The avian retroviral receptor Tva mediates the uptake of transcobalamin bound vitamin B12 (cobalamin).

The Avian sarcoma and leukosis viruses (ASLVs) are important chicken pathogens. Some of the virus subgroups, including ASLV-A and K, utilize the Tva receptor for cell entrance. Though Tva was identified three decades ago, its physiological function remains unknown. Previously, we have noted an intriguing resemblance and orthology between the chicken gene coding for Tva and the human gene coding for CD320, a receptor involved in cellular uptake of transcobalamin (TC) in complex with vitamin B12/cobalamin (Cbl).Here we show that both the transmembrane and the glycosylphosphatidylinositol (GPI)-anchored form of Tva in the chicken cell line DF-1 promotes the uptake of Cbl with help of expressed and purified chicken TC. The uptake of TC-Cbl complex was monitored using an isotope- or fluorophore-labeled Cbl. We show that (i) TC-Cbl is internalized in chicken cells; and (ii) the uptake is lower in the Tva-knockout cells and higher in Tva-overexpressing cells when compared with wild type chicken cells. The relation between physiological function of Tva and its role in infection was elaborated by showing that infection with ASLV subgroups (targeting Tva) impairs the uptake of TC-Cbl, while this is not the case for cells infected with ASLV-B (not recognized by Tva). In addition, exposure of the cells to a high concentration of TC-Cbl alleviates the infection with Tva-dependent ASLV.IMPORTANCE: We demonstrate that the ASLV receptor Tva participates in the physiological uptake of TC-Cbl, because the viral infection suppresses the uptake of Cbl and vice versa. Our results pave the road for future studies addressing the issues: (i) whether a virus infection can be inhibited by TC-Cbl complexes in vivo; and (ii) whether any human virus employs the human TC-Cbl receptor CD320. In broader terms, our study sheds light on the intricate interplay between physiological roles of cellular receptors and their involvement in virus infection.

Vitamin B12 and its binding proteins in milk from cow and buffalo in relation to bioavailability of B12.

Milk is an important source of highly bioavailable vitamin B12 (cobalamin) in human nutrition. In most animal products, vitamin B12 is strongly bound to various specific protein carriers. The 2 vitamin B12-specific proteins, predominantly transcobalamin (TC) and haptocorrin (HC), were earlier found in milk from Holstein Friesian cows and in human or sow milk, respectively. As the type of vitamin B12 binders may influence bioavailability of the vitamin, we examined vitamin B12 carriers in pooled milk specimens derived from European and Indian cow and buffalo herds. The total endogenous vitamin B12 concentration was comparable in all milk pools (≈3 nM), but the vitamin carriers varied considerably: TC + caseins in Danish cows, TC + HC in Indian cows and buffaloes, and mainly HC in Italian buffaloes. Danish cow milk contained half as much TC as vitamin B12, and the surplus vitamin was all attached via a single coordination bond to abundantly available histidine residues of casein. The specific binding proteins in Indian cow milk (TC + HC) approximately matched the molar content of vitamin B12. Milk from the 2 buffalo breeds contained more specific binders than vitamin B12, and the surplus proteins included the unsaturated TC ≈ 3 nM (Indian stock), or both TC ≈ 4 nM and HC ≈ 23 nM (Italian stock). The abundant HC of the latter sample bound nearly all endogenous vitamin B12. We tested (in vitro) the transfer of vitamin B12 from milk proteins to human carriers, involved in the intestinal uptake. The bovine TC-vitamin B12 complex rapidly dissociated at pH 2 (time of half reaction, τ1/2 < 1 min, 37°C) and was susceptible to digestion with trypsin + chymotrypsin (pH 7.5). Transfer of vitamin B12 from the precipitated bovine casein (pH 2) to human carriers proceeded with τ1/2 ≈ 7 min (37°C) and τ1/2 ≈ 35 min (20°C). Liberation of vitamin B12 from buffalo HC was hampered because of its pH stability and slow proteolysis. Nutritional availability of vitamin B12 is expected to be high in cow milk (with TC-vitamin B12 and casein-vitamin B12 complexes) but potentially constrained in buffalo milk (with HC-vitamin B12). This especially concerns the Italian buffalo milk, where a high excess of HC was found. We speculate whether the isolated stock of Italian buffalo maintained the ancestral secretion of carriers (HC ≫ vitamin B12, TC ≈ 0), whereas intensive crossbreeding of cows and buffaloes from other regions caused a change to TC ≤ vitamin B12, with low or absent HC. The substitution of HC by less sturdy carriers is apparently more beneficial to human consumers as far as vitamin B12 bioavailability is concerned.

