Ultrasensitive detection of holoTC for analysis of Vitamin B12 levels using Ag2MoO4 deposited PEDOT sensing platform.

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 (Ag2MoO4 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 ΔRct 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.

Folate and cobalamin status, indicators, modulators, interactions and reference ranges from early pregnancy until birth: The Reus-Tarragona Birth Cohort Study.

BACKGROUND: Folate and cobalamin status, although essential for pregnancy, are not routinely monitored in prenatal care. OBJECTIVE: To investigate folate and cobalamin status and determinants throughout pregnancy, in the absence of mandatory fortification with folic acid (FA). METHODS: In a cohort study of 831 women recruited at <12 gestational weeks (GW), plasma folate, total homocysteine (tHcy), cobalamin, holotranscobalamin (holoTC), methylmalonic acid (MMA), red blood cell folate (RBCF) concentrations and the combined cobalamin status indicator (cB12) were determined at ≤12, 15, 24-27, 34 GW, labor and in the cord. Single nucleotide polymorphisms affecting folate and cobalamin status were determined. FA, cobalamin, micronutrient supplement use and dietary folate and cobalamin intake (food frequency questionnaire) were recorded. Folate and cobalamin status predictors were assessed by multiple linear regression analysis. RESULTS: Only 36.1% of the participants took FA preconceptionally and 47.4% and 7.3% had suboptimal RBCF (<906 nmol/L) and plasma cobalamin status (≤221 pmol/L), respectively, at ≤12 GW. RBCF status was principally determined by planned pregnancy, FA supplementation, plasma cobalamin and methylenetetrahydrofolate (MTHFR) 677C>T genotype. Cobalamin supplement use was positively associated, while smoking and BMI were inversely associated with plasma cobalamin and holoTC. None of these were associated with plasma MMA. Only participants with the MTHFR 677TT genotype exceeding FA supplement recommendations improved their folate status (interaction term: B (95% CI):0.015 (0.01, 0.29), p: 0.032). Smoking was inversely associated with plasma cobalamin status in participants with the methionine synthase reductase (MTRR) 524CC genotype only(interaction term:0.07 (0.01, 0.04), p: 0.014). Mothers with low early pregnancy cobalamin status and also those with bigger newborns, had lower cobalamin status at labor. CONCLUSIONS: Suboptimal early pregnancy folate or cobalamin status affected47.4% and 7.3% of the participants, respectively. The MTHFR 677TT genotype consistently predicted folate status throughout pregnancy. Smoking and BMI were negatively associated with cobalamin status throughout pregnancy. CLINICAL TRIAL REGISTRY NUMBER AND WEBSITE WHERE IT WAS OBTAINED: NCT01778205. www. CLINICALTRIALS: gov.

Vitamin B12 status and the risk of developing sepsis in patients with bacterial infection: a prospective observational cohort study.

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.

Vitamin B12-Related Biomarkers.

BACKGROUND: Adult vitamin B12 (B12) deficiency may present itself with nonspecific mainly neurological symptoms, and thus plasma biomarkers are often judged to be of major importance in the further diagnostic process. Four biomarkers are of special relevance: total B12, holotranscobalamin (the part of B12 bound to the active transport protein, transcobalamin, also named holoTC or active B12) and the 2 so-called metabolic markers that accumulate if B12 is lacking, methylmalonic acid (MMA) and homocysteine. OBJECTIVE: This article briefly reviews the inherent limitation of biomarkers, discusses its use in establishing the diagnosis and cause of B12 deficiency, and when following or discontinuing treatment with B12. METHODS: The review is based on published papers, but also on knowledge gained from working within the area. CONCLUSION: It is concluded that a combination of a B12 and a metabolic marker, for example, total B12 and MMA, may prove most useful in daily practice. An unexpectedly high level of total B12 is most often of no clinical importance, though sometimes related to the presence of underlying cancer. Measurement of total B12 is of limited value in patients on treatment with pharmacological doses of B12 but may be helpful if B12 treatment is discontinued.

