Vitamin B12 deficiency

Abstract

Vitamin B12 (B12; also known as cobalamin) is a B vitamin that has an important role in
cellular metabolism, especially in DNA synthesis, methylation and mitochondrial metabolism. Clinical
B12 deficiency with classic haematological and neurological manifestations is relatively uncommon.
However, subclinical deficiency affects between 2.5% and 26% of the general population depending
on the definition used, although the clinical relevance is unclear. B12 deficiency can affect individuals
at all ages, but most particularly elderly individuals. Infants, children, adolescents and women
of reproductive age are also at high risk of deficiency in populations where dietary intake of
B12‑containing animal-derived foods is restricted. Deficiency is caused by either inadequate intake,
inadequate bioavailability or malabsorption. Disruption of B12 transport in the blood, or impaired
cellular uptake or metabolism causes an intracellular deficiency. Diagnostic biomarkers for B12 status
include decreased levels of circulating total B12 and transcobalamin-bound B12, and abnormally
increased levels of homocysteine and methylmalonic acid. However, the exact cut-offs to classify
clinical and subclinical deficiency remain debated. Management depends on B12 supplementation,
either via high-dose oral routes or via parenteral administration. This Primer describes the current
knowledge surrounding B12 deficiency, and highlights improvements in diagnostic methods as well
as shifting concepts about the prevalence, causes and manifestations of B12 deficiency.

Full text

VitaminB12 (B12; also known as cobalamin) is one of
eight B vitamins and its role in cellular metabolism is
closely intertwined with that of folate, another B vitamin
(FIG.1). Since the discovery and characterization of B12
more than 60years ago and the recognition of its central
role in preventing the serious disease known as perni-
cious anaemia, much has become known about B12
deficiency1,2. Pernicious anaemia originally acquired
its appropriate eponym because of the ultimately fatal
haemato logical and devastating neurological manifesta-
tions of the disease and was later shown to be caused by
autoimmune destruction of gastric parietal cells and their
product, intrinsic factor (also known as gastric intrinsic
factor), which is required for B12 absorption. Previously
considered to be a nutritional deficiency disease that
was largely caused by malabsorption of the vitamin and
restricted to older people, particularly those of North
European descent, B12 deficiency is now considered to
be a problem of global dimensions, frequently caused by
dietary inadequacy, particularly among children and in
women of reproductiveage3.
The effects of B12 deficiency are mainly seen in the
blood and nervous system. The classic manifestations of
B12 deficiency were first identified in pernicious anae-
mia, the cause of which was then unknown4,5. Since then,
the spectrum has shifted considerably, starting with the
renewed recognition that neurological manifestations
(such as sensory and motor disturbances (particularly in
the lower extremities), ataxia, cognitive decline leading
to dementia and psychiatric disorders) often predomin-
ate and can frequently occur in the absence of haemato-
logical complications6. In addition, the identification of
subtler degrees of B12 deficiency7, made possible by the
introduction of assays for the metabolites methyl malonic
acid (MMA) and homocysteine in clinical practice6,8
(FIG.1), has broadened the landscape of what might be
attributable to B12 deficiency, but has opened a Pandora’s
box of controversy regarding what could be considered
actual, clinical B12 deficiency as opposed to states of
metabolic inadequacy of the vitamin7,9. Progression from
normalcy to clinical deficiency passes through a stage of
inadequacy during which biochemical evidence of B12
insufficiency in the form of increased blood and tissue
levels of MMA and homocysteine and declining levels
of the portion of B12 bound to transcobalamin (known
as holotranscobalamin) precede the appearance of any
morbid manifestations of deficiency. This condition has
also been referred to as ‘subclinical’ B12 deficiency and is
associated with low or marginal B12 levels10. Individuals
with biomarkers indicating a subclinical deficiency are
Correspondence to R.G.
Department of Pathology
andLaboratory Medicine,
University of California Davis,
4400 V Street, PATH Building,
Davis, California95817, USA.
rgreen@ucdavis.edu
Article number: 17040
doi:10.1038/nrdp.2017.40
Published online 29 Jun 2017
VitaminB12 deficiency
Ralph Green1, Lindsay H.Allen2, Anne-Lise Bjørke-Monsen3, Alex Brito2,
Jean-LouisGuéant4, Joshua W.Miller5, Anne M.Molloy6, Ebba Nexo7, Sally Stabler8,
Ban-Hock Toh9, Per MagneUeland3,10 and Chittaranjan Yajnik11
Abstract | VitaminB12 (B12; also known as cobalamin) is a B vitamin that has an important role in
cellular metabolism, especially in DNA synthesis, methylation and mitochondrial metabolism. Clinical
B12 deficiency with classic haematological and neurological manifestations is relatively uncommon.
However, subclinical deficiency affects between 2.5% and 26% of the general population depending
on the definition used, although the clinical relevance is unclear. B12 deficiency can affect individuals
at all ages, but most particularly elderly individuals. Infants, children, adolescents and women
ofreproductive age are also at high risk of deficiency in populations where dietary intake of
B12‑containing animal‑derived foods is restricted. Deficiency is caused by either inadequate intake,
inadequate bioavailability or malabsorption. Disruption of B12 transport in the blood, or impaired
cellular uptake or metabolism causes an intracellular deficiency. Diagnostic biomarkers for B12 status
include decreased levels of circulating total B12 and transcobalamin‑bound B12, and abnormally
increased levels of homocysteine and methylmalonic acid. However, the exact cut‑offs to classify
clinical and subclinical deficiency remain debated. Management depends on B12 supplementation,
either via high‑dose oral routes or via parenteral administration. This Primer describes the current
knowledge surrounding B12 deficiency, and highlights improvements in diagnostic methods as well
asshifting concepts about the prevalence, causes and manifestations of B12 deficiency.
NATURE REVIEWS
|
DISEASE PRIMERS VOLUME 3
|
ARTICLE NUMBER 17040
|
1
PRIMER

particularly challenging for the clinician because it is
not known whether these individuals will progress to
overt deficiency or remain in a chronic but stable low
B12 status of no clinical relevance11–13.
In this Primer, we give a comprehensive description of
the epidemiology of B12 deficiency and dietary recom-
mendations to prevent its occurrence, as well as the
pathophysiology, diagnosis, preventive and public health
issues, and management surrounding the condition.
Epidemiology
Risk factors
In higher-income countries, the major risk factor for
developing B12 deficiency (BOX1) is the well- characterized
autoimmune disease pernicious anaemia, which is caused
by a lack of production of intrinsic factor by gastric
parietal cells that is needed for the intestinal absorption
of B12 and eventually leads to the development of anae-
mia and/or severe neurological symptoms. Pernicious
anaemia can affect people of all ages, but its incidence
rises with age1,5,14. Conservative estimates indicate that
perni cious anaemia affects 2–3% of individuals ≥65years
of age1,14. In addition, important risk factors are gastro-
intestinal surgery, such as gastric bypass or removal of
the terminal ileum, which compromise the absorption
ofB12. However, in low-income countries, B12 deficiency
is largely due to a low intake of B12-rich foods of animal
origin, but possibly also to gastrointestinal infections and
infestations, and host–microbiota interactions15.
Contributing risk factors include Helicobacter pylori
infection, intestinal bacterial overgrowth, poor food
intake, alcoholism, smoking and long-term use of drugs,
such as proton pump inhibitors, histamine H2 receptor
antagonists and metformin. Various diseases, includ-
ing malaria, HIV infection and tuberculosis16 might
contribute to deficiency through a combination of fac-
tors. Conversely, although not the cause of the primary
disease, concomitant B12 deficiency may affect the pro-
gression of the particular condition, for example, in HIV
infection17. Surprisingly, in one study, slum dwellers had
better B12 status than urban dwellers, possibly related to
poorer hygiene in the slums, which putatively exposes
individuals to ingestion of B12-containing micro-
organisms15. Both individual-level and population-level
factors (such as socioeconomic status, religion, cultural
practices and public health policies) contribute to the
general health status and B12 status of a population.
Prevalence
General population. Clinical B12 deficiency, with classic
haematological or neurological manifestations, is rela-
tively uncommon. Low or marginal B12 status, in the
absence of overt haematological or neurological impair-
ments, is much more common, particularly in popu-
lations with a low intake of B12-rich, animal-sourced
foods18. B12 status in the United States has been exten-
sively assessed in the National Health and Nutrition
Examination Survey (NHANES). Using NHANES data
from 1999 to 2004 (REF.19), the prevalence of B12 status
defined as low was estimated to be 2.9%, 10.6% or 25.7%
based on serum B12 cut-off values of <148, <200 and
<256 pmol per litre, respectively. Using these cut-off
values, the prevalence of low B12 status increased with
age from young adults (19–39years of age) to older adults
(≥60years of age), and was generally higher in women
than in men (prevalence of 3.3% versus 2.4% with a serum
B12 level of <148 pmol per litre, respectively). Using
instead increased levels of MMA as a functional indicator
of B12 status19 (TABLE1), the prevalence of low B12 status
was 2.3% or 5.8% based on cut-off values of >0.376and
>0.271 μmol per litre, respectively. The prevalence of
increased levels of MMA increased with age and was not
different between men and women. Notably, only 50–75%
of participants in NHANES with low levels of serum B12
had increased levels of MMA19. Individuals with low lev-
els of total serum B12 and increased levels of MMA might
better reflect the true prevalence of actual B12 deficiency
(defined biochemically) than those with only one of the
markers outside the cut-off limits. Data from NHANES
2003–2006 indicate that the consumption of B12-fortified
foods and supplements improved B12 status20.
National studies from various countries have shown
that prevalence estimates for low B12 status (<148 pmol
per litre) and marginal deficiency (148–221 pmol per litre)
far exceed those in the United States, particularly in South
America, Africa and Asia (FIG.2). Notably, a high preva-
lence of low B12 status is not confined to older adults,
with some countries exceeding >40% prevalence in dif-
ferent subpopulations (children, young adults, women of
childbearing age, pregnant women and older adults).
B12 status, as assessed by blood levels of B12 and its
biomarkers, varies throughout an individual’s lifetime,
asdoes the prevalence of B12 deficiency. When inter-
preting these markers it is, therefore, important to take
age and physiological circumstances of the individual
intoconsideration.
Author addresses
1Department of Pathology and Laboratory Medicine,
University of California Davis, 4400 V Street,
PATHBuilding, Davis, California 95817, USA.
2USDA, ARS Western Human Nutrition Research Center,
University of California Davis, Davis, California, USA.
3Laboratory of Clinical Biochemistry, Haukeland University
Hospital, Bergen, Norway.
4Inserm UMRS 954 N‑GERE (Nutrition Génétique
etExposition aux Risques Environnementaux),
Universityof Lorraine and INSERM, Nancy, France.
5School of Environmental and Biological Sciences,
RutgersUniversity, New Brunswick, New Jersey, USA.
6School of Medicine and School of Biochemistry and
Immunology, Trinity College Dublin, University of Dublin,
Dublin, Ireland.
7Department of Clinical Medicine, Clinical Biochemistry,
Aarhus University Hospital, Aarhus, Denmark.
8Department of Medicine, University of Colorado Denver,
Denver, Colorado, USA.
9Centre for Inflammatory Diseases, Monash Institute of
Medical Research, Clayton, Victoria, Australia.
10Section for Pharmacology, Department of Clinical
Science, University of Bergen, Bergen, Norway.
11Diabetes Unit, King Edward Memorial Hospital, Pune,
Maharashtra, India.
PRIMER
2
|
ARTICLE NUMBER 17040
|
VOLUME 3 www.nature.com/nrdp

Elderly individuals. As noted, B12 deficiency is more
prevalent among older people, generally increasing
beyond 60years of age21–23. Underlying causes of defi-
ciency are diverse and extend well beyond the 2–3%
prevalence of pernicious anaemia24 (BOX1). Among older
people, 10–15% have subclinical B12 deficiency12,19,
which can often, but not always, be normalized with
B12 therapy19,25,26. The prevalence is even higher in the
‘oldest-old’, with reports of 23–35% of individuals over
80years of age having B12 deficiency27. Variable suscep-
tibility across different population and racial groups has
also been reported14,23.
Pregnancy and lactation. Pregnancy and lactation alter
maternal B12 status in a manner that facilitates the trans-
fer of B12 to the fetus and infant. Profound physiological
and anatomical changes occur in virtually every organ
system during pregnancy with considerable conse-
quences on biochemical markers, thus complicating the
evaluation of micronutrient status and limiting the use of
established reference ranges determined in non- pregnant
women28. Most markers of B12 status (circulating levels
of total B12, holotranscobalamin, MMA and homo-
cysteine) are lower during pregnancy than pre-pregnancy
levels. Total B12 levels in the mother gradually decrease
during pregnancy28–30, whereas circulating levels of
holotranscobalamin decrease or remain relatively stable
(FIG.3). Homocysteine and MMA levels increase during
the latter part of pregnancy compared with the first tri-
mester, which may be indicative of a degree of metabolic
intracellular B12 depletion in pregnant women, despite
the fact that both homocysteine and MMA are lower
than the established cut-off levels defining deficiency in
non-pregnant wom en28,30–32. Indeed, this increase depends
on maternal B12 status28 and is lower in women taking
B12 supplements31. This indicates a need for specific
reference ranges based on B12-replete (supplementedto
normal levels) pregnant and lactating women. Owing
tothese changes, the true prevalence of B12 deficiency in
pregnancy is difficult to quantify, but is reported to occur
in <10% (in Canada and Brazil)33,34 to >70% (in parts of
India and Turkey) of pregnant women35–37.
In one study, postpartum, maternal circulating B12
levels were reported to be significantly higher than in
non-pregnant women31. This might represent a physio-
logical adaptation to enhance mobilization of maternal
B12 stores for transfer to the infant through increasing
levels in breast milk (BOX2).
Figure 1 | VitaminB12 and folate metabolism and function. VitaminB12 (B12) and folate are required for the methionine
synthase reaction in which a methyl group is transferred from methyltetrahydrofolate (methyl‑H4‑folate; also known
aslevomefolic acid) to homocysteine by methionine synthase, with methyl‑B12 as a coenzyme to form methionine.
Theresulting H4‑folate can then be returned to the folate pool and made available for the generation of methylene‑ H4‑
folate, the form required for denovo synthesis of thymidine, which is essential for DNA replication and repair. Hence,
either folate or B12 deficiency results in the same biochemical perturbation in thymidine synthesis and DNA replication.
In the case of B12 deficiency, folate is ‘trapped’ in the unusable methyl‑form5,194. B12 is also involved in the conversion
ofmethylmalonyl‑CoA (methylmalonic acid bound to coenzyme A) to succinyl‑CoA by the enzyme methylmalonyl‑CoA
mutase with adenosyl‑B12 as a cofactor; succinyl‑CoA is a major intermediary of the tricarboxylic acid (TCA) cycle.
InB12 deficiency, substrates of both B12‑dependent reactions accumulate, which leads to increased levels of
methylmalonic acid and homocysteine in the plasma. A combination of low levels of B12 and increased levels of
folatewas associated with higher concentrations of methylmalonic acid and total plasma homocysteine156,157.
Majorcomplications of B12 deficiency are megaloblastic anaemia, as a result of inhibition of DNA synthesis,
andneurological manifestations.
Nature Reviews | Disease Primers
Nucleus
Folate
cycle
Methylation
Methionine
cycle
TCA cycle
Deoxyuridine
Cytoplasm
Folic acid
H2-folate
H4-folate
Methylene-
H4-folate
Methyl-
H4-folate
Dietary folate
Methyl-B12
Methionine
synthase
MethionineHomocysteine
S-adenosyl-
methionine
S-adenosyl-
homocysteine
Methylmalonic
acid
Methylmalonyl-CoA
Succinyl-CoA
Mitochondrion
Methylmalonyl-CoA
mutase
Adenosyl-B12
DNA
synthesis
Thymidine
PRIMER
NATURE REVIEWS
|
DISEASE PRIMERS VOLUME 3
|
ARTICLE NUMBER 17040
|
3

