Diagnostic algorithms for the investigation of disorders of vitamin B₁₂: clinical practice review

Introduction

Background

Cobalamin (vitamin B₁₂) is an essential cofactor in two key metabolic pathways (1). Deficiency can cause diverse clinical symptoms and can sometimes result in irreversible disability if not treated promptly (2). In general, these patients will often first present to primary care (3). Clinical features can be very non-specific (3), and first-line laboratory tests may not always reflect the true clinical state. Additional follow-up testing is therefore frequently required (4), and as a result, investigation and diagnosis can be challenging.

Rationale and knowledge gap

Whilst the use of vitamin B₁₂ markers and algorithms has been reviewed previously (5), investigation of vitamin B₁₂ deficiency remains complex in clinical practice. Standardised diagnostic pathways may not apply to all patient groups, and guidance on alternative investigative strategies is limited. Unexpectedly raised vitamin B₁₂ concentrations are also not an uncommon finding and can present a diagnostic puzzle, yet are less frequently addressed in existing reviews.

Objective

This clinical practice review summarises current knowledge on how to undertake investigations into vitamin B₁₂ deficiency and presents diagnostic algorithms to support clinicians. This review is intended as a practical clinical resource for general practitioners and other primary care clinicians. We also highlight common situations where standardised pathways may not apply, outlining alternative strategies that may be required in these groups of patients. We also discuss the situation of unexpectedly raised vitamin B₁₂ concentrations, as this is also not an uncommon finding, and can be a diagnostic puzzle.

History

The essential nature of vitamin B₁₂ was first described in 1926, when George Whipple, George Minot, and William Murphy showed that a liver-rich diet could treat pernicious anaemia (PA), a historically fatal condition (6). This ‘anti-pernicious anaemia factor’ was later identified as vitamin B₁₂ by Karl Folkers and Earnest Lester Smith in 1948 (1). Eventually, its structure was fully elucidated by Dorothy Hodgkin in 1956 using X-ray crystallography, and its roles in DNA synthesis and neuronal function were further realised (7).

Cobalamin biochemistry

Vitamin B₁₂ is a structurally complex vitamin which is characterized by a central cobalt atom and a unique metal-carbon bond that plays a role in enzymatic catalysis (8). Its two biologically active forms, methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl), act as cofactors in two key metabolic reactions (1).

MeCbl serves as the cofactor for methionine synthase, which catalyses the formation of methionine from homocysteine (Figure 1). In this reaction, 5-methyltetrahydrofolate is converted back to tetrahydrofolate (THF), which re-enters the folate cycle, ultimately contributing to DNA synthesis (10). Methionine is then converted to S-adenosyl-L-methionine, which acts as a universal methyl donor in methylation reactions involving DNA, proteins, and other cellular molecules (11).

Figure 1 A diagram illustrating the role of MeCbl as a cofactor for methionine synthase in the formation of methionine. Created in BioRender (9). MeCbl, methylcobalamin.

AdoCbl acts as a cofactor for methylmalonyl-coenzyme A (CoA) mutase, which converts methylmalonyl-CoA from the metabolism of branched-chain amino acids, fatty acids, and cholesterol to succinyl-CoA, which can then enter the tricarboxylic acid (TCA) cycle (Figure 2) (12).

Figure 2 A diagram illustrating the role of AdoCbl as a cofactor for methylmalonyl-CoA mutase in the conversion of methymalonyl-CoA to succinyl-CoA. Created in BioRender (9). AdoCbl, adenosylcobalamin; CoA, coenzyme A; TCA, tricarboxylic acid.

Vitamin B₁₂ also has antioxidant effects, including scavenging of reactive oxygen species, preserving glutathione, and altering cytokines involved in oxidative stress (11,13,14).

Although vitamin B₁₂ is synthesised by gut bacteria and functions as a cofactor for over a dozen bacterial enzymes (15), the endogenously produced vitamin in the human colon is not available for absorption, since uptake occurs in the ileum (16). As a result, humans rely on dietary vitamin B₁₂ and have evolved complex absorption pathways involving binding proteins and receptors to ensure its efficient uptake from the diet (1).

