12/26/23, 12:13 AM Clinical manifestations and diagnosis of vitamin B12 and folate deficiency - UpToDate

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Clinical manifestations and diagnosis of vitamin B12 and

folate deficiency
AUTHORS: Robert T Means, Jr, MD, MACP, Kathleen M Fairfield, MD, DrPH
SECTION EDITOR: Clifford M Takemoto, MD
DEPUTY EDITORS: Jennifer S Tirnauer, MD, Jane Givens, MD, MSCE

All topics are updated as new evidence becomes available and our peer review process is complete.

Literature review current through: Nov 2023.

This topic last updated: Jun 16, 2023.

INTRODUCTION

This topic review discusses the symptoms, clinical manifestations, and diagnosis of vitamin B12
and folate deficiency, vitamins that are required for normal hematopoiesis and neurologic
function. Vitamin B12 and folate deficiencies are often considered together, although folate
deficiency has become less common in individuals who are living in developed countries and
consuming a normal diet.

Separate topic reviews discuss the treatment of these deficiencies, their causes and
pathophysiology, and other causes of macrocytic anemia:

● Treatment – (See "Treatment of vitamin B12 and folate deficiencies".)

● Pathophysiology – (See "Causes and pathophysiology of vitamin B12 and folate
deficiencies".)
● Other macrocytic anemias – (See "Macrocytosis/Macrocytic anemia".)

TERMINOLOGY

Vitamin B12 and folate are both water-soluble B vitamins required for formation of
hematopoietic cells (red blood cells, white blood cells, and platelets) ( table 1). (See "Overview
of water-soluble vitamins".)

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● Vitamin B12 – Vitamin B12 is also called cobalamin (Cbl). It is present in foods derived
from animal products. (See "Causes and pathophysiology of vitamin B12 and folate
deficiencies", section on 'Dietary sources and RDI'.)

The endogenous forms include cobalamin and holotranscobalamin, which represent the
active fraction of plasma cobalamin. The supplemental forms used to treat vitamin B12
deficiency include cyanocobalamin, which contains a cyanide (CN) group introduced
during chemical synthesis, and hydroxocobalamin.

Cyanocobalamin is predominantly used in the United States, while hydroxocobalamin is

predominantly used in Europe; both are effective in treating vitamin B12 deficiency. The
major difference between the two preparations is in the pharmacokinetics and dosing
interval, as discussed separately. (See "Treatment of vitamin B12 and folate deficiencies",
section on 'Available therapeutic preparations'.)

● Pernicious anemia – Pernicious anemia (PA) refers to vitamin B12 deficiency caused by
autoantibodies that interfere with vitamin B12 absorption by targeting intrinsic factor (IF),
gastric parietal cells, or both. At the time it was described, PA was associated with
continuous worsening of symptoms and even death without an available treatment.

● Folate – Folate is also called vitamin B9. The terms "folate" and "folic acid" are sometimes
used interchangeably. Technically, the vitamin is found in nature as a folate, while folic acid
is the synthetic form used therapeutically; it is an oxidized, water-soluble form that does
not exist in nature [1]. Dietary folates are also called folate polyglutamates [2]. Dietary
folate is found in plant-based foods and fortified grains. (See "Causes and pathophysiology
of vitamin B12 and folate deficiencies", section on 'Dietary sources and RDI'.)

Folinic acid (leucovorin, N5-formyl-tetrahydofolate [THF], 5-formylTHF) and 5-

methyltetrahydrofolate (5-MTHF) are naturally occurring forms of reduced folate [1]. The
roles of vitamin B12 and folates in this pathway are illustrated in the figure ( figure 1).
Folinic acid is typically used to prevent toxicities of methotrexate and to potentiate
cytotoxicity of fluorouracil (FU) in chemotherapy regimens for colon cancer. This is because
folinic acid is rapidly converted to the metabolically active form of folate required in cells
(tetrahydrofolate) without the need for dihydrofolate reductase, which is inhibited by
methotrexate. Folic acid, folinic acid, and 5-MTHF are all effective in treating folate
deficiency.

● Homocysteine and methylmalonic acid – Homocysteine is methylated to create

methionine in a reaction that requires folate and vitamin B12 (folate is a methyl donor)

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( figure 2). This is why both folate and vitamin B12 deficiencies result in accumulation of
homocysteine ( figure 3).

Methylmalonic acid (MMA) is converted to succinyl-CoA in a reaction that requires vitamin

B12 but not folate. This is why vitamin B12 deficiency causes accumulation of MMA but
folate deficiency does not.

Use of metabolite testing in diagnosis is discussed below. (See 'Metabolite testing (MMA
and homocysteine)' below.)

● Megaloblastic versus macrocytic anemia – Both vitamin B12 and folate deficiencies
cause megaloblastic anemia. Megaloblastic anemia is a term that refers to anemia in
which the process of nucleic acid metabolism is impaired, resulting in nuclear-cytoplasmic
dyssynchrony, reduced number of cell divisions in the bone marrow, and nuclear
abnormalities in both myeloid and erythroid precursors ( picture 1).

