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Background: The detection of deficiencies in B 12 and folate children is important. However, despite the availability of various markers to assess B 12 and folate metabolism, there are limited studies describing the reference intervals (RIs) and changes during growth and development for these markers in healthy children., Methods: Using samples collected from 378 children aged 30 days-< 18 years, we derived continuous RIs for holotranscobalamin, homocysteine and red cell folate., Results: The lower RI for holotranscobalamin was lowest at birth, rising during early childhood and then declining following ages 4-6 years whereas red cell folate was highest early in life and then declined steadily towards adulthood. Total homocysteine, reflective of both B 12 and folate status was elevated early in life, reaching a nadir at age 2 and then increasing towards adulthood., Conclusions: Continuous central 95 th percentile RI for holotranscobalamin, homocysteine and red cell folate for children ages 30 days to <18 years were established. Each marker shows dynamic changes throughout childhood and adolescence which will assist clinicians in more appropriately assessing B 12 and folate status in this population., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: Healthy plant-based diets, such as the lacto-ovo-vegetarian and the vegan diet, offer numerous benefits to human health. Poorly designed plant-based diets, however, bear the risk for vitamin- and micronutrient deficiencies. Vitamin B12 (B12, cobalamin) is a nutrient of particular concern in both diets, and should be readily supplemented on a continuous basis to ensure adequate B12 levels and to prevent deficiencies., Case Report: This case reports describes the history of a healthy man in his mid-30s who adopted a vegan diet approximately 10 y ago. Well informed about the risks of vitamin B12 deficiency on a plant-based diet, he regularly supplemented methylcobalamin for years (single oral dose: 500 µg, 3-4 times a week) in order to maintain an adequate vitamin B12 status. In late 2023, however, he decided to cease B12 supplementation for undisclosed reasons. Subsequent to this decision, we closely monitored his B12 status and longitudinally measured serum B12, homocysteine, and holotranscobalamin (holo-TC). Total serum folate was also determined as it is a modifier of homocysteine concentration. A gradual decrease in holo-TC and vitamin B12 levels was observed after 4 weeks and supplements had to be re-introduced after 16 weeks. Homocysteine increased concomitantly up to 18.2 μmol/L after 20 weeks., Conclusions: While a short-term B12 supplement intake cessation might be well tolerated by vegans with an adequate B12 status, an interruption of more than 8 weeks could signify B12 loss approaching suboptimal status. This case report reiterates the need for continuous B12 supplementation in persons following an unfortified plant-based diet., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Vitamin B12 is an essential micronutrient required for the proper functioning of the human body. Vitamin B12 deficiency is primarily causative of various neurolological disorders alongwith recurrence of oral ulcers and burning sensations which are early signs of condition such as pernicious anemia. Other complications associated with Vitamin B12 deficiency include risk of heart failure due to anemia, risk of developing autoimmune disorders and gastric cancer. Therefore, to obstruct these communal health issues, early detection of Vit B12 is highly needed. However, screening of vitamin B12 insufficiency is hindered by the low sensitivity of the conventional vitamin B12 test. Holotranscobalamin (holoTC) is an early indicator of the negative vitamin B12 balance as it is the first protein to decline in the serum. We report a novel impedimetric immunosensor based on flower-like poly (3,4-ethylenedioxythiophene) (PEDOT) nanostructural film impregnated with silver molybdate nanoparticles (Ag 2 MoO₄ NPs) deposited on fluorine-doped tin oxide electrode. The prepared electrodes were characterized by Field emission scanning electron microscopy (FE-SEM) with energy-dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD), and electrochemical studies. The activated anti-holoTC antibody was immobilized and optimized to capture the target in a response time of 15 min. The electrochemical performance of the sensor was carried out by using the electrochemical impedance spectroscopy technique (EIS) and a good linear relationship between ΔR ct and holoTC was obtained in the range from 0.1 pg mL -1 to 100 ng mL -1 with a detection limit of 0.093 pg mL -1 . The proposed sensor was successfully applied in human serum samples for holoTC detection. The experimental results showed that the immunosensor is highly selective towards holoTC and presented an acceptable stability of 20 days with reproducibility RSD ≤4%. To the best of our knowledge, this is the first developed electrochemical immunosensor for holoTC detection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Purpose: To investigate the bioavailability of vitamin B 12 from nori and to evaluate the required dosage for improving vitamin B 12 nutritional status in vegetarians not using supplements., Methods: The study design is an open-label, parallel, dose-response randomized controlled trial. Thirty vegetarians were assigned to control (no nori), low-dose (5 g nori, aiming to provide 2.4 µg vitamin B 12 per day), or high-dose (8 g nori, aiming to provide 4 µg vitamin B 12 per day) groups. The primary outcome was changes in vitamin B 12 status as measured by serum vitamin B 12 , holotranscobalamin (holoTC), homocysteine (Hcy), and methylmalonic acid (MMA), and a combined score of these four markers (4cB12 score) during the four-week intervention. Dietary vitamin B 12 intakes were assessed at baseline and end of the trial with a 17-item food frequency questionnaire designed for vitamin B 12 assessment. General linear model was used to compare least square means of changes in each biomarker of vitamin B 12 status, among the three groups, while adjusting for respective baseline biomarker., Results: After adjusting for baseline status, nori consumption led to significant improvement in serum vitamin B 12 (among-group P-value = 0.0029), holoTC (P = 0.0127), Hcy (P = 0.0225), and 4cB12 (P = 0.0094). Changes in MMA did not differ significantly across groups, but showed within-group pre-post improvement in the low-dose group (median [p25, p75] = -339 [-461, -198] nmol/L). Vitamin B 12 status appeared to plateau at low dose (5 g of nori), which compared with control group, improved serum vitamin B 12 (lease square mean [95% CI] = + 59 [25, 93] pmol/L, P = 0.0014); holoTC (+ 28.2 [10.1, 46.3] pmol/L, P = 0.0035); Hcy (-3.7 [-6.8, -0.6] µmol/L, p = 0.0226); and 4cB12 score (+ 0.67 [0.24, 1.09], p = 0.0036). High-dose resulted in similar improvements. There was no significant difference between low-dose and high-dose groups in all biomarkers of vitamin B 12 ., Conclusions: Consuming 5 g of nori per day for 4 weeks significantly improved vitamin B 12 status in vegetarians. A higher dose (8 g) may not confer additional benefits., Clinical Trial Registration: ClinicalTrials.gov Identifier: NCT05614960. Date of registration: November 14th 2022., (© 2024. The Author(s).)

