Vitamin Biomarkers: Vitamin B12

Optimal Takeaways  

Vitamin B12 (cobalamin) is an essential nutrient primarily found in animal-based foods. It is involved in many critical metabolic functions, including the production of red blood cells, protein, DNA, myelin, and energy, as well as the processing of homocysteine. Gastrointestinal absorption of B12 is complex, and reduced stomach acid or gastrointestinal surgery may lead to a deficiency. An insufficiency of B12 is associated with anemia, fatigue, birth defects, neurological abnormalities, mood disorders, and the accumulation of homocysteine. Elevated B12 can also be detrimental and may be related to liver or kidney disease, autoimmune disorders, cancer, and functional B12 deficiency.

Standard Range: 200.00 – 1100.00 pg/mL (147.56 – 811.58 pmol/L

The ODX Range: 545.00 – 1100 pg/mL (402.01 – 811.58 pmol/L) 

Low levels of vitamin B12 are associated with low hydrochloric acid, atrophic gastritis, gastrectomy, intestinal worms, malabsorption, inflammatory bowel disease, terminal ileum resection, pernicious anemia, megaloblastic anemia, deficiency of vitamins B12, C, or folate (Pagana 2021), use of metformin (Bell 2022), chronic pancreatitis, and use of proton pump inhibitors, H2 blockers, antibiotics, and colchicine (Sanz-Cuesta 2020).

Low B12 is also associated with polyneuropathy (Warendorf 2021), muscle cramps, dizziness, depression (Wolffenbuttel 2020), infertility, hyperhomocysteinemia, functional folate deficiency, hypersegmented neutrophils, transcobalamin deficiency, fatigue, cognitive and mood impairment (Allen 2018), chronic alcoholism, bacterial overgrowth, insomnia, disorientation, irreversible neurological damage (Butola 2020), elevated mean corpuscular volume (unless iron deficiency anemia is also present), neuropsychiatric changes, paresthesia, ataxia, impaired vibration sense, pallor, weakness, edema, skin pigment changes, jaundice (Harrington 2017), bursitis, neuritis, neuralgia, brain atrophy, dementia (Travica 2015), forgetfulness, numbness, vertigo (Jatoi 2020), oxidative stress (van de Lagemaat 2019), low alkaline phosphatase (Ray 2017), decreased iron uptake (Kotze 2009), and strict vegetarian diets (Paul 2017). 

High levels of vitamin B12 are associated with parenteral B12 administration (Wolffenbuttel 2020), excess B12 intake, liver dysfunction, myeloproliferative disease, leukemia, polycythemia vera (Pagana 2021), increased immunoglobulin-bound B12, increased transport proteins, reduced cellular uptake, autoimmune disorders, hematological disorders, renal failure (Vollbracht 2020), functional B12 deficiency (decreased intracellular B12), liver metastases, and solid neoplasms- primarily hepatocellular carcinoma and cancer of the colon, pancreas, breast, and stomach (Andres 2013).

Overview

Vitamin B12 (cobalamin) is an essential cobalt-containing vitamin produced by bacteria and consumed primarily through animal-based foods. The most important bioactive forms of B12 are methylcobalamin, adenosylcobalamin, and hydroxocobalamin. Methylcobalamin is abundant in milk and eggs, while adenosylcobalamin is found primarily in meat which also contains small amounts of methylcobalamin and hydroxocobalamin. Vegetarian sources of B12, such as seaweed and yeast, actually contain inactive B12 analogs that can interfere with bioavailable forms of B12 and exacerbate a B12 deficiency. Cyanocobalamin is a manufactured form that can be converted to methyl- or adenosylcobalamin in the body, producing cyanide as a byproduct. Cyanocobalamin may be contraindicated in certain circumstances, especially in those exposed to cigarette smoke which is contaminated with cyanide (Paul 2017).