The term megaloblastic should be confined to nucleated red blood cell (RBC) precursors
when used to describe red blood cell morphology. Macrocytic anemia is purely a
morphologic term that describes large RBCs in the peripheral blood and includes all
anemias with a high mean corpuscular volume (MCV). Megaloblastic anemia is a specific
subtype of macrocytic anemia. (See "Macrocytosis/Macrocytic anemia", section on
'Megaloblastic anemia'.)

The Centers for Disease Control in the United States maintains dietary fact sheets for vitamin
B12 and folate that discuss the amount of these vitamins in selected food sources as well as
recommended intakes and populations at risk for deficiency. Additional information about
dietary intake and absorption of these vitamins is presented separately. (See "Causes and
pathophysiology of vitamin B12 and folate deficiencies", section on 'Overview of intake and
metabolism'.)

EPIDEMIOLOGY

The prevalence of vitamin B12 and folate deficiencies is likely to vary among different
populations and depending on the threshold used to define deficiency. Examples include the
following:

● General population – In a 2016 series of 3324 patients with anemia in a general practice
population in the Netherlands, 249 had macrocytosis [3]. Of these, there were 46 cases of
vitamin B12 deficiency (1.4 percent of all anemic individuals; 18 percent of those with

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macrocytic anemia) and 16 cases of folate deficiency (0.5 percent of all anemic; 6 percent
of macrocytic anemia). A 2014 examination of folate testing performed on outpatients in a
single center in Boston, Massachusetts (United States) found folate deficiency in only 47 of
84,187 (0.06 percent); another 166 (0.2 percent) had low-normal values (3.0 to 3.9 ng/mL)
[4].

Pernicious anemia (vitamin B12 deficiency due to autoantibodies) (see 'Terminology'

above) is most common in White people from northern Europe; the incidence is lower in
individuals with African or non-northern European ancestry.

● Older adults – Multiple studies of older adults have shown higher prevalences of vitamin
B12 deficiency or insufficiency than in younger adults, with prevalences in older adults
ranging from 5 to 14 percent [5-8].

● Hospitalized patients – In a 2015 report of hospitalized inpatients in Canada who had

testing for vitamin B12 and folate levels, vitamin B12 deficiency was observed in 98 of
3154 (3.1 percent); an additional 425 individuals (13.5 percent) had vitamin B12 levels in
the low normal range (138 to 221 pmol/L), the clinical manifestations of which are
uncertain [9]. Folate deficiency (assessed using red blood cell folate) (see 'RBC folate'
below) was observed in 4 of 2563 (0.16 percent). It should be noted that these prevalences
apply to the subset of patients in whom testing was performed; the prevalences in all
hospitalized patients is expected to be lower.

A report from Israel that measured serum folate in 726 patients hospitalized in the
Internal Medicine Department over a one-year period found 97 (13.4 percent) to be folate
deficient [10]. While the folate-deficient patients had higher in-hospital mortality than non-
deficient patients (18.6 percent versus 12.1 percent), mortality did not differ between mild
folate deficiency and severe folate deficiency, suggesting that deficiency occurred in the
setting of underlying significant comorbidity.

● Infants and young children

• Maternal B12 deficiency – Vitamin B12 deficiency is rare in infants but may be seen in
newborns and breast-fed infants of vitamin B12-deficient mothers [8]. This neonatal
vitamin B12 deficiency, if not identified and treated, can result in developmental delay
or permanent neurologic damage [11]. Newborn screening programs evaluate for
metabolic disorders including methylmalonic acidemia and homocystinuria and can
also detect vitamin B12 deficiency in newborns. In the United States, the rate of
nutritional vitamin B12 deficiency from 2003 to 2007 was reported to be 0.88 in 100,000
births (1 in 113,636) [12]. However, using more sensitive and specific second-tier testing
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in 176,702 infants in Germany, the rate of vitamin B12 deficiency was 1 in 5355 [13]. In
another study of 258,637 infants in Spain, screening identified 130 with acquired
vitamin B12 deficiency (1 in 1989) [14].

• Lack of dietary folate – Folate deficiency may occur in a child unable to tolerate a
normal diet or in severely malnourished infants or children. Deficiencies are also
reported in infants drinking goats milk, which is deficient in folate [15,16]. (See
"Micronutrient deficiencies associated with malnutrition in children".)

The prevalence of folate deficiency in healthy people with normal dietary intake has declined
progressively in countries that have implemented routine folic acid supplementation of foods
(including grains, breads, cereals, pastas and rice), which began in the late 1990s and early
2000s in many parts of the world [17]. The countries that use fortification of foods with folic acid
are illustrated on a website from the Food Fortification Initiative [18]. This phenomenon is
illustrated by the following examples:

● In the 2015 report from Canada that evaluated hospitalized patients for folate deficiency
and found a prevalence of 0.16 percent, the only causes of folate deficiency were alcohol
use, a malabsorption syndrome, decreased oral intake due to schizophrenia, and a
spurious value [9].