Background: Data have shown that vitamin B12 has immunomodulatory effects via different pathways, which could influence the pathophysiology of sepsis. The objective of this study was to investigate whether vitamin B12 levels, assessed by the measurement of holotranscobalamin (HTC), total vitamin B12 (B12), and methylmalonic acid (MMA, which accumulates in case of B12 deficiency), are associated with the development of sepsis in patients with onset of bacterial infection., Methods: This was a single-center, prospective observational pilot study. Adult patients who presented to the emergency department with bacterial infection confirmed by a positive microbiological culture result were included in the study and followed up for 6 days to assess whether they developed sepsis or not. The primary objective was to compare HTC concentration in patients who developed sepsis to those who did not develop sepsis. Secondary objectives were the evaluation of B12 and MMA concentrations in those two groups. Multiple logistic regression models were used, with presence of sepsis as the outcome variable, and HTC, B12, and MMA concentrations as predictor variables, separately, and adjusted for potential confounders., Results: From 2019 to 2022, 2131 patients were assessed for eligibility, of whom 100 met the inclusion criteria. One patient was excluded from the analysis due to missing data. Of the 99 patients, 29 developed sepsis. There was no evidence for an association between HTC or B12 concentration and the development of sepsis (OR 0.65, 95% CI 0.31-1.29, p = 0.232, OR 0.84, 95% CI 0.44-1.54, p = 0.584, respectively). There was an association between MMA concentration and the development of sepsis, with a positive effect, i.e. with increasing MMA, the odds for sepsis increased (OR 2.36, 95% CI 1.21-4.87, p = 0.014). This association remained significant when adjusted for confounders (OR 2.72, 95% CI 1.23-6.60, p = 0.018)., Conclusions: Our study found an association between elevated MMA concentration and the development of sepsis. We did not find an association between HTC and B12 concentrations and the development of sepsis. Further, larger studies are warranted, as it could lead to interventional trials investigating whether B12 supplementation provides a clinical benefit to patients with infection or sepsis., Trial Registration: The study was registered on ClinicalTrials.gov under the identifier NCT04008446 on June 17, 2019., (© 2024. The Author(s).)

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.

Background: Haptocorrin (HC) and holotranscobalamin (holoTC) carry vitamin B12 (B12) in the circulation and can be useful biomarkers for evaluating B12 status. The concentration of both proteins depends on age, but data on reference intervals for children and the elderly are sparse. Similarly, not much is known about the effect of preanalytical factors., Methods: HC plasma samples from healthy elderly > 65 years (n = 124) were analysed, and both HC and holoTC were analysed in paediatric serum samples ≤ 18 years (n = 400). Furthermore, we investigated assay precision and stability., Results: HC and holoTC were effected by age. We established reference intervals for HC: 2-10 years, 369-1237 pmol/L; 11-18 years, 314-1128 pmol/L; 65-82 years, 242-680 pmol/L and for holoTC: 2-10 years, 46-206 pmol/L; 11-18 years, 30-178 pmol/L. Analytical coefficients of variations of 6.0-6.8% and 7.9-15.7% were found for HC and holoTC, respectively. HC were affected when stored at room temperature and by freeze/thaw. HoloTC was stable at room temperature and after delayed centrifugation., Conclusion: We present novel 95% age-related reference limits for HC and HoloTC in children, and for HC both in children and elderly. Moreover, we found HoloTC to be fairly stable when stored, whereas HC was more vulnerable to preanalytical factors., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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.

Background: Whether holotranscobalamin (holoTC) indicates B12 deficiency more sensitively than total vitamin B12 (B12) is unclear. This study is aimed at determining the impact of serum holoTC level as a risk factor for ischemic stroke and investigating its association with disease severity and short-term outcomes., Methods: Serum holoTC, total B12, and homocysteine levels were compared between 130 stroke patients and 138 healthy controls. Biomarker level correlations with disease severity and stroke functional outcomes were investigated., Results: holoTC levels were lower and homocysteine levels were higher in stroke patients than in healthy controls ( P < 0.05). The holoTC/total B12 ratio and homocysteine level significantly predicted ischemic stroke in the multivariable regression analysis ( P < 0.05). Along with hyperhomocysteinemia, patients more often had holoTC than total B12 deficiency (6.2% vs. 3.1%). holoTC levels negatively correlated with homocysteine levels (partial R -0.165, P < 0.05) in stroke patients in multiple linear regression analyses, but not total B12 levels. The holoTC level and holoTC/total B12 ratio, but not homocysteine and total B12 levels, negatively correlated with the National Institute of Health Stroke Scale (partial R , -0.405 and -0.207, respectively, P < 0.01)., Conclusions: Measurements of serum holoTC levels combined with total B12 and homocysteine levels may provide valuable information for predicting ischemic stroke and its severity and short-term outcomes of ischemic stroke patients., Competing Interests: The authors have declared that no competing interest exists., (Copyright © 2021 Oh Joo Kweon et al.)

Background: This study aimed to investigate the effect of cobalt transporter II gene (rs1801198, rs2301957, rs9606756) polymorphism on serum homocysteine level and its correlation with young and middle recurrent cerebral infarction., Methods: A total of 348 young and middle-aged patients with cerebral infarction admitted to The Third Affiliated Hospital of Qiqihar Medical University from January 2017 to March 2018 were enrolled. The patients were divided into recurrent and non-recurrent groups according to follow-up. DNA was extracted from the peripheral blood of patients, and the DNA samples were genotyped by IlluminaBeadArray technology to detect the gene polymorphisms of cobalt transporter II (TCN2) sites (rs1801198, rs2301957, rs9606756), and the homocysteine (hcy) level was determined by cyclic enzymatic method. VitB12 and folate levels were measured by chemiluminescence immunoassay, and holo transcobalamin (holoTC) expression levels were detected by enzyme-linked immunosorbent assay., Results: The frequency of alleles of rs9606756 mutation in the recurrent group was higher than that in the non-recurrent group (P<0.05), and the Hcy level in rs9606756 locus genotype AG+GG was significantly higher than the AA genotype in the recurrent group (P=0.031). Pearson correlation analysis showed that Hcy levels were associated with different genotypes of rs9606756 in the recurrent group (r=0.483, P=0.0003). The rs9606756 allele AA in SH-SY5Y cells was replaced with GG by point mutation experiment. The Hcy metabolism levels of wild and mutant cells were detected. The accumulation level of Hcy in the mutant group was significantly increased (P=0.007). The holoTC in the supernatant was significantly reduced in the mutant (P=0.032)., Conclusions: The TCN2 gene rs9606756 mutation is closely related to the level of Hcy metabolism in young and middle-aged patients, which may affect the recurrence of cerebral infarction. It is of great significance to further understand the pathogenesis, prevention and treatment of recurrent cerebral infarction in young and middle-aged patients.

We investigated the effect of long-term whey supplementation on biomarkers of B12 status in healthy older adults subjected to different schemes of supplements and exercise. The total study population examined at baseline consisted of 167 healthy older adults (age ≥ 65 year) who were randomized to 1-y intervention with two daily supplements of (1) whey protein (3.1 µg B12/day) (WHEY-ALL), (2) collagen (1.3 µg B12/day) (COLL), or (3) maltodextrin (0.3 µg B12/day) (CARB). WHEY-ALL was comprised of three groups, who performed heavy resistance training (HRTW), light resistance training (LITW), or no training (WHEY). Dietary intake was assessed through 3-d dietary records. For the longitudinal part of the study, we included only the participants ( n = 110), who met the criteria of ≥ 50% compliance to the nutritional intervention and ≥ 66% and ≥ 75% compliance to the heavy and light training, respectively. Fasting blood samples collected at baseline and 12 months and non-fasting samples collected at 6 and 18 months were examined for methylmalonic acid, B12 and holotranscobalamin. At baseline, the study population ( n = 167) had an overall adequate dietary B12 intake of median (range) 5.3 (0.7-65) µg/day and median B12 biomarker values within reference intervals. The whey intervention (WHEY-ALL) caused an increase in B12 ( P < 0.0001) and holotranscobalamin ( P < 0.0001). In addition, methylmalonic acid decreased in the LITW group ( P = 0.04). No change in B12 biomarkers was observed during the intervention with collagen or carbohydrate, and the training schedules induced no changes. In conclusion, longer-term daily whey intake increased plasma B12 and holotranscobalamin in older individuals. No effect of intervention with collagen or carbohydrate or different training regimes was observed. Interestingly, the biomarkers of B12 status appeared to be affected by fasting vs. non-fasting conditions during sample collection.