The tissue profile of metabolically active coenzyme forms of vitamin B12 differs in vitamin B12-depleted rats treated with hydroxo-B12 or cyano-B12.

Recent rat studies show different tissue distributions of vitamin B12 (B12), administered orally as hydroxo-B12 (HO-B12) (predominant in food) and cyano-B12 (CN-B12) (common in supplements). Here we examine male Wistar rats kept on a low-B12 diet for 4 weeks followed by a 2-week period on diets with HO-B12 (n 9) or CN-B12 (n 9), or maintained on a low-B12 diet (n 9). Plasma B12 was analysed before, during and after the study. The content of B12 and its variants (HO-B12, glutathionyl-B12, CN-B12, 5'-deoxyadenosyl-B12 (ADO-B12), and methyl-B12 (CH3-B12)) were assessed in the tissues at the end of the study. A period of 4 weeks on the low-B12 diet reduced plasma B12 by 58 % (from median 1323 (range 602-1791) to 562 (range 267-865) pmol/l, n 27). After 2 weeks on a high-B12 diet (week 6 v. week 4), plasma B12 increased by 68 % (HO-B12) and 131 % (CN-B12). Total B12 in the tissues accumulated differently: HO-B12>CN-B12 (liver, spleen), HO-B12

Tissue distribution of oral vitamin B12 is influenced by B12 status and B12 form: an experimental study in rats.

PURPOSE: Hydroxocobalamin (HOCbl) is the dominating Cbl form in food, whereas cyanocobalamin (CNCbl) is common in vitamin pills and oral supplements. This study compares single-dose absorption and distribution of oral HO[57Co]Cbl and CN[57Co]Cbl in Cbl-deficient and normal rats. METHODS: Male Wistar rats (7 weeks) were fed a 14-day diet with (n = 15) or without (n = 15) Cbl. We compared the uptakes of HO[57Co]Cbl (free or bound to bovine transcobalamin) and free CN[57Co]Cbl administered by gastric gavage (n = 5 in each diet group). Rats were sacrificed after 24 h. Blood, liver, kidney, brain, heart, spleen, intestines, skeletal muscle, 24-h urine and faeces were collected, and the content of [57Co]Cbl was measured. Endogenous Cbl in tissues and plasma was analysed by routine methods. RESULTS: Mean endogenous plasma-Cbl was sevenfold lower in deficient vs. normal rats (190 vs. 1330 pmol/L, p < 0.0001). Cbl depletion increased endogenous Cbl ratios (tissue/plasma = k in/k out) in all organs except for the kidney, where the ratio decreased considerably. Twenty-four-hour accumulation of labelled Cbl showed that HOCbl > CNCbl (liver) and CNCbl > HOCbl (brain, muscle and plasma). CONCLUSIONS: The Cbl status of rats and the administered Cbl form influence 24-h Cbl accumulation in tissues and plasma.

The Human Serum Metabolome of Vitamin B-12 Deficiency and Repletion, and Associations with Neurological Function in Elderly Adults.