B12 works with other B vitamins and supports several physiological activities, including mitochondrial energy generation, protein metabolism, DNA synthesis, red blood cell production, folate metabolism, and the conversion of homocysteine to methionine. Insufficiency of B12 will disrupt these functions and contribute to anemia, fatigue, neuropathy, cognitive impairment (especially with the ApoE4 genotype), and reproductive impairment (Allen 2018). Signs and symptoms, including anemia, may only sometimes be present with B12 deficiency, and underlying neurological damage may go undetected without comprehensive workup and intervention (Wolffenbuttel 2020).

The coenzyme forms of B12 include methionine synthase, which uses methylcobalamin in the methylation reaction that converts homocysteine back to methionine, and methylmalonyl-CoA mutase, which uses adenosylcobalamin and converts methylmalonyl-CoA to succinyl-CoA in the energy-generating citric acid cycle (Harrington 2017). Vitamin B12 also appears to function as an antioxidant. It may help scavenge superoxide, preserve glutathione, protect against the oxidative stress associated with homocysteine and advanced glycosylation end products, and modulate the oxidation and inflammation associated with the immune response (van de Lagemaat 2019). Oxidative stress and glutathione insufficiency, in turn, contribute to intracellular functional B12 deficiency (Vollbracht 2020).

The absorption of vitamin B12 is complex; it mainly depends on hydrochloric acid and intrinsic factor (IF) produced in the stomach. However, it also depends on absorption of the B12-IF complex at the terminal ileum. Low stomach acid, lack of intrinsic factor (i.e., pernicious anemia), or resection or inflammation of the terminal ileum can lead to a B12 deficiency (Pagana 2021). Intramuscular, sublingual, and nasal routes for B12 supplementation bypass the GI tract and may be preferred in such conditions (Thakkar 2015). Pernicious anemia is an autoimmune disorder characterized by anti-intrinsic factor antibodies, and it often coexists with other autoimmune disorders, including type 1 diabetes and thyroid disease. Some passive diffusion of B12 can result in adequate B12 absorption without intrinsic factor when high doses above 1,000 ug are used (Devalia 2014). Although enterohepatic circulation recycles some of the B12 excreted in the bile, those with pernicious anemia can become B12 depleted very quickly due to impaired intestinal absorption (Allen 2018).

Homocysteine and methylmalonic acid accumulation may indicate B12 insufficiency, especially if serum B12 falls below 406.61 pg/mL (300 pmol/L). Clinical signs of overt B12 deficiency include macrocytic/megaloblastic anemia, neuronal demyelination, and neuropathy. However, subclinical cobalamin deficiency (SCCD) may be characterized by inhibition of methylation, infertility, miscarriage, psychosis, memory loss, dementia, and depression. Excess synthetic folic acid found in supplements and fortified processed foods may make B12 insufficiency worse. The drug metformin can reduce serum levels of B12 due to the accumulation of B12 in the liver, though this may not reflect a true deficiency (Allen 2018).

Researchers recommend combining two to four biomarkers when assessing B12 status, i.e., measurement of serum B12, holotranscobalamin (holoTC), methylmalonic acid (MMA), and total homocysteine (Fedosov 2015). Levels of homocysteine and MMA begin to rise as serum B12 drops below 542 pg/mL (400 pmol/L) (Smith 2018). Measuring holotranscobalamin can help assess B12 status as it is immediately bioavailable for cellular uptake, and it is considered a more reliable indicator of B12 status than serum B12. Serum B12 alone may miss up to 45% of those with B12 deficiency. A more comprehensive functional approach is needed (Harrington 2017).

Folate status should also be assessed since B12 is required for folate activation and the incorporation of folate in cells (Pagana 2021). Therefore, a B12 deficiency can lead to a functional folate deficiency despite adequate folate intake. Macrocytic/megaloblastic anemia may be caused by either B12 or folate insufficiency, or both, and the cause must be determined before proceeding with intervention (Allen 2018).

