Clinical insight into vitamin B12


            It was once thought that vitamin B12 (B12) deficiency is rare and occurs sporadically among some strict vegetarians.(1) Recent studies have shown that B12 deficiency and depletion are a world-wide problem and may occur in a number of individuals including the elderly, vegetarians and those with limited intake of animal products, patients receiving metformin, patients taking non-steroidal anti-inflammatory medications, anti-convulsant drugs, protein pump inhibitors, β2 blockers, bile acid sequestrants, tetracycline, calcium channel blockers, those who had an upper gastrointestinal surgery and/or anyone after surgery with general anesthesia, chemotherapy patients, and individuals infected by Helicobacter pylori infection.(2)

Insight Regarding What Constitutes Adequate B12 Intake

Although the 1998 Institute of Medicine’s (IOM) Recommended Dietary Allowance (RDA) calls for intake of 2.4 µg/day by non-pregnant adults, 2.6 µg/day and 2.8 µg/day for pregnant and lactating women, respectively, newer research studies have shown that these recommendations have been underestimated.(3) This underestimation is likely a function of the underlying basis to set the RDA based on hematological status. Abnormal hematological status, such as megaloblastic anemia constitutes a late indicator of B12 deficiency. Developmental delays and neurologic complications develop prior to the occurrence of hematological symptoms.(2)

Bor et al. estimated that, in healthy individuals between 18 and 50 y of age with normal absorption, an intake of 4–7 µg/day is associated with an adequate vitamin B-12 status.(3) According to Doets et al. intake of 3.8 to 20.7 μg is needed to replete daily B12 loses in healthy adults and elderly.(4) Evatt et al. concluded that intake of 75 µg/day may nearly eliminate low B12 and biochemical B12 deficiency and that intake of 6 or 25 µg/day, in some individuals, is inadequate to prevent biochemical B12 deficiency.(5) B12 recommendations issued recently by the European Food Safety Authority (EFSA) of 4 µg/day for non-pregnant adults, 4.5 µg/day and 5 µg/day, respectively, for pregnant and lactating women, are more consistent with the new findings.(6)

Insight into B12 Assessment Methods And Interpretation of Laboratory Findings

Several B12 assessment methods are available for providers. They include serum/plasma B12, mean corpuscular volume (MCV), homocysteine (Hcy), holo transcobalamin II (holoTCII) and serum and urinary methylmalonic acid (MMA). Table 1 includes the list of biochemical B12 assessments along with the traditional and evidence-based deficiency cutoffs.

Table 1. Traditional and evidence-based vitamin B12 deficiency reference values.

Assessment Traditional assessment cutoffs Evidence-based assessment cutoffs
Serum B12 <148 pmol/L (<200 pg/ml) <400 pmol/L (<540 pg/ml)
Homocysteine >15 µmol/L ≥10 µmol/L
Holo TC II <35 pmol/L <50 pmol/L
Serum MMA >260 nmol/L or >271 nmol/L >260 nmol/L or >271 nmol/L
Urinary MMA >4.3 µmol/L >4.0 µmol/L
Urinary MMA/


>4.8 mmol/L creatinine >3.5 mmol/L creatinine
MCV >98 µm3/cell >98 µm3/cell


Serum/plasma B12

Many lab results use serum B12 <148 pmol/L (<200 pg/ml, <200 ng/L) as cutoff for deficiency status. Recent studies have shown that this cutoff is insufficient since symptoms of B12 deficiency are often seen among patients with considerably higher serum/plasma B12 (7). Herbert suggested that a level of 300 pg/mL or less (<227 pmol/L) should be considered deficient.(8) Herbert’s suggestion is supported by other authors, some of whom suggested even higher cutoff criteria, as noted by Gröber et al. who stated that B12 serum below 200 ng/L (<150 pmol/L) are sure signs of a B12 deficiency and that a functional B12 deficiency may also occur when serum B12 is less than 450 ng/L (~332 pmol/L).(9) Similarly, Schwarz et al. suggested that B12 deficiency cutoff values should be elevated to 304 or 368 ng/L.(10) In fact, more sensitive assessment methods of B12 status, described below, showed values indicative of a biochemical deficiency among individuals whose serum vitamin B12 concentration was as high as about 400 pmol/L (540 pg/mL).(11,12,13)