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

Vitamin B12 (cobalamin) is an essential cofactor in the one-carbon metabolism. One-carbon metabolism is a set of complex biochemical reactions, through which methyl groups are utilised or generated, and thus plays a vital role to many cellular functions in humans. Low levels of cobalamin have been associated to metabolic/reproductive pathologies. However, cobalamin status has never been investigated in morbid obesity in relation with the reduced semen quality. We analysed the cross-sectional data of 47-morbidly-obese and 21 lean men at Careggi University Hospital and evaluated total cobalamin (CBL) and holotranscobalamin (the active form of B12; holoTC) levels in serum and semen. Both seminal and serum concentrations of holoTC and CBL were lower in morbidly obese compared to lean men, although the difference did not reach any statistical significance for serum holoTC. Seminal CBL and holoTC were significantly higher than serum levels in both groups. Significant positive correlations were observed between seminal holoTC and total sperm motility ( r = 0.394, p = 0.012), sperm concentration ( r = 0.401, p = 0.009), total sperm number ( r = 0.343, p = 0.028), and negative correlation with semen pH ( r = -0.535, p = 0.0001). ROC analysis supported seminal holoTC as the best predictor of sperm number (AUC = 0.769 ± 0.08, p = 0.006). Our findings suggest that seminal rather than serum levels of holoTC may represent a good marker of semen quality in morbidly obese subjects.

Background & Aims: Bariatric surgery increases the risk of micronutrient deficiencies, including vitamin B12 (B12) deficiency. We analysed early changes in biomarkers of B12 status following bariatric surgery., Methods: We prospectively included adult patients (n = 27) referred for either Roux-en-Y Gastric Bypass (RYGB) (n = 19) or Sleeve Gastrectomy (SG) (n = 8). Blood samples were drawn before surgery and 2 and 6 months following surgery for measurement of B12, holotranscobalamin (holoTC), and methylmalonic acid (MMA). The B12 absorption capacity was estimated from the increase in plasma holoTC two days after a standardised oral B12 challenge., Results: B12 status decreased following both RYBG and SG. While a decrease in plasma B12 was not evident until 6 months postoperatively, we observed a statistically significant decrease in plasma holoTC and increase in MMA already 2 months postoperatively. These changes were more pronounced at 6 months post surgery. Correspondingly, the B12 absorption capacity was decreased following surgery., Conclusions: HoloTC and MMA were superior to B12 to detect early changes in B12 status following bariatric surgery. Our data challenge the current concept that liver B12 stores secure long-term maintenance of B12 status. They indicate that B12 treatment in pharmacological doses may be warranted immediately after surgery., (Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

There is clear evidence that proton-pump inhibitors (PPIs), H2-receptor antagonists (H2RAs), and metformin can reduce serum vitamin B-12 concentrations by inhibiting the absorption of the vitamin. However, it is unclear if the effects of these drugs on serum vitamin B-12 are associated with increased risk of biochemical or functional deficiency (as is indicated by elevated blood concentrations of homocysteine and methylmalonic acid) or clinical deficiency (including megaloblastic anemia and neurologic disorders such as peripheral neuropathy and cognitive dysfunction). This review provides an overview of vitamin B-12 absorption and biochemistry and the mechanisms by which PPIs, H2RAs, and metformin affect these functions. It also summarizes the literature relating the use of these drugs to the risk of vitamin B-12 deficiency. Also discussed is that strategies for assessing vitamin B-12 status and diagnosing vitamin B-12 deficiency have evolved in recent years beyond solely measuring serum total vitamin B-12. Multiple analyte testing, a strategy in which ≥2 of 4 biomarkers of vitamin B-12 status-serum total vitamin B-12, holotranscobalamin, homocysteine, and methylmalonic acid-are measured, increases sensitivity and specificity for diagnosing vitamin B-12 deficiency. It is concluded that randomized controlled trials are now needed that use the strategy of multiple analyte testing to determine if PPIs, H2RAs, and metformin do indeed increase the risk of vitamin B-12 deficiency. Until these studies are conducted, a reasonable recommendation for physicians and their patients who are taking these drugs is to monitor vitamin B-12 status and to provide vitamin B-12 supplements if altered blood biomarkers or clinical signs consistent with low or deficient vitamin B-12 status develop.

To investigate the association between plasma concentration of vitamin B 12 and B 12 intake from supplements, fortified foods, and animal source foods among vegetarians and non-vegetarians, we conducted a cross-sectional analysis among 728 participants of the Adventist Health Study 2 (AHS-2) calibration study. The median age of participants was 58 years, 65.4% were female, and 50.3% were White. We used six 24 h dietary recalls to measure B 12 intake, serum vitamin B 12 , and holotranscobalamin (holoTC) concentration. B 12 supplements had a significantly positive association with plasma B 12 among all subjects ( p trend < 0.001), especially among vegans and lacto-ovo vegetarians ( p trend < 0.001). Among non-users of B 12 supplements, B 12 intake from milk substitutes was significantly positively associated with holoTC ( p trend < 0.004) and serum B12 ( p trend < 0.030). In non-vegetarians, holoTC was significantly positively associated with B 12 intake from eggs, while serum B 12 was significantly positively associated with B 12 intake from milk in the upper tertile compared to the lower, and B 12 intake from meat in the middle compared to the lower tertile intake ( p < 0.011). Supplements containing B 12 followed by B 12 intake from milk substitutes were significant contributors of plasma vitamin B 12 concentration., Competing Interests: The authors declare no conflict of interest.

The aim of this study is to determine the prevalence and incidence of vitamin B12 deficiency after esophagectomy for cancer. It is unknown if patients after esophagectomy with gastric tube reconstruction are at an increased risk for vitamin B12 deficiency. A cross-sectional cohort (group A) and a prospective cohort (group B) of patients who underwent esophagectomy for cancer in two tertiary referral centers in the Netherlands were included. Serum levels of holo-transcobalamin (Holo-TC) and methyl malonic acid (MMA) were determined. Vitamin B12 deficiency was defined as Holo-TC < 21 pmol/L and/or MMA > 0.45 μmol/L. Vitamin B12 status was assessed in group A at a single time point between one and three years postoperatively and before and every three months after resection in group B. Ninety-nine patients were analyzed in group A. The median time between surgery and analysis of vitamin B12 deficiency was 19.3 months. In 11 of 99 (11%) patients, vitamin B12 deficiency was detected. In group B, 5 of 88 (5.6%) patients had vitamin B12 deficiency preoperatively, and another 9 (10.2%) patients developed vitamin B12 deficiency after the operation at a median time of 6 months postoperatively. The estimated one-year incidence of vitamin B12 deficiency was 18.2%. None of the patients with vitamin B12 deficiency had a megaloblastic anemia. Vitamin B12 deficiency can be anticipated in 18% of patients after esophagectomy with gastric tube reconstruction for cancer. During follow-up, Holo-TC and MMA levels should be measured to detect vitamin B12 deficiency and commence treatment timely.

