga Chlamydomonas reinhardtii and the addition of an N-terminal ARS2 signal peptide resulted in efficient IF secretion to the medium. Co-abundance of B12 and the secreted IF suggests the algal produced IF protein is functional and B12-binding. Utilizing IF expression could be an efficient tool to generate B12-enriched microalgae in a controlled manner that is suitable for vegetarians and, potentially, more bioavailable for humans.

Live cell imaging of vitamin B12 dynamics by genetically encoded fluorescent nanosensor

Vitamin B12 (cobalamin) is a co-factor of various enzymes and involved in the metabolism of every cell of human body. During infancy, low level of vitamin B12 is associated with negative consequences on the developing brain. Vitamin B12 deficiency causes various neurological abnormalities and pernicious anemia. Different methods such as, isotopic labeling, mass spectrocopy and Nuclear Magnetic Resonance spectroscopy have been used for measuring the level of cellular metabolites or signaling molecules, but these methods require the disruption and fractionation of tissues which suffer from contamination. Fluorescence Resonance Energy Transfer (FRET)-based genetically encoded nanosensors have been evolved as an ideal mean to determine the metabolite concentration in live cells. Here, we report the designing of FRET-based nanosensor for direct visualization of changes in vitamin B12 concentration in intact living cells. Initially, a construct was designed by using the vitamin B12 binding protein (BtuF), cyan (CFP) and yellow (YFP) variants of green fluorescent protein. This construct was then shuttled in different expression vectors. The constructed FRET sensor was termed as SenVitAL (Sensor for Vitamin Anemia Linked) which is found to be very specific for vitamin B12. This sensor is stable to pH changes, and measures the vitamin B12 in a concentration-dependent manner with an apparent affinity, Kd of ∼157μM. In case of E. coli cells, an increase in the emission intensity ratio was specifically detected after exposure to vitamin B12. In both in vitro and in vivo measurements, FRET ratio increases after the addition of vitamin B12. Furthermore, the results show that the SenVitAL can measure the vitamin B12 concentration in the cytosol of yeast and mammalian cells which proves its potential in eukaryotic system. Thus, the sensor can serve as novel indicator to investigate the vitamin B12 import and metabolism and, would help to elucidate their complex roles in biology.

Elucidation of roles for vitamin B12 in regulation of folate, ubiquinone, and methionine metabolism

Only a small fraction of vitamin B12-requiring organisms are able to synthesize B12 de novo, making it a common commodity in microbial communities. Initially recognized as an enzyme cofactor of a few enzymes, recent studies have revealed additional B12-binding enzymes and regulatory roles for B12 Here we report the development and use of a B12-based chemical probe to identify B12-binding proteins in a nonphototrophic B12-producing bacterium. Two unexpected discoveries resulted from this study. First, we identified a light-sensing B12-binding transcriptional regulator and demonstrated that it controls folate and ubiquinone biosynthesis. Second, our probe captured proteins involved in folate, methionine, and ubiquinone metabolism, suggesting that it may play a role as an allosteric effector of these processes. These metabolic processes produce precursors for synthesis of DNA, RNA, and protein. Thereby, B12 likely modulates growth, and by limiting its availability to auxotrophs, B12-producing organisms may facilitate coordination of community metabolism.

Application of a Multi-Marker Approach to Assess Vitamin B12 Status at the End of the First Trimester of Pregnancy

