Vitamin B12 deficiency: testing and treatment.

Background: Serum Vitamin B12 level is routinely analyzed using total Vitamin B12 assay. It can also be analyzed by active B12 assay. In this study, we compared the association of total and active Vitamin B12 levels with maternal medical complications associated with Vitamin B12 in pregnancy. Materials and Methods: Blood sera of 217 pregnant women in their first trimester were included to conduct this retrospective study. The levels of active B12 and total B12 were analyzed by Roche cobas e 801. The distribution of active and total B12 among the anemic groups was analyzed using the Mann–Whitney U -test. Categorical associations of complications with active and total B12 were done using Chi-square statistics. The discriminating ability of active and total B12 was analyzed by the receiver operating characteristic (ROC) curve. Results: The hemoglobin levels and the maternal medical complications associated with Vitamin B12, in the total and active Vitamin B12-deficient groups were found to be similar when compared to the corresponding nondeficient groups. There is a positive correlation between total and active Vitamin B12 assay ( r = 0.52, P < 0.01). The predictive ability of total and active B12 for both neonatal and maternal outcomes assessed using the ROC curve reported an AUC <0.70. Conclusions: Total B12 assay was strongly positively correlated with active B12 assay. However, neither of these predicted maternal medical complications or neonatal complications associated with B12 deficiency. Therefore, we suggest a continuation of the standard practice of total Vitamin B12 assay over active B12 assay for the estimation of B12 deficiency in pregnancy especially in low- and middle-income countries like India.

Utility and Patterns of Vitamin B12 and Folate Testing in Patients with Isolated Thrombocytopenia

Dear Sir, I appreciated the paper by Scarpa et al.1 on vitamin B12 deficiency in subjects with apparent normal serum levels and the comment by Cosar et al.2, also published in Blood Transfusion. Both authors correctly mentioned the potential relevance of low tissue levels and/or functional deficiencies and highlighted the importance of determining the levels of plasma total homocysteine (tHcY) and also methylmalonic acid, which is considered the “reference” indicator in order to evaluate the complete metabolic pattern of B12. As the difficulties of performing and interpreting the latter assay are well known2, an alternative way to ascertain the presence of subclinical or clinical deficiency of vitamin B12 is to determine the amount of its active fraction, holotranscobalamin (HoloTC, or active B12) i.e. the cobalamin–transcobalamin II complex released into the portal circulation and recognised by ubiquitous specific receptors. HoloTC is the biologically active form of vitamin B12 and represents only a fraction (10–30%) of total circulating vitamin B12, while the remaining fraction is bound to haptocorrin3 and is not metabolically active. The two circulating forms of vitamin B12 have complementary clinical significance: while it has been reported that a relevant fraction of individuals whose total B12 concentration is low show no clinical or biochemical evidence of cobalamin deficiency, it has also been shown that both neurological and metabolic abnormalities may be present when circulating levels of total B12 are within the normal range4 when active B12 levels are low. Furthermore, studies carried out in populations with supposedly low dietary intake of vitamin B12, such as vegetarians, vegans and elderly people, have indicated that HoloTC may be considered as an earlier indicator of vitamin B12 deficiency, and that its levels are modified quite rapidly after adequate dietary intake or supplementation of vitamin B123. From an analytical standpoint the correlation between the two forms of B12 is not linear; for example, when holoTC concentration was measured in 250 selected serum specimens from patients with low-intermediate levels (<221 pmol/L) of total B12, only a weak correlation (r2=0.420) was found between the two parameters and no correlation between holoTC and other metabolically correlated parameters (serum folate, total homocysteine and creatinine)5. The importance of establishing a correct cut-point, or cut-off, to be used to diagnose a status of active B12 deficiency stems from the following observations: in the already mentioned study by Bamonti et al.5, the optimal threshold for HoloTC in subjects with suboptimal total B12 values was estimated to be 40 pmol/L (maximum phi correlation) and this threshold was verified by receiver operating characteristic curve analysis to have a sensitivity of 0.86 and a specificity of 0.66. It is of note that HoloTC values and the estimated cut-off were not affected by gender or age (p=0.54 and p=0.30, respectively) and that the area under the curve showed a better predictive ability for vitamin B12 deficiency through HoloTC determination than other “classical” cobalamin deficiency predictors (serum folate and total homocysteine). Thus, HoloTC concentrations, in addition to total circulating B12 levels, may guarantee a more accurate evaluation of the metabolic status of cobalamin4,5.