Introduction: Vitamin B12 deficiency is mostly caused by insufficient gastro-intestinal absorption and in rare conditions by Transcobalamin (TC) deficiency. Unsaturated Transcobalamin (apoTC) can be measured by a binding assay using radiolabeled cobalamin. The Active B12 test analyzes saturated Transcobalamin (holoTC) and we hypothesize that this test can be used to measure total TC by additional in vitro saturation with cobalamin., Methods: Serum was saturated in vitro (16 times dilution) with a cyanocobalamin solution and total TC was selectively measured with the Abbott Active B12 test. ApoTC was calculated by subtracting endogenous holoTC from total TC after correction for dilution. Linearity was determined with a pool serum dilution series. Precision was investigated according to the CLSI EP15 protocol. Method comparison was performed against a binding assay using radiolabeled cobalamin. Reference values were determined in 100 healthy controls., Results: The method was linear in the range of 240 to 1933pmol/L (R 2 =0.997, lack of fit F=1.61). Precision of low- and high-pool total TC in serum were; 5.2% and 4.3% respectively. Method comparison against a radiolabeled cobalamin binding assay showed a proportional bias of 30% (y=0.70x+126). Total TC reference values were determined at 500-1276pmol/L., Conclusion: We describe a rapid method to quantify total TC, which can be implemented on routine platforms using commercial Active B12 tests. In addition, apoTC can be assessed by subtracting endogenous holoTC concentration which can be measured in the same run, securing the same calibration level for all three parameters (holoTC, apoTC and total TC). This method is applicable in clinical diagnostics and in larger epidemiological studies., (Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Data on biological variation for cobalamin and holotranscobalamin (holoTC) are limited. The aim of this study was to determine within-subject (CV I ) and between-subject (CV G ) biological variations for these analytes in a healthy population. We collected blood samples from 15 healthy volunteers (12 women and three men, 22-66 years) on the same weekday for 10 consecutive weeks. Serum samples were stored at -80 °C until analysis in duplicate in a single analytical run. The CV I and CV G were estimated by nested ANOVA. The CV I (95% CI) for cobalamin and holoTC was 6.7% (5.7-7.7) and 13.0% (11.5-15.0), respectively. The corresponding CV G was 24.1% (16.4-36.0) and 24.6% (16.3-37.7). The analytical variation (CV A ) (95% CI) was 3.5% (3.2-4.0) for cobalamin and 2.4% (2.1-2.6) for holoTC. The index of individuality (II) was low (<0.6) for both cobalamin and holoTC and the reference change value (RCV) was 20.1% for cobalamin and 36.6% for holoTC. Our study describes the components of biological variation of cobalamin and holoTC in a healthy population, contributing to a better clinical interpretation of these biomarkers.

Aim: We investigated cross-sectional associations between circulating homocysteine, folate, biomarkers of vitamin B 12 status and brain volumes. We furthermore compared brain volumes of participants who received daily folic acid and vitamin B 12 supplementation with participants who did not., Methods: Participants of the B-PROOF study ( n = 2919) were assigned to 400 µg folic acid and 500 µg vitamin B 12 , or a placebo. After two years of intervention, T₁-weighted magnetic resonance imaging (MRI) scans were made in a random subsample ( n = 218) to obtain grey and white matter volume, and total brain volume (TBV). Plasma homocysteine, serum folate, vitamin B 12 , holotranscobalamin, and methylmalonic acid concentrations were measured., Results: Multiple linear regression analyses showed inverse associations between plasma homocysteine with TBV (β = -0.91, 95% CI -1.85-0.03; p = 0.06) and between serum folate and TBV (β = -0.20, 95% CI -0.38, -0.02; p = 0.03). No significant associations were observed for serum vitamin B 12 and holotranscobalamin. Fully adjusted ANCOVA models showed that the group that received B-vitamins had a lower TBV (adjusted mean 1064, 95% CI 1058-1069 mL) than the non-supplemented group (1072, 95% CI 1067-1078 mL, p = 0.03)., Conclusions: Results were contradictory, with higher Hcy levels associated with lower TBV, but also with higher folate levels associated with lower TBV. In addition, the lack of a baseline measurement withholds us from giving recommendations on whether folic acid and vitamin B 12 supplementation will be beneficial above and beyond normal dietary intake for brain health., Competing Interests: The authors declare no conflict of interest.

Background: Investigation of possible B12 and folate deficiencies requires measurement of these vitamins in serum. There is evidence that holotranscobalamin (holoTC), the active portion of B12 available to cells, is a more specific marker of early B12 deficiency than total B12. The availability of immunoassays for holoTC prompted an international collaborative study to assign a holoTC value to the World Health Organization (WHO) 1st International Standard (IS) for vitamin B12 and serum folate, 03/178., Methods: The IS, 03/178, and three serum samples with different holoTC levels were assayed by 12 laboratories in eight countries using manual and automated immunoassays for holoTC; one laboratory additionally performed an in-house assay. Fourteen sets of data were analysed., Results: Overall, the IS, 03/178, and the three serum samples demonstrated assay linearity and parallelism. An overall geometric mean (GM) holoTC value of 106.8 pmol/L was obtained for 03/178, with an inter-laboratory geometric coefficient of variation (GCV) of 10.5%. There was a reduction in inter-laboratory variability when the holoTC levels in the serum samples were determined relative to the IS with an assigned holoTC value rather than to the assays' calibration. Accelerated degradation studies showed that 03/178 was sufficiently stable to serve as an IS for holoTC., Conclusions: The WHO Expert Committee on Biological Standardization endorsed the proposal to assign a holoTC value of 107 pmol/L to 03/178, corresponding to 0.107 pmol per ampoule, for use as the 1st IS for vitamin B12, serum folate, and holoTC.

Coronary artery disease (CAD) has been increasing alarmingly in India. We had earlier shown that vitamin B12 deficiency is associated with CAD in Indian population. However, only about a quarter of the total vitamin B12 is internalised in the cells by the proteins transcobalamin II. Vitamin B12-bound transcobalamin II (holotranscobalamin, holoTC) is thus referred to as biologically active B12. In this study, we ascertained the levels of holoTC in 501 CAD cases and 1253 healthy controls and for the first time show that holoTC levels are significantly lower (p = 2.57E-4) in CAD (26.81 pmol/l) cases as compared to controls (29.97 pmol/l).

Background: Crohn's disease (CD) is a risk factor for vitamin B12 deficiency due to frequent involvement of the terminal ileum. Conventional screening for B12 deficiency with serum B12 is relatively insensitive and measures total B12 concentration, of which a minority is present in a biologically active form. Holotranscobalamin (holoTC) combined with methylmalonic acid (MMA) is believed to be more accurate in identifying impaired B12 status. We evaluated the prevalence and risk factors for B12 deficiency using holoTC supported by MMA among patients with CD., Methods: We performed a single-center service evaluation of 381 patients with CD who underwent B12 assessment (holoTC/MMA) and compared them with 141 patients with ulcerative colitis. Eighty-nine patients with CD underwent paired serum B12 and holoTC. Among patients with CD, risk factors including terminal ileal resection length, ileal inflammation on endoscopy, and disease characteristics on magnetic resonance imaging were recorded., Results: Prevalence of B12 deficiency among patients with CD was 33% compared with 16% in ulcerative colitis (P < 0.0001). In 89 patients who underwent paired tests, conventional testing identified B12 deficiency in 5% of patients with CD, which increased to 32% using holoTC/MMA. Independent risk factors for B12 deficiency were ileal resection length ≤20 cm (odds ratio: 3.0, 95% confidence interval, 1.5-6.0, P = 0.002) and >20 cm (odds ratio: 6.7, 95% confidence interval, 3.0-14.7, P < 0.0001) and ileal inflammation (odds ratio: 3.9, 95% confidence interval, 2.2-6.9, P < 0.0001). On magnetic resonance imaging, active terminal ileal inflammation (P = 0.02) and an increased disease burden (≥1 skip lesion, P = 0.01 and prestenotic dilatation >3 cm, P = 0.01) were associated with B12 deficiency., Conclusions: Vitamin B12 deficiency is common in patients with CD. holoTC supported by MMA identifies patients with B12 deficiency considered replete on conventional testing.