BACKGROUND: The specific metabolomic perturbations that occur in vitamin B-12 deficiency, and their associations with neurological function, are not well characterized. OBJECTIVE: We sought to characterize the human serum metabolome in subclinical vitamin B-12 deficiency and repletion. METHODS: A before-and-after treatment study provided 1 injection of 10 mg vitamin B-12 (with 100 mg pyridoxine and 100 mg thiamin) to 27 community-dwelling elderly Chileans (∼74 y old) with vitamin B-12 deficiency, as evaluated with serum vitamin B-12, total plasma homocysteine (tHcy), methylmalonic acid (MMA), and holotranscobalamin. The combined indicator of vitamin B-12 status (cB-12) was computed. Targeted metabolites [166 acylcarnitines, amino acids, sugars, glycerophospholipids, and sphingolipids (liquid chromatography-tandem mass spectrometry)], and untargeted metabolites [247 chemical entities (gas chromatography time-of-flight mass spectrometry)] were measured at baseline and 4 mo after treatment. A peripheral nerve score was developed. Differences before and after treatment were examined. For targeted metabolomics, the data from 18 individuals with adequate vitamin B-12 status (selected from the same population) were added to the before-and-after treatment data set. Network visualizations and metabolic pathways are illustrated. RESULTS: The injection increased serum vitamin B-12, holotranscobalamin, and cB-12 (P < 0.001), and reduced tHcy and serum MMA (P < 0.001). Metabolomic changes from before to after treatment included increases (P < 0.001) in acylcarnitines, plasmalogens, and other phospholipids, whereas proline and other intermediaries of one-carbon metabolism-that is, methionine and cysteine-were reduced (P < 0.001). Direct significant correlations (P < 0.05 after the false discovery rate procedure) were identified between acylcarnitines, plasmalogens, phospholipids, lyso-phospholipids, and sphingomyelins compared with vitamin B-12 status and nerve function. Multiple connections were identified with primary metabolites (e.g., an inverse relation between vitamin B-12 markers and tryptophan, tyrosine, and pyruvic, succinic, and citric acids, and a direct correlation between the nerve score and arginine). CONCLUSIONS: The human serum metabolome in vitamin B-12 deficiency and the changes that occur after supplementation are characterized. Metabolomics revealed connections between vitamin B-12 status and serum metabolic markers of mitochondrial function, myelin integrity, oxidative stress, and peripheral nerve function, including some previously implicated in Alzheimer and Parkinson diseases. This trial was registered at www.controlled-trials.com as ISRCTN02694183.

False low holotranscobalamin levels in a patient with a novel TCN2 mutation.

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.

The extracellular heme-binding protein HbpS from the soil bacterium Streptomyces reticuli is an aquo-cobalamin binder.

The extracellular protein HbpS from Streptomyces reticuli interacts with iron ions and heme. It also acts in concert with the two-component sensing system SenS-SenR in response to oxidative stress. Sequence comparisons suggested that the protein may bind a cobalamin. UV-visible spectroscopy confirmed binding (Kd = 34 µm) to aquo-cobalamin (H2OCbl(+)) but not to other cobalamins. Competition experiments with the H2OCbl(+)-coordinating ligand CN(-) and comparison of mutants identified a histidine residue (His-156) that coordinates the cobalt ion of H2OCbl(+) and substitutes for water. HbpS·Cobalamin lacks the Asp-X-His-X-X-Gly motif seen in some cobalamin binding enzymes. Preliminary tests showed that a related HbpS protein from a different species also binds H2OCbl(+). Furthermore, analyses of HbpS-heme binding kinetics are consistent with the role of HbpS as a heme-sensor and suggested a role in heme transport. Given the high occurrence of HbpS-like sequences among Gram-positive and Gram-negative bacteria, our findings suggest a great functional versatility among these proteins.

Combined indicator of vitamin B12 status: modification for missing biomarkers and folate status and recommendations for revised cut-points.

BACKGROUND: A novel approach to determine vitamin B12 status is to combine four blood markers: total B12 (B12), holotranscobalamin (holoTC), methylmalonic acid (MMA) and total homocysteine (tHcy). This combined indicator of B12 status is expressed as cB12=log10[(holoTC·B12)/(MMA·Hcy)]-(age factor). Here we calculate cB12 in datasets with missing biomarkers, examine the influence of folate status, and revise diagnostic cut-points. METHODS: We used a database with all four markers (n=5211) plus folate measurements (n=972). A biomarker Z (assumed missing) was plotted versus X (a combination of other markers) and Y (age). Each chart was approximated by a function Ztheor, which predicted the potentially absent value(s). Statistical distributions of cB12 were aligned with physiological indicators of deficiency and used to determine cut-offs. RESULTS: The predictive functions Ztheor allowed assessment of the "incomplete" indicators, 3cB12 (three markers known) and 2cB12 (two markers known). Predictions contained a systematic deviation associated with dispersion along two axes Z and X (and unaccounted by the least squares fit). Increase in tHcy at low serum folate was corrected (cB12+Δfolate) based on the function of Δfolate=log10(Hcyreal/Hcytheor) versus folate. Statistical distributions of cB12 revealed the boundaries of groups with B12 deficiency, i.e., cB12<-0.5. CONCLUSIONS: We provide equations that combine two, three or four biomarkers into one diagnostic indicator, thereby rescaling unmatched data into the same coordinate system. Adjustment of this indicator is required if serum folate is <10 nmol/L and tHcy is measured. Revised cut-points and guidelines for using this approach are provided.