Mean Corpuscular Volume

MCV is an assessment of erythrocyte volume.(14) Theoretically, B12 deficiency should result in megaloblastic (macrocytic) anemia, which should be manifested in elevated MCV. However, MCV is also affected by intake and status of folate and iron. In case of high folate intake, megaloblastic anemia may not develop even in cases of severely low B12. Also, iron deficiency results in microcytic anemia. Thus, if a patient has both B12 and iron deficiency, MCV may be shown to be normal, since the impact of B12 deficiency is offset by the microcytic effect of iron deficiency. Thus, normal MCV should never be used to rule out B12 deficiency. Normal MCV has been determined to be 87 ± 7 µm3.(14)


B12, along with several other B-vitamins, is essential in Hcy metabolism via methylene tetrahydrofolate reductase and methionine synthase enzymes. Studies have shown that in the post-folic acid-fortification era, vitamin B12 is the predominant cause of hyperhomocysteinemia (HHcy).(15) Considering that HHcy has been associated with an increased risk of several health conditions, utilizing Hcy in clinical practice may have important implications in reducing the risk of health problems among patients.

Although Hcy concentration of ≥15 µmol/L is extensively used as indicative of HHcy, this cutoff is inadequate. To illustrate, Jacobsen stated that, risk for coronary artery disease is represented by a continuum of total Hcy concentration, with a substantial risk occurring between 10 and 15 µmol/L.(16) Thus, patients with Hcy ≥10 μmol/L may require B12 therapy.

Holotranscobalamin II

HoloTCII constitutes the fraction of serum B12 that, with the help of the intrinsic factor, is absorbed into enterocytes. HoloTCII is the carrier of B12 into cells.(17) Thus, holoTCII is affected by intake and absorption of B12. HoloTCII is considered an early marker of inadequate B12 status.(18) According to Herbert, all cells have receptors for holoTCII while only the liver has receptors for haptocorrin.(8) Since B12 can only be delivered to cells via holoTCII, low holoTCII indicates functional B12 deficiency. Low holoTCII may not always be accompanied by overt B12 deficiency symptoms. However, persistently inadequate holoTCII may eventually lead to the onset of B12 deficiency symptoms.

Low holoTCII may occur even when liver B12 stores are not depleted and serum/plasma B12 is above normal. Thus, using holoTCII will help in detecting functional B12 deficiency regardless of serum B12 values. The majority of researchers have used holoTCII <35 pmol/L as indicative of inadequate B12 supply/functional B12 deficiency. However, Herrmann and Obeid suggested that individuals with holoTCII between 23 pmol/L and 75 pmol/L should be checked for MMA concentration to confirm B12 status, while Lloyd-Wright et al. suggested that B12 deficiency is unlikely when holoTCII is >50 pmol/L.(19,20)

Methylmalonic Acid

MMA is a sensitive and specific marker of B12 status. Serum/plasma MMA may be affected by renal failure, thyroid disease, and small bowel bacteria overgrowth. Further, it can also be affected by an inborn error of metabolism that affects methylmalonate CoA. Thus, although it is possible to assess MMA concentration in serum/plasma, urinary MMA (uMMA) is considered more reliable and less invasive. (21,22). uMMA is affected by food intake, so fasting urine samples should be evaluated in order to obtain the most reliable results.(23)

A value of serum/plasma MMA >271 nmol/L or >260 nmol/L is considered indicative of elevated MMA. Normal uMMA is <4.0 µmol/L. More than one cutoff for normal uMMA/creatinine has been proposed (e.g. <4.8 mmol/mol creatinine, <3.2 mmol/mol creatinine, 2.0 mmol/mol creatinine and 1.5 mmol/mol creatinine). Research findings have shown that urinary MMA >3.5 mmol/mol creatinine correlates with diabetic polyneuropathy.(21)

Indirect Indicators of B12 Status

Vitamin B12 deficiency causes pancytopenia.(24) Thus, low normal or below normal platelet, and/or white blood cell count may be indicative of B12 deficiency. Such manifestations are often seen even among patients with “normal” values of vitamin B12 (often around or slightly above 200 pg/mL). Patients with B12 deficiency also can have low hemoglobin and hematocrit values, while simultaneously having relatively high ferritin.