Absorption

Ingested vitamin B₁₂ is bound to food proteins, and upon reaching the stomach, gastric acidity and pepsin release cobalamin from these proteins (Figure 3) (1). Haptocorrin (also known as R-protein or R-binder) is secreted in saliva and present in gastric juice and binds to the liberated cobalamin, which protects it from acidic degradation (1). In the small intestine, pancreatic proteases degrade the haptocorrin, releasing free cobalamin once more (1). Intrinsic factor (IF), secreted by gastric parietal cells (GPCs), binds this free vitamin to form the IF-cobalamin complex (1). This process is pH dependent, with haptocorrin binding being most efficient at the acidic pH of the stomach, whereas the relatively higher pH of the small intestine facilitates the preferential binding of vitamin B₁₂ to IF (1). The B₁₂-IF complex is then absorbed in the distal ileum via the cubam receptor complex (cubilin-amnionless) (1,17). Within enterocytes, cobalamin is released into the circulation. Circulating cobalamin is bound to both transcobalamin for systemic delivery to cells and bound to haptocorrin, serving mainly as a storage pool (1).

Figure 3 A diagram illustrating the absorption pathway of B₁₂ in humans. B₁₂ is released from dietary protein in the stomach where it then binds with haptocorrin. In the small intestine, pancreatic proteases release cobalamin from haptocorrin allowing it to bond to IF for absorption in the terminal ileum. In the enterocyte cobalamin then binds to transcobalamin II for delivery to cells. Created in BioRender (9). IF, intrinsic factor.

Global prevalence

The prevalence of vitamin B₁₂ deficiency differs markedly across populations. It is affected by age, diet, and geography, and is complicated by varying biomarker thresholds (18). National surveys in higher income countries such as the USA and UK show that deficiency is relatively rare in younger adults but increases with age, affecting approximately 5% of adults by age 70 years and >10% of those over 75 years (18,19). In contrast, deficiency is far more prevalent in low- and middle-income countries, at least in part driven by a reliance on plant-based diets with limited intake of animal products (20,21). In Mexico, a large national survey reported that more than 45% of women and children had either deficient or marginal vitamin B₁₂ status (22). In sub-Saharan Africa, studies in Kenyan schoolchildren reported deficiency in 40–70% depending on the cohort (23,24), while in India, 67% of adult men, and 81% in an urban subgroup, were deficient (20).

Vitamin B₁₂ deficiency is therefore uncommon in younger adults in wealthy nations but rises with age, while much higher burdens persist in resource-limited settings. Both population diet and methodological differences in defining deficiency contribute to the wide variation observed between studies. In particular, commonly used diagnostic thresholds for serum vitamin B₁₂ vary substantially, with different cut-offs applied across studies and guidelines, typically ranging from approximately 148 pmol/L to ~200 pmol/L. Similar variability exists for functional biomarkers, with reported methylmalonic acid (MMA) cut-offs ranging from ~210 to 480 nmol/L. These differences markedly influence prevalence estimates and limit direct comparability between studies (18).

Signs and symptoms of vitamin B₁₂ deficiency

Historically, vitamin B₁₂ deficiency was considered a haematological disorder. Neurological symptoms are now commonly recognised as being caused by vitamin B₁₂ deficiency, and patients may present with anaemia, neurological disease, or both. Importantly, not every symptom may be present in an individual with vitamin B₁₂ deficiency (25).

The haematological effects of vitamin B₁₂ deficiency have been covered extensively by others (26,27), but the defective DNA synthesis that occurs as a result of vitamin B₁₂ deficiency can impair haematopoiesis, leading to the development of a megaloblastic anaemia with the presence of macrocytic red blood cells. Pancytopenia and hypersegmented neutrophils are other haematological features that may be noted (26,28). Symptoms of anaemia associated with vitamin B₁₂ deficiency can include weakness and fatigue, shortness of breath, and exercise intolerance (29).

Neurological disease associated with vitamin B₁₂ deficiency can be diverse. Symmetrical paraesthesia, typically occurring in the hands or feet and often described as burning or tingling (30), is a commonly reported symptom. Loss of vibration sense and ataxia have also been reported and may be associated with a positive Romberg sign (poor balance when eyes are closed) (30). Lhermitte’s phenomenon (tingling or buzzing radiating down the spine) has also been reported (31) in addition to peripheral neuropathy and motor weakness (30).

Severe neurological consequences include subacute combined degeneration of the spinal cord, which can occur with both chronic, untreated vitamin B₁₂ deficiency as well as acutely after nitrous oxide-induced inactivation of cobalamin (32). This can cause spasticity and gait disturbance (33), and bladder or bowel dysfunction (34).

Rarer clinical manifestations include optic neuropathy with vision loss and central scotoma (35), tinnitus and hearing changes (36), and psychiatric effects such as hallucinations (37), catatonia, and delirium (38). Vitamin B₁₂ deficiency can also produce cognitive deficits, which in older adults, or those with diagnosed neurodegenerative disease, may be misattributed to primary dementia (39).