Infancy and childhood. Infancy, childhood and adoles-
cence are times of rapid growth, during which demand for
B12 is high and B12 status undergoes marked changes38,39.
Low maternal B12 status, extended breastfeeding and
alow intake of animal food after weaning are major risk
factors for B12 deficiency. During the first months of life,
B12 levels decrease, whereas homocysteine and MMA
levels increase, with the lowest B12 concentrations and
the highest homocysteine and MMA levels seen between
4 and 6months in exclusively breastfed infants39–41. From
6months, serum B12 levels increase and peak between
3and 7years of age and then decrease throughout adoles-
cence. Plasma homo cysteine levels decrease and remain
low (<6 μmol per litre) up to 7years of age, and then start
to increase; the median plasma MMA levels decrease
after 6months and remain low throughout childhood
(<0.26 μmol per litre)39.
During the first 2years of life, the homocysteine
level is a reliable marker of B12 status, whereas in
older children and adults in high-income countries,
homocysteine levels mainly reflect folate status in non-
folic-acid- fortified populations39. In breastfed infants
(BOX2),MMA levels are inversely related to B12 levels,
but the MMAconcentrations are relatively high through-
outthe B12 distribution than in older children39. The
higher MMA levels may be due to intestinal absorption of
propionate and other MMA precursors that are produced
by intestinal bacteria or by degradation of odd chain fatty
acids that are present in breast milk39,42.
A biochemical profile with low levels of B12 and high
levels of homocysteine and MMA, which is indicative of
impaired B12 functional status, is seen in more than two-
thirds of mainly breastfed Norwegian infants of 6weeks
to 4months of age43, a finding that is also observed in
other infant populations41,44. Intervention studies with
a single intramuscular dose of 400 μg of hydroxy-B12
given at 6weeks have demonstrated that it is possible
to improve this profile, with a 39% reduction in homo-
cysteine levels and a 66% reduction in MMA levels, indi-
cating that the profile commonly seen in breastfed infants
does indeed reflect a modifiable state of B12 inadequacy
and not simply organimmaturity43,45.
In studies from Pune, India, there was a precipitous
fall in circulating B12 concentrations from 6 to 12 and
18years of age, and deficiency (<150 pmol per litre) was
found in 16%, 24% and 58% at those ages, respectively
(C.Y., unpublished observations). B12 concentrations
were associated with a genetic risk score for B12-related
morbidity based on eight independently associated
single-nucleotide polymorphisms in genes encoding
proteins involved in B12 metabolism, maternal vitamin
concentrations in pregnancy and weight gain during
childhood (C.Y., unpublished observations). In addition,
astrong intra-familial correlation for B12 status has been
reported, indicating that the ‘family environment’ (com-
prising socioeconomic factors, food habits, hygiene, and
religious and cultural practices) is important. Across-
sectional study showed similar intra- familial associations
of B12 status in Amazonian children (4.5% deficient)46.
Mechanisms/pathophysiology
B12 deficiency relates to a series of pathophysiological
mechanisms that can occur in infancy, childhood, ado-
lescence and adult life, all of which can affect the B12
supply, the demand or both. Cellular deficiency in B12is
caused by inadequate intake, malabsorption, chem-
ical inactivation, or inherited disruption of either B12
transport in the blood or intracellular metabolism1,4,5.
Inadequate intake and bioavailability
Microorganisms (that is, bacteria and archaea) are the
ultimate source of B12 in nature, and, in conventional
diets, B12 is exclusively available from animal food
sources, such as meat, liver, fish, eggs and dairy prod-
ucts. The daily B12 requirement of 2–3 μg is easily met
in those who consume large amounts of animal products
or who take supplements, but not in vegans who obtain
only 0–0.25 μg daily18. Low intake of animal-sourced
foods may be involuntary owing to limited accessibility
in the food supply, or voluntary owing to cultural, reli-
gious or personal restrictions. In addition, demands and
needs vary depending on age and physiological status,
such as pregnancy.
Although the Dietary Recommended Intakes for B12
have been defined for children and adolescents (by age),
for adults, pregnant and breastfeeding women and elderly
individuals aggregate figures and various sources have
been used38. In addition, the Dietary Recommended
Intakes do not take factors that influence bioavail-
ability into account, which seem to vary widely within
these demographic groups, conditions of the gastro-
intestinal tract, overall total amount of B12 ingested
and food sources1,3. Bioavailability is in part related to
the need to release B12 from its protein carriers in food.
Bioavailability of B12 from milk is better than from other
Box 1 | Causes of vitamin B12 deficiency
Conditions that cause vitamin B12 (B12) deficiency and their associated pathogenetic
bases:
• Pernicious anaemia: insufficient B12 absorption caused by a deficiency of intrinsic
factor (usually owing to an autoimmune disease)
• Gastric disease or surgery (partial or complete gastrectomy or gastric reduction
surgery): a deficiency of intrinsic factor
• Chronic atrophic gastritis (chronic inflammation causing loss of the gastric
acid‑producing cells) and an intake of drugs that affect gastric acid secretion or
gastric pH (that is, proton pump inhibitors, histamine receptor 2 antagonists and
antacids): B12 is not released from the food matrix owing to insufficient hydrochloric
acid and low pepsin activity
• Pancreatic disease or pancreatectomy: B12 is not released from the haptocorrin
complex owing to insufficient pancreatic enzyme activity
• Other intestinal diseases, ileal resection, parasitic infestations and bacterial
overgrowth: impaired absorption of the B12–intrinsic factor complex
• Medications that affect B12 absorption or metabolism: reduction of serum B12 levels
via known mechanisms (for example, cholestyramine) and unknown mechanisms
(forexample, metformin)
• Dietary factors such as general malnutrition, vegetarian or vegan diet, and chronic
alcoholism: reduced B12 consumption
• Inherited disorders: decreased expression, binding activity or affinity of receptors
andproteins involved in B12 trafficking and metabolism
• Miscellaneous: including HIV infection and nitrous oxide anaesthesia
PRIMER
4
|
ARTICLE NUMBER 17040
|
VOLUME 3 www.nature.com/nrdp

animal food sources38. B12 from cow’s milk may be more
bioavailable than B12 from human breast milk, although
this may relate, to some extent, to the higher concentra-
tion of B12 in cow’s milk44. Transcobalamin from cow’s
milk promotes intestinal cellular uptake of B12 invitro47.
The possibility that transcobalamin or other factors
present in cow’s milk might enhance B12 absorption
could have implications for both the prevention and the
management of B12deficiency.
Chemical inactivation of B12
The anaesthetic gas nitrous oxide chemically inactivates
B12 through irreversible oxidation of its coenzyme form,
methylcobalamin, at the active site of the B12-dependent
methionine synthase reaction48. Depending on the B12
status of the individual exposed to the gas, as well as
the frequency and duration of its use, deficiency may be
precipitous or gradual49,50.
Absorption of B12
The main steps in B12 bioavailability, absorption, blood
transport and intracellular metabolism are summarized
in FIG.4.
Pernicious anaemia. The virtually complete absence
of intrinsic factor in patients with pernicious anaemia
causes B12 deficiency through malabsorption of both
dietary and recycled biliary B12, resulting in progressive
exhaustion of B12 reserves in the body. Pernicious anae-
mia arises as a consequence of autoimmune gastritis,
which is a chronic inflammatory disease of the fundus
and body of the stomach, sparing the antrum. Initially
asymptomatic and often associated with circu lating
parietal cell antibody directed against the gastric proton
pump (gastric H+/K+ ATPase), the gastritis progresses
over many years to type A chronic atrophic gastritis
with destruction of gastric parietal cells, which produce
hydrochloric acid and intrinsic factor51. The gastritis
may present initially as iron deficiency anaemia from
loss of gastric acid, which is required for facilitating iron
absorption52. Ultimately, loss of intrinsic factor together
with production of neutralizing antibody against intrin-
sic factor leads to B12 malabsorption, mega loblastic
anaemia (anaemia caused by defective DNA synthe-
sis) and neurological complications, including neuro-
pathy and subacute combined degeneration of the
spinal cord. FIGURE5 summarizes the key mechanistic
pathways involved in pernicious anaemia. Mutations
in GIF, encoding intrinsic factor, can also lead to an
inherited form of B12 malabsorption and deficiency,
which resembles pernicious anaemia, but without
autoantibodiesinvolvement 2.
Clustering of autoimmune gastritis with other
organ-specific autoimmune diseases, including auto-
immune thyroiditis and type1 diabetes mellitus,
suggests genetic predisposition to the development of
gastritis. However, the precise predisposing genes have
not been identified. A report of molecular mimicry by
H.pylori antigens of proton pump antigens has raised
the suggestion of a microbial trigger for the initiation
of auto immune gastritis53, but this remains unproved.
However, this observation raises important questions
regarding the role of this ubiquitous microorganism in
the causation of B12 malabsorption.
Impaired protein degradation. Digestion of food-
derived proteins and haptocorrin derived from the saliva
and bile (previously known as R binder) are essential
for the transfer of B12 to intrinsic factor in the duo-
denum (FIG.4). Haptocorrin is a B12-binding protein
present in many body fluids. It protects B12 during its
passage through the acidic environment in the stomach.
Table 1 | Biomarkers of vitamin B12 status
Biomarker; unit Assay principle Tentative
reference
interval*
Tentative
cut‑offvalue
for B12
deficiency*
Tentative
cut-off value
for B12
repletion*
Major confounding factors
B12; pmol per litre Protein‑binding assay 200–600 <148 >221 Alterations in the plasma‑binding
proteins, haptocorrin or transcobalamin
Holotranscobalamin
(transcobalamin‑
bound, active B12);
pmolper litre
Immunological 40–100 <35 >40 Genetic variation in TCN2 (REFS73,209)
and kidney function
Homocysteine;
μmolper litre‡
Immunological, high‑performance
liquid chromatography or gas
chromatography mass spectrometry
8–15 >15 <8 Folate and B6 deficiency, kidney
andthyroid function, sex and age
Methylmalonic
acid; μmolper litre
Liquid chromatography–
mass spectrometry or gas
chromatography mass spectrometry
0.04–0.37 >0.37 <0.27 Kidney function and HIBCH
polymorphisms117
4cB12§See formula below –2.5–1.5 <–0.5 >0.5 Can be corrected for folate status and age
Blood tests are used to confirm a diagnosis of vitamin B12 (B12) deficiency or to rule out the presence of poor B12 status. Most often, plasma B12 concentration — or
as a more recent alternative holotranscobalamin — is used as the initial test. If the result is within the grey zone, analysis of the methylmalonic acid level is required.
Homocysteine can replace methylmalonic acid in folate‑repleted populations. *Reference intervals cover 95% of B12‑replete individuals. Deficiency includes both
clinical and subclinical deficiency. The exact values (except for 4cB12) for both reference intervals and cut‑off limits may deviate in local settings37. The values
indicated are based on the cited literature and the experience of the authors, and define the cut‑off value for subclinical B12 deficiency. ‡In populations not
receiving folic acid fortification. §Combined indicator of B12 status involving four parameters (4cB12) is a combination of B12, holotranscobalamin, homocysteine
and methylmalonic acid, and is quantified as log10((B12 x holotranscobalamin)/(methylmalonic acid x homocysteine))test106.
PRIMER
NATURE REVIEWS
|
DISEASE PRIMERS VOLUME 3
|
ARTICLE NUMBER 17040
|
5

Reduced acid secretion in the stomach associated with
chronic gastritis or long-term treatment with proton
pump inhibitors impairs the release of dietary B12
from food proteins and may produce deficiency, despite
sufficient intrinsic factor secretion54–56.
Conversely, both Zollinger–Ellison syndrome (caused
by a gastrin-producing tumour and associated with
gastric hyperacidity) and exocrine pancreatic insuffi-
ciency are rare causes of B12 malabsorption due to low
pH in the small intestine and impaired degradation
of haptocorrin by pancreatic enzymes; both limit B12
transfer from haptocorrin to intrinsic factor2.
Bacterial overgrowth. Some microbial intestinal flora,
such as Pseudomonas spp. and Klebsiella spp., are B12
providers, others transform B12 into other corrinoids
(B12 analogues), whereas the majority are B12 con-
sumers57. This explains how B12 can be a modulator of
microbiota, whereas its bioavailability to the host can,
inturn, be influenced by the microflora57. The high
specifi city of intrinsic factor for B12 limits the assimi-
lation of analogues produced by the microflora58.
Agenome-wide association study (GWAS) of B12
deficiency in India identified single-nucleotide poly-
morphisms in genes encoding proteins involved in the
glycan pathway, which could influence intestinal absorp-
tion and also influence the gut microbiota59. Extreme
conditions of bacterial overgrowth, as arise particularly
in blind loop syndrome (resulting from surgical anasto-
moses that create gastrointestinal cul de sacs), produce
a state akin to malabsorption of B12 through extensive
heterotrophic consumption of the nutrient. These syn-
dromes may occur following gastrectomies, segmentary
and ileocolic intestinal resection, inflammatory bowel
diseases, diverticulosis as well as prolonged gastric
achlor hydria (impaired gastric acid secretion). In addi-
tion, poly morphisms in FUT2, which encodes fucosyl-
transferase (an enzyme that catalyses fucose addition
to form H-type antigens in exocrine secretions and red
blood cell membranes60), are involved in susceptibility to
H.pylori infection. Fucosyltransferase can also influence
the secretion of intrinsic factor, which is needed for the
absorption ofB12 (REFS59,61,62).
Impaired intestinal internalization. Two distinct
mechanisms are involved in the intestinal uptake of
B12: specific and efficient uptake via receptor- mediated
endocytosis of the intrinsic factor–B12 complex in the
terminal ileum mediated by a receptor complex known
as cubam, which consists of the proteins cubilin and
amnionless (encoded by CUBN and AMN, respec-
tively)2 (FIG.4), and an inefficient, passive diffusion path-
way that takes place throughout the intestine. Megalin
(also known as low-density lipoprotein receptor-related
protein 2 (LRP2)) — a multiligand transmembrane pro-
tein — can contribute to the internalization of B12 in
the intestine, in a manner that is analogous to its role
in the reabsorption of transcobalamin in the kidney2,63.
However, patients with mutations that affect LRP2 have
not been found to have any abnormality in B12 absorp-
tion. Once internalized into the enterocyte, there is
Figure 2 | Prevalence of low and marginal vitamin B12. Global prevalence of low
andmarginal serum or plasma vitamin B12 (B12) concentrations from selected national
surveys or large studies. Studies selected are nationally representative or large, with
apparently healthy individuals categorized as having low or marginal serum or plasma
B12 concentrations defined as <148 pmol per litre and 148–221 pmol per litre,
respectively. These figures are based on data extracted from three systematic reviews
that focused on population‑based studies assessing B12 status33,195,196, complemented
with individual studies197–199 and ongoing studies. Data presented from Denmark, the
United Kingdom, Turkey, Australia, South India and Jordan are limited to the prevalence
of low B12 status because marginal prevalence was not reported22,200–205.
Nature Reviews | Disease Primers
North America
Canada (6–79 years of age)
Canada (adult women; Vancouver)
NHANES III (<4 years of age)
NHANES III (12–19 years of age)
NHANES III (20–29 years of age)
NHANES III (30–39 years of age)
NHANES III (40–49 years of age)
NHANES III (50–59 years of age)
NHANES III (60–69 years of age)
NHANES III (>70 years of age)
SALSA (elderly Latino individuals)
Latin America
Brazil (1st trimester pregnancy)
Brazil (2nd trimester pregnancy)
Brazil (3rd trimester pregnancy)
Chile (adult women)
Chile (elderly individuals)
Colombia (<18 years of age)
Colombia (pregnant women)
Colombia (adult women)
Costa Rica (adults)
Ecuador (elderly individuals)
Guatamala (lactating women)
Guatamala (school children)
Guatamala (infants)
Guatamala (adult women)
Mexico (preschoolers)
Mexico (school children)
Mexico (adolescent girls)
Mexico (adult women)
Venezuela (elderly individuals)
Europe
Denmark (adults)
UK OHAP (elderly individuals)
UK MRC (elderly individuals)
UK NDNC (elderly individuals)
Italy (elderly individuals)
Oceania
Australia (refugees)
New Zealand (elderly individuals)
Africa
Botswana (school children)
Cameroon (children)
Cameroon (adult women)
Kenya (school children)
Niger (pregnant women)
The Gambia (1st trimester pregnancy)
The Gambia (3rd trimester pregnancy)
The Gambia (12 weeks postpartum)
The Gambia (infants 12 weeks of age)
The Gambia (infants 24 weeks of age)
Asia
Jordan (>19 years of age)
Turkey (adult women)
Bangladesh (adult women)
Bangladesh (pregnant women)
India (preschoolers)
India (adults)
South India (adults)
China (adult women)
40 10020 60
Prevalence of low and marginal serum B12 (%)
0 80
Low B12
concentration
<148 pmol per litre
Marginal B12
concentration
148–221 pmol per litre
PRIMER
6
|
ARTICLE NUMBER 17040
|
VOLUME 3 www.nature.com/nrdp