Evaluation of MMA levels, when serum B12 is at the low end of the conventional range, can help confirm a metabolic B12 deficiency and help guide repletion. One retrospective study of 331 polyneuropathy patients found that assessing MMA in those with a serum B12 below 412 pg/mL (304 pmol/L) helps confirm metabolic B12 deficiency and best identifies those who may benefit from B12 supplementation. Researchers found elevated MMA above 290 nmol/L (0.28 umol/L) to be a better indicator of functional B12 status than homocysteine which can be confounded by outside factors, including folate or B6 deficiency, smoking and alcohol use, and hypothyroidism (Warendorf 2021).

Total serum B12 reflects freely circulating B12 and immunoglobulin-transcobalamin complexes, which do not provide bioavailable B12 but can contribute to increased B12 levels. Elevated serum B12 may also be due to increased transport proteins, decreased cellular uptake, autoimmune or hematological disorders, renal failure, liver disease, and cancer. A decrease in intracellular B12 is considered a functional B12 deficiency. It can be caused by insufficient uptake or impairment of B12 activation within the cell, which can occur with oxidative stress and has been observed in diabetes and Alzheimer's. Providing glutathione or vitamin C, which regenerates glutathione, may be therapeutic in such cases (Vollbracht 2020). One clinical study found that individuals with serum B12 below 211 pg/mL (156 pmol/L) had significantly lower glutathione and total antioxidant levels and significantly elevated malondialdehyde levels indicative of oxidative stress (Misra 2017).

A functional intracellular deficit of B12 can occur at any serum level. Though counterintuitive, elevated serum B12 can lead to decreased cellular uptake, functional deficiency, and related complications, including high homocysteine and MMA. Elevated B12 may be associated with liver disease, kidney failure, inflammatory disease, and cancer (Andres 2013).

Vitamin B12 insufficiency may be common. Data analysis from a population health check program revealed less than optimal levels of vitamin B12, vitamin D, and iodine and above optimal homocysteine, which was inversely correlated with B12. A serum B12 below 500 pg/mL (368.90 pmol/L) may be associated with depleted cerebrospinal B12 and neurological disorders. The brain atrophy and dementia associated with B12 deficiency may be reversed with the repletion of serum B12. Some researchers suggest an optimal range of 500 – 1300 pg/mL (368.90 - 959.14 pmol/L) for serum B12 (Travica 2015).

B12 insufficiency must be identified early as insufficiency without overt deficiency is associated with neural tube defects, infantile tremor, cognitive deficits, white matter damage, impaired memory, whole brain atrophy, stroke, depression, macular degeneration, low bone mineral density, and DNA damage (Smith 2018).

A B12 insufficiency can be corrected with the active forms of B12, adenosyl- and methyl-cobalamin. Supplements may be given orally if GI absorption is normal, intramuscularly, or sublingually if absorption is impaired. Active forms are preferred over cyanocobalamin, especially in smokers who are already exposed to excess cyanide (Thakkar 2015). Hydroxocobalamin is also a preferred alternative to cyanocobalamin as it is more effectively utilized and retained in the body (Jatoi 2020).

Vegetarian or vegan diets may be insufficient in B12 and lead to deficiency over time as tissue and liver stores become depleted. Microwaving food may compromise B12 absorption and contribute to insufficiency as well (Travica 2015).

References

Allen, Lindsay H et al. “Biomarkers of Nutrition for Development (BOND): Vitamin B-12 Review.” The Journal of nutrition vol. 148,suppl_4 (2018): 1995S-2027S. doi:10.1093/jn/nxy201

Andres, E et al. “The pathophysiology of elevated vitamin B12 in clinical practice.” QJM : monthly journal of the Association of Physicians vol. 106,6 (2013): 505-15. doi:10.1093/qjmed/hct051

Bell, David S H. “Metformin-induced vitamin B12 deficiency can cause or worsen distal symmetrical, autonomic and cardiac neuropathy in the patient with diabetes.” Diabetes, obesity & metabolism vol. 24,8 (2022): 1423-1428. doi:10.1111/dom.14734