Background: Cellular uptake of vitamin B12 (B12) demands binding of the vitamin to transcobalamin (TC) and recognition of TC-B12 (holoTC) by the receptor CD320. Recently, we identified a soluble form of CD320 (sCD320) in human plasma. Here we present data on the occurrence of this soluble receptor in cerebrospinal fluid (CSF) and show its correlations to dementia-related biomarkers tau proteins and amyloid-beta., Methods: We collected 223 cerebrospinal fluid samples and corresponding plasma samples (n = 46). We measured CSF and plasma sCD320, holoTC and total TC employing in-house ELISA methods and CSF phospho-tau (181P) (p-tau), total tau (t-tau) and amyloid-beta 1-42 (Aβ) (n = 177) employing commercial ELISA kits (Innogenetics Company). Size exclusion chromatography was performed on a Superdex 200 column., Results: The median sCD320 concentration in CSF (14 pmol/L) is around five times lower than in plasma (72 pmol/L). No correlation was observed between plasma and CSF sCD320 levels (n = 46), while the behavior upon size exclusion chromatography was the same. In CSF, sCD320 correlates to holoTC and total TC (Spearman's correlation (Rs) = 0.325, 0.232 (n = 218, 217) respectively, p < 0.01). Interestingly, sCD320 correlates to p-tau and t-tau (Rs = 0.599, 0.569 (n = 173, 176) respectively, p < 0.001) and to Aβ (Rs = 0.265, p < 0.001 (n = 177))., Conclusion: We document for the first time the occurrence of sCD320 in human CSF. We report that the concentration of sCD320 correlates to the dementia-related biomarkers p-tau, t-tau and Aβ.

Background: The vitamin B12 and folate status in nonanaemic healthy older persons needs attention the more so as decrease in levels may be anticipated from reduced haematinic provision and/or impaired intestinal uptake., Methods: A total of 1143 subjectively healthy Swiss midlands participants (637 females and 506 males), ≥60 years of age were included in this study. Levels of vitamin B12, holotranscobalamin (holoTC), methylmalonic acid (MMA), homocysteine (Hcy), serum folate, red blood cell (RBC) folate were measured. Further, Fedosov's wellness score was determined. Associations of age, gender, and cystatin C/creatinine-based estimated kidney function, with the investigated parameters were assessed. Reference intervals were calculated. Further, ROC analysis was done to assess accuracy of the individual parameters in recognizing a deficient vitamin B12 status. Finally, decision limits for sensitive, specific and optimal recognition of vitamin B12 status with individual parameters were derived., Results: Three age groups: 60-69, 70-79 and ≥ 80 had median B12 (pmol/L) levels of 237, 228 and 231 respectively (p = 0.22), holoTC (pmol/L) of 52, 546 and 52 (p = 0.60) but Hcy (μmol/L) 12, 15 and 16 (p < 0.001), MMA (nmol/L) 207, 221 and 244 (p < 0.001). Hcy and MMA (both p < 0.001), but not holoTC (p = 0.12) and vitamin B12 (p = 0.44) were found to be affected by kidney function. In a linear regression model Fedosov's wellness score was independently associated with kidney function (p < 0.001) but not with age. Total serum folate and red blood cell (RBC) folate drift apart with increasing age: whereas the former decreases (p = 0.01) RBC folate remains in the same bandwidth across all age groups (p = 0.12) A common reference interval combining age and gender strata can be obtained for vitamin B12 and holoTC, whereas a more differentiated approach seems warranted for serum folate and RBC folate., Conclusion: Whereas the vitamin B12 and holoTC levels remain steady after 60 years of age, we observed a significant increment in MMA levels accompanied by increments in Hcy; this is better explained by age-related reduced kidney function than by vitamin B12 insufficiency. Total serum folate levels but not RBC folate levels decreased with progressing age.

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.

Background: A vitamin B12 deficiency can be an underlying cause or a deteriorating factor in several diseases. Nevertheless, early identification of such a deficiency remains a problem. Holotranscobalamin (HTC) is presently considered to be the gold standard. We tested the predictive power of other B12 parameters by comparing them with HTC., Methods: The blood of 77 patients from a medical office was tested for HTC, total B12 (CLIA [chemiluminescent immunoassay] and MTP [microbiological test with microtitre plates]), MMA (methylmalonic acid), HCY (homocysteine), and MCV (mean cell volume). The parameters were correlated and sensitivity, specificity, PPV (positive predictive value), NPV (negative predictive value), LR+ (positive likelihood ratio), and LR- (negative likelihood ratio) in comparison to HTC were determined. A ROC analysis was also performed., Results: At a cutoff value of 35 pmol/L for HTC, the total B12 CLIA (cutoff 211 ng/L) qualified 53% of individuals as having a B12 deficiency. The total B12 MTP (cutoff 288 ng/L) classified 71% as having a B12 deficiency. Specificity was similar in both cases (CLIA, 93%; MTP, 95%). With a cutoff value of 10 µmol/L for homocysteine, the best negative prediction was achieved. MVA has a low sensitivity (41%) and a high specificity (90%). Based on the ROC analysis, which indicated superiority of the B12-MTP, the reference levels of B12-CLIA and B12-MTP were raised to 304 and 368 ng/L, respectively. Thus, a probable B12 deficiency was identified in 94% of cases with either method and with a comparable specificity., Conclusions: If total B12 is applied to identify B12 deficiency, the cutoff values should be elevated to 304 (B12-CLIA) and 368 ng/L (B12-MTP) to improve the predictive power. The negative-predictive power of HCY can be useful in daily routine. HTC has a broad grey area of uncertainty and MMA should only be applied as a confirmatory test.

A combination of high folate with low vitamin B12 plasma status has been associated with cognitive impairment in a population exposed to mandatory folic acid fortification. The objective of the present study was to examine the interactions between plasma concentrations of folate and vitamin B12 markers in relation to cognitive performance in Norwegian elderly who were unexposed to mandatory or voluntary folic acid fortification. Cognitive performance was assessed by six cognitive tests in 2203 individuals aged 72-74 years. A combined score was calculated using principal component analysis. The associations of folate concentrations, vitamin B12 markers (total vitamin B12, holotranscobalamin (holoTC) and methylmalonic acid (MMA)) and their interactions in relation to cognitive performance were evaluated by quantile regression and least-squares regression, adjusted for sex, education, apo-ɛ4 genotype, history of CVD/hypertension and creatinine. Cross-sectional analyses revealed an interaction (P= 0·009) between plasma concentrations of folate and vitamin B12 in relation to cognitive performance. Plasma vitamin B12 concentrations in the lowest quartile ( < 274 pmol/l) combined with plasma folate concentrations in the highest quartile (>18·5 nmol/l) were associated with a reduced risk of cognitive impairment compared with plasma concentrations in the middle quartiles of both vitamins (OR 0·22, 95 % CI 0·05, 0·92). The interaction between folate and holoTC or MMA in relation to cognitive performance was not significant. In conclusion, this large study population unexposed to mandatory folic acid fortification showed that plasma folate, but not plasma vitamin B12, was associated with cognitive performance. Among the elderly participants with vitamin B12 concentrations in the lower range, the association between plasma folate and cognitive performance was strongest.