Since, as described above, some of the biomarkers of B12 status are affected by factors other than B12 status, it would be prudent, in clinical practice, to follow the advice Herbert (as well as others) suggested to utilize more than one assessment method in order to obtain reliable B12 status.(8,25) Practically, with the exception of serum/plasma B12 and MCV, any combination of two of the above described assessment methods (e.g. serum B12 and Hcy or serum B12 and uMMA) should give a more reliable picture of B12 status than any single assessment method.

Insight into Clinical Significance of B12 Status

            B12 deficiency may result in a number of different symptoms listed in table 2. In addition, according to Van Campenhout et al. there is strong epidemiological data linking Hcy with atherosclerosis. Hcy increases inflammation and apoptosis of the endothelial cells.(26) Other mechanisms by which Hcy may detrimentally impact risk of cardiovascular events include impairment of nitric oxide synthesis, increased LDL cholesterol oxidation, and foam cell formation.(27) Humphrey et al. estimated that each increase of 5 µmol/L is associated with about a 20% increased in the risk of coronary heart disease (CHD), which is independent of the traditional CHD risk factors.(28)

            Epidemiological data have shown an association between elevated Hcy and lowered plasma B12 with risk of bone fractures.(29) Van Meurs suggested that HHcy is a strong and independent risk factor for osteoporotic bone fractures in older men and women.(30) Elevated Hcy increases bone fracture risk in more than one way, including obstructing collagen cross-links formation, increasing osteoclasts activity, reducing osteoblasts activity, impairing taurine synthesis, increasing C-terminal telopeptides of collagen I, and impairing IGF-1 synthesis.(31)

Van Tiggelen et al. suggested that low B12 in the cerebrospinal fluid is, at least a contributing, if not a causative factor in organic mental disorder, including depression.(32) Hcy status is also associated with brain atrophy rate. Smith et al. showed that, in older individuals with mild cognitive impairment, Hcy lowering with B-vitamins (folate, B12 and vitamin B6) slowed down atrophy rate among the treatment group in comparison to controls.(33) According to Jernerén et al. B-vitamin treatment (folate, B12 and vitamin B6) slowed down brain atrophy rate only in individuals with high (> 590 µmol/L) baseline omega-3 fatty acids. The same effect was not found among individuals with low (< 390 µmol/L) baseline omega-3 fatty acids. Thus, the efficacy of B12 (plus folate and vitamin B6) therapy to prevent brain atrophy, and perhaps associated cognitive manifestations, may depend on omega-3 fatty acids status.(34)

Results of a recent meta-analysis, based on 31 studies with a total of 6,394 patients, type 2 diabetic patients with HHcy had a 93% higher risk of developing retinopathy (OR = 1.93, 95% CI 1.46-2.53).(35) Similarly, patients with type 1 diabetes with HHcy had an elevated risk (OR = 1.83, 95% CI 1.28-2.62).(35) HHcy have also been associated with other diabetic co-morbidities, including macular edema, nephropathy, and neuropathy.(36)

Table 2. Selected symptoms of vitamin B12 deficiency and/or hyperhomocysteinemia.