Background:During pregnancy, vitamin B12 (B12) utilisation increases, with deficiency being associated with adverse outcomes (Murphy 2007, VanderJagt 2011). It remains unclear however at what level vitamin B12 status becomes detrimental, and the correlation with fetal harm.Establishing B12 status during pregnancy is complicated by physiological changes such as haemodilution due to expanded blood volume, altered renal function, alterations in the vitamin B12 -binding proteins and transfer of materno-fetal B12 yet laboratories typically rely on the total abundance of B12 in serum (serum B12) asthe sole laboratory status indicator, interpreting results against reference range cut offs derived from non-pregnant populations, including men.Serum B12 assays measure the sum of holohaptocorrin and holotranscobalamin (holoTC). Other markers of B12 status are also available and are increasingly being adopted: holoTC (active B12) accounts 6-20% of bound B12, which is the only form of B12 presented for cellular uptake and used to satisfy metabolic demand; methylmalonic acid (MMA) is a by-product of methylmalonyl-CoA metabolism, the concentration of which in serum correlates inversely with tissue B12 utilisation.Methods:Over a 2 monthperiod in 2015 we used an extended panel of laboratory tests consisting of serum B12, holoTC, and MMA to determine B12 status, and assess appropriateness of our assay reference ranges, in 100 pregnant women with normal renal function at the end of the first trimester.Laboratory AnalysisSerum B12 and holoTC were determined by an Abbott Architect analyzer. The serum B12 assay has a reference range of 187 - 883 ng/L. The holoTC assay has ²10% cross-reactivity from B12-binding proteins haptocorrin and apotranscobalamin. Values >128 pmol/L are above the dynamic assay range. In our laboratory holoTC results <25 pmol/L are defined as deficient and those >70 pmol/L as replete. Results 25-70 pmol/L are classified as indeterminate and a serum MMA assay subsequently performed (Sobczyńska-Malefora A et al 2014). We determined serum MMA using a Gerstel Multipurpose Sampler coupled to a liquid chromatography tandem mass spectrometer with electrospray ionization with results >270 nmol/L considered indicative of suboptimal B12 status (Harrington DJ 2016).Results:All three assays, figure 3, were performed for 92 women (insufficient sample available n=8).HoloTCand MMA results are shown in Table 1. The distribution ofholoTCand MMA concentrations are shown in Figure 1.One woman had a serum B12 below the reference range with a corresponding indeterminate holoTC and normal MMA. This set of results suggests questionable abundance of B12 that has not translated to functional deficiency.One woman had a holoTCconcentration <25pmol/L, however the corresponding serum B12 and MMA were not indicative of sub optimal B12 status. Fourteen women had aholoTC result above the dynamic assay range.Two women had an elevated MMA concentration with corresponding holoTCresults indicating questionable abundance of B12 but serum B12 within our reference limit. This triad of values indicates possible emerging suboptimal B12 status that would have been overlooked if serum B12 had been used in isolation.Discussion:B12 requirement increases in pregnancy and diet alone is unlikely to meet requirements (Murphy et al 2007). Deficiency has a detrimental impact on maternal and infant development and therefore it is useful to explore the relationship between different laboratory markers of B12 status in pregnancy (Ramirez-Velez et al 2016, Siddiqua et al 2014). The comparison of serum B12,holoTC and MMA levels at the end of the first trimester with reference ranges derived from non-pregnant adults suggests that they are broadly applicable.Serum B12 is the least sensitive marker and its use in isolation to assess B12 status is limiting. The application of holoTC and MMA in tandem can reveal previously missed deficient states. Using these markers two women in this small study were identified as possibly deficient although the true significance of a mildly elevated MMA in pregnancy requires further investigation. These data highlight the benefit of adopting a panel of markers ahead of a single test to assess B12 status. Identifying women at the end of the first trimester that may benefit from B12 replacement potentially protects from deficient states. [Display omitted] [Display omitted] [Display omitted] DisclosuresRobinson:Pharmacosmos: Honoraria; Amgen: Honoraria; Novartis: Honoraria.

Forms and Amounts of Vitamin B12 in Infant Formula: A Pilot Study.

PurposeInfant formula is based on cow’s milk and designed to mimic breast milk for substitution. Vitamin B12 (B12) is bound to proteins in both breast milk and cow’s milk, and in milk from both species the vitamin occurs mainly in its natural form such as hydroxo-B12 with little or no synthetic B12 (cyano-B12). Here we test commercially available infant formulas.MethodsEleven commercially available infant formulas were measured for content of B12 and analyzed for the presence of B12-binding proteins and forms of B12 using size exclusion chromatography and HPLC.ResultsAll infant formulas contained B12 by and large in accord with the informations given on the package inserts. None of the formulas contained protein-bound B12, and cyano-B12 accounted for 19–78% of the total amount of B12 present, while hydroxo-B12 constituted more or less the rest.ConclusionsThis pilot study shows that infant formula differs from breast milk in providing the infant with free B12, rather than protein-bound B12, and by a relative high content of cyano-B12. The consequence of supplying the infant with synthetic cyano-B12 remains to be elucidated.

MtrA of the sodium ion pumping methyltransferase binds cobalamin in a unique mode.

In the three domains of life, vitamin B12 (cobalamin) is primarily used in methyltransferase and isomerase reactions. The methyltransferase complex MtrA–H of methanogenic archaea has a key function in energy conservation by catalysing the methyl transfer from methyl-tetrahydromethanopterin to coenzyme M and its coupling with sodium-ion translocation. The cobalamin-binding subunit MtrA is not homologous to any known B12-binding proteins and is proposed as the motor of the sodium-ion pump. Here, we present crystal structures of the soluble domain of the membrane-associated MtrA from Methanocaldococcus jannaschii and the cytoplasmic MtrA homologue/cobalamin complex from Methanothermus fervidus. The MtrA fold corresponds to the Rossmann-type α/β fold, which is also found in many cobalamin-containing proteins. Surprisingly, the cobalamin-binding site of MtrA differed greatly from all the other cobalamin-binding sites. Nevertheless, the hydrogen-bond linkage at the lower axial-ligand site of cobalt was equivalently constructed to that found in other methyltransferases and mutases. A distinct polypeptide segment fixed through the hydrogen-bond linkage in the relaxed Co(III) state might be involved in propagating the energy released upon corrinoid demethylation to the sodium-translocation site by a conformational change.