Plain language titleVitamin B12-Related Blood TestsPlain language summaryBlood-testing is considered an important part of the diagnostic procedure in patients suspected to suffer from B12 deficiency. A deficiency is supported by a low level of plasma B12, and confirmed by a high level of methylmalonic acid, judged according to age and kidney function. Alternatively, a high level of homocysteine may support the diagnosis. Treatment of B12 deficiency is mainly guided by improvement of symptoms, with a very limited need for further blood testing. If B12-treatment is discontinued, B12 status should be judged every 6 months for approximately 2 years to detect a possible reoccurrence of a deficient state. An unexpected high level of plasma B12 is most often of no clinical implication.

Excess Folic Acid and Vitamin B12 Deficiency: Clinical Implications?

BACKGROUND: In the 1940s to 1950s, high-dose folic acid supplements (>5 mg/d) were used clinically to reverse the megaloblastic anemia of vitamin B12 deficiency caused by pernicious anemia. However, this treatment strategy masked the underlying B12 deficiency and possibly exacerbated its neuropathological progression. The issue of masking and exacerbating B12 deficiency has recently been rekindled with the institution of folic acid fortification and the wide-spread use of folic acid supplements. OBJECTIVES: The objectives of this review are to describe clinical and epidemiological evidence that excess folic acid exacerbates B12 deficiency, to summarize a hypothesis to explain this phenomenon, and to provide guidance for clinicians. RESULTS: Cognitive function test scores are lower and blood homocysteine and methylmalonic acid concentrations are higher in people with low B12 and elevated folate than in those with low B12 and nonelevated folate. High-dose folic acid supplementation in patients with pernicious anemia or epilepsy cause significant reductions in serum B12. It is hypothesized that high-dose folic acid supplements cause depletion of serum holotranscobalamin and thus exacerbate B12 deficiency. CONCLUSION: The evidence for excess folic acid exacerbating B12 deficiency is primarily correlative or from uncontrolled clinical observations, and the hypothesis to explain the phenomenon has not yet been tested. Nonetheless, the evidence is sufficiently compelling to warrant increased vigilance for identifying B12 deficiency in at risk individuals, including older adults and others with low B12 intake or conditions that are associated with B12 malabsorption, who also ingest excessive folic acid or are prescribed folic acid in high doses.

Plain language titleExcess Folic Acid and Vitamin B12 Deficiency: Clinical Implications?Plain language summaryIt has been known for many decades that high doses of the B vitamin supplement, folic acid, can alleviate the anemia of vitamin B12 deficiency, at least temporarily. However, by alleviating the anemia, such folic acid supplements were said to "mask" the underlying vitamin B12 deficiency, thus allowing neurological damage to continue or possibly be exacerbated. Consequently, treating vitamin B12 deficiency with high dose folic acid was discontinued in the 1970s. The issue of whether folic acid supplements can exacerbate vitamin B12 deficiency reemerged in the 1990s with folic acid fortification of cereals and grains in the United States and Canada (and now in over 80 countries around the world) to prevent spina bifida and other birth defects. This narrative review summarizes the results of studies that have assessed the relationships between folic acid and folate and vitamin B12 status in patients and in populations. A recent hypothesis on how folic acid might exacerbate vitamin B12 deficiency is summarized, and recommendations to clinicians are made for increased vigilance in assessing vitamin B12 status in certain groups at risk of vitamin B12 deficiency, including older adults, people with gastrointestinal issues and other factors that cause vitamin B12 malabsorption, people with unexplained neurological problems, and people who follow vegan or vegetarian diets which are naturally low in vitamin B12.

Serum Vitamin B12 and Holotranscobalamin Levels in Subclinical Hypothyroid Patients in Relation to Thyroid-Stimulating Hormone (TSH) Levels and the Positivity of Anti-thyroid Peroxidase Antibodies: A Case-Control Study.