Background: Vitamin B12 insufficiency/deficiency is common in mixed patient populations. However there is no single marker which can reliably diagnose B12 insufficiency/deficiency. Elevated concentrations of methylmalonic acid (MMA) are considered the most representative marker of metabolic vitamin B12 insufficiency, but poor assay availability limits clinical utility. Low concentrations of serum vitamin B12 are often used to assess vitamin B12 status but this approach generates a high rate of false negative results. Emerging evidence indicates that holotranscobalamin (holoTC) may be a more reliable indicator of vitamin B12 status., Aims and Methods: We substituted serum vitamin B12 measurement with holoTC, supported by MMA in patients referred for assessment of vitamin B12 status. A service evaluation was undertaken of the pattern of MMA values obtained for patients with holoTC 25-50 pmol/L (an indeterminate result). MMA cut-offs of 280 and 360 nmol/L were applied for patients ≤ 65 or >65 years respectively., Results: A total of 4,175 consecutive patients were investigated and MMA was analysed for 19% of patients. The incidence of elevated MMA was 41% (holoTC, 25-29 pmol/L), 32% (30-34 pmol/L), 33% (35-39 pmol/L), 30% (40-44 pmol/L), and 26% (45-50 pmol/L)., Conclusions: Our results indicate that in the clinical setting a holoTC between 25 and 50 pmol/L is a poor predictor for the concentration of MMA provided the goal is to identify patients with MMA values above the limits used in the present study. Further studies are needed to evaluate to what extent holoTC <25 and >50 pmol/L reflect circulatory MMA concentrations., (© 2013.)

Background: Subclinical cobalamin deficiency is common in the elderly, but the sensitivity and specificity of serum total cobalamin for this diagnosis is poor. Serum holotranscobalamin (holoTC), a measure of biologically available cobalamin, is considered a better marker for early cobalamin depletion than total cobalamin. However, in elderly populations, health-related reference intervals for holoTC and correlations to renal function are not entirely clear., Methods: HoloTC was determined with an automated microparticle enzyme immunoassay (AxSYM®) in 790 elderly non-vitamin-supplemented Swedish men, median age 75.3 years. Renal function was assessed with creatinine, cystatin C and estimated glomerular filtration rate (eGFR calculated from creatinine)., Results: Median holoTC was 51.8 pmol/L, the health-related reference interval 19.6-132.3 pmol/L. There was no significant difference in mean holoTC in probands with normal compared to high creatinine (P = 0.80) and cystatin C (P = 0.82). No significant differences between the quartiles of creatinine or cystatin C in mean of log holoTC were seen. HoloTC correlated strongly with total cobalamin (r = 0.69, P < 0.001), weaker with eGFRcreatinine (r = -0.09, P < 0.05) and creatinine (r = 0.09, P < 0.05), the latter correlation was only seen in subjects with creatinine <100 µmol/L. HoloTC correlated negatively with plasma total homocysteine (r = -0.24, P < 0.001), but not with cystatin C and age., Conclusions: Serum holoTC in healthy elderly men shows the same distribution as earlier described for a younger reference population. In this group of elderly subjects, holoTC did not correlate to reduced renal function. Thus, holoTC appears to be a promising tool for evaluating cobalamin status also in elderly populations.

Background: Asymptomatic Indian lacto vegetarians, who make up more than half of the Indian population in different geographic regions, have distinctly low vitamin B-12 concentrations than non- vegetarians. Vegetarians consume milk but it seems that the amount is not enough to improve vitamin B-12 status or vitamin B-12 concentration in milk itself may be low. The aim of this study was to determine if daily milk consumption can improve vitamin B-12 status., Methods: Fifteen male and 36 female, young healthy post-graduate volunteers participated. Blood from ten participants (4 males and 6 females) was collected (day-1). They continued their regular diet for next fourteen days and on day-15, blood of all 51 participants was collected, plasma vitamin B-12 concentration was measured and were divided into two groups; Normal (vitamin B-12 >148 pmol/L, n = 22) and Vitamin B-12 deficient (<148 pmol/L, n = 29), the remaining plasma was stored. All participants consumed 600 ml. of non-enriched buffalo milk (200 × 3) during the day along with their usual diet. Next day blood was collected for plasma holotranscobalamin II measurement. Subjects from deficient group continued to drink 400 ml of milk daily for next 14 days and blood was collected on day-30. Plasma holotranscoabalamin II (day-1, 15, 16, 30), vitamin B-12, folate, total homocysteine, creatinine and hematoloical parameters (day-1, 15, 30), and milk vitamin B-12 concentrations (day-15, 16, 30) were measured., Results: Fifty seven per cent of the participants were vitamin B-12 deficient and 65% were hyperhomocysteinemic. No significant difference in biomarkers was observed when there was no intervention. Plasma holotranscobalamin II concentration increased from 19.6 to 22.27 pmol/L (p < 0.0001) 24 hrs after milk load in the whole group. Plasma vitamin B-12 increased from 92.5 to 122 pmol/L and tHcy concentrations decreased from 31.9 to 24.9 μ mol/L (p < 0.0001 for both) 14 days after regular milk intake in vitamin B-12 deficient subjects., Conclusions: Regular intake of milk improved vitamin B-12 status of vitamin B-12 deficient vegetarians indicating a potential dietary strategy to improve the vitamin status.

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

Aim: The aim of the study is to compare the effects of metformin and insulin treatment for gestational diabetes mellitus (GDM) on vitamin B12 and homocysteine (Hcy) status., Methods: Women with GDM, who met criteria for insulin treatment, were randomly assigned to metformin (n = 89) or insulin (n = 91) in the Adelaide cohort of the metformin in gestational diabetes (MiG) trial. Fasting serum total vitamin B12 (TB12), holotranscobalamin (HoloTC), a marker of functional B12 status and plasma Hcy concentrations were measured at 20-34 weeks (at randomization) and 36 weeks gestation, then at 6-8 weeks postpartum., Results: Circulating TB12, HoloTC and Hcy were similar in both treatment groups at each time point. Women who were taking dietary folate supplements at randomization had higher serum TB12 and HoloTC at randomization than those not taking folate. Overall, serum TB12 fell more between randomization and 36 weeks gestation in the metformin group than in the insulin group (metformin: -19.7 ± 4.7 pmol/l, insulin: -6.4 ± 3.6 pmol/l, p = 0.004). The decrease in serum TB12 during treatment was greater with increasing treatment duration in metformin-treated (p < 0.001), but not in insulin-treated women., Conclusions: Total, but not bioavailable, vitamin B12 stores were depleted during pregnancy to a greater extent in metformin-treated than in insulin-treated women with GDM, but neither analyte differed between groups at any stage. This adds further evidence supporting metformin as a safe alternative treatment to insulin in GDM. Further investigation is needed to evaluate whether women treated with metformin for longer periods in pregnancy require additional B12 or other supplementation., (© 2013 Blackwell Publishing Ltd.)

