Vitamin B₁₂ (cobalamin) is an essential cofactor for two metabolic pathways. It is obtained principally from food of animal origin. Cobalamin becomes bioavailable through a series of steps pertaining to its release from dietary protein, intrinsic factor-mediated absorption, haptocorrin or transcobalamin-mediated transport, cellular uptake, and two enzymatic conversions (via methionine synthase and methylmalonyl-CoA-mutase) into cofactor forms: methylcobalamin and adenosylcobalamin. Vitamin B₁₂ deficiency can masquerade as a multitude of illnesses, presenting different perspectives from the point of view of the hematologist, neurologist, gastroenterologist, general physician, or dietician. Increased physician vigilance and heightened patient awareness often account for its early presentation, and testing sometimes occurs during a phase of vitamin B₁₂ insufficiency before the main onset of the disease. The chosen test often depends on its availability rather than on the diagnostic performance and sensitivity to irrelevant factors interfering with vitamin B₁₂ markers. Although serum B₁₂ is still the most commonly used and widely available test, diagnostics by holotranscobalamin, serum methylmalonic acid, and plasma homocysteine measurements have grown in the last several years in routine practice. The lack of a robust absorption test, coupled with compromised sensitivity and specificity of other tests (intrinsic factor and gastric parietal cell antibodies), hinders determination of the cause for depleted B₁₂ status. This can lead to incorrect supplementation regimes and uncertainty regarding later treatment. This review discusses currently available knowledge on vitamin B₁₂, informs the reader about the pitfalls of tests for assessing its deficiency, reviews B₁₂ status in various populations at different disease stages, and provides recommendations for interpretation, treatment, and associated risks. Future directions for diagnostics of B₁₂ status and health interventions are also discussed.

Folate (vitamin B₉) plays a crucial role in fundamental cellular processes, including nucleic acid biosynthesis, methyl group biogenesis and amino acid metabolism. The detection and correction of folate deficiency prevents megaloblastic anaemia and reduces the risk of neural tube defects. Coexisting deficiencies of folate and vitamin B₁₂are associated with cognitive decline, depression and neuropathy. Folate deficiency and excess has also been implicated in some cancers. Excessive exposure to folic acid, a synthetic compound used in supplements and fortified foods, has also been linked to adverse health effects. Of at least three distinct laboratory markers of folate status, it is the total abundance of folate in serum/plasma that is used by the majority of laboratories. The analysis of folate in red cells is also commonly performed. Since the folate content of red cells is fixed during erythropoiesis, this marker is indicative of folate status over the preceding ~4 months. Poor stability, variation in polyglutamate chain length and unreliable extraction from red cells are factors that make the analysis of folate challenging. The clinical use of measuring specific folate species has also been explored. 5-Methyltetrahydrofolate, the main form of folate found in blood, is essential for the vitamin B₁₂-dependent methionine synthase mediated remethylation of homocysteine to methionine. As such, homocysteine measurement reflects cellular folate and vitamin B₁₂use. When interpreting homocysteine results, age, sex and pregnancy, specific reference ranges should be applied. The evaluation of folate status using combined markers of abundance and cellular use has been adopted by some laboratories. In the presence of discordance between laboratory results and strong clinical features of deficiency, treatment should not be delayed. High folate status should be followed up with the assessment of vitamin B₁₂status, a review of previous results and reassessment of folic acid supplementation regime.

Vitamin K is required for the ɣ-carboxylation of specific glutamic acid residues within the Gla domain of the 17 vitamin K-dependent proteins (VKDPs). The timely detection and correction of vitamin K deficiency can protect against bleeding. Vitamin K also plays a role in bone metabolism and vascular calcification. Patients at increased risk of vitamin K deficiency include those with a restricted diet or malnutrition, lipid malabsorption, cancer, renal disease, neonates and the elderly. Coagulation assays such as the prothrombin time have been used erroneously as indicators of vitamin K status, lacking sufficient sensitivity and specificity for this application. The measurement of phylloquinone (K₁) in serum is the most commonly used marker of vitamin K status and reflects abundance of the vitamin. Concentrations <0.15 µg/L are indicative of deficiency. Disadvantages of this approach include exclusion of the other vitamin K homologues and interference from recent dietary intake. The cellular utilisation of vitamin K is determined through measurement of the prevalence of undercarboxylated VKDPs. Most commonly, undercarboxylated prothrombin (Protein Induced by Vitamin K Absence/antagonism, PIVKA-II) is used (reference range 17.4–50.9 mAU/mL (Abbott Architect), providing a retrospective indicator of hepatic vitamin K status. Current clinical applications of PIVKA-II include supporting the diagnosis of vitamin K deficiency bleeding of the newborn, monitoring exposure to vitamin K antagonists, and when used in combination with α-fetoprotein, as a diagnostic marker of hepatocellular carcinoma. Using K₁ and PIVKA-II in tandem is an approach that can be used successfully for many patient cohorts, providing insight into both abundance and utilisation of the vitamin.