Devalia, Vinod et al. “Guidelines for the diagnosis and treatment of cobalamin and folate disorders.” British journal of haematology vol. 166,4 (2014): 496-513. doi:10.1111/bjh.12959

Fedosov, Sergey N et al. “Combined indicator of vitamin B12 status: modification for missing biomarkers and folate status and recommendations for revised cut-points.” Clinical chemistry and laboratory medicine vol. 53,8 (2015): 1215-25. doi:10.1515/cclm-2014-0818

Harrington, Dominic J. “Laboratory assessment of vitamin B12 status.” Journal of clinical pathology vol. 70,2 (2017): 168-173. doi:10.1136/jclinpath-2015-203502

Jatoi, Shazia et al. “Low Vitamin B12 Levels: An Underestimated Cause Of Minimal Cognitive Impairment And Dementia.” Cureus vol. 12,2 e6976. 13 Feb. 2020, doi:10.7759/cureus.6976

Kotze, M J et al. “Pathogenic Mechanisms Underlying Iron Deficiency and Iron Overload: New Insights for Clinical Application.” EJIFCC vol. 20,2 108-23. 25 Aug. 2009

Misra, Usha Kant et al. “Oxidative Stress Markers in Vitamin B12 Deficiency.” Molecular neurobiology vol. 54,2 (2017): 1278-1284. doi:10.1007/s12035-016-9736-2

Pagana, Kathleen Deska, et al. Mosby's Diagnostic and Laboratory Test Reference. 15th ed., Mosby, 2021.

Paul, Cristiana, and David M Brady. “Comparative Bioavailability and Utilization of Particular Forms of B12 Supplements With Potential to Mitigate B12-related Genetic Polymorphisms.” Integrative medicine (Encinitas, Calif.) vol. 16,1 (2017): 42-49.

Ray, Chinmaya Sundar, et al. "Low alkaline phosphatase (ALP) in adult population an indicator of zinc (Zn) and magnesium (Mg) deficiency." Current Research in Nutrition and Food Science Journal 5.3 (2017): 347-352.

Sanz-Cuesta, Teresa et al. “Oral versus intramuscular administration of vitamin B12 for vitamin B12 deficiency in primary care: a pragmatic, randomised, non-inferiority clinical trial (OB12).” BMJ open vol. 10,8 e033687. 20 Aug. 2020, doi:10.1136/bmjopen-2019-033687

Smith, A David et al. “Vitamin B12.” Advances in food and nutrition research vol. 83 (2018): 215-279. doi:10.1016/bs.afnr.2017.11.005

Thakkar, K, and G Billa. “Treatment of vitamin B12 deficiency-methylcobalamine? Cyancobalamine? Hydroxcobalamin?-clearing the confusion.” European journal of clinical nutrition vol. 69,1 (2015): 1-2. doi:10.1038/ejcn.2014.165

Travica, Nikolaj, et al. "Integrative Health Check reveals suboptimal levels in a number of vital biomarkers." Advances in integrative medicine 2.3 (2015): 135-140.

van de Lagemaat, Erik E et al. “Vitamin B12 in Relation to Oxidative Stress: A Systematic Review.” Nutrients vol. 11,2 482. 25 Feb. 2019, doi:10.3390/nu11020482

Vollbracht, C et al. “Supraphysiological vitamin B12 serum concentrations without supplementation: the pitfalls of interpretation.” QJM : monthly journal of the Association of Physicians vol. 113,9 (2020): 619-620. doi:10.1093/qjmed/hcz164

Warendorf, Janna K et al. “Clinical relevance of testing for metabolic vitamin B12 deficiency in patients with polyneuropathy.” Nutritional neuroscience, 1-11. 25 Oct. 2021, doi:10.1080/1028415X.2021.1985751

Wolffenbuttel, B H R et al. “Association of vitamin B12, methylmalonic acid, and functional parameters.” The Netherlands journal of medicine vol. 78,1 (2020): 10-24.