Risk factors/causes

Vegan, vegetarian, and plant-based diets

Dietary vitamin B₁₂ is provided almost exclusively by animal-derived foods such as meat, milk, eggs, and fish, since plant foods do not naturally contain bioactive cobalamin. Consequently, individuals consuming diets low in or devoid of animal products are at increased risk of vitamin B₁₂ deficiency unless they use fortified foods or supplements (40). Global prevalence of reduced meat diets is on the increase as this is seen to be both a healthy lifestyle choice and a way to minimise human environmental impact (41). A large meta-analysis of vitamin B₁₂ deficiency among those who follow plant-based diets identified that whilst vegans’ vitamin B₁₂ intake is lower than that of vegetarians and lacto-ovo-vegetarians, vegans are more likely to be aware of the risks of vitamin B₁₂ deficiency and take supplements compared with vegetarians (42). It is therefore of importance when taking a clinical history in patients suspected of vitamin B₁₂ deficiency, to consider these dietary factors as a potential cause.

PA/autoimmune gastritis

PA is an end-stage complication of autoimmune gastritis and is the most common cause of vitamin B₁₂ deficiency throughout the world, with prevalence increasing with age (43,44). Chronic gastritis ultimately leads to the destruction of GPCs, which are the producers of hydrochloric acid and IF and are located primarily in the fundus and body of the stomach (45). Pathogenesis of PA is typically autoreactive CD4⁺ T cell-mediated. Studies have shown that pro-inflammatory TH1 CD4 T cells directed against gastric H⁺/K⁺ ATPase are present in the gastric mucosa of patients with PA (46). The reduction in GPCs leads to a deficiency in IF (47), which in turn results in dietary vitamin B₁₂ malabsorption and therefore leads to clinical consequences such as megaloblastic anaemia (48).

Generalised malabsorption

In addition to autoimmune gastritis, any other condition leading to generalised malabsorption can lead to vitamin B₁₂ deficiency. A very common cause of malabsorption leading to vitamin B₁₂ deficiency is coeliac disease (49). Other causes of malabsorption, which can lead to deficiencies in vitamin B₁₂, include Crohn’s disease and other inflammatory bowel conditions, with the prevalence further increasing if the patients have undergone resection of the terminal ileum (50).

There is also a significant risk in those patients who have undergone bariatric surgery, in particular Roux-en-Y gastric bypass surgery (51). Other forms of bariatric surgery still carry risks, even gastric banding, but this is less common (52). Typically, patients undergoing these surgeries will be advised to take supplementation post-surgery (53).

Older adults

Vitamin B₁₂ deficiency is more common in older adults, reflecting both age-related physiological changes and medication effects (18). The first step of vitamin B₁₂ absorption is gastric acid- and pepsin-mediated release of cobalamin from food proteins (Figure 3) (1). Achlorhydria, which becomes more prevalent with advancing age, impairs this process and is associated with reduced circulating vitamin B₁₂ levels and a higher risk of clinical deficiency (54). Polypharmacy is also common in this population, including the use of medications that can impair vitamin B₁₂ absorption (55).

Medications associated with alterations in vitamin B₁₂

Proton pump inhibitors (PPIs) and histamine-2 receptor antagonists (H₂RAs), both widely used for dyspepsia and gastro-oesophageal reflux disease, are associated with an increased risk of vitamin B₁₂ deficiency (56,57). By lowering gastric acidity, these drugs interfere with the first step of absorption, which is the release of vitamin B₁₂ from food proteins. This results in food-bound cobalamin malabsorption. Metformin has also been linked to vitamin B₁₂ deficiency (58). The most likely mechanism is interference with the calcium-dependent uptake of the IF—cobalamin complex by the cubilin—amnionless receptor in the terminal ileum, and calcium supplementation has been shown to reverse this effect (59).

Vitamin B₁₂ and human immunodeficiency virus (HIV)

The relationship between vitamin B₁₂ and HIV infection is complex. Well-treated HIV likely has a limited impact on vitamin B₁₂ and its biochemical markers (60); however, in poorly controlled or uncontrolled HIV, assessment of vitamin B₁₂ status becomes more complicated. In untreated patients, total vitamin B₁₂ decreases proportionally to decreases in CD4 counts (61). One of the major sources of haptocorrin production is immune cells, and in immunodeficient states, haptocorrin can be decreased. This may lead to falsely low total vitamin B₁₂ results (62), prompting unnecessary follow-up functional testing. Conversely, because haptocorrin is also an acute-phase protein, its synthesis may increase in systemic inflammation, complicating interpretation (25). HIV patients, therefore, present a diagnostic challenge. In patients with HIV, therefore, the use of holotranscobalamin (holoTC) instead of total vitamin B₁₂ may provide additional diagnostic benefit (25).