Background Subclinical hypothyroidism (SCH) is characterized by elevated thyroid-stimulating hormone (TSH) levels, while thyroid hormones (free thyroxine (T4) and free triiodothyronine (T3)) remain within the reference ranges. Vitamin B12 (cobalamin) deficiency is common in patients with autoimmune disorders, including autoimmune hypothyroidism. The study was aimed at evaluating serum vitamin B12 levels and holotranscobalamin (HoloTC) levels in SCH patients and ascertaining their association with a risky level of TSH and the positivity of anti-thyroid peroxidase (anti-TPO) antibodies. Methodology A case-control study was conducted at Azadi Teaching Hospital, Duhok, a city in the Kurdistan region of Iraq, involving 153 participants, including 72 newly diagnosed SCH patients and 81 healthy controls. Serum levels of vitamin B12, HoloTC, TSH, free T4, free T3, and anti-TPO antibodies were measured based on different principles. Results The mean age of patients with SCH was 32.87±8.7 years, with predominantly females comprising 75% and 77.8% being less than 40 years of age. Moreover, the mean levels of serum TSH (6.96±2.68 µIU/L), anti-TPO antibodies (53.31±81.32 IU/ml), and HoloTC (41.93±19.42 pmol/l) were significantly higher in patients with SCH compared to healthy control participants (p < 0.05), whereas there was a non-significantly higher level of vitamin B12(320.72±98.42 pg/ml) among SCH patients compared to healthy control participants (p = 0.220). The mean levels of vitamin B12 (345.33±103.22 pg/ml) and HoloTC (40.14±18.16 pmol/l) were insignificantly lower in SCH patients with TSH levels more than 7 µIU/L (p > 0.05), as well as the mean levels of vitamin B12 (308.82±96.12 pg/ml) and HoloTC (41.14±19.29 pmol/l) insignificantly lower in SCH patients with positive anti-TPO antibodies (p > 0.05).  Conclusions This study highlights the potential association between SCH and altered vitamin B12 status, particularly evident in HoloTC levels. The presence of positive anti-TPO antibodies and the degree of elevation in TSH levels may exacerbate vitamin B12 deficiency in SCH patients.

Holo-transcobalamin is not associated with the risk of all-cause mortality in the general population.

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

Preliminary evaluation of the diagnostic performance of Roche Elecsys® active vitamin B12 versus total vitamin B12 for vitamin B12 deficiency screening.

INTRODUCTION: The prevalence of vitamin B12 deficiency is high in at-risk populations with sometimes irreversible consequences. Beside total B12 (TVB12), active B12 (AVB12) is a promising first-line marker. Only Abbott AVB12 assays were largely evaluated and generally demonstrated benefit in clinical practice. More recently developed Roche AVB12 still requires some investigations. OBJECTIVES: Our study aimed to evaluate the Roche Elecsys® AVB12 immunoassay performance versus Roche Elecsys® TVB12 competition assay. DESIGN: and Methods: We included 175 patients at Rouen University Hospital who had a TVB12 value <300 pmol/L. We evaluated performance of AVB12 by comparing the results with TVB12 and MMA values in case of disagreement. RESULTS: Positive correlation was found between the AVB12 and TVB12. We found a disagreement between TVB12 and AVB12 in 18.8% of cases. Among 33 cases of disagreement, 76% had normal AVB12 but low TVB12, whereas 24% had low AVB12 and normal TVB12. Thirty-one MMA determinations were performed: 71% showed agreement between MMA and AVB12, versus 29% between MMA and TVB12. TVB12 reported a sensitivity (Se) at 66.7%, specificity (Sp) at 20%, positive predictive value (PPV) at 16.7% and negative predictive value (NPV) at 71.4% for the prediction of MMA elevation. We determined an optimized cut-off value of 45.5 pmol/L for AVB12, which reported a Se 66.7%, Sp 60%, PPV 30.7%, and NPV 88.9%. CONCLUSIONS: Our results provide preliminary evidence that Roche AVB12 may offer better discrimination than Roche TVB12 in the diagnosis of vitamin B12 deficiency. Further more detailed evaluation is warranted.

Intake of vitamin B12 in relation to vitamin B12 status in groups susceptible to deficiency: a systematic review.