Macro-B12 and Unexpectedly High Levels of Plasma B12: A Critical Review.

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.

Kinetics of Cellular Cobalamin Uptake and Conversion: Comparison of Aquo/Hydroxocobalamin to Cyanocobalamin.

Cyanocobalamin (CNCbl) and aquo/hydroxocobalamin (HOCbl) are the forms of vitamin B12 that are most commonly used for supplementation. They are both converted to methylcobalamin (MeCbl) and 5'-deoxyadenosylcobalamin (AdoCbl), which metabolize homocysteine and methylmalonic acid, respectively. Here, we compare the kinetics of uptake and the intracellular transformations of radiolabeled CNCbl vs. HOCbl in HeLa cells. More HOCbl was accumulated over 4-48 h, but further extrapolation indicated similar uptake (>90%) for both vitamin forms. The initially synthesized coenzyme was MeCbl, which noticeably exceeded AdoCbl during 48 h. Yet, the synthesis of AdoCbl accelerated, and the predicted final levels of Cbls were MeCbl ≈ AdoCbl ≈ 40% and HOCbl ≈ 20%. The designed kinetic model revealed the same patterns of the uptake and turnover for CNCbl and HOCbl, apart from two steps. First, the "activating" intracellular processing of the internalized HOCbl was six-fold faster. Second, the detachment rates from the cell surface (when the "excessive" Cbl-molecules were refluxed into the external medium) related as 4:1 for CNCbl vs. HOCbl. This gave a two-fold faster cellular accumulation and processing of HOCbl vs. CNCbl. In medical terms, our data suggest (i) an earlier response to the treatment of Cbl-deficiency with HOCbl, and (ii) the manifestation of a successful treatment initially as a decrease in homocysteine.

Physiological and molecular aspects of cobalamin transport.

Minute doses of a complex cofactor cobalamin (Cbl, vitamin B12) are essential for metabolism. The nutritional chain for humans includes: (1) production of Cbl by bacteria in the intestinal tract of herbivores; (2) accumulation of the absorbed Cbl in animal tissues; (3) consumption of food of animal origin. Most biological sources contain both Cbl and its analogues, i.e. Cbl-resembling compounds physiologically inactive in animal cells. Selective assimilation of the true vitamin requires an interplay between three transporting proteins - haptocorrin (HC), intrinsic factor (IF), transcobalamin (TC) - and several receptors. HC is present in many biological fluids, including gastric juice, where it assists in disposal of analogues. Gastric IF selectively binds dietary Cbl and enters the intestinal cells via receptor-mediated endocytosis. Absorbed Cbl is transmitted to TC and delivered to the tissues with blood flow. The complex transport system guarantees a very efficient uptake of the vitamin, but failure at any link causes Cbl-deficiency. Early detection of a negative B12 balance is highly desirable to prevent irreversible neurological damages, anaemia and death in aggravated cases. The review focuses on the molecular mechanisms of cobalamin transport with emphasis on interaction of corrinoids with the specific proteins and protein-receptor recognition. The last section briefly describes practical aspects of recent basic research concerning early detection of B12-related disorders, medical application of Cbl-conjugates, and purification of corrinoids from biological samples.

Low methylcobalamin in liver tissues is an artifact as shown by a revised extraction procedure.