Nitrous oxide use

Nitrous oxide is a colourless gas that has been used both therapeutically and recreationally. Recreational use is particularly prevalent amongst young people, and its use may be increasing (63). Nitrous oxide oxidises cob(I)alamin (Co¹⁺), the transient reduced intermediate in the methionine synthase cycle, to cob(III)alamin (Co³⁺). In this oxidised state, the cofactor can no longer participate in methyl transfer, resulting in inactivation of methionine synthase (Figure 1), and the oxidised cobalamin is effectively lost from the functional pool (64). This produces a rapid functional block in one-carbon metabolism (25). Clinically, nitrous oxide exposure can precipitate acute neurological manifestations of functional vitamin B₁₂ deficiency and, with chronic use, increases the risk of true deficiency (63).

Vitamin B₁₂ and folate interactions

Vitamin B₁₂ and folate metabolism intersect at the enzyme methionine synthase (Figure 1). In states of vitamin B₁₂ deficiency, its activity is impaired, leading to the “methyl trap” of folate in the form of 5-methyl-THF, which produces a secondary functional folate deficiency (26). Both vitamin deficiencies therefore result in indistinguishable haematological findings, most notably megaloblastic anaemia (4,26). Clinically, it is important to distinguish them apart, as folate supplementation may correct the anaemia while allowing irreversible neurological damage from unrecognised vitamin B₁₂ deficiency to progress (25,26). Observational evidence suggests that folate supplementation in the setting of vitamin B₁₂ deficiency may exacerbate neurological injury. One proposed mechanism is that increased folate availability promotes erythropoiesis, increasing bone-marrow demand for vitamin B₁₂ and diverting limited B₁₂ away from the brain; additionally, high folate intake may increase urinary excretion of vitamin B₁₂, further reducing its availability to neural tissue (65). For this reason, it is critical to identify and treat vitamin B₁₂ deficiency whenever folate deficiency is suspected or confirmed.

Assessment of B₁₂ status

Serum total vitamin B₁₂ levels

Serum total vitamin B₁₂ is usually measured with automated competitive binding protein assays, which use purified IF as the capture molecule. The sample is first pre-treated with cyanide, which denatures binding proteins, liberates vitamin B₁₂, and converts the various circulating forms of cobalamin to stable cyanocobalamin, which provides a common analyte for consistent quantification (25).

Total serum cobalamin assays do have a significant limitation, however, as these assays measure all circulating vitamin B₁₂, which includes both biologically active holoTC (cobalamin bound to transcobalamin) and the storage haptocorrin-bound forms. Only holoTC is available for cellular uptake, so total vitamin B₁₂ levels can be misleading (25).

While a very low total serum vitamin B₁₂ level (<150 pmol/L) is strongly suggestive of deficiency, there is a substantial “grey zone” (typically 150–300 pmol/L) where deficiency cannot be excluded. Population reference ranges often show a skewed distribution toward the lower end, meaning that up to one-third of individuals in some cohorts fall into this diagnostic uncertainty zone (3,4,25).

HoloTC “active B₁₂” assays

holoTC, often referred to as “active B₁₂”, represents the fraction of cobalamin which is bound to transcobalamin and therefore available for cellular uptake by tissues throughout the body. In contrast, haptocorrin-bound cobalamin is largely taken up by the liver. Unlike total vitamin B₁₂, holoTC reflects only the biologically relevant pool and is not confounded by haptocorrin-bound cobalamin (25). Measurement of holoTC has been proposed as a more sensitive marker of early deficiency; however, comparative studies show inconsistent results, with many studies only showing modest improvements in diagnostic performance (18,25).

Functional markers

An alternative to direct measurement of cobalamin is the use of functional markers to look at disturbances in the metabolic pathways in which vitamin B₁₂ is utilised.

Homocysteine

As already discussed, cobalamin is an essential cofactor for methionine synthase and the remethylation of homocysteine to methionine (Figure 1). Deficiency of cobalamin impairs this pathway and leads to elevated circulating homocysteine concentrations.

Although homocysteine is a sensitive marker for vitamin B₁₂ deficiency, it has relatively low specificity. Several other conditions can also cause elevated homocysteine, most significantly folate deficiency (66). Additional causes of elevated homocysteine include renal impairment, hypothyroidism, heart failure, and certain inherited metabolic disorders (66). Because of this, homocysteine is best used as a supportive marker (25).

Homocysteine can be measured by several techniques, most commonly immunoassays or liquid chromatography-mass spectrometry (LC-MS) (66).