Objective: To systematically review the evidence for whether habitual or different levels of experimental intake of vitamin B12 from diet and supplements is sufficient to ensure adequate B12 status in groups most susceptible to vitamin B12 deficiency. Methods: We searched MEDLINE, Embase, Cochrane Central Register of Controlled Trials and Scopus up to 21 May 2021, for intervention studies, prospective cohort studies and case-control studies assessing B12 intake from diet and/or supplements in relation to B12 status (s/p-B12, holotranscobalamin, methylmalonic acid, homocysteine or breastmilk B12). Cross-sectional studies were eligible for studies conducted during pregnancy and lactation. Included populations were children (0-18 years), young adults (18-35 years), pregnant or lactating women, older adults (≥65 years) and vegans or vegetarians. Study selection, data extraction and risk of bias assessment were conducted by two assessors independently. The evidence was synthesized qualitatively and classified according to the World Cancer Research Fund. Results: The searches yielded 4855 articles of which 89 were assessed in full text and 18 included. Three studies were conducted during pregnancy and three during lactation or infancy - all observational. Eight studies were conducted among older adults; most were interventions among B12-deficient participants. Four studies were eligible for vegetarian and vegans, all interventions. The strength of evidence that habitual B12 intake or an intake in line with the current Nordic recommended intake (RI) is sufficient to ensure adequate status was considered Limited - no conclusion for all included populations. Conclusion: Evidence is insufficient to assess if or which level of B12 intake is sufficient to maintain adequate status for all included populations. Population-based cohort studies and low-to-moderate dose interventions that address this question are highly warranted.

Vitamin B12 Status in Recreational Users of Nitrous Oxide: A Systematic Review Focusing on the Prevalence of Laboratory Abnormalities.

The recreational use of nitrous oxide (N2O) as "laughing gas" is a growing problem. The chronic toxicity of N2O is mainly due to its ability to oxidize vitamin B12, making it dysfunctional as a cofactor in metabolic pathways. This mechanism plays a major role in the development of neurological disorders in N2O users. The assessment of vitamin B12 status in N2O users is important but challenging due to the lack of decrease in total vitamin B12 in most cases despite genuine vitamin B12 functional deficiency. Other biomarkers, such as holotranscobalamin (holoTC), homocysteine (tHcy) and methylmalonic acid (MMA), are interesting candidates to properly assess vitamin B12 status. Here, we conducted a systematic review of case series in order to assess the prevalence of abnormal values of total vitamin B12, holoTC, tHcy and MMA in recreational N2O users, which is an important prerequisite for determining the best screening strategy in future guidelines. We included 23 case series (574 N2O users) from the PubMed database. Total circulating vitamin B12 concentration was low in 42.2% (95% confidence interval 37.8-46.6%, n = 486) of N2O users, while 28.6% (7.5-49.6%, n = 21) of N2O users had low circulating concentrations of holoTC. tHcy levels were elevated in 79.7% (75.9-83.5%, n = 429) of N2O users, while 79.6% (71.5-87.7%, n = 98) of N2O users had increased concentrations of MMA. In summary, the increases in tHcy and MMA were the most prevalent abnormalities, and should be measured alone or in combination in symptomatic N2O users rather than total vitamin B12 or holoTC.

Reference intervals and stability of haptocorrin and holotranscobalamin in Danish children and elderly.

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.

Association of maternal folate and B12 vitamin status with gestational diabetes mellitus: Still an open issue.

OBJECTIVES: There is conflicting evidence about the role of folate and B12in gestational diabetes mellitus (GDM) onset. The association of vitamin status with GDM was therefore revalued, also measuring the B12active form holotranscobalamin. METHODS: 677 women were evaluated at 24-28 weeks of gestation when OGTT was carried out. The 'one-step' strategy was employed for GDM diagnosis. Odds ratio (OR) of having GDM was estimated to quantify the association with vitamin levels. RESULTS: 180 women (26.6%) had GDM. They were older (median, 34.6 vs. 33.3 years, p = 0.019) and had higher body mass index (BMI) (25.8 vs. 24.1 kg/m2, p < 0.001). Multiparous women had lower levels of all evaluated micronutrients, while overweight lowered both folate and total B12, but not holotranscobalamin. Lower total B12(270 vs. 290 ng/L, p = 0.005), but not holotranscobalamin, was observed in GDM, being weakly negatively correlated with fasting glycemia (r = -0.11, p = 0.005) and 1-h OGTT serum insulin (r = -0.09, p = 0.014). At multivariate analysis, age, BMI and multiparity remained the strongest GDM predictors, while total B12(but not holotranscobalamin and folate) showed a slight protective effect (OR = 0.996, p = 0.038). CONCLUSIONS: A weak association between total B12 levels and GDM risk was shown, but it was not confirmed when holotranscobalamin was measured.