some evidence that multidrug resistance protein1 may
be involved in the process to export the vitamin into the
bloodstream64. B12 is exported into the bloodstream
where it binds to transcobalamin for delivery of the
vitamin to all cells in thebody.
Imerslund–Gräsbeck syndrome (megaloblastic anae-
mia1; Online Mendelian Inheritance in Man (OMIM)
#261100) is a rare recessive disorder caused by muta-
tions in CUBN or AMN that result in intestinal B12
mal absorption, anaemia and deficient renal protein
reabsorption65,66. Various phenotypes exist, including
decreased binding activity or affinity by the receptor or
expression of an unstable receptor67.
Inflammatory bowel diseases, infection and drugs.
Coeliac disease or Crohn’s disease affecting the ileum
results in B12 malabsorption through villous atrophy
and mucosal injury. Two parasitic infestations prod-
uced by a fish tapeworm, Diphyllobothrium latum, and
the protozoan Giardia lamblia can lead to malabsorp-
tion through B12 trapping by the parasite. Tropical
sprue results in malabsorption through intestinal villous
atrophy. B12 malabsorption may also be due to medical
interventions. Cholestyramine (a bile acid resin used
to treat hypercholesterolaemia) can chelate intrinsic
factor, whereas colchicine (used for acute gout) and
several antibiotics (including the anti-tuberculosis drug
para-aminosalicylic acid) can act as inhibitors of intrin-
sic factor–B12 endocytosis. The duration or frequency
of use of these drugs is usually insufficient to result in
clinical B12 deficiency, in contrast to the long-term use
of drugs such as proton pump inhibitors, histamine H2
receptor antagonists and metformin, as noted above1,5.
Disrupted transport and intracellular metabolism
About 20% of total plasma B12 levels are bound to
transcobalamin and are, therefore, available to the cells
via receptor-mediated uptake. The major, remaining,
fraction is bound to a circulating form of haptocorrin.
The transcobalamin-bound B12 fraction (holotrans-
cobalamin) is internalized by receptor-mediated endo-
cytosis via the receptor CD320 antigen (in the brain and
other tissues) or LRP2 (in the luminal portion of the
kidney tubule) and is subsequently degraded in lyso-
somes68. Intracellularly, B12 is reduced and converted into
its coenzymatically active forms for use in two intra cellular
processes: the cytoplasmic conversion of homocysteine
into methionine and the mitochondrial metabolism of
methylmalonyl-CoA to succinyl-CoA.There are several
inherited disorders that affect the sequential steps in the
assimilation, transport and intracellular processing of B12
(REFS69,70) (FIG.4b). The crucial role of transco balamin
in B12 transport and delivery is exemplified by clinical
reports that >20 inherited mutations in TCN2 (encoding
transco balamin2) mani fest as failure to thrive and severe
disease in infancy. These mutations are associated with
severe megalo blastic anaemia and neurological problems
with fatal outcome if not recog nized and promptly treated
with high doses of B12 (REFS70–72). Other TCN2 poly-
morphisms have less-profound effects on B12 metabo-
lism, including possible differences in the expression level
and blood concentration of transcobalamin and indices
of B12 status in healthyindividuals73,74.
The inherited disorders of intracellular B12 metabo-
lism are classified as complementation groups cblA–J by
complementation phenotyping of fibroblasts70,75 (FIG.4b).
They encompass several genes, which, respectively,
encode or regulate the transcription of proteins involved
in the intracellular trafficking and metabolic activation
of B12. Mutations that block the lysosomal release of B12
or the cytoplasmic function of methylmalonic aciduria
and homocystinuria typeC protein (MMACHC) lead
to impaired synthesis of adenosyl- B12 and methyl-B12
with consequent increase in the levels of homo cysteine
and MMA. These dis orders are character ized by megalo-
blastic anaemia, neurological involvement, stunted
growth and sometimes retino pathy. Mutations that block
only the synthesis of adenosyl- B12 result in increased
levels of MMA and produce acute encephalopathy with
ketoacidosis, hyperammonaemia and hyper glycaemia.
Mutations that block the synthesis of methyl-B12
increase only the levels of homocysteine and are mani-
fested by megalo blastic anaemia and encephalopathy
during the first few months oflife.
Complications of B12 deficiency
Cellular and molecular consequences. The common
consequence of B12 deficiency and genetic disorders
affecting B12 metabolism is a cellular deficit in one or
both of the coenzyme forms of B12 (that is, adenosyl-B12
and methyl-B12). At the molecular level, B12 defi-
ciency leads to an impaired methylation and impaired
metabo lism of methylmalonate, which is derived from
the catabolism of certain amino acids and fatty acids
(FIG.1). Methyl-B12 deficiency results in homocysteine
Figure 3 | Biomarkers of vitaminB12 status during pregnancy and lactation. Levels of
vitaminB12 (B12) and associated markers presented as percentage of normal levels based
on a population of 207 non‑pregnant, non‑lactating women 18–40years of age with
serum B12 levels of 225–750 pmol per litre, serum folate levels of >10 nmol per litre and
normal renal function (a glomerular filtration rate of >75). Percentage change during
pregnancy and lactation was calculated using the following median levels: B12: 359 pmol
per litre, homocysteine: 9.3 μmol per litre and methylmalonic acid (MMA): 0.12 μmol per
litre. Data during pregnancy for B12 and total homocysteine levels are based on REF.29;
data on MMA levels are based on REF.28; and data on B12 status from the lactational
period are based on REF.43. Percentage change of holotranscobalamin levels during
pregnancy is based on REF.28 and during lactation on REF.31. Errors bars represent the
10th and 90th percentiles from the geometric mean.
Nature Reviews | Disease Primers
Normal adult level (%)
200
150
100
50
0
Phase
Third trimesterSecond trimester Lactation
First trimester
B12
Total homocysteine
MMA
Holotranscobalamin
PRIMER
NATURE REVIEWS
|
DISEASE PRIMERS VOLUME 3
|
ARTICLE NUMBER 17040
|
7

accumulation and reduced synthesis of methionine and
S-adenosylmethionine. Homocysteine accumulation can
induce cellular stress, apoptosis and homocysteinylation
of functional proteins in the blood and tissues (the for-
mation of covalent adducts with ε-amino group of their
lysine residues)76. S-adenosylmethionine is the methyl
donor that is required for epigenetic reactions, includ-
ing methylation of DNA, histones and other regulators
of gene expression. Adenosyl-B12 deficiency leads to an
accumulation of MMA, the consequences of which are
not clear, as discussedbelow.
Recent data also indicate a strong link between
B12 deficiency and cellular stress through the reduced
expression of NAD+-dependent protein deacetylase sir-
tuin1 (hSIRT1), the subsequent increased acetylation of
heat shock factor1 (HSF1) and the impaired expression
of heatshock proteins77. Consistently, fibroblasts from
patients with cblC, caused by mutations in MMACHC
(FIG.4b), showed expression changes in genes encoding
key proteins of cellular endoplasmic reticulum that were
involved in oxidative stress (such as heat shock proteins,
ubiquitins and proteins involved in the glutathione path-
way)78. The increased production of reactive oxygen
species (ROS) triggers endoplasmic reticulum stress
andapoptosis79.
The causes of the neurological complications remain
to be determined. The decreased synthesis of succinyl-
CoA and the accumulation of MMA could, theoretically,
contribute to the neurological manifestations of B12 defi-
ciency through the formation of odd chain and methyl-
branched chain fatty acids49, but evidence to support this
theory is scant. Studies of inherited defects have led to
the conclusion that a lack of methyl-B12 or methionine
synthase inhibition is the major cause of the neurological
lesions in B12 deficiency. In addition, the neurological
complications might be due to inflammation80,81, oxid-
ative stress82 and microvascular disease associated with
hyperhomocysteinaemia83.
Low B12 status and increased levels of homocysteine
are also associated with reduced methylation of the pro-
moters of the genes encoding amyloid precursor protein
and γ-secretases, leading to increased amyloid-β produc-
tion. S-adenosylmethionine administration reverses these
effects and improves spatial memory performance84.
B12deficiency induces an increased expression of protein
phosphatase 2A, nerve growth factor and tumour necro-
sis factor, and decreased expression of epidermal growth
factor in cell and animal models85,86. These changes are
consistent with an influence of B12 deficiency on mye-
lin homeostasis and on the amyloid and tau pathways of
neurodegenerative disorders.
Neurological manifestations. B12 deficiency affects the
nervous system, resulting in demyelination of peripheral
and central neurons1,4, which is generally considered to
be the mechanism underlying the classic myeloneuro-
pathy of B12 deficiency. The long tracts of white matter
in the posterior and lateral columns of the spinal cord
containing sensory neurons that are responsible for the
conduction of vibration and position are particularly sus-
ceptible to demyelination, but motor neuron myelination
can also be affected. The neurological manifestations of
B12 deficiency can precede the appearance of haemato-
logical changes and may even occur in the absence of any
haematological complications6.
Adequate B12 status is crucial for normal neuro-
development, as demonstrated by the clinical picture
presented in children with inherited disorders of B12
metabolism87. The first postnatal months are the most
dynamic and vulnerable period of brain development.
The signs and symptoms of B12 deficiency in childhood
depend on theage of the child and on the severity and
duration of the deficit88 (BOX3). In addition, a random-
ized, double-blind B12 intervention study provided evi-
dence of functional motor impairment in infants with
a biochemical profile that was indicative of moderate
B12 deficiency45. A single intramuscular dose of 400 μg
of hydroxy-B12 resulted in biochemical evidence of
B12 repletion and improvement in motor function and
regurgi tations, suggesting that adequate B12 status is
important for the rapidly developing nervous system45.
Although B12 treatment in deficient infants and toddlers
commonly causes rapid progress in motor development
and improvement in clinical symptoms45,89, prolonged
deficiency may result in permanent developmental
disabilities, even after optimal treatment89.
As with folate deficiency90, maternal low B12 status
and B12 deficiency during pregnancy and lactation can
have consequences for the offspring (BOX3), including
neural tube defects91,92. Although there is little clarity on
the consequences of subclinical low B12 status in adults,
there is evidence that, if allowed to persist, it may increase
the risk of initiation or rate of progression of several
chronic diseases associated with ageing (BOX3).
Low B12 status has been shown to be the primary
modifiable cause of increased plasma levels of homocys-
teine in a folic-acid-fortified population residing in a high-
income country93. This is particularly important for older
adults in whom increased levels of homocysteine associ-
ated with low B12 status predict accelerated brain atrophy
and incident clinical cognitive impairment94–97. Analysis
of NHANES data has shown a higher risk of anaemia
and cognitive impairment in older adults who have low
serum levels of B12 and increased serum levels of folate
compared with those with low B12 but without increased
Box 2 | Vitamin B12 in breast milk
Vitamin B12 (B12) levels in breast milk are highly correlated with maternal serum B12
levels31,178. Although maternal B12 levels tend to increase postpartum, the level of
increase depends on pre‑pregnancy stores, B12 intake (diet and supplements) and
depletion of the stores during pregnancy. Reported B12 concentrations in human milk
vary substantially from 150 to 700 pmol per litre41,179. Breast milk concentrations in
American women are reported to be 300 pmol per litre31 to >600 pmol per litre180,
whereas the levels in India and Guatemala are very low (<100 pmol per litre)146,178
oreven undetectable in those with very low dietary B12 intake. Levels in human milk
fallprogressively during the lactation period41. The estimated B12 intake of the
breastfed infant is maximal at 12weeks and is reduced by 50% at week 24 (REF.179).
Most commercially prepared infant formulas are enriched with B12 up to
concentrations of 800–1,200 pmol per litre179,181, and better B12 status (higher levels
ofB12 and lower levels of homocysteine and methylmalonic acid) is reported in
formula‑fed than in breastfed infants40,41,44.
PRIMER
8
|
ARTICLE NUMBER 17040
|
VOLUME 3 www.nature.com/nrdp