Category Symptoms
Neurological Deterioration of the myelin, cognitive decline (e.g. memory loss), confusion, speech impairment (slurring), difficulty walking, inability to feel the ground, tingling, paresthesia, difficulty concentrating, numbness in both legs, mood alteration/swings, muscle cramps, paralysis, electric shock sensations, jerking movements of abdominal muscles, anxiety, depression, clumsiness, visual impairment, gait, shooting pain in calves, difficulty falling asleep, restless leg syndrome, optic neuropathy, subacute degeneration of the spine
Psychiatric Disorientation, hyperactivity, decreased need for sleep, reckless and agitated behavior, social withdrawal, decreased interest, apathy, suspiciousness, hearing voices, hallucinations, anhedonia
Developmental (in infants and children) Failure to thrive, falling off the growth curves (e.g. <5th percentile on different growth charts, delays in fine and gross motor skills development, delays in receptive and expressive language development, unable to sit and/or walk, involuntary movements
Oral Glossitis, pain and burning sensation in tongue, burning mouth syndrome, gradually progressive hoarseness, difficulty eating, red stains on inside of cheeks and tongue/glossitis/beefy tongue with U-shape streaks, oral epithelial dysplasia, cheilosis
Dermatological Hyperpigmentation (blackish discoloration of the skin on knuckles, darkening of hands, feet, and tongue), skin lesions on feet, neck and upper and lower limbs, vitiligo foci (white patches on skin)
Hematological Pancytopenia (low count of all blood cell types), macrocytic anemia, hyperhomocysteinemia
Other/rare Anorexia, exercise intolerance, urinary incontinence, persistent watery diarrhea, normal blood pressure in supine position and rapid blood pressure drop in standing up position


Insight into B12 Replacement Therapy

            Treatment of B12 deficiency consists of either intramuscular injections or the use of B12 supplements. Nasal B12 sprays and toothpastes fortified with B12 are also available but not nearly as commonly used. The most frequently applied intramuscular injection dose is 1,000 µg.(37) Lower doses (e.g. 100 µg or 250 µg) are sometimes used to treat children. There is wide variation in the frequency of intramuscular injections, from daily to weekly in the onset of treatment and monthly as follow up. Injections given daily or even every other day are seldom used for more than the first week of treatment. A supplemental B12 dose of 1,000 µg/day is just as effective as therapy with intramuscular injection.(37) However, when oral supplements are utilized, patients need to use them daily for a longer period of time compared to therapy with B12 injections. Several forms of B12 are available as intramuscular preparations and oral supplements, including cyanocobalamin, methylcobalamin, and hydroxocobalamin. There is not clinical evidence of one form of B12 having advantage over the other in terms of bioavailability or clinical significance. However, findings from a recent study showed that administration of cyanocobalamin resulted in more than two-fold increase in holoTCII level in individuals with low and normal B12 compared to administration of hydroxocobalamin.(38) Cyanocobalamin is the most stable form of B12 and it is the least expensive one. It contains small amounts of cyanide, which is excreted via the kidneys. Cyanide excretion may be impaired in individuals with renal failure who should be treated with other B12 forms.


Inadequate B12 status is a major public health problem in both industrial and developing countries. Adequate intake of B12 is higher than current IOM recommendations. To make a reliable assessment of B12 status more than one assessment methods should be utilized. B12 deficiency and depletion are associated with an increased risk of several health conditions, including atherosclerosis, bone fractures, and brain atrophy. Intramuscular injections or the use of supplements constitute reliable treatments or B12 deficiency.


Author has no conflict of interest of any kind.


Roman Pawlak, PhD, RDN received his PhD from the University of Southern Mississippi (2003). Associate Professor at East Carolina University. Author: “Vitamin B12. Combating the epidemic of deficiency,” “Forever young. Secrets of delaying aging and living disease free,” “Vegan/vegetarian mother and her baby,” “In defense of vegetarianism,” “Healthy diet without secrets,” and “I am the Lord who heals you.” Member of the Vegetarian Practice Group at the Academy of Nutrition and Dietetics. He owns and manages a website