Comparison of Serum Holotranscobalamin with Serum Vitamin B12 in Population Prone to Megaloblastic Anemia and their Correlation with Nerve Conduction Study.

Early detection of megaloblastic anemia and associated neurological complications is crucial for management. This study was conducted to compare serum holotranscobalamin level with serum vitamin B12 level as early biomarker in people prone to megaloblastic anemia and to evaluate co-relation between these biomarkers and nerve conduction study in study patients. 83 adult patients (Hb > 12 gm/dl) prone to megaloblastic anemia were studied for basic haematological investigations, random blood sugar, thyroid function test, liver function test, kidney function test, serum vitamin B12, serum holotranscobalamin and serum folic acid levels. 45 patients among them underwent nerve conduction studies. All study patients were classified in 6 groups on the basis of risk factors for megaloblastic anemia. 29 patients (34.9%) were on antiepileptic drugs, 26 (31.3%) were chronic alcoholic, 10 patients (12%) each, had malabsorption and ileal tuberculosis, 6 (7.22%) had chronic pancreatitis and 2 (2.4%) had ileal resection. 30 patients (36.14%) had low serum holotranscobalamin, including 7 patients (8.43%) with low serum vitamin B12 level also, unmasking vitamin B12 deficiency in 23 patients (27.7%). 7 patients (8.43%) had mean corpuscular volume (MCV) > 100fL and 8 patients (9.63%) had vitamin B12 deficiency related changes on peripheral smear. Serum vitamin B12 and holotranscobalamin levels were significantly low in patients with peripheral smear changes, with p value 0.039 and 0.041 respectively, while no such association seen with MCV. Subclinical peripheral neuropathy was detected in 18 (40%) out of 45 patients on nerve conduction study. Serum holotranscobalamin levels were significantly lower (p = 0.031) than serum vitamin B12 levels (p = 0.2) in patients with neuropathic changes. Rest investigations and serum folic acid levels were normal in all patients. Holotranscobalamin levels can be considered early and reliable marker for vitamin B12 deficiency and deficiency associated peripheral neuropathy, even in patients who are prone to megaloblastic anemia, and not yet anemic or symptomatic for neuropathy.

Age- and ethnicity-related reference intervals for serum vitamin B12.

BACKGROUND: Age and ethnicity are known to influence serum vitamin B12 (B12) concentration, yet universal reference intervals (RIs) are typically applied by laboratories. Both lower and upper RI limits for B12 are clinically relevant. Low values suggest deficiency leading to anemia and/or neurological impairment, while high values are not always an innocuous consequence of high B12 intake but are associated with some cancers, autoimmune, liver, and renal diseases. This work aimed to establish age- and ethnicity-related RIs for B12 using a modified indirect method based on Hoffmann's approach. METHODS: A total of 72,091 anonymized B12 results (Jan 2018-Nov 2019) were analyzed from an ethnically-diverse South-East London general practice patient population. Patients belonged to five ethnic groups: Asian, Black, White, Mixed, or Other. Multiple records for the same patient and results with missing ethnicity were excluded from the analysis of adult RIs. B12 analyses were performed using ARCHITECT® (Abbott Diagnostics). RESULTS: B12 was significantly higher in Black compared with Asian and White adults. There were no differences in B12 between Asian and White adults. Children (all ethnicities) between 2 and 5 years old had the highest B12. Because of the small number of children (up to the age of 13) in each ethnic-related age category, all ethnic groups were combined to obtain age-related RIs. The children's RIs ranged from 159 to 1025 pmol/L for 0-1-year-olds to 276-1102 pmol/L for 2-5-year-olds. The RIs for Black and White/Asian people >13 years of age were 166-805 pmol/L and 134-511 pmol/L respectively. CONCLUSIONS: The application of age- and ethnicity-appropriate RIs into diagnostic practice will provide a more accurate evaluation of B12 status when using the B12 test alone or in combination with other markers.

Diagnostic reliability of serum active B12 (holo-transcobalamin) in true evaluation of vitamin B12 deficiency: Relevance in current perspective.