folate levels98. In addition, folic acid supplementation
in patients who are B12 deficient partially or tempor-
arily restores haematological complications that arise
from impaired DNA synthesis1,4,5, whereas neurological
complications are unaffected or aggravated98,99.
Haematological manifestations. The haematological
effect of B12 deficiency is megaloblastic anaemia, which
results from disruption of DNA synthesis. When B12 is
deficient, H4-folate synthesis is impaired (FIG.1), which
limits the supply of the required form of folate for the syn-
thesis of thymidylate and DNA. This causes misincorpor-
ation of dUTP in place of thymidine triphosphate during
DNA synthesis1,4,5. DNA synthesis in tissues undergoing
rapid cellular turnover, such as the haematopoietic sys-
tem, is particularly affected. Unbalanced growth in divid-
ing bone marrow cells produces abnormally large cells
with fine, immature-looking nuclear chromatin. This
predominantly affects erythroid precursors, giving rise
to anaemia with abnormally large red cells (macrocytes).
Other haematopoietic cells are also affected, result-
ing in gigantic granulocyte precursors in the marrow
and neutro phils with more than the normal number
of nuclear lobes (hypersegmented neutrophils) in the
blood (FIG.6). In addition to anaemia, there may also be a
decrease in the numbers of all blood cells (pancytopenia).
Figure 4 | Absorption, enterohepatic circulation and intracellular metabolism of vitamin B12. a|VitaminB12 (B12)
ismainly derived from animal sources. Following intake, it is released from its food carrier proteins by proteolysis in the
acidic environment of the stomach, where it binds to haptocorrin58. Haptocorrin is produced by the salivary glands and
protects B12 from acid degradation. Degradation of haptocorrin and the pH change in the duodenum favour B12 binding
to gastric intrinsic factor, which is produced by gastric parietal cells. The intrinsic factor–B12 complex binds to the cubam
receptor (consisting of cubilin and amnionless2,65,66). This receptor mediates the uptake of the intrinsic factor–B12 complex
in the enterocytes of the distal ileum via receptor‑mediated endocytosis. After lysosomal release, B12 exits via the
basolateral membrane of the enterocyte, facilitated by multidrug resistance protein 1 (MDR1), and binds to transcobalamin,
the blood carrier of B12 that is responsible for cellular delivery of B12 (REFS2,58,64). The majority of B12 is stored in the liver;
some B12 is excreted in bile and undergoes enterohepatic circulation167,206. b|Cellular uptake of B12 involves the CD320
receptor present on all cell types. Studies on CD320‑knockout mice suggest that other yet to be identified receptors for
transcobalamin may exist207. Several inherited disorders — designated CblA to CblJ — are associated with mutations in
genes encoding proteins involved in intracellular B12 metabolism. These include (indicated with *): two lysosomal proteins
(lysosomal cobalamin transporter (LMBD1) and the lysosomal membrane transporter ABCD4); the cytoplasmic chaperones,
methylmalonic aciduria and homocystinuria type C protein (MMACHC) and MMADHC, whose function has not been
established; the mitochondrial enzyme methylmalonyl‑CoA (MUT) as well as two mitochondrial proteins (cblA and cblB)
involved in the transfer and maintenance of the adenosyl group to adenosylcobalamin, mutations of which result in
methylmalonic aciduria cblA and cblB type (MMAA and MMAB), respectively; and the two respective target enzymes
ofmethyl‑B12 (methionine synthase reductase (MTRR) and methionine synthase (MTR))69. HCl, hydrochloric acid.
a
Liver
Stomach
Gall
bladder
b
Dietary protein
Transcobalamin
Intrinsic factor
Haptocorrin
Cubam receptor
B12
Ileal
mucosal
cell
Lysosome
Faeces:
60–80% of
ingested B12
Blood
vessel
Bile excretion:
~0.15% of body
store per day
HCI and pepsin
secretion
Dietary B12 intake (5–10 μg per day)
From the salivary gland
Nature Reviews | Disease Primers
CD320*
Lysosome
LMBD1*
ABCD4*
MMACHC*
MMADHC*
Methylmalonyl-CoA
Succinyl-CoA
MUT*
MMAA*
MMAB*
Methyl-B12
Methionine
Homocysteine
MTRR*
MTR*
MDR1
Adenosyl-B12
Mitochondrion
PRIMER
NATURE REVIEWS
|
DISEASE PRIMERS VOLUME 3
|
ARTICLE NUMBER 17040
|
9

Diagnosis, screening and prevention
Diagnosis
The clinical manifestations of B12 deficiency are varied
and may mimic or are mimicked by various other diseases.
Indeed, the clinical presentation of severe megaloblastic
anaemia can closely mimic haemolytic anaemia, throm-
botic thrombocytopenic purpura and myelodysplastic
syndromes100–103. Delay in diagnosing B12 deficiency may
be life-threatening, and inappropriate treatment of the
mimicker entities in a patient with underlying B12 defi-
ciency is a major clinical error. Making a specific diagnosis
of deficiency is paramount. Before committing a patient
to lifetime B12 treatment, the deficiency should be docu-
mented by pre-treatment increases in the levels of MMA
or homocysteine, further underscored by the presence of
anti-intrinsic factor antibodies or a history of the relevant
post-surgical states that could lead to B12 deficiency,
such as gastrectomy, gastric reduction or ileal resection.
Empirically, treatment with high-dose B12 while awaiting
the results of more specific testing does not causeharm.
Who to test? Two groups of patients should be consid-
ered for diagnostic testing for B12 deficiency. The first
group comprises those with clinical evidence of B12
deficiency, including macrocytic anaemia and/or neuro-
logical symptoms. In such patients, laboratory tests may
help to confirm the diagnosis, but a firm clinical sus-
picion of B12 deficiency warrants immediate treatment
with B12 (REF.10 4).
The other, much larger group of patients who experi-
ence nonspecific symptoms, quite often not including
anaemia, presents a greater challenge. This group
includes elderly individuals, individuals on a diet with
limited amounts of B12, such as vegans, individuals
withimpaired fertility, patients with gastrointestinal
diseases, and patients with non-characteristic and
unexplained haematological or neurological symptoms4.
Within this group, individuals with poor B12 status are
mainly identi fied by biomarker measurements (TABLE1).
However, this approach does have its limitations. Taken
individually, serum B12 or holotranscobalamin values
Figure 5 | Mechanism and complications of autoimmune gastritis. Autoimmune gastritis and type A chronic atrophic
gastritis can develop in genetically predisposed individuals. Environmental risk factors (such as Helicobacter pylori
infection) might also have a role. Apoptosis of parietal cells might result in the release of gastric H+/K+ ATPase
constituents,which are taken up by gastric dendritic cells that then migrate to the draining gastric lymph nodes to
activate naive CD4+ Tcells. The gastric H+/K+ ATPase‑activated CD4+ Tcells then migrate to the gastric mucosa to initiate
tissue damage by binding to MHC classII molecules and by activating FAS‑dependent mechanisms208. Gastritis is marked
by the presence of autoantibodies targeting the gastric H+/K+ ATPase. Chronic atrophic gastritis also represents a risk
factor for gastric cancer arising from polypeptide‑expressing intestinal metaplasia, as well as enterochromaffin‑like cell
hyperplasia arising from gastrin hypersecretion by antral G cells predisposing to gastric carcinoid. Pernicious anaemia
arises from intrinsic factor deficiency as a result of loss of intrinsic factor‑producing gastric parietal cells and the presence
of intrinsic factor autoantibodies. Loss of parietal cells that also produce acid via the gastric H+/K+ ATPase can lead to iron
deficiency anaemia.
Nature Reviews | Disease Primers
Pernicious anaemia Iron deficiency
Type A chronic atrophic gastritis
Increased gastrin secretion
Failure of intrinsic
factor production
Parietal cell destruction
Impaired gastric acid secretion
Enterochromaffin-like
cell hyperplasia
Presence of
anti-intrinsic factor
autoantibody Intestinal
polypeptide-expressing
metaplasia
Parietal cell death
Uptake of the gastric H+/K+ ATPase by dendritic cells
Activated CD4+ T cells directed at the gastric H+/K+ ATPase
Autoimmune gastritis
Genetic predisposition and environmental factors
Parietal cell autoantibody against gastric H+/K+ ATPase
Gastric cancerGastric carcinoid
PRIMER
10
|
ARTICLE NUMBER 17040
|
VOLUME 3 www.nature.com/nrdp

below the lower level of the reference interval have
low specificity for identifying true vitamin deficiency.
Poor sensitivity of these tests also means that true B12
deficiency may often go unrecognized9,105. In general,
ifunequivocal or profound B12 deficiency is present,
itis important that it be diagnosed and treated as swiftly
as possible. Indeed, once B12-related neurological
damage has occurred, it might not completely reverse
followingtreatment.
Diagnostic tests and biomarkers. Despite the limita-
tions, biomarkers — total B12, holotranscobalamin,
MMA and homocysteine, as well as combinations of
these measurements — are increasingly used106. Assay
characteristics and confounding factors are summarized
in TABLE1.
Serum B12 level is usually the first-line test. In gen-
eral, a value well below the lower limit of the reference
interval is indicative of probable B12 deficiency, whereas
a value well above this limit indicates a sufficient B12
status. Important exceptions may occur in the pres-
ence of circulating antibodies against intrinsic factor in
patients with pernicious anaemia, in whom spuriously
normal B12 levels have been reported107. In daily prac-
tice, the same reference interval is used across age and
sex, even though the reference interval broadens with
age108. The reference interval is method dependent and,
therefore, no universal cut-off limits can be stated. The
level of B12 is influenced by the concentration of the
two plasma-binding proteins. High levels of hapto corrin
are associated with liver diseases, myelo proliferative
diseases, such as chronic myeloid leukaemia, and other
malignancies, explaining why an increased level of B12
may be encountered in theseconditions109.
Active, transcobalamin-bound B12 (holotransco-
balamin) should, theoretically, be a more-sensitive
marker for B12 deficiency than total serum B12 levels110.
Measurement of holotranscobalamin is gradually being
incorporated into the clinical laboratory, but so far, the
biomarker has proved only marginally better than total
B12 levels111–113.
Homocysteine and MMA accumulations in plasma
marks B12 deficiency4, but homocysteine levels are also
influenced by other parameters (TABLE1). The method
of blood collection and plasma separation also pre-
sents some pre-analytical challenges for homocysteine
measurements114. MMA level is the most specific and
sensitive single marker for B12 deficiency, and is often
used as the ‘gold standard’ for defining B12 status115,116.
The major drawbacks are availability and cost of the
assay. Amajor advantage is that the analyte is very well
standardized, which allows for a uniform interval of ref-
erence of ≤0.27 μmol per litre, and a cut-off point that
indicates poor B12 status of >0.37 μmol per litre106,115.
However, genetic determinants of MMA cut-offs have
recently been challenged in a GWAS of genetic factors
that influence plasma MMA levels117. HIBCH, which
encodes a protein involved in valine catabolism, was
found to contain a single-nucleotide polymorphism
(rs291466) that was the most common genetic driver
of plasma MMA levels. This common polymorphism
is widespread in populations (with a minor allele fre-
quency of 0.43), and people who are homozygous for
the common allele have plasma MMA concentrations
that are approximately 46% higher than those with
the rarer allele117. This B12-independent determinant
of plasma MMA may influence its usefulness as a B12
status indicator, when used as a single determinant.
Box 3 | Consequences and symptoms of vitamin B12 deficiency or low vitamin B12 status*
Symptoms of B12 deficiency in infants
• Feeding difficulties88
• Regurgitations88
• Constipation88
• Apathy88
• Irritability88
• Twitching, tremors and myoclonic jerks88
• Slow growth, small head circumference and brain lesions
• Developmental delay, including reduced gross motor development,
smiling and babbling88
• Pancytopenia
Consequences of low maternal B12 status or deficiency in infants
• Neural tube defects reported in infants of mothers with low B12 status
in folic acid‑fortified and non‑fortified populations91,92
• Effects on infant development, including stunting, cerebral atrophy,
hypotonia, lethargy, developmental delays and abnormal
electroencephalogram88
• Contribution to adult cardiovascular disease, neurodegenerative
disorders and psychiatric illness through increased levels of circulating
homocysteine1
• Contribution to the development of diabetes mellitus through effects
on insulin and lipid metabolism37
Symptoms of B12 deficiency in older children
• Lower school performance
• Reduced weight182
• Reduced height and head circumference182
• Macrocytic anaemia182
• Normal or increased weight183,184
• Normal haematological values184
• Impaired mental and social development, short‑term memory
andattention185–187
Consequences of low B12 status or deficiency in adults
• Megaloblastic anaemia or macrocytic anaemia1,4,5,123
• Subacute combined degeneration of the spinal cord4,6,168
• Impaired sensory and peripheral nerve function188
• Cognitive impairment127,130,189,190
• Depression126
• Bone disease135,191
• Hearing loss192
• Macular degeneration193
The severity of vitamin B12 (B12) deficiency varies and is defined in the
sourcereferences. *The data are derived from several sources as indicated
bythereferences.
PRIMER
NATURE REVIEWS
|
DISEASE PRIMERS VOLUME 3
|
ARTICLE NUMBER 17040
|
11

This is mainly relevant in older adults who have higher
levels of MMA that are unrelated to B12 status than
younger individuals. Elderly individuals who have the
genotype that leads to higher levels of MMA could
easily have MMA concentrations of >0.37 μmol per
litre, without being deficient in B12. The relevance
of this polymorphism in the clinical setting warrants
furtherstudy.
Many laboratories use a diagnostic strategy that
involves more than one biomarker, most often using
B12 levels as the initial test and MMA or homocysteine
levels as the second-line test118. Recently, this approach
has been further improved by the development of an
equation that includes two to four biomarkers. This
newly introduced B12 index is termed cB12 (combined
indicator of B12 status)106. The advantage of cB12 is that
it is independent of local reference intervals and can be
adjusted to correct for folate status and age. This index
shows a stronger association with haemoglobin con-
centrations, cognitive function and peripheral nerve
conductivity than single markers. At this time, the
prospective clinical usefulness of the index remains to
beconfirmed.
Diagnosing B12 deficiency in elderly individuals.
B12 deficiency is particularly difficult to identify in
elderly individuals because the typical haematological
and neuro logical manifestations of clinical B12 defi-
ciency are frequently not present or are easily confused
with similar manifestations of other common diseases
of older age, most notably dementia. Moreover, the
sensitiv ity of the biomarkers MMA and plasma homo-
cysteine lessens with older age because of impaired
kidney function8,119–121. Haematological symptoms occur
in <50% of individuals, and, even when present, macro-
cytic anaemia in older individuals is frequently due to
other haematological disorders, such as myelo dysplasia,
or the use of medications that have no bearing on B12
status122,123. As noted above, neuropsychiatric and
neuro logical symptoms are more-frequently observed
in elderly individuals who are B12 deficient6,124–130, but
these symptoms generally tend to be nonspecific and
similar to the symptoms encountered in many other
chronic diseases of ageing. As a result, management
relies heavily on clinical judgement. Several intervention
studies have focused on the effect of correcting hyper-
homocysteinaemia with B12 supplementation. Some,
but not all, studies have demonstrated improvements
in disease state95,131–136. In one study, supplementation
with homocysteine-lowering vitamins that included
B12 was effective in reducing the rate of brain atrophy
among patients with increased levels of homocysteine
at baseline94,96.
Diagnosing B12 deficiency in infants. As stated above,
B12 deficiency caused by inherited defects may occur
early in life. Typically, infants present with pancyto-
penia and failure to thrive. In addition, nutritional
B12 deficiency should be considered in young, mainly
breastfed infants with feeding difficulties, subtle neuro-
logical symptoms and delayed motor development,
particularly if the B12 intake of the mother has been
low. Owing to the subtle symptoms of B12 deficiency
and the large variations in normal development dur-
ing infancy (BOX3), B12 deficiency is reported to have a
median diagnostic delay of 4months in this age group88.
A plasma homocysteine level of ≥6.5 μmol per litre has
been suggested as a cut-off level for defining B12 defi-
ciency in infants45. This represents the 97.5 percentile
in 4-month-old infants who are given a single intra-
muscular dose of 400 μg of hydroxy-B12 at 6weeks, to
render them B12 replete43.
Determining the cause of B12 deficiency. In terms
ofoptimal patient management, establishing a causeof
B12 deficiency is highly desirable. To achieve this, a key
step is to determine whether the cause is low intake or
malabsorption, and, if the latter, whether the defect is
gastric or intestinal. Over the past two decades, use
of the gold-standard test for assessing B12 absorptive
capacity — known as the Schilling test — has dwindled
to essentially zero owing to the lack of availability of
radiolabelled B12 that is required for the test and high
cost, among other factors. No substitute test has been
validated for widespread clinical use. The CobaSorb
test, which is based on an increment in the serum
holotranscobalamin level following oral dosing with
B12, shows promise in that no increase in the level of
holotranscobalamin occurs in patients who lack intrinsic
factor as well as in patients with Imerslund–Gräsbeck
syndrome137,138, but the test has the limitation that it can-
not be used once the patient is treated with B12 (REF.139).
A 14C-labelled form of B12 produced through microbial
synthesis offers the possibility of being used to measure
B12 absorption, but this has not yet been developed into
a clinical test140.
In the absence of a robust and reliable absorption test,
other approaches can be used. To diagnose classic perni-
cious anaemia141, the detection of autoantibodies against
intrinsic factor and gastric H+/K+ ATPase can be used, as
well as the levels of pepsinogen or gastrin142. Although
a negative result for anti-intrinsic factor autoantibodies
does not exclude pernicious anaemia, a positive result
Figure 6 | Blood and bone marrow morphological changes in vitamin B12 deficiency.
a|Blood smear showing nuclear hypersegmentation (more than five lobes) of a
neutrophil with several larger than normal oval erythrocytes present. b|Bone marrow
aspirate smear showing abnormally large erythroid precursor cells with fine,
immature‑looking nuclear chromatin and large ‘giant’ granulocytic band form with
horseshoe‑shaped nucleus. Nuclear maturation is retarded in both the erythroid and
thegranulocytic precursors owing to faulty DNA synthesis.
Nature Reviews | Disease Primers
a b
PRIMER
12
|
ARTICLE NUMBER 17040
|
VOLUME 3 www.nature.com/nrdp