Background: Haptocorrin (HC) carries cobalamin analogues (CorA), but whether CorA are produced in the body is unknown. All cobalamins (Cbl) to the foetus are delivered by the Cbl-specific protein transcobalamin (TC), and therefore analysis of cord serum for CorA may help to clarify the origin of CorA., Methods: HC-CorA were quantified in paired samples of cord serum from newborns and serum from mothers (n = 69)., Results: The CorA-concentration was higher in cord serum (median = 380, range: 41-780 pmol/L) than in serum from the mothers (median = 160, range: 64-330 pmol/L), (p<0.0001). HPLC-analysis showed CorA-peaks with retention times of 13.5, 14,5 and 16.5 min in samples from both the mother and cord serum. The peak with retention time 16.5 min constituted 24% (mother) and 45% (cord serum) of the total amount CorA, and eluted as does dicyanocobinamide., Conclusion: Our results support that CorA in the human body are derived from Cbl.

Alopecia areata has been associated with many autoimmune diseases. There is a common belief that the prevalence of pernicious anemia is increased in patients with alopecia areata. In this study, we sought to investigate vitamin B12, folate and homocysteine metabolism in alopecia areata. We measured holotranscobalamine (holoTC), vitamin B12, folate and homocysteine levels in 75 patients with alopecia areata and 54 controls. We did not find any significant differences in these parameters between these groups. We think that alopecia areata may not be associated with vitamin B12 deficiency. The co-occurrence of pernicious anemia and alopecia areata in rare autoimmune syndromes, may not justify routine measurements of these parameters in alopecia areata patients.

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

Background: Many randomized controlled trials (RCTs) and observational studies have provided information on the association between vitamin B-12 intake and biomarkers. The use of these data to estimate dose-response relations provides a useful means to summarize the body of evidence., Objective: We systematically reviewed studies that investigated vitamin B-12 intake and biomarkers of vitamin B-12 status and estimated dose-response relations with the use of a meta-analysis., Design: This systematic review included all RCTs, prospective cohort studies, nested case-control studies, and cross-sectional studies in healthy adult populations published through January 2010 that supplied or measured dietary vitamin B-12 intake and measured vitamin B-12 status as serum or plasma vitamin B-12, methylmalonic acid (MMA), or holotranscobalamin. We calculated an intake-status regression coefficient ( ) for each individual study and calculated the overall pooled and SE ( ) by using random-effects meta-analysis on a double-log scale., Results: The meta-analysis of observational studies showed a weaker slope of dose-response relations than the meta-analysis of RCTs. The pooled dose-response relation of all studies between vitamin B-12 intake and status indicated that a doubling of the vitamin B-12 intake increased vitamin B-12 concentrations by 11% (95% CI: 9.4%, 12.5%). This increase was larger for studies in elderly persons (13%) than in studies in adults (8%). The dose-response relation between vitamin B-12 intake and MMA concentrations indicated a decrease in MMA of 7% (95% CI: -10%, -4%) for every doubling of the vitamin B-12 intake. The assessment of risk of bias within individual studies and across studies indicated risk that was unlikely to seriously alter these results., Conclusion: The obtained dose-response estimate between vitamin B-12 intake and status provides complementary evidence to underpin recommendations for a vitamin B-12 intake of populations.

Plasma vitamin B-12 is the most commonly used biomarker of vitamin B-12 status, but the predictive value for low vitamin B-12 status is poor. The urinary methylmalonic acid (uMMA) concentration has potential as a functional biomarker of vitamin B-12 status, but the response to supplemental vitamin B-12 is uncertain. A study was conducted to investigate the responsiveness of uMMA to supplemental vitamin B-12 in comparison with other biomarkers of vitamin B-12 status [plasma vitamin B-12, serum holotranscobalamin (holoTC), plasma MMA] in elderly people with moderately poor vitamin B-12 status. A double-blind, placebo-controlled, randomized 8-wk intervention study was carried out using vitamin B-12 supplements (500 μg/d, 100 μg/d, and 10 μg/d cyanocobalamin) in 100 elderly people with a combined plasma vitamin B-12 <250 pmol/L and uMMA ratio (μmol MMA/mmol creatinine) >1.5. All biomarkers had a dose response to supplemental vitamin B-12. Improvements in plasma vitamin B-12 and serum holoTC were achieved at cobalamin supplements of 10 μg/d, but even 500 μg/d for 8 wk did not normalize plasma vitamin B-12 in 8% and serum holoTC in 12% of people. The response in uMMA was comparable with plasma MMA; 15-25% of people still showed evidence of metabolic deficiency after 500 μg/d cobalamin for 8 wk. There was a differential response in urinary and plasma MMA according to smoking behavior; the response was enhanced in ex-smokers compared with never-smokers. uMMA offers an alternative marker of metabolic vitamin-B12 status, obviating the need for blood sampling.

Objective: To investigate the cross-sectional relation between metabolic markers of vitamin B(12) status and cognitive performance, and possible effect modification by the presence of depression and apolipoprotein E (ApoE) ε4., Methods: This is a population-based study of 1935 participants, aged 71 to 74 years, from Norway. Participants were administered a cognitive test battery, and vitamin B(12) status was assessed by measurements of plasma vitamin B(12), holotranscobalamin (holoTC), methylmalonic acid (MMA), and total homocysteine., Results: The geometric mean (95% confidence interval) for vitamin B(12) was 348 pM (341-354), whereas 5.9% of participants had vitamin B(12) levels lower than 200 pM. In linear regression analyses, holoTC (p = .039) and the holoTC/vitamin B(12) ratio (p = .013) were positively related, whereas MMA (p = .010) was inversely related, to global cognition, after adjustment for sex, education, ApoE status, plasma creatinine, and history of diabetes, cardiovascular disease, hypertension, and depression. Among those positive for ApoE ε4, but not among those without the ε4 allele, plasma vitamin B(12) was positively associated with global cognition (p = .015), whereas MMA was inversely related to global cognition (p = .036) and executive function (p = .014). In participants with depression, MMA was inversely associated with global cognition (p < .001) and episodic memory (p = .001)., Conclusions: Among the well-nourished elderly, low vitamin B(12) status is associated with cognitive deficit, particularly in those with the ApoE ε4 allele or with depression.

Poor vitamin B₁₂ status may lead to the development of cognitive decline and dementia but there is a large variation in the quality, design of and results reported from these investigations. We have undertaken a systematic review of the evidence for the association between vitamin B₁₂ status and cognitive decline in older adults. A database search of the literature to 2011 was undertaken, using keywords related to vitamin B₁₂ and cognition. All prospective cohort studies assessing the association of serum vitamin B₁₂ or biomarkers were included. Quality assessment and extraction of the data were undertaken by two researchers. The quality assessment tool assigns a positive, neutral or negative rating. Of 3772 published articles, thirty-five cohort studies (n 14 325 subjects) were identified and evaluated. No association between serum vitamin B₁₂ concentrations and cognitive decline or dementia was found. However, four studies that used newer biomarkers of vitamin B₁₂ status (methylmalonic acid and holotranscobalamin (holoTC)) showed associations between poor vitamin B₁₂ status and the increased risk of cognitive decline or dementia diagnosis. In general, the studies were of reasonable quality (twenty-one positive, ten neutral and four negative quality) but of short duration and inadequate subject numbers to determine whether an effect exists. Future studies should be of adequate duration (at least 6 years), recruit subjects from the seventh decade, choose markers of vitamin B₁₂ status with adequate specificity such as holoTC and/or methylmalonic acid and employ standardised neurocognitive assessment tools and not screening tests in order to ascertain any relationship between vitamin B₁₂ status and cognitive decline.