BACKGROUND: Vitamin B12 (cobalamin, Cbl) is represented by several molecular variants distinguished by the exchangeable ligand X coordinated to cobalt ion (XCbl). The most typical XCbl-forms are cyanocobalamin (CNCbl), hydroxocobalamin (HOCbl), methylcobalamin (MeCbl) and 5'-deoxydeoxyadenosylcobalamin (AdoCbl). Cells convert the "inactive" vitamins CNCbl and HOCbl to the two critically important coenzymes AdoCbl or MeCbl. Surprisingly, little or no MeCbl is usually uncovered in the tissue samples, as compared to AdoCbl and HOCbl. We hypothesized that a low level of MeCbl is an artifact of "harsh" extractions, leading to degradation of MeCbl and/or its conversion to other XCbl-forms. METHODS: We designed a "mild" extraction protocol, including homogenization of rat liver in ammonium acetate (pH 4.6), dilution with EtOH (final 60%) and heating for 10 min at 70 °C. The XCbls were separated by HPLC and quantified by isotope dilution assays. RESULTS: A "mild" extraction revealed the following composition of Cbls: 37% AdoCbl, 35% MeCbl, 15% HOCbl and 13% CNCbl. The usual "harsh" protocol (pH 7, 20 min at 80 °C) changed this balance to 33%, 5%, 43% and 17%, respectively. A model assay revealed that MeCbl underwent demethylation and conversion to HOCbl at pH 3 and pH > 7, when heated with thiols. Other changes included decyanation of CNCbl and destruction of HOCbl. CONCLUSIONS: Our procedure reveals a high content of MeCbl in rat liver. GENERAL SIGNIFICANCE: This result challenges previous data and pinpoints the need for new studies to characterize the endogenous Cbl-forms in health and disease.

Protein binding assays for an accurate differentiation of vitamin B12 from its inactive analogue. A study on edible cricket powder.

Inactive analogues of vitamin B12 (cobalamin, Cbl) can mimic the active Cbl in food if using the traditional microbiological measurements. Thus, overestimated Cbl was recently revealed in edible insects employing immunoaffinity adsorption, HPLC-separation and mass spectrometry (https://doi.org/10.1016/j.foodchem.2021.129048). Here we demonstrate the utility of a convenient binding assay to evaluate Cbl in edible cricket powders. The assay employed the Cbl-specific protein intrinsic factor (IF) and the analogue-detecting protein haptocorrin. The excessive analogues had a weak affinity for IF, resulting in a modest overestimate of Cbl. This overestimate was corrected by a novel mathematical procedure, based on the ratio of analogue/Cbl in the sample and their relative affinities for IF. We found that 100 g of cricket powders contained 40-60 µg of analogues and 0.75-2.2 µg of Cbl. This result was confirmed by HPLC. A correct approach to Cbl-measurements is essential for nutritional assessment of any analogue-containing food.

A blue corrinoid from partial degradation of vitamin B12 in aqueous bicarbonate: spectra, structure, and interaction with proteins of B12 transport.

Cobalamin (Cbl) is a complex cofactor produced only by bacteria but used by all animals and humans. Cyanocobalamin (vitamin B(12), CNCbl) is one commonly isolated form of cobalamin. B(12) belongs to a large group of corrinoids, which are characterized by a distinct red color conferred by the system of conjugated double bonds of the corrin ring retaining a Co(III) ion. A unique blue Cbl derivative was produced by hydrolysis of CNCbl in a weakly alkaline aqueous solution of bicarbonate. This corrinoid was purified and isolated as dark blue crystals. Its spectroscopic analysis and X-ray crystallography revealed B-ring opening with formation of 7,8-seco-cyanocobalamin (7,8-sCNCbl). The unprecedented structural change was caused by cleavage of the peripheral C-C bond between saturated carbons 7 and 8 of the corrin macrocycle accompanied by formation of a C═C bond at C7 and a carbonyl group at C8. Additionally, the C-amide was hydrolyzed to a carboxylic acid. The extended conjugation of the π-system caused a considerable red shift of the absorbance spectrum. Formation and degradation of 7,8-sCNCbl were analyzed qualitatively. Its interaction with the proteins of mammalian Cbl transport revealed both a slow binding kinetics and a low overall affinity. The binding data were compared to those of other monocarboxylic derivatives and agreed with the earlier proposed scheme for two-step ligand recognition. The obtained results are consistent with the structural models of 7,8-sCNCbl and the transport proteins intrinsic factor and transcobalamin. Potential applications of the novel derivative for drug conjugation are discussed.