has 100% specificity. However, a positive result for
anti-H+/K+ ATPase autoantibodies is nonspecific as it
may be seen also in patients without pernicious anae-
mia143. The finding of atrophic gastritis during upper
endoscopy may provide an important lead for suspecting
pernicious anaemia, as all patients with pernicious anae-
mia have atrophic gastritis; when atrophic gastritisis
found, other non-immune causes of atrophic gastritis
should be excluded.
Curiously, a decline in MMA levels following B12
intake or injection has so far not been developed into
a standardized functional test, although this parameter
may prove very valuable for confirming the presence of
B12 deficiency and also for adjusting treatment.
When B12 deficiency is found or suspected in the
paediatric age group, inborn errors of metabolism affect-
ing cobalamin processing should be considered in the
differential diagnosis, and suitable tests, either through
complementation phenotyping or genetic analysis,
should be carried out69–71,75.
Prevention
Pregnant women, infants and children. Deficiency of
B12 is emerging as a public health concern in many
low-income countries. A WHO Consultation iden-
tified infants, preschool children and pregnant and
lactating women as the most vulnerable groups144. The
strategies for prevention of B12 deficiency and its pub-
lic health significance may be discussed in a life-course
model145. FIGURE7 summarizes the factors influenc-
ing B12 status across the life course and stresses the
importance of direct transfer of B12 from the mother
to the baby during pregnancy and lactation. Maternal
B12 status during pregnancy and cord blood B12 con-
centration predict B12 status of the offspring well into
early adulthood, highlighting a crucial role for mater-
nal vitamin status to prevent B12 deficiency in the next
generation. Importantly, this transgenerational cycle
becomes self-perpetuating if the fetus is female. This
suggests that improving the nutrition of young girls has
the potential of improving the ‘legacy’ B12 status of the
population for many generations and reducing associ-
ated morbidity. This novel idea could have a substan-
tial long-term benefit in public health. Maternal B12
status is negatively affected by a combination of diets
poor in animal-sourced foods, high fertility rates and
short inter-pregnancy intervals, and further aggrav-
ated by sociocultural factors such as early marriage with
adoles cent pregnancies, as well as dietary taboos during
pregnancy and lactation.
Oral supplementation of urban Indian women with
B12 (50 μg daily) throughout pregnancy and early lac-
tation significantly increased the B12 status of mothers
(median: 184 versus 105 pmol per litre; P < 0.001) and
infants (199 versus 139 pmol per litre;P = 0.01) compared
with non-supplemented controls. Supplementation
improved median breast milk B12 concentration 6weeks
post-partum (136 versus 87 pmol per litre; P < 0.001)
and was also associated with a drop in the incidence
of intrauterine growth retardation146. In Bangladesh,
maternal supplementation with 250 μg of B12 daily dur-
ing most of the pregnancy and 3months of lactation
resulted in improved maternal and infant status, sub-
stantially increased B12 in colostrum and breast milk,
and improved influenza H1N1 vaccine-specific response
in the mothers147.
General prevention strategies. The overall long-term
strategy for controlling B12 deficiency is to promote
consumption of foods rich in B12. Vegetarians will
always have difficulty achieving this because the natural
dietary source of B12 is animal-origin foods, unless they
consume eggs, milk and other dairy products, or foods
fortified with the vitamin. In addition to ethics, religion
and culture, socioeconomic factors may limit the intake
of animal-origin foods (FIG.7).
B12-containing plant-derived food sources con-
sumed during pregnancy include green and purple
lavers (Nori) and blue-green algae or cyanobacteria
(Spirulina). Nori is considered suitable for humans, but
it is not widely available, whereas the more widely avail-
able Spirulina may contain pseudo-B12, a biologically
Figure 7 | Determinants of vitaminB12 status. Important factors that affect the demand
and supply of vitaminB12 (B12) on the individual and population levels and throughout
the patient’s lifetime are highlighted. It is striking that the mother exerts a triple
influence on the B12 status of the offspring: genetics, intrauterine and lactational B12
transfer, and postnatal family environment (socioeconomic status, hygiene, diet, religion
and culture). The father exerts a double influence: genetic and family environment.
Afemale child will continue to propagate the direct maternal influences into the next
generation. The suspected role of epigenetics in all three processes awaits elucidation.
The nutritional status of the population and, therefore, the public health measures
to improve it depend on the socioeconomic factors, religious and cultural practices,
andthe public health policies of the local, national and international regulatory systems.
Although targeting the B12 deficiency on an individual level is needed, more widespread
interventions targeting the population are needed to prevent B12 deficiency.
Nature Reviews | Disease Primers
H
e
a
l
t
h
c
a
r
e
s
y
s
t
e
m
(
s
u
p
p
l
e
m
e
n
t
a
t
i
o
n
a
n
d
f
o
r
t
i
fi
c
a
t
i
o
n
)
R
e
l
i
g
i
o
n
a
n
d
c
u
l
t
u
r
e
D
i
e
t
,
h
y
g
i
e
n
e
,
e
d
u
c
a
t
i
o
n
a
n
d
s
o
c
i
o
e
c
o
n
o
m
i
c
s
t
a
t
u
s
Biology
• Genetics
• Absorption
• Transport
Maternal influences
including placental
transfer and
lactation
PRIMER
NATURE REVIEWS
|
DISEASE PRIMERS VOLUME 3
|
ARTICLE NUMBER 17040
|
13

inactive analogue148, which would not be beneficial and
might even be harmful. Thus, dairy products and eggs
remain the only acceptable animal source of B12 for
vegetarians. A study from Pune reported improvement
in B12 status in young, healthy, B12-deficient vegetari-
ans with regular intake of non-fortified milk (600 ml per
day)149, whereas a food-based micronutrient-rich snack
(dried fruits, leafy vegetables and milk powder) failed to
improve B12 status in a large trial150,151.
Use of widely available and regularly consumed
foods as vehicles to deliver B12 is another preventive
strategy. Fortification of wheat flour, bread, milk, break-
fast cereal, nutrient bars, energy drinks and mineral
water have been used with success152–154. Probiotics have
also been recently used with limited success to improve
B12 status155. Trials are essential in countries where B12
deficiency is a public health problem. Population-wide
fortification of the diet with folic acid has been effective
in reducing the incidence of neural tube defects in many
countries90. However, in individuals with a low intake of
B12-consuming foods that are fortified with folic acid,
data are emerging that excessively high levels of folate,
occurring primarily as a result of additional folic acid
consumption through supplement use, may aggravate
their B12 deficiency98,99,156,157. The mechanism under-
lying this adverse effect of high levels of folate on B12
status is notknown.
Management
Treatment
In target groups that are at higher risk of B12 insuffi-
ciency, the goals are to prevent B12 deficiency in the
first place and to treat such deficiency through reple-
tion when it occurs. B12 deficiency is mainly caused
by inadequate intake. Once B12 is repleted in these
patients, B12 stores are generally well conserved
because of an intact intrinsic factor-dependent absorp-
tive mechanism and enterohepatic circulation of B12.
This is not the case in those individuals who have
some form of malabsorption caused by failure of the
intrinsic factor-dependent conservation of biliary B12.
Thisfundamental difference in the capacity to conserve
bili ary B12 or not is an important determinant of both
the rapidity of onset of depletion of body B12 stores
and the severity ofthe B12 deficiency that supervenes
when left untreated.
Severe clinical abnormalities should be treated
intensively with B12, cyano-B12 or hydroxy-B12
(BOX4). After an intramuscular injection of 1,000 μg of
cyano-B12, about 150 μg is ultimately retained in the
body, mainly through storage in the liver, although
the variability in retention was large in the several
original studies from which this figure was derived158.
Hydroxy-B12 is commonly used in Europe, often
at intervals of 2–3months, as it seems to have better
retention than cyano-B12. There is no advantage to
using the light-sensitive forms of cobalamin, such
as methyl-B12 or adenosyl-B12, instead of the stable
cyano or hydroxy forms, which are readily converted
in the body into the coenzyme forms, methyl-B12 and
adenosyl-B12(REF.159).
High-dose oral supplementation is an effective
alternative to parenteral treatment. Radioactive B12
was used to show that 0.5–4% of an oral dose was
absorbed; thus, high-dose tablets of 1,000 μg will deliver
on average 5–40 μg of B12 (REF.160). Randomized stud-
ies of daily high oral doses of 1,000–2,000 μg have
shown equivalence or superiority to injected B12
(REFS161–163). Oral doses of >500 μg daily have been
necessary to correct MMA levels in elderly individuals,
despite considerations that they have milder forms of
B12malabsorption164,165.
Patients with true pernicious anaemia, lacking
intrinsic factor, are unable to reabsorb the B12 lost in
bile, which varies from 3 to 9 μg daily141. To maintain
tissue stores, between 100 and 300 μg of B12 should
therefore be retained monthly. The daily requirement
for individuals without malabsorption has been set at
2.4 μg, although an intake of 4–7 μg resulted in lower
serum MMA values166. People who are B12 deficient
due to low dietary intake will require loading with high-
dose B12 to establish or restore tissue levels, after which
time smaller supplements will suffice as conservation of
biliary B12 is possible through enterohepatic recycling
and the normally highly efficient reabsorption of biliary
B12 (REF.167). Detection of B12 malabsorption using the
CobaSorb test can help to determine whether patients
will respond to low-dose B12 supplements or whether
patients will require treatment with pharmacological
doses of the vitamin, either orally or by intramuscular
injection139. Establishment or restoration of adequate
B12 stores is particularly important in pregnant and
lactatingwomen.
Severe neurological abnormalities should be treated
aggressively with daily injections for a week and then
weekly treatment until stabilized. Some patients request
more-frequent injections, claiming subjective improve-
ment in symptoms and mood. The basis for this is not
understood, but as B12 is considered harmless with no
defined tolerable upper intake level38, there is no harm
in a ‘personalized’ approach to meet the need of the
individual patient.
Box 4 | Treatment of vitaminB12 deficiency
Malabsorption
• Parenteral vitamin B12 (B12) administration: 1,000 μg cyano‑B12 or hydroxy‑B12
intramuscular injection daily or every other day for 1week followed by weekly
injections up to 8weeks then every 3–4weeks*.
• Oral administration: high‑dose cyano‑B12 (2,000 μg) oral tablets daily until remission
then 1,000–2,000 μg daily‡.
Dietary deficiency
• Oral: consider a daily high dose to replace stores over 3–4months then at least 6 μg daily.
Infants
• Parenteral: 250–1,000 μg intramuscular cyano‑B12 or hydroxy‑B12 daily, then weekly
until recovery followed by oral 1–2 μg daily or B12‑containing formulas, and treatment
of the mother to correct breast milk.
*Hydroxocobalamin can be given every 2–3months after the initial intensive treatment.
‡Lowerdoses of 600 ug have been shown to be adequate165; elderly and post‑gastric bypass
patients should continue daily high‑dose treatment.
PRIMER
14
|
ARTICLE NUMBER 17040
|
VOLUME 3 www.nature.com/nrdp

Response to treatment
Megaloblastic anaemia caused by B12 deficiency
responds to B12 treatment with reticulocytosis (increased
numbers of immature red blood cells) in approximately
5days and usually complete correction of the red blood
cell count within 4–6weeks141. If the patient is also iron
deficient, then microcytic anaemia (anaemia with smaller
than normal red cells) will be unmasked; other coexist-
ing conditions such as chronic renal disease, inflamma-
tory disorders, chronic liver disease or even coexisting
myelodysplasia will also limit theresponse.
The response to neurological abnormalities is slower
with the exception of mental symptoms such as emo-
tional lability, paranoia and irritability, which may
improve rapidly 168,169. There are occasional patients who
have a transient exacerbation or new presentation of
para esthesia (a prickling sensation) or other complaints
in the first week of treatment. The duration and severity
of the neurological deficits before treatment generally
predict the ultimate outcome169. Paraesthesias with-
out sensory loss or motor weakness are the most likely
symptoms to completely correct. Imaging studies of the
spinal cord show rapid correction of demyelination170.
If neurological symptoms progress after the patient has
been satisfactorily treated, then they are not due to B12
deficiency and another cause should besought.
Quality of life
Patients with true pernicious anaemia are at risk for
other autoimmune disorders, particularly autoimmune
thyroid disease. The chronic gastritis seen in patients
with B12 malabsorption increases the risk of gastric
carcinoid tumours and adenocarcinoma (FIG.5), as well
as malabsorption of other micronutrients; thus, iron
deficiency and/or abdominal symptoms should be
evalu ated promptly171. Patients with severe demyelin-
ating central nervous system disease may have perma-
nent impairments in proprioception, sensation and
muscle weakness that impair quality of life169. Generally,
improvement will not continue after a year of adequate
therapy. B12-deficient infants and young children may
have permanent impairment in brain development and
function172. A challenging aspect in the management of
non-nutritional B12 deficiency is to ensure the lifelong
treatment of the patient. Patients must be educated that
the requirement for treatment is ongoing. An advantage
of high-dose oral treatment in the United States is the
easy over-the-counter availability of the supplements.
Thus, patients are not dependent on health care provid-
ers for their replacement. However, patient or caregiver
education is important to ensure continuedcompliance.
Outlook
The topic of B12 deficiency has attracted and still attracts
considerable scientific attention, from physicians, scien-
tists, the nutritional supplement and diagnostics industry
and the public. Despite the body of knowledge on B12
deficiency that has accumulated, many key questions
remain unanswered and many issues are unresolved
(TABLE2). The advent of ever more sophisticated and
accurate analytical methods has refined the capacity to
identify B12 status more precisely and to draw important
distinctions between clinically evident B12 deficiency
with its associated disease consequences and a preclinical
state of B12 insufficiency that confers an increased risk or
susceptibility for the development of certain disease states.
Furthermore, from the public health perspective, under-
standing the conditions that predispose to B12 deficiency
is key in order to implement the appropriate measures to
prevent such deficiency in populations atrisk.
Table 2 | Research needs relating to vitaminB12 (B12) deficiency
Requirement Current situation Potential advantages
Better understanding of factors
that influence B12 bioavailability
Bioavailability seems to vary widely, but the reasons are unclear Improves prevention and management
strategies
Improved knowledge about the
role of the gut microbiota in B12
requirements
Studies suggest that variable degradation of B12 in the gut
lumen might be influenced by the microbiota and that this
might affect B12 bioavailability
Probiotics might be effective for prevention
ortreatment
Identification of obscure causes
of B12 deficiency
In some patients who partially or fully respond to B12 treatment,
testing does not identify the underlying metabolic defect
Improves prevention and management
strategies
Better understanding of the
relationship between folate
andB12
Several epidemiological studies have found that high
folate levels exacerbate the metabolic, haematological
andneurological effects of B12 deficiency
Informs supplementation strategies, particularly
in regions with both a high frequency of B12
deficiency and a population‑based folic acid
supplementation programme
Investigation of potential adverse
effects of high B12 intake
No adverse effects have been identified, so there is no safe
upper limit of B12 dose or level
Informs future large‑scale, population
fortification programmes
Improvements in the detection
of B12 deficiency
Available tests have generally poor specificity and sensitivity More‑accurate identification of B12‑deficient
individuals
Development of reliable
methods to identify B12
malabsorption
The current test (CobaSorb) does not discriminate between
gastric and intestinal cause of B12 malabsorption and is only
useful in patients who have not been treated with B12
Ability to confirm underlying absorptive defect
and discriminate between gastric and ileal
causes
Better understanding of the
factors that determine variable
manifestations of B12 deficiency
Why some patients with B12 deficiency show haematological
features of B12 deficiency, whereas others show neurological
features is not clear
Genetic differences for these variations in
disease phenotype should be explored using
whole‑genome sequencing
PRIMER
NATURE REVIEWS
|
DISEASE PRIMERS VOLUME 3
|
ARTICLE NUMBER 17040
|
15