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

Cobalamin status is negatively correlated with vascular endothelial-cadherin in vegetarian and vegan women with vitamin B12 deficiency.

Optimal vitamin B12 status is important for vascular health. Vascular endothelial (VE) cadherin is an adherent junction protein involved in the maintenance of a functional endothelium. We hypothesized that vitamin B12 deficiency can negatively affect markers of vascular function, such as VE-cadherin. Within a human intervention study, we explored the possible association between cobalamin status (i.e., vitamin B12, holotranscobalamin, and homocysteine) and VE-cadherin (as marker of vascular health) in vegetarians/vegans (VEG) with B12 deficiency. The associations were evaluated at baseline and after 90-day supplementation with 2000 µg/wk of vitamin B12. On the whole, an inverse association between VE-cadherin and holotranscobalamin (P = .014) and a positive association between VE-cadherin and homocysteine (P = .041) was documented at baseline. VEG women showed higher levels of VE-cadherin compared with VEG men (P = .044), suggesting an increase in endothelial permeability. The intervention with vitamin B12 restored serum vitamin levels and improved the overall cobalamin status, whereas it did not affect VE-cadherin levels. The inverse association between holotranscobalamin and VE-cadherin was also maintained after intervention in women, corroborating the strong correlation between these 2 parameters. The results obtained seem to suggest a possible association between cobalamin status and VE-cadherin even if the intervention with B12 failed to positively affect VE-cadherin levels. Thus, further studies are needed to corroborate these findings and clarify the contribution of a vitamin B12 intervention on VE-cadherin levels in this target population. This trial was registered at ISRCTN registry (ISRCTN75099618).

Liver and vitamin B12 parameters in patients with anorexia nervosa before and after short-term weight restoration.

Hepatic involvement in anorexia nervosa (AN) has been previously reported, but a link to elevated vitamin B12 concentrations, which can be a sign for liver damage, has not been thoroughly examined. We measured liver enzymes (alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase) and vitamin B12 parameters (total B12, holotranscobalamin, methylmalonic acid) in the plasma of young female patients with acute AN (n=77) and after short-term weight restoration (n=58, median body mass increase=25%), in comparison to healthy control participants (n=63). For a comprehensive assessment of vitamin B12 status, the combined marker cB12 was calculated. In acute AN, activities of alanine aminotransferase and gamma-glutamyltransferase as well as holotranscobalamin concentrations were elevated, and alanine aminotransferase activities positively correlated with total B12, holotranscobalamin and cB12 in patients with elevated liver enzyme activities. After weight restoration, alanine aminotransferase activities and holotranscobalamin concentrations were elevated, and cB12 increased above the level of the healthy control group. The present study provides further evidence for a hepatic involvement in acute AN in concert with vitamin B12 parameters and points to refeeding-associated alterations of liver and vitamin B12 parameters. Future studies should include non-invasive methods to characterize hepatic involvement and evaluate vitamin B12 status as a potential marker of liver damage/irritation.

Perspective: The High-Folate-Low-Vitamin B-12 Interaction Is a Novel Cause of Vitamin B-12 Depletion with a Specific Etiology-A Hypothesis.

Vitamin B-12 is a water-soluble vitamin that plays important roles in intermediary metabolism. Vitamin B-12 deficiency has many identifiable causes, including autoimmune and other gastrointestinal malabsorption disorders, dietary deficiency, and congenital defects in genes that are involved in vitamin B-12 trafficking and functions. Another putative cause of vitamin B-12 deficiency is the high-folate-low vitamin B-12 interaction, first suspected as the cause for observed relapse and exacerbation of the neurological symptoms in patients with pernicious anemia who were prescribed high oral doses of folic acid. We propose that this interaction is real and represents a novel cause of vitamin B-12 depletion with specific etiology. We hypothesize that excessive intake of folic acid depletes serum holotranscobalamin (holoTC), thereby decreasing active vitamin B-12 in the circulation and limiting its availability for tissues. This effect is specific for holoTC and does not affect holohaptocorrin, the inert form of serum vitamin B-12. Depletion of holoTC by folic acid in individuals with already low vitamin B-12 status further compromises the availability of vitamin B-12 coenzymes to their respective enzymes, and consequently a more pronounced state of biochemical deficiency. This hypothesis is drawn from evidence of observational and intervention studies of vitamin B-12-deficient patients and epidemiological cohorts. The evidence also suggests that, in a depleted state, vitamin B-12 is diverted to the hematopoietic system or the kidney. This most likely reflects a selective response of tissues expressing folate receptors with high affinity for unmetabolized folic acid (UMFA; e.g., hematopoietic progenitors and renal tubules) compared with those tissues (e.g., liver) that only express the reduced folate carrier, which is universally expressed but has poor affinity for UMFA. The biochemical and physiological mechanisms underlying this interaction require elucidation to clarify its potential public health significance.