e20639 Purpose: Chemotherapy-induced damage to the stomach and small intestine may inhibit the absorption of vitamin B12. To assess the risk of chemotherapy causing acute vitamin B12 deficiency, we primarily measured the active form of vitamin B12, holotranscobalamin, but also total serum B12, methylmalonic acid, and total homocysteine. Experimental Design: We studied 21 patients that were actively on chemotherapy and recorded values for holotranscobalamin, total serum B12, methylmalonic acid, total homocysteine, and cystatin-c. Measurements were taken both before and after four doses of chemotherapy. T-tests were employed to determine statistical significance. Results: There was a statistically significant drop in holotranscobalamin after chemotherapy in eighteen out of twenty-one patients (p = 2.64x10–3). Three patients were vitamin B12 deficient as defined by total B12 &lt; 300 pg/ml. Neither methylmalonic acid (p = 3.95x10–1), nor total homocysteine (p = 4.34x10–1), showed any significant changes. Conclusions: Chemotherapy caused an acute deficiency of the only metabolically active form of vitamin B12, holotranscobalamin, despite B12 supplementation. We suggest monitoring patients for changes in holotranscobalamin to ensure that prolonged deficiency does not occur. The clinical implications of acute lowering of holotranscobalamin remain unknown. No significant financial relationships to disclose.

Vitamin B12 deficiency is a serious disorder that can lead to severe neurological symptoms, especially if not detected and treated effectively. Nutritional deficiency due to dietary restrictions, deranged metabolism, and subsequent vitamin loss during dialysis are important causes of vitamin B12 deficiency in CKD patients. Hyperhomocysteinemia, a complication of vitamin B12 deficiency, has grown as an important risk factor for cardiovascular disease and the leading cause of mortality in patients with CKD. Serum samples were randomly selected from 124 patients (46 females, 78 males; age range 18-65 years) referred to the Dept. of Biochemistry, Institute of Nephrourology, Bangalore, India for the assessment of vitamin B12 status. For each patient, serum total vitamin B12 level and active B12 (holoTC) level were determined by chemiluminescent microparticle immunoassay on Architect ci1000 analyzer. Out of the total 124 patients, 17 CKD patients were excluded from the study, and in the remaining 107 patients, 13.08% showed a deficiency of both Total vitamin B12 and Active B12. In the 107 patients, the mean total vitamin B12 level was 604.85 ± 495.2 pg/mL, and the mean Active B12 level (holoTC) was 67.1 ± 32.75 pmol/L, with a strong positive correlation (=0.501, &amp;#60;0.01) between total B12 and active B12 levels. A significant deficient level of B12 was found in the patients on hemodialysis for more than three years.: Active B12 can aid vitamin B12 measurements for diagnosis of B12 deficiency and can be a potential indicator of B12 deficiency in patients with CKD.

Serum concentrations of homocysteine (Hcy) and methylmalonic acid (MMA) become increased in B12-deficient subjects and are therefore, considered specific markers of B12 deficiency. Serum level of holotranscobalamin (holoTC) becomes decreased before the development of the metabolic dysfunction. We investigated the usefulness of holoTC in diagnosing B12 deficiency in some clinical settings. We measured serum concentrations of holoTC, MMA, Hcy and total B12 in omnivores, vegetarians, elderly people and haemodialysis patients. Our results indicated that the incidence of holoTC <35 pmol/L was highest in the vegans (76%). Low holoTC and elevated MMA were detected in 64% of the vegans and 43% of the lacto- and lacto-ovovegetarians. An elevated MMA and a low holoTC were found in subjects with total serum B12 as high as 300 pmol/L. The distribution of holoTC in elderly people was similar to that in younger adults (median holoTC 55 pmol/L in both groups). A low holoTC and an elevated MMA were found in 16% of the elderly group. An elevated MMA and a normal holoTC were found in 20% of the elderly group who had a relatively high median serum concentration of creatinine (106.1 micromol/L). Serum concentrations of holoTC in dialysis patients were considerably higher than all other groups (median 100 pmol/L). This was also associated with severely increased serum levels of MMA (median 987 nmol/L). From these results it can be concluded that serum concentration of holoTC is a much better predictor of B12 status than total B12. This was particularly evident in case of dietary B12 deficiency. Serum concentrations of holoTC as well as MMA can be affected by renal dysfunction. Elevated MMA and normal holoTC in patients with renal insufficiency may not exclude vitamin B12 deficiency. HoloTC seems not to be a promising marker in predicting B12 status in renal patients.