MMA

MMA is formed during the breakdown of odd-chain fatty acids, certain branched-chain amino acids, propionate and cholesterol to form propionyl-CoA. It is then converted to succinyl-CoA by methylmalonyl-CoA mutase and this requires AdoCbl as a cofactor (Figure 2). In vitamin B₁₂ deficiency states, this reaction is impaired, resulting in accumulation of MMA in serum and urine. MMA is considered the more specific functional marker of vitamin B₁₂ deficiency compared to homocysteine (25). MMA, however is not without confounders, as renal impairment can elevate concentrations independently of vitamin B₁₂ status due to reduced excretion (67). Like homocysteine, several methods have been proposed for measurement, with LC-MS being the most widely used in clinical laboratories (25).

Analytical and pre-analytical considerations for vitamin B₁₂ testing

All current assays for vitamin B₁₂ have a few methodological limitations. Binding protein-based assays have been shown to be susceptible to interference from anti-IF antibodies (68), high-dose biotin supplementation (69), and macro-vitamin B₁₂ complexes (70), all of which can produce spurious results. Assays may also be poorly standardised between manufacturers, and data from external quality assessment schemes have consistently highlighted substantial inter-assay variability (25). As a result, reference intervals and diagnostic cut-offs are method-specific.

If carrying out functional marker testing, sample handling must be considered. Homocysteine metabolism continues within whole blood samples that are stored at room temperature. Therefore, prompt separation of plasma/serum from red blood cells is required to prevent an artefactual rise in homocysteine (71). Serum samples are particularly sensitive as they require a period of time at room temperature to allow the sample to clot. MMA is generally considered more stable and is less sensitive to delayed separation (25). Beyond these analytical issues, a major challenge remains the absence of harmonisation across assays.

Further investigations

Once vitamin B₁₂ deficiency has been confirmed biochemically, further investigation to identify the underlying cause may be required. This serves two key purposes as it can influence the choice of treatment (oral vs. intramuscular) and it may reveal an associated condition that requires management beyond simple correction of the vitamin B₁₂ deficiency (4).

IF/GPC antibodies

Testing for antibodies associated with PA is recommended to determine the underlying cause of the deficiency. National Institute for Health and Care Excellence (NICE) guideline titled ‘Vitamin B₁₂ deficiency in over 16s: diagnosis and management’ (NG239) recommends testing for IF antibodies as a first line and then if negative and suspicion remains, further testing for GPC antibodies (4). Identifying the underlying cause will guide treatment options (72). Antibodies are detected against IF in 70% of cases of PA (73,74) and target either the binding site of cobalamin to IF or the site at which IF binds the ileal mucosa. Antibodies to GPCs target the H⁺/K⁺ ATPase (proton pump) and are present in 80–90% of people with PA (44). However, on progression of the disease and subsequent loss of GPCs, incidence of GPC antibodies may decrease (75). It is proposed that GPC antibodies are present in earlier stages of disease (76) and may precede symptomatic disease (77).

Due to the fact that none of these tests have 100% sensitivity for autoimmune gastritis/PA, diagnosis usually relies on a combination of clinical, serological, and histological findings (78).

Both of the target antibodies can be identified in the laboratory using enzyme-linked immunoassay, indirect immunofluorescent techniques, or radioimmunoassay (79). Their presence in combination or isolation is not always indicative of PA and are not present in all cases of PA (80). This is part due to difficulties around establishing true reference ranges in immunological diagnostic tests (81).

Serological testing for coeliac disease could be considered if PA can be ruled out, but there is vitamin B₁₂ deficiency with unknown cause (4).

Other tests

Several additional tests for assessing vitamin B₁₂ absorption have been described, but they are now rarely used in routine practice. The Schilling test, which employed radiolabelled cobalamin administered with and without IF, is now considered obsolete (26). A more recent alternative, the CobaSorb test, uses a non-radioactive oral vitamin B₁₂ load alongside measurement of serum concentrations to infer absorption (82). While simpler and safer, it has not been widely adopted and has not entered mainstream diagnostic use.

Treatment

Differing treatment algorithms and the benefits and drawbacks of each are not within the scope of this review, and have been thoroughly reviewed elsewhere (83). However, there are some considerations when it comes to investigating disorders of vitamin B₁₂.

Because neurological damage caused by vitamin B₁₂ deficiency may be irreversible, treatment should be initiated at the earliest opportunity (4). In individuals with convincing symptoms and clear risk factors, this may be appropriate even before diagnostic testing has been completed (4). Ideally, serum total vitamin B₁₂ and at least one functional marker, with MMA being the most useful, should be measured prior to starting therapy if feasible (25).