Efficacy of supplementation with methylcobalamin and cyancobalamin in maintaining the level of serum holotranscobalamin in a group of plant-based diet (vegan) adults.

Plant-based nutrition has become extremely popular in the contemporary era. Its positive effects are sustained by many studies, but one of its risks is that it is almost completely devoid of vitamin B12. In the present study, we analyzed the effects of two types of vitamin B12 supplements, cyancobalamin and methylcobalamin on the level of active serum vitamin (holotranscobalamin) in a group of Romanian individuals (n=42) following a (vegan) plant-based diet. The results revealed that cyancobalamin gives better results in maintaining B12, as quantified by the holotranscobalamin value (median=150 pcg/l) when compared with methylcobalamin (median=78.5 pcg/l). The frequency of administration, regardless of the quantity in one dose, is another important factor in maintaining the holotranscobalamin level within suitable limits. More frequent intakes give more optimal results. Vegans trying to supplement with alternative products (algae, kombucha, other fermented products), had the lowest levels of holotranscobalamin, always bellow the recommended level of 35 pcg/l (median=29 pcg/l). Vegans must be educated on B12 supplementation, about the pharmaceutical forms on the market and their performances and on choosing the optimal plan in order to avoid the onset of B12 deficiency.

Associations of circulating folate, vitamin B12 and homocysteine concentrations in early pregnancy and cord blood with epigenetic gestational age: the Generation R Study.

BACKGROUND: Circulating folate, vitamin B12 and homocysteine concentrations during fetal development have been associated with health outcomes in childhood. Changes in fetal DNA methylation may be an underlying mechanism. This may be reflected in altered epigenetic aging of the fetus, as compared to chronological aging. The difference between gestational age derived in clinical practice and gestational age predicted from neonatal DNA methylation data is referred to as gestational age acceleration. Differences in circulating folate, vitamin B12 and homocysteine concentrations during fetal development may be associated with gestational age acceleration. RESULTS: Up to 1346 newborns participating in the Generation R Study, a population-based prospective cohort study, had both cord blood DNA methylation data available and information on plasma folate, serum total and active B12 and plasma homocysteine concentrations, measured in early pregnancy and/or in cord blood. A subgroup of 380 newborns had mothers with optimal pregnancy dating based on a regular menstrual cycle and a known date of last menstrual period. For comparison, gestational age acceleration was calculated based the method of both Bohlin and Knight. In the total study population, which was more similar to Bohlin's training population, one standard deviation score (SDS) higher maternal plasma homocysteine concentrations was nominally associated with positive gestational age acceleration [0.07 weeks, 95% confidence interval (CI) 0.02, 0.13] by Bohlin's method. In the subgroup with pregnancy dating based on last menstrual period, the method that was also used in Knight's training population, one SDS higher cord serum total and active B12 concentrations were nominally associated with negative gestational age acceleration [(- 0.16 weeks, 95% CI - 0.30, - 0.02) and (- 0.15 weeks, 95% CI - 0.29, - 0.01), respectively] by Knight's method. CONCLUSIONS: We found some evidence to support associations of higher maternal plasma homocysteine concentrations with positive gestational age acceleration, suggesting faster epigenetic than clinical gestational aging. Cord serum vitamin B12 concentrations may be associated with negative gestational age acceleration, indicating slower epigenetic than clinical gestational aging. Future studies could examine whether altered fetal epigenetic aging underlies the associations of circulating homocysteine and vitamin B12 blood concentrations during fetal development with long-term health outcomes.