Contrary to previous beliefs, B12 deficiency is not
confined to elderly individuals, to white individ uals and
to individuals with intestinal malabsorption. Inadequacy
of this nutrient, ranging from varying degrees of insuffi-
ciency to outright deficiency, has a wide prevalence and
affects individuals of all ages, but most particularly infants,
children, adolescents and women of reproductive age in
populations in which dietary intake of B12-containing
animal- derived foods is restricted. It is becoming increas-
ingly clear that neuro logical consequences among the very
young and the old are due, at least in part, to inadequate
B12 status. There is clear evidence that low B12 status is
a risk factor for cognitive decline and cerebral atrophy
associated with ageing173 and is associated with incident
dementia97. Moreover, vitamin supplements containing
B12 delay these changes96. Recent findings indicate that
there is a progressive decrease in B12 levels in the brain
across the lifespan, which parallels with an increased
demand for antioxidants with age174. At the other end of
the age scale, in infancy, B12 deficiency is associated with
neuro logical problems ranging from neuro muscular dif-
ficulty with swallowing to verbal development87, and these
problems have been ameliorated by administration of a
B12 supplement43,45. Universal improvement of B12 status,
therefore, seems to be a nutritional imperative with pos-
sibly profound beneficial effects on the nervous system,
particularly at the bookends oflife.
One of the most pressing and enduring questions
about B12 deficiency is how and to what extent it is
influenced by the supply, and particularly excesses of
its closely affiliated vitamin, folate. Another enigma in
B12 deficiency is the often widely differing manifesta-
tions among patients with regard to the dominance of
either haematological or neurological complications. The
spectrum of the B12-deficient phenotype is broad1,4–6, yet
the reasons for this remain unknown. It is possible that
genetic factors or nutrient–nutrient interactions may
explain these differences in susceptibility175–177.
Much has been learned about B12 since its isolation
and characterization almost 70years ago. As the pace of
scientific progress continues to accelerate, it is likely that
there will be answers to most of the questions posed in
this Primer, but other scientific questions are bound to
arise. Above all, the anticipation that the global burden
of B12 deficiency will be considerably alleviated is an
outcome that is both reasonable and desirable.
1. Green,R. & Miller,J.W. in Handbook of Vitamins
5thedn (eds Zempleni,J. etal.) 447–489
(Taylor&Francis, 2014).
A comprehensive review of B12 biochemistry,
nutrition and metabolism.
2. Nielsen,M.J. etal. VitaminB12 transport from
foodto the body’s cells — a sophisticated, multistep
pathway. Nat. Rev. Gastroenterol. Hepatol. 9,
345–354 (2012).
A thorough review of B12 absorption, cellular
uptake and intracellular metabolism in health
anddisease.
3. Allen,L.H. How common is vitaminB-12 deficiency?
Am. J.Clin. Nutr. 89
, 693S–696S
(2009).
4. Stabler,S.P. Clinical practice. VitaminB12 deficiency.
N.Engl. J.Med. 368, 149–160 (2013).
An authoritative review of B12 deficiency from
theclinical perspective.
5. Green,R. VitaminB12 deficiency from the perspective
of a practicing hematologist. Blood 129, 2603–2611
(2017).
A recent and authoritative up-to-date summary
ofthe clinical aspects of B12 deficiency with
emphasis on the haematological perspective.
6. Lindenbaum,J. etal. Neuropsychiatric disorders
caused by cobalamin deficiency in the absence
ofanemia or macrocytosis. N.Engl. J.Med. 31 8,
1720–1728 (1988).
7. Carmel,R. Subtle and atypical cobalamin deficiency
states. Am. J.Hematol. 34, 108–114 (1990).
8. Green,R. Metabolite assays in cobalamin and folate
deficiency. Baillieres Clin. Haematol. 8, 533–566
(1995).
9. Green,R. Screening for vitaminB12 deficiency:
caveat emptor. Ann. Intern. Med. 124, 509–511
(1996).
10. Carmel,R. Subclinical cobalamin deficiency.
Curr.Opin. Gastroenterol. 28, 151–158 (2012).
A detailed description of the puzzling but common
entity of B12 inadequacy without frank
manifestations of deficiency.
11. Carmel,R. Cobalamin, the stomach, and aging.
Am.J.Clin. Nutr. 66, 750–759 (1997).
12. Carmel,R. & Sarrai,M. Diagnosis and management of
clinical and subclinical cobalamin deficiency: advances
and controversies. Curr. Hematol. Rep. 5, 23–33
(2006).
13. Carmel,R. etal. Update on cobalamin, folate, and
homocysteine. Hematology Am. Soc. Hematol. Educ.
Program 2003, 62–81 (2003).
14. Carmel,R. Prevalence of undiagnosed pernicious
anemia in the elderly. Arch. Intern. Med. 156,
1097–1100 (1996).
15. Yajnik,C.S. etal. VitaminB12 deficiency and
hyperhomocysteinemia in rural and urban Indians.
J.Assoc. Physicians India 54, 775–782 (2006).
16. Premkumar,M. etal. Cobalamin and folic acid status
in relation to the etiopathogenesis of pancytopenia
inadults at a tertiary care centre in north India.
Anemia 2012, 707402 (2012).
17. James,J.S. Low vitaminB-12 blood levels associated
with faster progression to AIDS. AIDS Treat. News
264, 3–4 (1997).
18. Allen,L.H. Causes of vitaminB12 and folate
deficiency. Food Nutr. Bull. 29 (Suppl. 2), S20–S34
(2008).
19. Bailey,R.L. etal. Monitoring of vitaminB-12
nutritional status in the United States by using plasma
methylmalonic acid and serum vitaminB-12.
Am.J.Clin. Nutr. 94, 552–561 (2011).
20. Yang,Q. etal. Folic acid source, usual intake, and
folate and vitaminB-12 status in US adults: National
Health and Nutrition Examination Survey (NHANES)
2003–2006. Am. J.Clin. Nutr. 91 , 64–72 (2010).
21. Pennypacker,L.C. etal. High prevalence of cobalamin
deficiency in elderly outpatients. J.Am. Geriatr. Soc.
40, 1197–1204 (1992).
22. Clarke,R. etal. VitaminB12 and folate deficiency
inlater life. Age Ageing 33, 34–41 (2004).
23. Carmel,R. etal. Serum cobalamin, homocysteine,
and methylmalonic acid concentrations in a
multiethnic elderly population: ethnic and sex
differences in cobalamin and metabolite
abnormalities. Am. J.Clin. Nutr. 70, 904–910
(1999).
24. Stabler,S.P., Lindenbaum,J. & Allen,R.H.
VitaminB-12 deficiency in the elderly: current
dilemmas. Am. J.Clin. Nutr. 66, 741–749 (1997).
25. Naurath,H.J. etal. Effects of vitaminB12, folate, and
vitaminB6 supplements in elderly people with normal
serum vitamin concentrations. Lancet 346, 85–89
(1995).
26. van Asselt,D.Z. etal. Cobalamin-binding proteins
innormal and cobalamin-deficient older subjects.
Ann.Clin. Biochem. 40, 65–69 (2003).
27. Johnson,M.A. etal. VitaminB12 deficiency in African
American and white octogenarians and centenarians
inGeorgia. J.Nutr. Health Aging 14, 339–345 (2010).
28. Murphy,M.M. etal. Longitudinal study of the effect
of pregnancy on maternal and fetal cobalamin status
in healthy women and their offspring. J.Nutr. 137,
1863–1867 (2007).
29. Greibe,E. etal. Uptake of cobalamin and markers
ofcobalamin status: a longitudinal study of healthy
pregnant women. Clin. Chem. Lab Med. 49,
1877–1882 (2011).
30. Milman,N. etal. Cobalamin status during normal
pregnancy and postpartum: a longitudinal study
comprising 406 Danish women. Eur. J.Haematol. 76,
521–525 (2006).
31. Bae,S. etal. VitaminB-12 status differs among
pregnant, lactating, and control women with equivalent
nutrient intakes. J.Nutr. 145, 1507–1514 (2015).
32. Obeid,R. etal. The cobalamin-binding proteins
transcobalamin and haptocorrin in maternal and cord
blood sera at birth. Clin. Chem. 52, 263–269 (2006).
33. McLean,E., de Benoist,B. & Allen,L.H. Review of the
magnitude of folate and vitaminB12 deficiencies
worldwide. Food Nutr. Bull. 29 (Suppl. 2), S38–S51
(2008).
34. Barnabe,A. etal. Folate, vitaminB12 and homocysteine
status in the post-folic acid fortification era in different
subgroups of the Brazilian population attended to
atapublic health care center. Nutr. J. 14, 19 (2015).
35. Yusufji,D., Mathan,V.I. & Baker,S.J. Iron, folate, and
vitaminB12 nutrition in pregnancy: a study of 1 000
women from southern India. Bull. World Health Organ.
48, 15–22 (1973).
36. Koc,A. etal. High frequency of maternal vitaminB12
deficiency as an important cause of infantile
vitaminB12 deficiency in Sanliurfa province of Turkey.
Eur. J.Nutr. 45, 291–297 (2006).
37. Yajnik,C.S. etal. VitaminB12 and folate
concentrations during pregnancy and insulin resistance
in the offspring: the Pune Maternal Nutrition Study.
Diabetologia 51, 29–38 (2008).
38. Tucker,K.L. etal. Plasma vitaminB-12 concentrations
relate to intake source in the Framingham Offspring
study. Am. J.Clin. Nutr. 71, 514–522 (2000).
39. Monsen,A.L. etal. Cobalamin status and its
biochemical markers methylmalonic acid and
homocysteine in different age groups from 4days
to19years. Clin. Chem. 49, 2067–2075 (2003).
40. Fokkema,M.R. etal. Plasma total homocysteine
increases from day 20 to 40 in breastfed but not
formula-fed low-birthweight infants. Acta Paediatr. 91,
507–511 (2002).
41. Greibe,E. etal. Cobalamin and haptocorrin in human
milk and cobalamin-related variables in mother and
child: a 9-mo longitudinal study. Am. J.Clin. Nutr. 98,
389–395 (2013).
42. Molloy,A.M. etal. Effects of folate and vitaminB12
deficiencies during pregnancy on fetal, infant,
andchilddevelopment. Food Nutr. Bull. 29 (Suppl.2),
S101–S111 (2008).
43. Bjorke-Monsen,A.L. etal. Common metabolic profile
in infants indicating impaired cobalamin status
responds to cobalamin supplementation. Pediatrics
122, 83–91 (2008).
PRIMER
16
|
ARTICLE NUMBER 17040
|
VOLUME 3 www.nature.com/nrdp