Elevated Plasma Vitamin B12 in Patients with Hepatic Glycogen Storage Diseases.

Background: Hepatic glycogen storage diseases (GSDs) are inborn errors of metabolism affecting the synthesis or breakdown of glycogen in the liver. This study, for the first time, systematically assessed vitamin B12 status in a large cohort of hepatic GSD patients. Methods: Plasma vitamin B12, total plasma homocysteine (tHcy) and methylmalonic acid concentrations were measured in 44 patients with hepatic GSDs and compared to 42 healthy age- and gender-matched controls. Correlations of vitamin B12 status with different disease markers of GSDs (including liver transaminase activities and triglycerides) as well as the vitamin B12 intake were studied. Results: GSD patients had significantly higher plasma vitamin B12 concentrations than healthy controls (p = 0.0002). Plasma vitamin B12 concentration remained elevated in GSD patients irrespective of vitamin B12 intake. Plasma vitamin B12 concentrations correlated negatively with triglyceride levels, whereas no correlations were detected with liver transaminase activities (GOT and GPT) in GSD patients. Merging biomarker data of healthy controls and GSD patients showed a positive correlation between vitamin B12 status and liver function, which suggests complex biomarker associations. A combined analysis of biomarkers permitted a reliable clustering of healthy controls versus GSD patients. Conclusions: Elevated plasma concentration of vitamin B12 (irrespective of B12 intake) is a common finding in patients with hepatic GSD. The negative correlation of plasma vitamin B12 with triglyceride levels suggests an influence of metabolic control on the vitamin B12 status of GSD patients. Elevated vitamin B12 was not correlated with GOT and GPT in our cohort of GSD patients. Merging of data from healthy controls and GSD patients yielded positive correlations between these biomarkers. This apparent dichotomy highlights the intrinsic complexity of biomarker associations and argues against generalizations of liver disease and elevated vitamin B12 in blood. Further studies are needed to determine whether the identified associations are causal or coincidental, and the possible impact of chronically elevated vitamin B12 on GSD.

Release of toxic Gd3+ ions to tumour cells by vitamin B12 bioconjugates.

Two probes consisting of vitamin B(12) (CNCbl) conjugated to Gd chelates by esterification of the ribose 5'-OH moiety, Gd-DTPA-CNCbl (1; DTPA = diethylenetriamine-N,N,N',N'',N''-pentaacetic acid) and Gd-TTHA-CNCbl (2; TTHA = triethylenetetramine-N,N,N',N'',N''',N'''-hexaacetic acid), have been synthesised and characterised. The crystal structure of a dimeric form of 1, obtained by crystallisation with an excess of GdCl(3), has been determined. The kinetics of binding to and dissociation from transcobalamin II show that 1 and 2 maintain high-affinity binding to the vitamin B(12) transport protein. Complex 2 is very stable with respect to Gd(3+) release owing to the saturated co-ordination of the Gd(3+) ion by four amino and five carboxylate groups. Hydrolysis of the ester functionality occurs on the time scale of several hours. The lack of saturation and the possible involvement of the ester functionality in co-ordination result in lower stability of 1 towards hydrolysis and in a considerable release of Gd(3+) in vitro. Gd(3+) ions released from 1 are avidly taken up by the K562 tumour cells to an extent corresponding to approximately 10(10) Gd(3+) per cell. The internalisation of toxic Gd(3+) ions causes a marked decrease in cell viability as assessed by Trypan blue and WST-1 tests. On the contrary, the experiments with the more stable 2 did not show any significant cell internalisation of Gd(3+) ions and any influence on cell viability. The results point to new avenues of in situ generation of cytotoxic pathways based on the release of toxic Gd(3+) ions by vitamin B(12) bioconjugates.

Kinetic analysis of transcellular passage of the cobalamin-transcobalamin complex in Caco-2 monolayers.