  1. Wax E. Vitamin B12 source. Accessed October 5, 2017.
  2. Pawlak R. Vitamin B12. Combating the epidemic of deficiency. 1st edition. 2016. Greenville NC.
  3. Institute of Medicine. Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline. Accessed October 5, 2017.
  4. Bor MV. von Castel-Roberts KM. Kauwell GP. Stabler SP. Allen RH. Maneval DR. Bailey LB. Nexo E. Daily intake of 4 to 7 microg dietary vitamin B-12 is associated with steady concentrations of vitamin B-12-related biomarkers in a healthy young population. Am J Clin Nutr. 2010;91(3):571-577.
  5. Doets EL. In ‘t Veld PH. Szczecińska A. Dhonukshe-Rutten RA. Cavelaars AE. van ‘t Veer P. Brzozowska A. de Groot LC. Systematic review on daily vitamin B12 losses and bioavailability for deriving recommendations on vitamin B12 intake with the factorial approach. Ann Nutr Metab. 2013;62(4):311-313.
  6. Evatt ML. Terry PD. Ziegler TR. Oakley GP. Association between vitamin B12-containing supplement consumption and prevalence of biochemically defined B12 deficiency in adults in NHANES III (Third National Health and Nutrition Examination Survey). Pub Health Nutr. 2010;13(1):25–31. 22.
  7. European Food Safety Authority. Scientific Opinion on Dietary Reference Values for cobalamin. Accessed October 5, 2017.
  8. Langan RC, Zawistowski KJ, DO. Update on Vitamin B12 Deficiency. Am Fam Physician. 2011;83(12):1425-1430.
  9. Herbert V. Vitamin B−12: plant sources, requirements, and assay. Am J Clin Nutr. l988;48(3suppl.):852−858.
  10. Gröber U. Kisters K. Schmidt J. Neuroenhancement with vitamin B12-underestimated neurological significance. Nutrients. 2013;12;5(12):5031-5045.
  11. Schwarz J. Morstadt E. Dura A. Wintgens KF. Hartmann K. Armbruster FP. Dschietzig T. Biochemical Identification of Vitamin B12 Deficiency in a Medical Office. Clin Lab. 2015;61(7):687-692.
  12. Vogiatzoglou A. Oulhaj A. Smith AD. Nurk E. Drevon CA. Ueland PM. Vollset SE. Tell GS. Refsum H. Determinants of plasma methylmalonic acid in a large population: implications for assessment of vitamin B12 status. Clin Chem. 2009;55(12):2198-2206.
  13. Smith AD. Refsum H. Do we need to reconsider blood level of vitamin B12? J Int Medic. 2012;271(2):179-182.
  14. Pawlak R. Parrott SJ. Raj S. Cullum-Dugan D. Lucus D. Understanding vitamin B12. Am J Lifestyle Med. 2012;7(1):59-65.
  15. Sarma RP. Red cell indices. In Walker HK, Hall WD, Hurst JW. Clinical Methods: The History, Physical, and Laboratory Examinations. 3rd edition. Boston: Butterworths; 1990. Accessed October 5, 2017.
  16. Green R. Miller JW. Vitamin B12 deficiency is the dominant nutritional cause of hyperhomocysteinemia in a folic acid-fortified population. Clin Chem Lab Med. 2005;43(10):1048-1051.
  17. Jacobsen DW. Homocysteine and vitamins in cardiovascular disease. Clin Chem. 1998;44:8(B):1833-1843.
  18. Quadros EV. Regec AL. Khan FK. Quadros L. Rpthenberg SP. Transcobalamin II synthesized in the intestinal villi facilitates transfer of cobalamin to the portal blood. Am J Physiol. 1999;277(1 Pt 1):G161-166.
  19. Herbert V. Fong W. Gulle V. Stopler T. Low holotranscobalamin II is the earliest serum marker for subnormal vitamin B12 (cobalamin) absorption in patients with AIDS. Am J Hematol. 1990;34(2):132-139.
  20. Herrmann W. Obeid R. Utility and limitations of biochemical markers of vitamin B12 deficiency. Eur J Clin Invest. 2013;43(3):231–237.
  21. Lloyd-Wright Z. Hvas AM. Møller J. Sanders TA. Nexø E. Holotranscobalamin as an indicator of dietary vitamin B12 deficiency. Clin Chem. 2003;49(12):2076–2078.