After intramuscular vitamin B₁₂ administration, serum total vitamin B₁₂ concentrations increase sharply, but this rise does not reflect functional recovery and therefore has little value for monitoring treatment response (4,84). Functional markers such as MMA and homocysteine decrease after effective therapy, often within days to weeks, although the time course may vary depending on baseline levels and comorbidities (84). By contrast, in patients receiving oral supplementation, a measurable rise in total vitamin B₁₂ can still provide useful confirmation of absorption (25).

Diagnostic algorithms

General investigations in uncomplicated adult patients

A number of diagnostic algorithms and guidelines for vitamin B₁₂ deficiency have been proposed globally (Table 1) (3,4,85,86). While these guidelines broadly follow a common strategy, there are differences between them. Specific cut-off values differ due to differences in methodologies and a lack of consensus in defining true vitamin B₁₂ deficiency. The tests that are available also differ, which will alter the strategy that is taken when investigating these patients (Table 1).

Screening asymptomatic individuals for vitamin B₁₂ deficiency is currently not recommended, therefore diagnosis starts with assessment of clinical presentation and recognition of symptoms, and evaluation of risk factors that may predispose to deficiency (see sections on clinical presentation and risk factors).

If both symptoms and risk factors are present, a first-line biochemical test (serum vitamin B₁₂ or holoTC if available) should be carried out (Figure 4). In symptomatic patients without clear risk factors, clinical judgement is required, as deficiency may still be present in the absence of obvious underlying risks.

Figure 4 Proposed algorithm for investigating B₁₂ deficiency. Created in BioRender (9). MMA, methylmalonic acid.

Interpretation of the initial result stratifies patients into three categories:

• Likely deficient: below the guideline-defined lower cut-off → proceed to treatment and investigate the cause.
• Indeterminate: between lower and upper cut-offs → proceed to functional testing MMA or homocysteine).
• Unlikely deficient: above the upper cut-off → vitamin B₁₂ deficiency not supported, consider alternative causes for the clinical presentation.

In patients with indeterminate results, functional testing can confirm the diagnosis. A normal MMA or homocysteine supports exclusion of deficiency, whereas an elevated result indicates functional vitamin B₁₂ deficiency and warrants treatment. Importantly, in patients with overt neurological or haematological features, treatment should not be delayed while awaiting second-line tests.

Follow-up testing can be carried out either sequentially or in parallel. Sequential testing (awaiting the first result, before collecting and carrying out the follow-up test) reduces unnecessary testing, however, may potentially require an additional phlebotomy appointment which carries with it a resource implication. Parallel testing may also identify patients with circulating vitamin B₁₂ concentrations in the sufficient range but with an elevated MMA, a scenario sometimes described as subclinical vitamin B₁₂ deficiency (discussed further in the “Research gaps” section). A combined approach, in which all required blood samples are collected at the initial visit, but cascade testing is performed by the laboratory based on predefined criteria, represents a pragmatic compromise, with the potential to balance diagnostic efficiency against cost and resource utilisation.

Mathematical models utilising multiple parameters have been proposed and are seeing increasing interest in helping improve both the sensitivity and specificity in detecting vitamin B₁₂ deficiency and are showing significant promise in validation studies (87,88).

These are not currently recommended as part of current clinical practice guidelines, however, their improvements in sensitivity and specificity may well lead them to being incorporated in the future.

Once a deficiency diagnosis has been made, further investigations may be appropriate to determine the underlying cause, particularly if autoimmune gastritis or generalized malabsorption is suspected. Options include IF or parietal cell antibody assays, serum gastrin, CobaSorb, and gastroscopy. Broader investigations, such as coeliac serology or pancreatic function tests may also be considered.

Exceptions

While the above algorithm applies to most patients, certain exceptions require special consideration in terms of testing strategy or cut-off interpretation.

Paediatrics

In patients under the age of 18 years, investigation can be complicated in several ways. Whilst clinical features of deficiency clearly overlap with the adult features, the way in which they present, particularly to a general practitioner, could be different. For example, early neurological dysfunction could present with poor school performance (89). Risk factors may also be less obvious, for example, a child may not report a vegetarian diet, but could perhaps have a very restricted “fussy” diet deficient in key nutrients (90). It is therefore important to keep an open mind when carrying out an initial assessment.

Biochemical investigation can also be complicated. Paediatric reference ranges for total vitamin B₁₂ have been shown to be higher than adult ranges, particularly in younger children, and holoTC has been shown to undergo significant age-related changes (91-93). Therefore, adult reference ranges should not be directly applied to this population.

In paediatrics, a lower upper threshold for carrying out functional testing should be considered, and if strong clinical suspicion is present, functional testing may be appropriate first line. Among second-line markers, MMA is generally preferred over homocysteine in paediatrics as it is less influenced by folate status or other confounders (93). MMA concentrations generally approximate adults, apart from in very young children (<1 year) who have markedly higher MMAs (91,93).