We suggest a novel kinetic approach to quantifying receptor-ligand interactions via the cellular transport and/or accumulation of the ligand. The system of cobalamin (Cbl, vitamin B12) transport was used as a model, because Cbl is an obligatory cofactor, taken up by animal cells with the help of a transport protein and a membrane receptor. Bovine transcobalamin (bTC) stimulated the cellular accumulation and transcytosis of radioactive [57Co]Cbl in polarized monolayers of Caco-2 cells. The bovine protein was much more efficient than human TC. The transport was inhibited in a dose-dependent manner by the unlabeled bTC-Cbl complex, the ligand-free bTC, and the receptor-associated protein (RAP). This inhibition pattern implied the presence of a megalin-like receptor. Quantitative assessment of kinetic records by the suggested method revealed the apparent concentration of receptors in vitro (≈15 nM), as well as the dissociation constants of bTC-Cbl ( Kd = 13 nM) and RAP ( Kd = 1.3 nM). The data were used to estimate the effective luminal concentrations of TC-specific receptors in kidneys (3.8 µM) and intestine (50 nM), the tissues resembling polarized Caco-2 cells.

Cyano-B12 or Whey Powder with Endogenous Hydroxo-B12 for Supplementation in B12 Deficient Lactovegetarians.

Lactovegetarians (n = 35) with low vitamin B12 (B12) status were intervened for eight weeks capsules containing cyano-B12 (CN-B12), (2 × 2.8 µg/day), or equivalent doses of endogenous B12 (mainly hydroxo-B12 (HO-B12)) in whey powder. Blood samples were examined at baseline, every second week during the intervention, and two weeks post-intervention. The groups did not differ at baseline in [global median (min/max)] plasma B12 [112(61/185)] pmol/L, holotranscobalamin [20(4/99)] pmol/L, folate [13(11/16)], the metabolites total homocysteine [18(9/52)] µmol/L and methylmalonic acid [0.90(0.28/2.5)] µmol/L, and the combined indicator of B12 status (4cB12) [-1.7(-3.0/-0.33)]. Both supplements caused significant effects, though none of the biomarkers returned to normal values. Total plasma B12 showed a higher increase in the capsule group compared to the whey powder group (p = 0.02). However, the increase of plasma holotranscobalamin (p = 0.06) and the lowering of the metabolites (p > 0.07) were alike in both groups. Thereby, the high total plasma B12 in the capsule group was not mirrored in enhanced B12 metabolism, possibly because the B12 surplus was mainly accumulated on an "inert" carrier haptocorrin, considered to be of marginal importance for tissue delivery of B12. In conclusion, we demonstrate that administration of whey powder (HO-B12) or capsules (CN-B12) equivalent to 5.6 µg of B12 daily for eight weeks similarly improves B12 status but does not normalize it. We document that the results for plasma B12 should be interpreted with caution following administration of CN-B12, since the change is disproportionately high compared to the responses of complementary biomarkers.

Comparative Bioavailability of Synthetic B12 and Dietary Vitamin B12 Present in Cow and Buffalo Milk: A Prospective Study in Lactovegetarian Indians.

We assessed improvements in the vitamin B12 status of Indian lactovegetarians receiving four weeks supplementation with natural B12 in milk versus cyano-B12 in capsules. Three groups (n = 22, 23, 22) received daily oral doses of cyano-B12 (2 × 0.76 µg) or milk (2 × 200 mL) from a cow or buffalo (amounting to B12 ≈ 2 × 0.76 µg). Their blood was examined at baseline and each following week. The baselines (median (min/max)) indicated a low B12 status: plasma B12 (116(51/314)) pmol/L, holotranscobalamin (holoTC) (30(7/119)) pmol/L, total homocysteine (Hcy) (24(10/118)) µmol/L, methylmalonic acid (MMA) (0.58(0.15/2.2)) µmol/L and combined B12 index (cB12) (-1.32 - (-3.12/+0.29)). Shifts from the baselines (B12, holoTC, cB12) and ratios to the baselines (Hcy, MMA) were analyzed over time. The cyano-B12 treatment gave more total B12 in plasma at week one (+29 pmol/L, p = 0.004) but showed no further increase. Other biomarkers changed more comparably between the three groups (p ≥ 0.05): holoTC showed a transient spike that leveled off, Hcy finally decreased to 0.8 × baseline, while MMA showed marginal changes. The combined indexes improved comparably (p = 0.6) in all groups (+0.2(-0.3/+0.9), p ≤ 0.002). In conclusion, the tested formulations similarly improved B12 status, but did not normalize it.