  22. Sun AL. Ni YH. Li XB. Zhuang XH. Liu YT. Liu XH. Chen SH. Urinary methylmalonic acid as an indicator of early vitamin B12 deficiency and its role in polyneuropathy in type 2 diabetes. J Diabetes Res. 2014;2014:921616. doi: 10.1155/2014/921616.
  23. Norman EJ. Urinary methylmalonic acid/creatinine ratio: a gold standard test for tissue vitamin B12 deficiency. J Am Geriatr Soc. 1999;47(9):1158-1159.
  24. Halfdanarson TR. Walker JA. Litzow MR. Hanson CA. Severe vitamin B12 deficiency resulting in pancytopenia, splenomegaly and leukoerythroblastosis. Eur J Haematol. 2008;80(5):448-451.
  25. Hannibal L. Lysne V. Bjørke-Monsen AL. Behringer S. Grünert SC. Spiekerkoetter U. Jacobsen DW. Blom HJ. Biomarkers and Algorithms for the Diagnosis of Vitamin B12 Deficiency. Front Mol Biosci. 2016;3:27. doi: 10.3389/fmolb.2016.00027.
  26. Van Campenhout A. Moran CS. Parr A. Clancy P. Rush C. Jakubowski H. MChir JG. Role of homocysteine in aortic calcification and osteogenic cell differentiation. Atherosclerosis. 2009;202(2):557–566.
  27. Pawlak R. Is vitamin B12 deficiency a risk factor for cardiovascular disease among vegetarians? Am J Prev Med 2015;48(6):e11–e26.
  28. Humphrey LL. Fu R. Rogers K. Freeman M. Helfand M. Homocysteine level and coronary heart disease incidence: A systemic review and meta-analysis. Mayo Clin Proc. 2008;83(11):1203-1212.
  29. van Wijngaarden JP. Doets EL. Szczecińska A. Souverein OW. Duffy ME. Dullemeijer C. Cavelaars AE. Pietruszka B. Van’t Veer P. Brzozowska A. Dhonukshe-Rutten RA. de Groot CP. Vitamin B12, Folate, Homocysteine, and Bone Health in Adults and Elderly People: A Systematic Review with Meta-Analyses. J Nutr Metab, 2013; Accessed October 6, 2017.
  30. van Meurs. Dhonukshe-Rutten RA. Pluijm SM. van der Klift M. de Jonge R. Lindemans J. de Groot LC. Hofman A. Witteman JC. van Leeuwen JP. Breteler MM. Lips P. Pols HA. Uitterlinden AG. Homocysteine Levels and the Risk of Osteoporotic Fracture. N Engl J Med, 2004;May 13;350(20):2042-2049.
  31. Babatunde T. Pawlak R. Vitamin B12 deficiency and Hyperhomocysteinemia: Risk factors for low bone density, bone turnover, and bone fractures among vegetarian adults. Unpublished.
  32. van Tiggelen CJ. Peperkamp CP. Tertoolen JF. Vitamin B12 Levels of Cerebrospinal Fluid in Patients with Organic Mental Disorder. J Orthomolec Psych. 1983;12(4):305-311.
  33. Smith AD. Smith SM. de Jager CA. Whitbread P. Johnston C. Agacinski G. Oulhaj A. Bradley KM. Jacoby R. Refsum H. Homocysteine-lowering by B vitamins slows the rate of accelerated brain atrophy in mild cognitive impairment: A randomized controlled trial. PLoS ONE 2010;5(9):e12244.
  34. Jernerén F. Elshorbagy AK. Oulhaj A. Smith SM. Refsum H. Smith AD. Brain atrophy in cognitively impaired elderly: the importance of long-chain ω-3 fatty acids and B vitamin status in a randomized controlled trial. Am J Clin Nutr. 2015;102(1):215-221.
  35. Xu C. Wu Y. Liu G. Liu X. Wang F. Yu J. Relationship between homocysteine level and diabetic retinopathy: a systematic review and meta-analysis. Diagn Pathol. 2014;9:167. doi: 10.1186/s13000-014-0167-y.
  36. Li J. Zhang H. Shi M. Yan L. Xie M. Homocysteine is linked to macular edema in type 2 diabetes. Curr Eye Res. 2014;39(7):730-735.
  37. Stabler SP. Vitamin B12 deficiency. New Engl J Med. 2013;Jan 10;368(2):149-160.
  38. Greibe E, Mahalle N, Bhide V, Heegaard CW, Naik S, Nexo E. Increase in circulating holotranscobalamin after oral administration of cyanocobalamin or hydroxocobalamin in healthy adults with low and normal cobalamin status. Eur J Nutr. 2017 Oct 16. doi: 10.1007/s00394-017-1553-5.




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