Effect of supplementation with folic-acid on relation between plasma homocysteine, folate, and vitamin B12.

Vitamin B12 Intake and Related Biomarkers: Associations in a Dutch Elderly Population.

BackgroundMegaloblastic anemia is characterized by abnormally large red blood cells caused by a deficiency in either vitamin B12 or folic acid, both of which are essential for DNA synthesis. Vitamin B12 insufficiency can lead to severe neurological damage, making early identification of vitamin B12 deficiency crucial to prevent irreversible harm. Vitamin B12 deficiency results in decreased levels of holotranscobalamin (Holo-TC) and increased levels of methylmalonic acid (MMA). Methylmalonic acid is considered the gold standard for diagnosing B12 deficiency because it is a specific marker that rises when B12 is insufficient, even when serum B12 levels appear normal. Elevated MMA levels reflect impaired B12 metabolism, making it a critical tool for early detection and intervention. Previous research indicates that Holo-TC, the active form of vitamin B12 available to cells, is a more specific diagnostic tool for early vitamin B12 deficiency than total B12. This study aims to determine the diagnostic validity of total vitamin B12 and Holo-TC using MMA as the gold standard in patients with megaloblastic anemia.MethodsA total of 95 megaloblastic anemia patients were selected from Jinnah Hospital and Lahore General Hospital, Lahore, Pakistan, after receiving approval from the ethical review committees. This was a cross-sectional study. Whole blood, serum, and urine samples were collected in ethylenediamine tetraacetic acid (EDTA) vials, gel vials, and urine containers, respectively. The EDTA samples were used for complete blood count measurements using a hematology analyzer (Sysmex-XT 1800i, Sysmex America, Inc., Mundelein, IL), while serum and urine samples were employed for the detection of serum folic acid, cobalamin, Holo-TC, and MMA levels through manual enzyme-linked immunosorbent assay (ELISA) techniques. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of Holo-TC and cobalamin were calculated.ResultsThe majority of patients fell within the age group of 20-70 years, with 57% of them being females and 43% males; Holo-TC exhibited a sensitivity of 98.9% and specificity of 50.00%, with a PPV of 98.90% and NPV of 50%. Vitamin B12 demonstrated a sensitivity of 63% and specificity of 50%, with a PPV of 98.33% and NPV of 2.85%. The diagnostic accuracy of Holo-TC and vitamin B12 was observed to be 97.8% and 63%, respectively. ConclusionsBetween the two, Holo-TC displays higher diagnostic accuracy than vitamin B12 and can serve as the primary test for patients suspected of having vitamin B12 deficiency.

Vitamin B₁₂Deficiency and Depression in the Elderly

To study the correlations between total vitamin B12(B12), holo-haptocorrin, and holo-transcobalamin II (holo-TCII) concentrations in human sera; the association between reduced holo-TCII concentrations and macrocytosis attributable to B12 deficiency. Serum samples from 38 healthy volunteers, 113 patients with normal total serum B12 concentrations and 93 patients with low total serum B12 were studied. Holo-TCII was removed from whole serum by adsorption with amorphous precipitated silica, and both whole serum and adsorbed serum were assayed for B12 using the Becton Dickinson vitamin B12 [57Co] radioassay kit. In all three groups of subjects studied there were strong correlations between the logarithms of the total serum B12 and the holo-haptocorrin concentrations with regression coefficients between 0.884 and 0.967. By contrast, the correlations between the logarithms of the total serum B12 and holo-TCII concentrations were weaker, especially in the patients with normal or low total serum B12, for whom the regression coefficients were 0.491 and 0.391, respectively. Analysis of the clinical records of a proportion of the patients studied indicated that there were many more patients with low holo-TCII concentrations than with haematological disturbances related to B12 deficiency. The total serum B12 concentration is a relatively poor indicator of holo-TCII concentrations and, therefore, of the ability of serum to deliver B12 to tissues. Additional information regarding B12 values can therefore be gleaned from measuring holo-TCII concentrations in the serum. Low holo-TCII concentrations, however, are an early sign of negative B12 balance and are frequently unassociated with haematological abnormalities caused by B12 deficiency.