44. Hay,G. etal. Folate and cobalamin status in relation
to breastfeeding and weaning in healthy infants.
Am.J.Clin. Nutr. 88, 105–114 (2008).
45. Torsvik,I. etal. Cobalamin supplementation improves
motor development and regurgitations in infants:
results from a randomized intervention study.
Am.J.Clin. Nutr. 98, 1233–1240 (2013).
46. Cobayashi,F. etal. Genetic and environmental factors
associated with vitaminB12 status in Amazonian
children. Public Health Nutr. 18, 2202–2210
(2015).
47. Hine,B. etal. Transcobalamin derived from bovine
milk stimulates apical uptake of vitaminB12 into
human intestinal epithelial cells. J.Cell. Biochem. 115 ,
1948–1954 (2014).
48. Chanarin,I. Cobalamins and nitrous oxide: a review.
J.Clin. Pathol. 33, 909–916 (1980).
49. Green,R. & Kinsella,L.J. Current concepts in the
diagnosis of cobalamin deficiency. Neurology 45,
1435–1440 (1995).
50. O’Leary,P.W., Combs,M.J. & Schilling,R.F.
Synergistic deleterious effects of nitrous oxide
exposure and vitaminB12 deficiency. J.Lab. Clin.
Med. 105, 428–431 (1985).
51. Toh,B.H., van Driel,I.R. & Gleeson,P.A. Pernicious
anemia. N.Engl. J.Med. 337, 1441–1448 (1997).
A landmark paper on the clinical and
immunological basis of the disease that is the
essential paradigm of B12 deficiency and its
immunological basis.
52. Hershko,C. etal. Variable hematologic presentation
ofautoimmune gastritis: age-related progression from
iron deficiency to cobalamin depletion. Blood 107,
1673–1679 (2006).
53. Amedei,A. etal. Molecular mimicry between
Helicobacter pylori antigens and H+, K+ —
adenosine triphosphatase in human gastric
autoimmunity. J.Exp. Med. 198, 1147–1156
(2003).
54. Doscherholmen,A., McMahon,J. & Ripley,D.
VitaminB12 assimilation from chicken meat.
Am.J.Clin. Nutr. 31 , 825–830 (1978).
55. Bellou,A. etal. Cobalamin deficiency with
megaloblastic anaemia in one patient under long-term
omeprazole therapy. J.Intern. Med. 240, 161–164
(1996).
56. Lam,J.R. etal. Proton pump inhibitor and histamine2
receptor antagonist use and vitaminB12 deficiency.
JAMA 310, 2435–2442 (2013).
57. Degnan,P.H., Taga,M.E. & Goodman,A.L.
VitaminB12 as a modulator of gut microbial ecology.
Cell Metab. 20, 769–778 (2014).
58. Fedosov,S.N. etal. Mechanisms of discrimination
between cobalamins and their natural analogues
during their binding to the specific B12-transporting
proteins. Biochemistry 46, 6446–6458 (2007).
59. Tanwar,V.S. etal. Common variant in FUT2 gene
isassociated with levels of vitaminB12 in Indian
population. Gene 515, 224–228 (2013).
60. Kelly,R.J. etal. Sequence and expression of a
candidate for the human Secretor blood group
alpha(1,2)fucosyltransferase gene (FUT2).
Homozygosity for an enzyme-inactivating nonsense
mutation commonly correlates with the non-secretor
phenotype. J.Biol. Chem. 270, 4640–4649 (1995).
61. Hazra,A. etal. Common variants of FUT2 are
associated with plasma vitaminB12 levels.
Nat.Genet. 40, 1160–1162 (2008).
62. Tanaka,T. etal. Genome-wide association study of
vitaminB6, vitaminB12, folate, and homocysteine
blood concentrations. Am. J.Hum. Genet. 84,
477–482 (2009).
63. Jensen,L.L. etal. Lack of megalin expression in adult
human terminal ileum suggests megalin-independent
cubilin/amnionless activity during vitaminB12
absorption. Physiol. Rep. 2, e12086 (2014).
64. Beedholm-Ebsen,R. etal. Identification of multidrug
resistance protein 1 (MRP1/ABCC1) as a molecular
gate for cellular export of cobalamin. Blood 115 ,
1632–1639 (2010).
65. Aminoff,M. etal. Mutations in CUBN, encoding the
intrinsic factor-vitaminB12 receptor, cubilin, cause
hereditary megaloblastic anaemia 1. Nat. Genet. 21,
309–313 (1999).
66. Tanner,S.M. etal. Amnionless, essential for mouse
gastrulation, is mutated in recessive hereditary
megaloblastic anemia. Nat. Genet. 33, 426–429
(2003).
67. Gueant,J.L. etal. Decreased activity of intestinal and
urinary intrinsic factor receptor in Grasbeck-Imerslund
disease. Gastroenterology 10 8, 1622–1628 (1995).
68. Quadros,E.V. & Sequeira,J.M. Cellular uptake of
cobalamin: transcobalamin and the TCblR/CD320
receptor. Biochimie 95, 1008–1018 (2013).
69. Gherasim,C., Lofgren,M. & Banerjee,R. Navigating
the B12 road: assimilation, delivery, and disorders
ofcobalamin. J.Biol. Chem. 288, 13186–13193
(2013).
An in-depth review of the fascinating molecular
ensemble that is involved in the handling of this
precious micronutrient in genetically normal and
mutated cells.
70. Froese,D.S. & Gravel,R.A. Genetic disorders of
vitaminB12 metabolism: eight complementation
groups — eight genes. Expert Rev. Mol. Med. 12, e37
(2010).
A well-illustrated, concise but complete summary
of the eight recognized inborn errors of B12
metabolism that uses the ‘experiments of nature’
to explain the complex network of intracellular
pathways involved in B12 processing.
71. Whitehead,V.M. Acquired and inherited disorders
ofcobalamin and folate in children. Br. J.Haematol.
134, 125–136 (2006).
72. Trakadis,Y.J. etal. Update on transcobalamin
deficiency: clinical presentation, treatment and
outcome. J.Inherit. Metab. Dis. 37, 461–473
(2014).
73. Miller,J.W. etal. Transcobalamin II 775G>C
polymorphism and indices of vitaminB12 status
inhealthy older adults. Blood 100, 718–720 (2002).
74. Namour,F. etal. Transcobalamin codon 259
polymorphism in HT-29 and Caco-2 cells and in
Caucasians: relation to transcobalamin and
homocysteine concentration in blood. Blood 97,
1092–1098 (2001).
75. Watkins,D. & Rosenblatt,D.S. Lessons in biology
from patients with inborn errors of vitaminB12
metabolism. Biochimie 95, 1019–1022 (2013).
76. Sharma,G.S., Kumar,T. & Singh,L.R.
N-homocysteinylation induces different structural and
functional consequences on acidic and basic proteins.
PLoS ONE 9, e116386 (2014).
77. Ghemrawi,R. etal. Decreased vitaminB12 availability
induces ER stress through impaired
SIRT1-deacetylation of HSF1. Cell Death Dis. 4, e553
(2013).
78. Hannibal,L., DiBello,P.M. & Jacobsen,D.W.
Proteomics of vitaminB12 processing. Clin. Chem.
Lab. Med. 51, 477–488 (2013).
79. Richard,E. etal. Oxidative stress and apoptosis in
homocystinuria patients with genetic remethylation
defects. J.Cell. Biochem. 114, 183–191 (2013).
80. Peracchi,M. etal. Human cobalamin deficiency:
alterations in serum tumour necrosis factor-alpha
andepidermal growth factor. Eur. J.Haematol. 67,
123–127 (2001).
81. Scalabrino,G. etal. High tumor necrosis factor-alpha
levels in cerebrospinal fluid of cobalamin-deficient
patients. Ann. Neurol. 56, 886–890 (2004).
82. Caterino,M. etal. The proteome of cblC defect: invivo
elucidation of altered cellular pathways in humans.
J.Inherit. Metab. Dis. 38, 969–979 (2015).
83. Troen,A.M. etal. B-vitamin deficiency causes
hyperhomocysteinemia and vascular cognitive
impairment in mice. Proc. Natl Acad. Sci. USA 10 5,
12474–12479 (2008).
84. Fuso,A. & Scarpa,S. One-carbon metabolism and
Alzheimer’s disease: is it all a methylation matter?
Neurobiol. Aging 32, 1192–1195 (2011).
85. Scalabrino,G. The multi-faceted basis of vitaminB12
(cobalamin) neurotrophism in adult central nervous
system: lessons learned from its deficiency.
Prog.Neurobiol. 88, 203–220 (2009).
86. Battaglia-Hsu,S.F. etal. VitaminB12 deficiency
reduces proliferation and promotes differentiation
ofneuroblastoma cells and up-regulates PP2A,
proNGF, and TACE. Proc. Natl Acad. Sci. USA 10 6,
21930–21935 (2009).
87. Rosenblatt,D.S. & Whitehead,V.M. Cobalamin
andfolate deficiency: acquired and hereditary disorders
in children. Semin. Hematol. 36, 19–34 (1999).
88. Dror,D.K. & Allen,L.H. Effect of vitaminB12
deficiency on neurodevelopment in infants: current
knowledge and possible mechanisms. Nutr. Rev. 66,
250–255 (2008).
89. Demir,N. etal. Clinical and neurological findings
ofsevere vitaminB12 deficiency in infancy and
importance of early diagnosis and treatment.
J.Paediatr. Child Health 49, 820–824 (2013).
90. Copp,A.J. etal. Spina bifida. Nat. Rev. Dis. Primers
1, 15007 (2015).
91. Molloy,A.M. etal. Maternal vitaminB12 status
andrisk of neural tube defects in a population with
high neural tube defect prevalence and no folic acid
fortification. Pediatrics 123, 917–923 (2009).
92. Suarez,L. etal. Maternal serum B12 levels and risk
for neural tube defects in a Texas–Mexico border
population. Ann. Epidemiol. 13, 81–88 (2003).
93. Green,R. & Miller,J.W. VitaminB12 deficiency is the
dominant nutritional cause of hyperhomocysteinemia
in a folic acid-fortified population. Clin. Chem. Lab.
Med. 43, 1048–1051 (2005).
94. Smith,A.D. etal. Homocysteine-lowering by
Bvitamins slows the rate of accelerated brain atrophy
in mild cognitive impairment: a randomized controlled
trial. PLoS ONE 5, e12244 (2010).
95. de Jager,C.A. etal. Cognitive and clinical outcomes
ofhomocysteine-lowering B-vitamin treatment in mild
cognitive impairment: a randomized controlled trial.
Int. J.Geriatr. Psychiatry 27, 592–600 (2012).
96. Douaud,G. etal. Preventing Alzheimer’s disease-
related gray matter atrophy by B-vitamin treatment.
Proc. Natl Acad. Sci. USA 110, 9523–9528 (2013).
97. Haan,M.N. etal. Homocysteine, B vitamins, and
theincidence of dementia and cognitive impairment:
results from the Sacramento Area Latino Study
onAging. Am. J.Clin. Nutr. 85, 511–517 (2007).
98. Morris,M.S. etal. Folate and vitaminB-12 status
inrelation to anemia, macrocytosis, and cognitive
impairment in older Americans in the age of folic acid
fortification. Am. J.Clin. Nutr. 85, 193–200 (2007).
99. Brito,A. etal. VitaminB-12 treatment of
asymptomatic, deficient, elderly Chileans improves
conductivity in myelinated peripheral nerves, but high
serum folate impairs vitaminB-12 status response
assessed by the combined indicator of vitaminB-12
status. Am. J.Clin. Nutr. 103 , 250–257 (2016).
100. Chhabra,N., Lee,S. & Sakalis,E.G. Cobalamin
deficiency causing severe hemolytic anemia:
apernicious presentation. Am. J.Med. 128, e5–e6
(2015).
101. Parmentier,S. etal. Severe pernicious anemia with
distinct cytogenetic and flow cytometric aberrations
mimicking myelodysplastic syndrome. Ann. Hematol.
91, 1979–1981 (2012).
102. Andres,E. etal. Current hematological findings in
cobalamin deficiency. A study of 201 consecutive
patients with documented cobalamin deficiency.
Clin.Lab. Haematol. 28, 50–56 (2006).
103. Green,R. Anemias beyond B12 and iron deficiency:
the buzz about other B’s, elementary, and
nonelementary problems. Hematology Am. Soc.
Hematol. Educ. Program 2012, 492–498 (2012).
104. Devalia,V., Hamilton,M.S. & Molloy,A.M. Guidelines
for the diagnosis and treatment of cobalamin and
folate disorders. Br. J.Haematol. 166, 496–513
(2014).
105. Lindenbaum,J. etal. Diagnosis of cobalamin
deficiency: II. Relative sensitivities of serum
cobalamin, methylmalonic acid, and total
homocysteine concentrations. Am. J.Hematol. 34,
99–107 (1990).
106. Fedosov,S.N. etal. Combined indicator of
vitaminB12 status: modification for missing
biomarkers and folate status and recommendations
for revised cut-points. Clin. Chem. Lab. Med. 53,
1215–1225 (2015).
107. Carmel,R. & Agrawal,Y.P. Failures of cobalamin
assays in pernicious anemia. N.Engl. J.Med. 367,
385–386 (2012).
108. Nexo,E. Variation with age of reference values for
P-cobalamins. Scand. J.Haematol. 30, 430–432
(1983).
109. Arendt,J.F. & Nexo,E. Unexpected high plasma
cobalamin: proposal for a diagnostic strategy.
Clin.Chem. Lab. Med. 51, 489–496 (2013).
110 . Herzlich,B. & Herbert,V. Depletion of serum
holotranscobalamin II. An early sign of negative
vitaminB12 balance. Lab. Invest. 58, 332–337
(1988).
111. Risch,M. etal. VitaminB12 and folate levels in
healthy Swiss senior citizens: a prospective study
evaluating reference intervals and decision limits.
BMCGeriatr. 15, 82 (2015).
112 . Miller,J.W. etal. Measurement of total vitaminB12
and holotranscobalamin, singly and in combination,
inscreening for metabolic vitaminB12 deficiency.
Clin.Chem. 52, 278–285 (2006).
113 . Nexo,E. & Hoffmann-Lucke,E. Holotranscobalamin,
amarker of vitaminB-12 status: analytical aspects
and clinical utility. Am. J.Clin. Nutr. 94, 359S–365S
(2011).
PRIMER
NATURE REVIEWS
|
DISEASE PRIMERS VOLUME 3
|
ARTICLE NUMBER 17040
|
17