Vitamin B ₁₂ (cobalamin; B ₁₂ ) is an essential micronutrient, but deficiency is common. The prompt diagnosis and treatment of B ₁₂ deficiency protects against megaloblastic anaemia, neuropathy and neuropsychiatric changes. Biomarkers of B ₁₂ status include the measurement of serum B ₁₂ (also known as total B ₁₂ or serum cobalamin), holotranscobalamin (holoTC or ‘active B12’), methylmalonic acid (MMA) and total plasma homocysteine (Hcy). There is no ‘gold standard’ test for deficiency and the sensitivity and specificity of each biomarker for the evaluation of B ₁₂ status is affected by analytical and biological factors that may confer a high degree of diagnostic uncertainty. Limited access to technical and clinical expertise can lead to an over-reliance on the serum B ₁₂ test, which is readily available and highly automated. In some cases, the sequential use of different B ₁₂ status biomarkers or the calculation of a composite B ₁₂ status score, derived from a panel of B ₁₂ biomarkers and adjusted for folate status and age, can be used to detect deficient states that may otherwise be overlooked when using a single biomarker approach. This review summarizes the utility of B ₁₂ -related biomarkers and describes approaches to their application and interpretation.

The impact of warfarin therapy on the functions of extrahepatic vitamin K-dependent proteins (VKDP) is less clearly understood and less widely recognised in clinical practice than that on the hepatic counterparts (clotting factors II, VII, IX and X). Warfarin inhibits osteocalcin, an abundant extrahepatic VKDP involved in the mineralisation and maturation of bone and thus, primarily by this mechanism, may have an adverse effect on bone health. Whilst some studies do link warfarin use to an increase in osteoporosis and fracture risk others have not. Warfarin also inhibits the extrahepatic VKDP matrix gla protein (MGP) which acts to prevent ectopic calcification of the vasculature. Studies have consistently found a correlation between warfarin use and vascular calcification with inhibition of MGP believed to be the main cause. Inhibition of MGP also appears to explain warfarin’s well established teratogenic effect. Further adverse effects may also arise from warfarin’s inhibition of other known extrahepatic VKDPs. The available evidence is intriguing, and suggests that the impact of warfarin on the extrahepatic functions of vitamin K-dependent proteins warrants further careful consideration.

Aims: A growing body of evidence suggests that ethnicity and race influence vitamin B ₁₂ metabolism and status yet clinical awareness of this is poor, causing doubts regarding diagnosis and treatment. Moreover, deficiency and insufficiency cut-offs are universally applied for this test in most diagnostic settings. The objective of this study was to assess serum vitamin B ₁₂ concentrations in Black, Asian and White primary care patients in London, UK, particularly in patients of Black or Black British ethnic origin and establish if there is a need for specific reference ranges. Methods: Serum B ₁₂ results from 49 414 patients were processed between January 2018 and November 2019 using the Architect assay (Abbott Diagnostics) at St. Thomas’ Hospital, London, UK. Age, sex and ethnicity data were collected from the laboratory Health Informatics Team. Results: Black patients (n=13 806) were found to have significantly higher serum vitamin B ₁₂ concentration across all age groups and both sexes, especially Nigerian patients (median B ₁₂ 505 pmol/L,IQR: 362–727, n=891), compared with Asian and White ethnic groups (p652 pmol/L) (adjusted OR 3.38, 95% CI 3.17 to 3.61, p<0.0001). Conclusions: It is likely that a combination of genetic and acquired/environmental factors are responsible for the ethnic differences in serum B ₁₂ . This suggests that there is a need for ethnic-specific reference ranges with indications for the incorporation of age and sex too.