Pregnancy and oral contraceptive pill

In pregnancy, investigation of vitamin B₁₂ deficiency is complicated by several physiological changes, including haemodilution and redistribution of vitamin B₁₂ to the foetus (94). Reference ranges, therefore, for total vitamin B₁₂ indicate a marked lowering of B₁₂ as pregnancy progresses, even in individuals who are vitamin B₁₂ replete. HoloTC has also been shown to change, however, the changes are much more subtle (94). Total vitamin B₁₂ may therefore be misleading in these patients, and therefore, HoloTC has been suggested as the better first-line marker in pregnancy (4). A further complication with regards to vitamin B₁₂ in pregnancy is its interaction with folate. Folate supplementation in pregnancy is widely recommended due to its marked impact on reducing neural tube defects. However, it may go some way in masking haematological features of vitamin B₁₂ deficiency, and therefore it is critical that if vitamin B₁₂ deficiency is present, that this is identified early on. Untreated vitamin B₁₂ deficiency in pregnancy alongside elevated homocysteine concentrations has been associated with adverse foetal outcomes including intrauterine growth restriction, neural tube defects, and preterm birth (95). Functional markers such as MMA and homocysteine can be used in pregnancy, but these too are affected by the physiological changes of gestation and therefore should always be interpreted with caution in this context (96,97). Use of the combined oral contraceptive pill has also been shown to lower total cobalamin concentrations, believed to be due to changes in binding proteins (98).

Renal disease

The relationship between vitamin B₁₂ status and chronic kidney disease (CKD) is complex. Patients with CKD, particularly end-stage CKD on haemodialysis, have a modestly increased risk of genuine deficiency (99). Potential mechanisms include reduced dietary intake, dialysis losses, and disturbances in cellular handling (99,100).

Diagnosis is also complicated by renal effects on biomarkers. Total vitamin B₁₂ and holoTC may be elevated in CKD, giving the false impression of sufficiency despite functional deficiency (100). Conversely, MMA and homocysteine are often raised because of reduced renal clearance, which can mimic vitamin B₁₂ deficiency (101). For this reason, interpretation of MMA should be adjusted for kidney function, and several estimated glomerular filtration rate (eGFR)-based correction tools have been proposed, although these are not yet in routine clinical use (102,103).

Nitrous oxide

Typical diagnostic algorithms break down in the context of nitrous oxide use, particularly heavy acute use. Total vitamin B₁₂ often remains normal, as N₂O inactivates MeCbl rather than reducing circulating vitamin B₁₂ (63). Functional markers may also be misleading, as MMA elevation is not reliably found in all cases. In this context, homocysteine is often the more dependable marker after exposure, as it is consistently elevated in N₂O users (104) and can rise very quickly after use. In cases of chronic nitrous oxide use, total vitamin B₁₂ and MMA become more helpful, as the inactivated vitamin B₁₂ is removed from the circulation, and generalised deficiency sets in.

High vitamin B₁₂

There is no clinical context in which vitamin B₁₂ is measured for any reason other than for investigating possible deficiency (outside of very specialist situations where it may be requested by a haematologist as a prognostic marker). Elevated serum vitamin B₁₂ is therefore almost always an incidental finding, although it is not uncommon in routine practice (105).

The most common explanation for an elevated vitamin B₁₂ concentration is recent treatment or supplementation. Once this has been excluded, the main pathological causes to consider are liver disease, renal disease, and haematological malignancy (106). In liver disease, hepatocellular injury can release stored cobalamin, and chronic liver damage may increase haptocorrin synthesis (107). Anorexia nervosa has been associated with raised vitamin B₁₂ concentrations, and this is thought to be due to liver autophagy (108).

Advanced CKD can lead to reduced clearance of cobalamin-binding proteins can elevate total vitamin B₁₂ (100). In haematological malignancies such as chronic myeloid leukaemia, polycythaemia vera, essential thrombocythaemia, and acute myeloid leukaemia, granulocyte-related haptocorrin overproduction is the primary mechanism (109).

A practical investigative approach, therefore, is to request renal function, liver function tests, and a full blood count. If these are normal and the patient is otherwise well, immediate further investigation is usually unnecessary. However, if the result persists on repeat testing, or if there are additional clinical concerns such as unexplained weight loss, anaemia, or systemic symptoms, broader evaluation should be considered. Solid organ malignancies have also been associated with persistently elevated vitamin B₁₂, and autoimmune or chronic inflammatory conditions may contribute in some cases (105). Population studies suggest that unexplained persistent elevation of vitamin B₁₂ may be associated with increased risks of cancer and all-cause mortality, although the underpinning mechanisms are not fully understood (110).

