Vitamin B12 and Folate

Vitamin B12 and folate are required for DNA synthesis, and their deficiency causes megaloblastic anaemia, macrocytosis and cytopenias. B12 deficiency also causes subacute combined degeneration of the cord, peripheral neuropathy, cognitive impairment and psychiatric disease, which can occur without anaemia or macrocytosis. The panel includes serum B12, holotranscobalamin (active B12), serum folate and red cell folate, with methylmalonic acid and homocysteine as functional markers.

B12 deficiency is common in older adults, vegans, people with gastric or ileal disease and long term users of metformin or acid suppression. Folate deficiency has become less common in countries that fortify food, and persists with alcohol dependence, malabsorption, haemolysis and pregnancy.

Indications. Macrocytosis, megaloblastic anaemia, pancytopenia, neuropathy, myelopathy, cognitive decline, glossitis, bariatric surgery, gastrectomy, ileal disease or resection, coeliac disease, long term metformin or proton pump inhibitor use, vegan or restrictive diet, alcohol dependence, and before and during pregnancy when risk is present.

Specimen. Serum for B12, holotranscobalamin and serum folate. A fasting sample is preferred for serum folate, since recent intake raises the result. Red cell folate needs an EDTA whole blood sample, protected from light and processed promptly. Methylmalonic acid is measured in serum or urine, and homocysteine in a chilled, rapidly separated sample.

Adult reference ranges are given. Local laboratory ranges take precedence.

Serum vitamin B12. Total B12, mostly bound to haptocorrin and not biologically available.

Range: 200 to 900 ng/L (148 to 664 pmol/L). Under 200 ng/L (148 pmol/L) indicates likely deficiency, and 200 to 300 ng/L (148 to 221 pmol/L) is indeterminate.

Raised: liver disease, myeloproliferative neoplasms, hypereosinophilic syndromes, solid tumours with liver metastases, renal failure, B12 supplementation.

Lowered: pernicious anaemia, gastrectomy, ileal disease or resection, Crohn disease, bacterial overgrowth, fish tapeworm, vegan diet, metformin, acid suppression.

A normal level does not exclude deficiency when symptoms are present, and an indeterminate level needs a second test. Nitrous oxide exposure causes functional deficiency with a normal serum level.

Holotranscobalamin. B12 bound to transcobalamin, the biologically active fraction taken up by cells. Range: assay specific, typically over 50 pmol/L normal, 25 to 50 pmol/L borderline, under 25 pmol/L deficient. Raised: renal failure, supplementation. Lowered: B12 deficiency of any cause.

It becomes abnormal earlier than total B12 and is more specific, which makes it useful for indeterminate serum B12 results.

Serum folate. Reflects recent folate intake. Range: about 3 to 20 microg/L (7 to 45 nmol/L), with under 3 microg/L (7 nmol/L) indicating deficiency. Raised: supplements, fortified food, haemolysis, B12 deficiency (the folate trap). Lowered: poor diet, alcohol, malabsorption, pregnancy, haemolysis, methotrexate, trimethoprim, phenytoin, valproate, dialysis.

It falls within days of reduced intake, so it may be low while tissue stores are adequate.

Red cell folate. Reflects tissue folate over the previous 3 to 4 months. Range: lower limit about 140 microg/L (about 320 nmol/L), assay specific. Raised: recent supplementation, reticulocytosis. Lowered: folate deficiency, and also B12 deficiency, since red cell folate depends on B12.

It is more stable than serum folate but is not reliable when B12 is deficient. It is no longer routinely requested in many laboratories.

Methylmalonic acid (MMA). Metabolite that accumulates in B12 deficiency, since B12 is a cofactor for methylmalonyl-CoA mutase. Range: serum up to about 0.4 micromol/L. Raised: B12 deficiency, chronic kidney disease, hypovolaemia, bacterial overgrowth, older age. Lowered: no clinical significance.

It is sensitive for tissue B12 deficiency and stays normal in isolated folate deficiency. In renal impairment it is raised independently of B12, so interpret with eGFR.

Homocysteine. Metabolite raised in B12, folate and B6 deficiency. Range: fasting 5 to 15 micromol/L. Raised: B12 deficiency, folate deficiency, B6 deficiency, chronic kidney disease, hypothyroidism, smoking, methotrexate, genetic defects (cystathionine beta synthase deficiency, MTHFR variants). Lowered: no clinical significance.

It is sensitive but non-specific. A normal level argues against both deficiencies.

• Low B12 with raised MMA (and homocysteine): B12 deficiency.
• Low folate with normal MMA and raised homocysteine: folate deficiency.
• Both low: combined deficiency, with malabsorption (coeliac disease, tropical sprue, small bowel disease) in mind.
• Low serum B12 with normal MMA and holotranscobalamin: a false low result (pregnancy, oral contraceptives, haptocorrin deficiency, folate deficiency, myeloma).
• Macrocytosis with normal B12 and folate: alcohol, liver disease, hypothyroidism, myelodysplasia, drugs (hydroxycarbamide, methotrexate, azathioprine, zidovudine), reticulocytosis.
• Raised serum B12 without supplementation: liver disease, myeloproliferative neoplasm, malignancy, renal failure, or an assay artefact from macro-B12.

Causes of B12 deficiency. Pernicious anaemia (autoimmune atrophic gastritis), gastrectomy and bariatric surgery, terminal ileal disease or resection, bacterial overgrowth, fish tapeworm, pancreatic insufficiency, dietary deficiency, metformin, acid suppression, nitrous oxide, and inherited disorders in infants.

Pernicious anaemia testing. Intrinsic factor antibodies are highly specific but positive in only about half of cases. Parietal cell antibodies are more sensitive but less specific, and are found in older adults and other autoimmune conditions.

Critical findings needing immediate action.

• Neurological signs (paraesthesia, ataxia, loss of vibration or position sense, cognitive decline) with low or borderline B12 need prompt specialist assessment, since delay risks irreversible damage.
• Severe pancytopenia with very high LDH and haemolysis can mimic thrombotic microangiopathy in megaloblastic marrow failure.

Interferences and pitfalls.

• Starting folate alone before B12 deficiency is excluded can correct the anaemia while neurological damage progresses.
• Serum folate falls with delay and light exposure, and haemolysis falsely raises it.
• Recent B12 supplementation or injection gives a falsely normal serum level.
• In rare cases, intrinsic factor antibodies interfere with B12 immunoassays and give a falsely normal result.
• Biotin interferes with some immunoassays.
• Alcohol, phenytoin and methotrexate lower serum folate independently of stores.
• Haemolysed or lipaemic samples distort results.

Special groups.

• Pregnancy: serum B12 and folate fall physiologically. Periconceptional folate status affects neural tube defect risk.
• Infants of B12-deficient mothers (including those who are vegan) can present with developmental regression and anaemia, and MMA is useful in this group.
• Older adults: symptomatic deficiency can occur with serum B12 in the indeterminate range, so MMA or holotranscobalamin is useful.
• Chronic kidney disease: MMA and homocysteine are raised independently of B12 and folate.

Follow up tests. Full blood count, blood film, reticulocyte count, methylmalonic acid, homocysteine, intrinsic factor and parietal cell antibodies, coeliac serology, Helicobacter pylori testing, thyroid function tests, lactate dehydrogenase, upper gastrointestinal endoscopy with gastric biopsy, bone marrow examination when cytopenias are unexplained.