Biological markers for the status of vitamins B12 and D: the importance of some analytical aspects in relation to clinical interpretation of results When vitamin B12 deficiency is expressed clinically, the diagnostic performance of total cobalamin is identical to that of holotranscobalamin II. In subclinical B12 deficiency, the two aforementioned markers perform less well. Additional analysis of a second, functional marker (methylmalonate or homocysteine) is recommended. Different analytical approaches for 25-hydroxyvitamin D quantification, the marker of vitamin D deficiency, are not yet standardized. Measurement biases of up to +/- 20% compared with the original method used to establish threshold values are still observed.

Background: Cobalamin deficiency is a common problem in the elderly. There is no consensus about adequate doses for supplementation., Subjects/methods: We performed an intervention study in order to establish the efficacy of a supplement providing 500 µg cyanocobalamin for four weeks in sixty-four institutionalized elderly residents, over 60 years of age, in Madrid (Spain). Before and after treatment, concentrations of serum cobalamin, serum holotranscobalamin, serum total homocysteine, and serum and red blood cell folate were analyzed. Clusters were built according to the initial cobalamin status and differences in the effect of supplementation were checked using a general linear model for repeated measures., Results: Cobalamin and holotranscobalamin increased highly significantly from 308 to 558 pmol/L and from 54 to 96 pmol/L (p < 0.001) in the whole study group as well as in each subgroup (clustered by initial cobalamin levels, all p < 0.01), with the highest relative change in the subgroup with the lowest initial cobalamin values. Total homocysteine decreased from 15 to 13 µmol/l, p < 0.001). Only the change of cobalamin (F = 4.61, p < 0.01), but not of holotranscobalamin nor total homocysteine, depended on the initial serum cobalamin status., Conclusions: A supplementation with an oral supplement solution of 500 µg cyanocobalamin daily for only four weeks, a shorter period than that found in former studies, may be considered suitable in institutionalized elderly.

Background: An age-related deterioration of vitamin B-12 status has been well documented. The early detection of deficiency may prevent the development of serious clinical symptoms, but plasma vitamin B-12 concentration is known to be an imperfect measure of vitamin B-12 status. Urinary methylmalonic acid (MMA) may be a more informative biomarker of vitamin B-12 status; however, biochemical, dietary, and other lifestyle determinants are not known., Objective: We identified determinants of urinary MMA concentrations in free-living men and women aged ≥65 y in the United Kingdom., Design: A cross-sectional study in 591 men and women aged 65-85 y, with no clinical evidence of vitamin B-12 deficiency, was conducted to determine the demographic, clinical, and lifestyle determinants of urinary MMA concentration expressed as the ratio of micromoles of MMA to millimoles of creatinine (uMMA ratio)., Results: Twenty percent of subjects had plasma vitamin B-12 concentrations <200 pmol/L. Seventeen percent of the variation in the uMMA ratio could be explained by plasma holotranscobalamin and sex; total vitamin B-12 intake and measures of renal function and gastric function made only a small contribution to the model. The uMMA ratio was lower in people with moderately impaired renal function., Conclusions: Plasma holotranscobalamin and sex were the most important determinants of uMMA ratio in elderly people with no clinical diagnosis of renal impairment. This biomarker might underestimate vitamin B-12 deficiency in a population in which renal impairment is prevalent. This trial was registered at www.controlled-trials.com as ISRCJN83921062.

A 28-year-old female vegetarian was referred to a specialist in internal medicine with persistent iron deficiency. Laboratory analysis revealed microcytic anaemia with low ferritin levels but normal total vitamin B12 levels. The red blood cell distribution width, however, showed a very wide variation in red blood cell sizes, indicating a coexisting vitamin B12 deficiency, which was confirmed by the low concentration of active vitamin B12. Another patient, a 69-year-old woman with a history of previous gastric surgery and renal insufficiency as a complication of diabetes mellitus, was suspected to be deficient in vitamin B12, as she had low total vitamin B12 levels and an accumulation of methylmalonic acid and homocysteine in her blood. Testing the total concentration of vitamin B12 alone has insufficient diagnostic accuracy and no accepted gold standard is available for diagnosing vitamin B12 deficiency. With the development of newer tests, such as measuring holotranscobalamin II (concentration of active vitamin B12), atypical and subclinical deficiency states can be recognized. A new approach to diagnosing vitamin B12 deficiency is presented, based upon these 2 case descriptions.

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

Few studies have investigated the role of vitamin B12 metabolism in vitiligo. We tested the hypothesis that vitamin B12 and folate metabolism might have an influence on the pathogenesis of vitiligo. Full blood count and levels of folic acid, vitamin B12, homocysteine and holotranscobalamine were examined for 69 patients with vitiligo and 52 controls. The vitiligo group had higher levels of homocysteine (P < 0.01) and haemoglobin (P < 0.01) levels, and lower levels of vitamin B12 (P < 0.01) and holotranscobalamine (P < 0.0001) than the control group. Folic acid levels were similar for both groups. In a risk analysis, hyperhomocysteinaemia (≥ 15 μmol/L, P < 0.01) and vitamin B12 deficiency (< 200 pg/mL, P < 0.01) were significant risk factors for vitiligo. Patients with holotranscobalamine levels in the lowest quartile had an increased risk for co-occurrence of vitiligo (P < 0.005). Vitamin B12 deficiency and hyperhomocysteinaemia may share a common genetic background with vitiligo., (© The Author(s). CED © 2011 British Association of Dermatologists.)

Isotretinoin (Iso) has been used for the treatment of acne. Some previous studies reported elevated homocysteine (Hcy) levels after treatment with Iso. Some side effects have clinical presentations similar to vitamin B12, folic acid deficiencies, and hyperhomocysteinemia. In the present study we evaluated the plasma Hcy levels, the vitamins involved in its metabolism (vitamin B12 and folic acid), and holotranscobalamin (HoloTC), a transport system for vitamin B12 absorption in patients receiving Iso treatment for acne vulgaris. A total of 66 patients with acne vulgaris between the ages of 18 and 40 years were included. Screening for hemoglobin, creatinine, SGOT, SGPT, total cholesterol, triglycerides, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and very low-density lipoprotein cholesterol (VLDL-C), folic acid, vitamin B12, Hcy, and HoloTC were done just before initiation (pretreatment) and after four months of Iso treatment (posttreatment). Posttreatment vitamin B12, folic acid, and HoloTC levels were significantly lower while Hcy levels were significantly higher compared with initial values. Posttreatment total cholesterol, LDL-C, triglycerides, VLDL-C, SGPT, and SGOT levels were also higher, and HDL-C levels were lower compared with initial values while there was no change in hemoglobin levels during Iso treatment. We found that Iso usage might cause decreased vitamin B12, folic acid, and HoloTC. These Iso side effects might contribute to the missing link between Iso usage, hyperhomocysteinemia, and neuropsychiatric disorders. Trials may be made with the aim of demonstrating (clearly) if starting vitamin B12 and folic acid replacement therapies with Iso treatment initialization could be useful for preventing hyperhomocysteinemia and possibly related disorders., (© 2011 The International Society of Dermatology.)