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. Our study aimed to evaluate the Roche Elecsys® AVB12 immunoassay performance versus Roche Elecsys® TVB12 competition assay. 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. 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%. 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.

Metformin is the most commonly prescribed oral treatment for type 2 diabetes mellitus (T2DM) in the UK. Long-term therapy has been linked to vitamin B12 deficiency, a concern recognised for decades but not consistently addressed. In June 2022, the UK Medicines and Healthcare products Regulatory Agency (MHRA) classified low vitamin B12 levels as a common adverse effect of metformin and advised clinicians to consider periodic testing in at-risk patients. Translating such regulatory advice into practice can be challenging, and the extent to which the guidance has influenced testing and diagnostic coding for vitamin B12 deficiency remains unclear. This study evaluated trends in vitamin B12 testing and deficiency coding in metformin-treated patients compared with the general population before and after the 2022 MHRA Drug Safety Update. A retrospective quantitative analysis was conducted using Read code data from 148,000 electronic medical records across three Primary Care Networks (PCNs) in the Southwest of England. Vitamin B12 testing and deficiency coding rates were compared in patients prescribed metformin and the general population across two periods: pre-guidance (2017-2021) and post-guidance (2022-2024). Welch's t-tests were used to determine statistical significance, with p < 0.05 considered significant. Among patients prescribed metformin, vitamin B12 testing rates rose from 34.5% (SD = 1.8) pre-guidance to 38.2% (SD = 0.4) post-guidance (p = 0.008). In the general population, testing rates increased from 12.2% to 14.7% (p = 0.009). However, coding for vitamin B12 deficiency remained unchanged at 0.25% in the metformin group and decreased from 0.072% to 0.060% in the general population, with no statistically significant difference. The post-guidance period included only two years of data, which limits the ability to assess longer-term or comparative trends between groups. This study demonstrated that the release of the MHRA Drug Safety Update was associated with a modest but statistically significant increase in vitamin B12 testing among patients prescribed metformin, paralleled by a smaller rise in testing within the general population. However, diagnostic coding practices did not change, suggesting limited translation of safety alerts into structured documentation. Further research is warranted to explore barriers and evaluate interventions to improve monitoring and coding compliance in primary care.

Measurement of Vitamin B12 and Serum Methylmalonic Acid Levels: Role of Stepwise Cascade Testing in Diagnosing Vitamin B12 Deficiency

Vitamin B12 (cobalamin, Cbl, B12) is an indispensable water-soluble micronutrient that serves as a coenzyme for cytosolic methionine synthase (MS) and mitochondrial methylmalonyl-CoA mutase (MCM). Deficiency of Cbl, whether nutritional or due to inborn errors of Cbl metabolism, inactivate MS and MCM leading to the accumulation of homocysteine (Hcy) and methylmalonic acid (MMA), respectively. In conjunction with total B12 and its bioactive protein-bound form, holo-transcobalamin (holo-TC), Hcy, and MMA are the preferred serum biomarkers utilized to determine B12 status. Clinically, vitamin B12 deficiency leads to neurological deterioration and megaloblastic anemia, and, if left untreated, to death. Subclinical vitamin B12 deficiency (usually defined as a total serum B12 of <200 pmol/L) presents asymptomatically or with rather subtle generic symptoms that oftentimes are mistakenly ascribed to unrelated disorders. Numerous studies have now established that serum vitamin B12 has limited diagnostic value as a stand-alone marker. Low serum levels of vitamin B12 not always represent deficiency, and likewise, severe functional deficiency of the micronutrient has been documented in the presence of normal and even high levels of serum vitamin B12. This review discusses the usefulness and limitations of current biomarkers of B12 status in newborn screening, infant and adult diagnostics, the algorithms utilized to diagnose B12 deficiency and unusual findings of vitamin B12 status in various human disorders.