114 . Refsum,H. etal. Facts and recommendations about
total homocysteine determinations: an expert opinion.
Clin. Chem. 50, 3–32 (2004).
115 . Rasmussen,K. etal. Age- and gender-specific
reference intervals for total homocysteine and
methylmalonic acid in plasma before and after vitamin
supplementation. Clin. Chem. 42, 630–636 (1996).
116 . Vogiatzoglou,A. etal. Determinants of plasma
methylmalonic acid in a large population: implications
for assessment of vitaminB12 status. Clin. Chem. 55,
2198–2206 (2009).
117 . Molloy,A.M. etal. A common polymorphism in
HIBCH influences methylmalonic acid concentrations
in blood independently of cobalamin. Am. J.Hum.
Genet. 98, 869–882 (2016).
118 . Hvas,A.M. & Nexo,E. Diagnosis and treatment of
vitaminB12 deficiency — an update. Haematologica
91, 1506–1512 (2006).
119 . Bailey,R.L. etal. Modeling a methylmalonic acid-
derived change point for serum vitaminB-12 for
adults in NHANES. Am. J.Clin. Nutr. 98, 460–467
(2013).
120. Morris,M.S. etal. Elevated serum methylmalonic
acid concentrations are common among elderly
Americans. J.Nutr. 132, 2799–2803 (2002).
121. Loikas,S. etal. Renal impairment compromises the
use of total homocysteine and methylmalonic acid
butnot total vitaminB12 and holotranscobalamin
inscreening for vitaminB12 deficiency in the aged.
Clin.Chem. Lab. Med. 45, 197–201 (2007).
122. Carmel,R. Anemia and aging: an overview of clinical,
diagnostic and biological issues. Blood Rev. 15, 9–18
(2001).
123. Green,R. & Dwyre,D.M. Evaluation of macrocytic
anemias. Semin. Hematol. 52, 279–286 (2015).
124. Lachner,C., Steinle,N.I. & Regenold,W.T. The
neuropsychiatry of vitaminB12 deficiency in elderly
patients. J.Neuropsychiatry Clin. Neurosci. 24, 5–15
(2012).
125. Reynolds,E. VitaminB12, folic acid, and the nervous
system. Lancet Neurol. 5, 949–960 (2006).
126. Biemans,E. etal. Cobalamin status and its relation
with depression, cognition and neuropathy in patients
with type2 diabetes mellitus using metformin.
ActaDiabetol. 52, 383–393 (2015).
127. Clarke,R. etal. Low vitaminB-12 status and risk
ofcognitive decline in older adults. Am. J.Clin. Nutr.
86, 1384–1391 (2007).
128. Doets,E.L. etal. VitaminB12 intake and status
andcognitive function in elderly people.
Epidemiol.Rev. 35, 2–21 (2013).
129. O’Leary,F., Allman-Farinelli,M. & Samman,S.
VitaminB12 status, cognitive decline and dementia:
asystematic review of prospective cohort studies.
Br.J.Nutr. 108, 1948–1961 (2012).
130. Vogiatzoglou,A. etal. Cognitive function in an elderly
population: interaction between vitaminB12 status,
depression, and apolipoprotein E ε4: the Hordaland
Homocysteine Study. Psychosom. Med. 75, 20–29
(2013).
131. Aisen,P.S. etal. High-dose B vitamin supplementation
and cognitive decline in Alzheimer disease:
arandomized controlled trial. JAMA 300, 1774–1783
(2008).
132. McMahon,J.A. etal. A controlled trial of
homocysteine lowering and cognitive performance.
N.Engl. J.Med. 354, 2764–2772 (2006).
133. Eussen,S.J. etal. Effect of oral vitaminB-12 with or
without folic acid on cognitive function in older people
with mild vitaminB-12 deficiency: a randomized,
placebo-controlled trial. Am. J.Clin. Nutr. 84,
361–370 (2006).
134. Durga,J. etal. Effect of 3-year folic acid
supplementation on cognitive function in older adults
in the FACIT trial: a randomised, double blind,
controlled trial. Lancet 369, 208–216 (2007).
135. Sato,Y. etal. Effect of folate and mecobalamin
on hip fractures in patients with stroke: a randomized
controlled trial. JAMA 293, 1082–1088 (2005).
136. Dangour,A.D. etal. A randomised controlled trial
investigating the effect of vitaminB12
supplementation on neurological function in healthy
older people: the Older People and Enhanced
Neurological function (OPEN) study protocol
[ISRCTN54195799]. Nutr. J. 10, 22 (2011).
137. Hvas,A.M., Morkbak,A.L. & Nexo,E. Plasma
holotranscobalamin compared with plasma
cobalamins for assessment of vitaminB12 absorption;
optimisation of a non-radioactive vitaminB12
absorption test (CobaSorb). Clin. Chim. Acta 376,
150–154 (2007).
138. Bor,M.V. etal. Nonradioactive vitaminB12
absorption test evaluated in controls and in patients
with inherited malabsorption of vitaminB12.
Clin.Chem. 51, 2151–2155 (2005).
139. Hvas,A.M. etal. The vitaminB12 absorption test,
CobaSorb, identifies patients not requiring
vitaminB12 injection therapy. Scand. J.Clin. Lab.
Invest. 71, 432–438 (2011).
140. Carkeet,C. etal. Human vitaminB12 absorption
measurement by accelerator mass spectrometry using
specifically labeled 14C-cobalamin. Proc. Natl Acad.
Sci. USA 103, 5694–5699 (2006).
141. Stabler,S.P. VitaminB12 deficiency. N.Engl. J.Med.
368, 2041–2042 (2013).
142. Toh,B.H. Diagnosis and classification of autoimmune
gastritis. Autoimmun. Rev. 13, 459–462 (2014).
143. Lahner,E. etal. Reassessment of intrinsic factor and
parietal cell autoantibodies in atrophic gastritis with
respect to cobalamin deficiency. Am. J.Gastroenterol.
104, 2071–2079 (2009).
144. de Benoist,B. Conclusions of a WHO Technical
Consultation on folate and vitaminB12 deficiencies.
Food Nutr. Bull. 29 (Suppl. 2), S238–S244 (2008).
145. World Health Organization. The implications for
training of embracing: a life course approach to health
(WHO, 2000).
146. Duggan,C. etal. VitaminB-12 supplementation
during pregnancy and early lactation increases
maternal, breast milk, and infant measures of
vitaminB-12 status. J.Nutr. 144, 758–764 (2014).
147. Siddiqua,T.J. etal. VitaminB12 supplementation
during pregnancy and postpartum improves B12
status of both mothers and infants but vaccine
response in mothers only: a randomized clinical trial
inBangladesh. Eur. J.Nutr. 55, 281–293 (2016).
148. Watanabe,F. VitaminB12 sources and bioavailability.
Exp. Biol. Med. (Maywood) 232, 1266–1274 (2007).
149. Naik,S. etal. Daily milk intake improves vitaminB-12
status in young vegetarian Indians: an intervention
trial. Nutr. J. 12, 136 (2013).
150. Potdar,R.D. etal. Improving women’s diet quality
preconceptionally and during gestation: effects
onbirth weight and prevalence of low birth weight
—arandomized controlled efficacy trial in India
(Mumbai Maternal Nutrition Project). Am. J.Clin. Nutr.
100, 1257–1268 (2014).
151. Kehoe,S.H. etal. Effects of a food-based intervention
on markers of micronutrient status among Indian
women of low socio-economic status. Br. J.Nutr. 113 ,
813–821 (2015).
152. Winkels,R.M. etal. Bread cofortified with folic acid
and vitaminB-12 improves the folate and vitaminB-12
status of healthy older people: arandomized controlled
trial. Am. J.Clin. Nutr. 88, 348–355 (2008).
153. Dhonukshe-Rutten,R.A. etal. Effect of
supplementation with cobalamin carried either by a
milk product or a capsule in mildly cobalamin-deficient
elderly Dutch persons. Am. J.Clin. Nutr. 82, 568–574
(2005).
154. Tapola,N.S. etal. Mineral water fortified with folic
acid, vitamins B6, B12, D and calcium improves folate
status and decreases plasma homocysteine
concentration in men and women. Eur. J.Clin. Nutr.
58, 376–385 (2004).
155. Mohammad,M.A. etal. Plasma cobalamin and folate
and their metabolic markers methylmalonic acid and
total homocysteine among Egyptian children before
and after nutritional supplementation with the
probiotic bacteria Lactobacillus acidophilus in yoghurt
matrix. Int. J.Food Sci. Nutr. 57, 470–480 (2006).
156. Miller,J.W. etal. Metabolic evidence of vitaminB-12
deficiency, including high homocysteine and
methylmalonic acid and low holotranscobalamin, is
more pronounced in older adults with elevated plasma
folate. Am. J.Clin. Nutr. 90, 1586–1592 (2009).
157. Selhub,J., Morris,M.S. & Jacques,P.F.
InvitaminB12 deficiency, higher serum folate is
associated with increased total homocysteine and
methylmalonic acid concentrations. Proc. Natl Acad.
Sci. USA 104, 19995–20000 (2007).
158. Carmel,R. How I treat cobalamin (vitaminB12)
deficiency. Blood 112 , 2214–2221 (2008).
159. Obeid,R., Fedosov,S.N. & Nexo,E. Cobalamin
coenzyme forms are not likely to be superior to cyano-
and hydroxyl-cobalamin in prevention or treatment
ofcobalamin deficiency. Mol. Nutr. Food Res. 59,
1364–1372 (2015).
160. Berlin,H., Berlin,R. & Brante,G. Oral treatment
ofpernicious anemia with high doses of vitaminB12
without intrinsic factor. Acta Med. Scand. 184,
247–258 (1968).
161. Kuzminski,A.M. etal. Effective treatment of
cobalamin deficiency with oral cobalamin. Blood 92,
1191–1198 (1998).
162. Castelli,M.C. etal. Comparing the efficacy and
tolerability of a new daily oral vitaminB12 formulation
and intermittent intramuscular vitaminB12 in
normalizing low cobalamin levels: a randomized,
open-label, parallel-group study. Clin. Ther. 33,
358–371.e2 (2011).
163. Kim,H.I. etal. Oral vitaminB12 replacement:
aneffective treatment for vitaminB12 deficiency
aftertotal gastrectomy in gastric cancer patients.
Ann.Surg. Oncol. 18, 3711–3717 (2011).
164. Rajan,S. etal. Response of elevated methylmalonic
acid to three dose levels of oral cobalamin in older
adults. J.Am. Geriatr. Soc. 50, 1789–1795 (2002).
165. Eussen,S.J. etal. Oral cyanocobalamin
supplementation in older people with vitaminB12
deficiency: a dose-finding trial. Arch. Intern. Med.
165, 1167–1172 (2005).
166. Bor,M.V. etal. Daily intake of 4 to 7 μg dietary
vitaminB-12 is associated with steady concentrations
of vitaminB-12-related biomarkers in a healthy young
population. Am. J.Clin. Nutr. 91 , 571–577 (2010).
167. Green,R. etal. Absorption of biliary cobalamin in
baboons following total gastrectomy. J.Lab. Clin. Med.
100, 771–777 (1982).
168. Green,R. in Advances in Thomas Addison’s Diseases
(eds Bhatt,R., James,V.H.T., Besser,G.M.,
Bottazzo,G.F. & Keen,H.) 377–390 (Society for
Endocrinology & The Thomas Addison Society, 1994).
169. Kumar,N. Neurologic aspects of cobalamin (B12)
deficiency. Handb. Clin. Neurol. 120, 915–926
(2014).
A complete description of the protean neurological
manifestations of B12 deficiency.
170. Pittock,S.J., Payne,T.A. & Harper,C.M. Reversible
myelopathy in a 34-year-old man with vitaminB12
deficiency. Mayo Clin. Proc. 77, 291–294 (2002).
171. Hirota,W.K. etal. ASGE guideline: the role of
endoscopy in the surveillance of premalignant
conditions of the upper GI tract. Gastrointest. Endosc.
63, 570–580 (2006).
172. Finkelstein,J.L., Layden,A.J. & Stover,P.J.
VitaminB-12 and perinatal health. Adv. Nutr. 6,
552–563 (2015).
173. Vogiatzoglou,A. etal. VitaminB12 status and rate
ofbrain volume loss in community-dwelling elderly.
Neurology 71, 826–832 (2008).
174. Zhang,Y. etal. Decreased brain levels of vitaminB12
in aging, autism and schizophrenia. PLoS ONE 11,
e0146797 (2016).
175. Stone,N. etal. Bioinformatic and genetic association
analysis of microRNA target sites in one-carbon
metabolism genes. PLoS ONE 6, e21851 (2011).
176. Joslin,A.C. etal. Concept mapping one-carbon
metabolism to model future ontologies for nutrient-
gene-phenotype interactions. Genes Nutr. 9, 419
(2014).
177. Grarup,N. etal. Genetic architecture of vitaminB12
and folate levels uncovered applying deeply
sequenced large datasets. PLoS Genet. 9, e1003530
(2013).
An informative and detailed description of the
genetic blueprint of the architecture of the genome
as it applies to B12 status as well as the closely
related vitaminB9 (folate).
178. Deegan,K.L. etal. Breast milk vitaminB-12
concentrations in Guatemalan women are correlated
with maternal but not infant vitaminB-12 status
at12months postpartum. J.Nutr. 142, 112–116
(2012).
179. Ford,C. etal. VitaminB12 levels in human milk during
the first nine months of lactation. Int. J.Vitam. Nutr.
Res. 66, 329–331 (1996).
180. Thomas,M.R. etal. The effects of vitaminC,
vitaminB6, vitaminB12, folic acid, riboflavin, and
thiamin on the breast milk and maternal status
of well-nourished women at 6months postpartum.
Am. J.Clin. Nutr. 33, 2151–2156 (1980).
181. Greibe,E. & Nexo,E. Forms and amounts of
vitaminB12 in infant formula: a pilot study. PLoS ONE
11, e0165458 (2016).
182. Strand,T.A. etal. VitaminB-12, folic acid, and
growthin 6- to 30-month-old children: a randomized
controlled trial. Pediatrics 135, e918–e926 (2015).
183. Hussein,L. etal. Serum vitaminB12 concentrations
among mothers and newborns and follow-up study
toassess implication on the growth velocity and the
urinary methylmalonic acid excretion. Int. J.Vitam.
Nutr. Res. 79, 297–307 (2009).
PRIMER
18
|
ARTICLE NUMBER 17040
|
VOLUME 3 www.nature.com/nrdp

184. Jenssen,H.B. etal. Biochemical signs of impaired
cobalamin function do not affect hematological
parameters in young infants: results from a double-
blind randomized controlled trial. Pediatr. Res. 74,
327–332 (2013).
185. Bhate,V.K. etal. VitaminB12 and folate during
pregnancy and offspring motor, mental and social
development at 2years of age. J.Dev. Orig. Health
Dis. 3, 123–130 (2012).
186. Bhate,V. etal. VitaminB12 status of pregnant
Indianwomen and cognitive function in their
9-year-old children. Food Nutr. Bull. 29, 249–254
(2008).
187. Strand,T.A. etal. Cobalamin and folate status
predicts mental development scores in North Indian
children 12–18 mo of age. Am. J.Clin. Nutr. 97,
310–317 (2013).
188. Leishear,K. etal. Relationship between vitaminB12
and sensory and motor peripheral nerve function in
older adults. J.Am. Geriatr. Soc. 60, 1057–1063
(2012).
189. Lildballe,D.L. etal. Association of cognitive
impairment with combinations of vitaminB12-related
parameters. Clin. Chem. 57, 1436–1443 (2011).
190. Smith,A.D. & Refsum,H. VitaminB-12 and cognition
in the elderly. Am. J.Clin. Nutr. 89, 707S–711S
(2009).
191. McLean,R.R. etal. Plasma B vitamins, homocysteine
and their relation with bone loss and hip fracture in
elderly men and women. J.Clin. Endocrinol. Metab.
93, 2206–2212 (2008).
192. Park,S. etal. Age-related hearing loss, methylmalonic
acid, and vitaminB12 status in older adults. J.Nutr.
Elder. 25, 105–120 (2006).
193. Gopinath,B. etal. Homocysteine, folate, vitaminB-12,
and 10-y incidence of age-related macular
degeneration. Am. J.Clin. Nutr. 98, 129–135 (2013).
194. Herbert,V. & Zalusky,R. Interrelations of vitaminB12
and folic acid metabolism: folic acid clearance studies.
J.Clin. Invest. 41, 1263–1276 (1962).
195. Allen,L.H. Folate and vitaminB12 status in the
Americas. Nutr. Rev. 62, S29–S33 (2004).
196. Brito,A. etal. Folate and vitaminB12 status in Latin
America and the Caribbean: an update. Food Nutr.
Bull. 36 (Suppl. 2), S109–S118 (2015).
197. Campbell,A.K. etal. Plasma vitaminB-12
concentrations in an elderly latino population
arepredicted by serum gastrin concentrations
andcrystalline vitaminB-12 intake. J.Nutr. 133,
2770–2776 (2003).
198. MacFarlane,A.J., Greene-Finestone,L.S. & Shi,Y.
VitaminB-12 and homocysteine status in a folate-
replete population: results from the Canadian Health
Measures Survey. Am. J.Clin. Nutr. 94, 1079–1087
(2011).
199. Quay,T.A. etal. High prevalence of suboptimal
vitaminB12 status in young adult women of South
Asian and European ethnicity. Appl. Physiol. Nutr.
Metab. 40, 1279–1286 (2015).
200. Clarke,R. etal. Detection of vitaminB12 deficiency
inolder people by measuring vitaminB12 or the
active fraction of vitaminB12, holotranscobalamin.
Clin. Chem. 53, 963–970 (2007).
201. Karabulut,A. etal. Premarital screening of 466
Mediterranean women for serum ferritin, vitaminB12,
and folate concentrations. Turk. J.Med. Sci. 45,
358–363 (2015).
202. Benson,J. etal. Low vitaminB12 levels among newly-
arrived refugees from Bhutan, Iran and Afghanistan:
amulticentre Australian study. PLoS ONE 8, e57998
(2013).
203. Sivaprasad,M. etal. Status of vitaminB12 and folate
among the urban adult population in South India.
Ann.Nutr. Metab. 68, 94–102 (2016).
204. El-Khateeb,M. etal. VitaminB12 deficiency in Jordan:
a population-based study. Ann. Nutr. Metab. 64,
101–105 (2014).
205. Thuesen,B.H. etal. Lifestyle and genetic
determinants of folate and vitaminB12 levels
inageneral adult population. Br. J.Nutr. 103,
1195–1204 (2010).
206. el Kholty,S. etal. Portal and biliary phases of
enterohepatic circulation of corrinoids in humans.
Gastroenterology 101, 1399–1408 (1991).
207. Lai,S.C. etal. The transcobalamin receptor knockout
mouse: a model for vitaminB12 deficiency in the
central nervous system. FASEB J. 27, 2468–2475
(2013).
208. Tateyama,M. etal. CD4 T lymphocytes are primed
toexpress Fas ligand by CD4 cross-linking and
tocontribute to CD8 T-cell apoptosis via Fas/FasL
deathsignaling pathway. Blood 96, 195–202 (2000).
209. Refsum,H. etal. Holotranscobalamin and total
transcobalamin in human plasma: determination,
determinants, and reference values in healthy adults.
Clin. Chem. 52, 129–137 (2006).
Acknowledgements
The authors thank N. DeGeorge and L. Texeira for their
administrative and editing support.
Author contributions
Introduction (R.G.); Epidemiology (L.H.A., A.B., A.M.M.,
A.-L.B.-M., J.W.M. and P.M.U.); Mechanisms/pathophysiology
(J.-L.G. and B.-H.T.); Diagnosis, screening and prevention
(E.N. and C.Y.); Management (S.S.); Quality of life (S.S.);
Outlook (R.G.); Overview of Primer (R.G.).
Competing interests
R.G. has previously served on speakers’ bureaus and as a
consultant for Emisphere Technologies. J.W.M. has served on
a scientific steering committee for Emisphere Technologies.
A.M.M. received an honorarium as a speaker at the Abbott
Transformation Forum, Manchester, UK. S.S. indirectly
benefits from the activities of a company formed by the
University of Colorado aimed at measuring vitaminB12-
related metabolites. Otherwise she does not have any conflict
of interest. All other authors declare no competinginterests.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional
claims in published maps and institutional affiliations.
How to cite this article
Green, R. etal. VitaminB12 deficiency. Nat. Rev. Dis. Primers
3, 17040 (2017).
PRIMER
NATURE REVIEWS
|
DISEASE PRIMERS VOLUME 3
|
ARTICLE NUMBER 17040
|
19