It is also important to recognise that often the rise in vitamin B₁₂ reflects increased haptocorrin-bound cobalamin, which is not biologically active. True deficiency can therefore be masked, and if clinical suspicion of deficiency remains, holoTC or functional markers such as MMA or homocysteine should be measured.

Genetic causes

There are numerous rare genetic causes of alterations in vitamin B₁₂ metabolism, which can lead to a very wide spectrum of clinical disease (111). A large number of these result in clinically overt presentations during childhood, producing phenotypes such as methylmalonic aciduria and homocystinuria (111). More subtle, clinically benign associations with genetic polymorphisms have also been described, and these can have significant effects on vitamin B₁₂ parameters while being of uncertain clinical significance (111).

Research gaps

Despite many advances in understanding and improvements in diagnostic strategies, key uncertainties persist in the clinical investigation of vitamin B₁₂ disorders (3,4,25).

The role of holoTC compared to total serum vitamin B₁₂ assays is still unclear. While holoTC has been proposed as a more sensitive early marker, its added value in clinical practice remains unclear, and evidence of population-level benefit is mixed (18,25). It is possible that holoTC may see more utility in specific sub-populations where binding protein disturbances are a recognised complication.

Although MMA and homocysteine are widely used functional markers, the clinical significance of “borderline” results in these assays remains unresolved. Cut-offs vary between studies, and it is uncertain whether mild elevations predict clinically meaningful deficiency or reflect non-specific metabolic disturbances. This is compounded by the numerous population studies which report abnormal vitamin B₁₂-related biomarkers in asymptomatic individuals, and the clinical relevance of this “subclinical deficiency” is controversial (18). It remains unclear whether this “sub-clinical” deficiency represents an early stage of true deficiency, a benign biochemical phenotype, or a risk factor for other unrelated chronic disease (9). This is one of the reasons that screening asymptomatic patients is broadly not recommended.

Other controversies exist too: following the adoption of the term “autoimmune gastritis” in place of “pernicious anaemia” by UK NICE, there is ongoing debate over the immunological basis of these definitions and their clinical implications. The most recent NICE guidelines (NG239) propose that true PA, defined as life-threatening anaemia associated with vitamin B₁₂ deficiency, is so rare due to treatment advancements that the autoimmune cause of vitamin B₁₂ deficiency should be autoimmune gastritis (4,112). The role of antibody testing (IF, parietal cell, and emerging markers) in distinguishing this continuum of autoimmune disease from other causes of malabsorption requires further clarification.

Finally, the interplay between vitamin B₁₂ and the gut microbiome represents a fascinating but under-explored field. Although humans synthesise vitamin B₁₂ in the colon, it is not absorbable at this site. The functional consequences of altered microbial vitamin B₁₂ metabolism for host health, and the reciprocal effects of host vitamin B₁₂ status on microbiome composition and possible effects on bacterial pathogenesis, remain poorly understood (113).

Strengths and weaknesses

Strengths

This clinical practice review is clinically focused and practice-orientated, intended to reflect the realities of common patient presentations. It has an emphasis on structured clinical decision-making approaches whilst balancing this with situations where algorithms may break down or mislead. It integrates core biochemical principles with clinical presentations and covers both low B₁₂ but also covered unexpectedly raised B₁₂.

Weaknesses

A key weakness of this review is that it is a narrative review rather than a systematic one. There is also limited discussion of treatment strategies, outside of those which could have an influence on diagnosis. It assumes that all relevant tests are available to clinicians, and given that the field is evolving, cut-offs and strategies presented here may need to be amended if new evidence is published.

Conclusions

Disorders of vitamin B₁₂, particularly deficiency, are a diverse range of conditions affecting wide populations, and has the potential to lead to severe clinical consequences if not identified and treated. Significant improvement in the understanding and testing for these conditions has really improved the diagnostic process, and these algorithms should help guide clinicians in approaching this diagnostic puzzle. However, there are numerous situations in which published algorithms break down, and alternative approaches are required, therefore it is important that any clinician reviewing patients is aware of what alternative strategies are available.

Acknowledgments

We would like to thank Erin Wearmouth for reviewing the manuscript and proposing minor edits.

Footnote

Provenance and Peer Review: This article was commissioned by the Guest Editor (Sukhbir Kaur) for the series “Trace Elements and Vitamins” published in Journal of Laboratory and Precision Medicine. The article has undergone external peer review.

Peer Review File: Available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-2025-1-61/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-2025-1-61/coif). The series “Trace Elements and Vitamins” was commissioned by the editorial office without any funding or sponsorship. The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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