● Direct measurement of population folate levels in the NHANES study showed that after
national fortification began, the mean serum folate level was 2.5 times higher than in the
pre-fortification period, and red blood cell folate was 1.5 times higher [19].

In contrast, the prevalence of vitamin B12 deficiency has remained stable over time. This is likely
because most cases of vitamin B12 deficiency are due to decreased absorption rather than
dietary lack; therefore, routine supplementation of vitamin B12 has not been implemented, and
measurement of vitamin B12 status is indicated in the evaluation of anemia.

ASSOCIATED CONDITIONS

Many individuals with vitamin B12 or folate deficiency have an associated condition that
predisposes them to one or more vitamin deficiencies. In some cases, these conditions may be
known, and in others they may be appreciated in retrospect after the diagnosis of vitamin B12
or folate deficiency has been made.

● Vitamin B12 deficiency – Conditions that may be associated with vitamin B12 deficiency
are listed in the table ( table 2). Examples include [20-23]:

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• Decreased intake (eg, reduced intake of animal products, strict vegan diet,
breastfeeding by a vitamin B12-deficient mother).

• Decreased absorption (eg, gastrectomy, bariatric surgery, resection of the terminal

ileum, Crohn disease, celiac disease, pancreatic insufficiency, bacterial overgrowth, fish
tapeworm infection, gastric atrophy associated with aging).

• Other autoimmune conditions, such as thyroid disease or vitiligo, in individuals with

pernicious anemia.

• Medications and drugs that interfere with absorption or stability (eg, metformin,
histamine receptor antagonists, proton pump inhibitors, nitrous oxide). (See "Causes
and pathophysiology of vitamin B12 and folate deficiencies", section on 'Metformin'.)

• Rare genetic disorders. (See "Causes and pathophysiology of vitamin B12 and folate
deficiencies".)

Annual monitoring for vitamin B12 deficiency is recommended for patients receiving
metformin; this is discussed in more detail separately. (See "Metformin in the treatment of
adults with type 2 diabetes mellitus", section on 'Monitoring'.)

● Folate deficiency – Conditions that may be associated with folate deficiency are listed in
the table ( table 3). Examples include [2]:

• Increased requirements due to pregnancy, hemolytic anemia.

• Decreased intake, especially in individuals with excessive alcohol use and

corresponding reductions in dietary intake of folate-rich foods such as fresh vegetables
and fortified grains.

• Use of goat's milk (which has low folate concentrations) as the main source of food in
infants and toddlers.

• Residence in a place where routine folate supplementation of foods does not occur.

• Decreased absorption in the setting of gastric bypass surgery.

• Loss during hemodialysis (along with other water-soluble vitamins), although patients
are routinely given a multivitamin containing folic acid. (See "Pathogenesis and
treatment of malnutrition in patients on maintenance hemodialysis".)

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• Increased requirements, such as occur with severe chronic hemolytic anemia or

exfoliative dermatitis.

• Medications and drugs that interfere with metabolism (eg, methotrexate,

sulfasalazine). (See "Major side effects of low-dose methotrexate", section on
'Myelosuppression'.)

• Rare genetic disorders. (See "Causes and pathophysiology of vitamin B12 and folate
deficiencies".)

Bariatric surgery places patients at risk of developing both vitamin B12 and folate deficiencies
(along with other deficiencies), requiring life-long dietary supplementation. Bariatric surgery
can also lead to iron deficiency, which causes microcytic anemia that may obscure the finding of
macrocytosis on the CBC (see 'CBC and blood smear' below). Specific bariatric procedures have
different risks depending on whether the absorptive surfaces were removed. As an example,
Roux-en-Y bypass is more likely to cause vitamin B12 deficiency than other procedures. Routine
vitamin supplementation is recommended, as shown in the table and discussed separately
( table 4). (See "Bariatric surgery: Postoperative nutritional management".)

The mechanisms by which these conditions cause deficiencies are discussed in more detail
separately. (See "Causes and pathophysiology of vitamin B12 and folate deficiencies".)

CLINICAL PRESENTATION

Typical presentation — Patients with vitamin B12 and/or folate deficiency most commonly
present with anemia, which may be associated with nonspecific symptoms (often fatigue, which
can occur in anemia of any etiology). Another common presentation is with an incidental
finding of mild anemia. In settings with frequent complete blood count (CBC) testing, most
patients will not have the classic findings previously associated with these deficiencies, such as
major neurologic changes with vitamin B12 deficiency.

The classic findings associated with vitamin B12 and folate deficiency include worsening
macrocytic anemia, yellowed skin (caused by combined anemia and jaundice), and variable
neurologic abnormalities more prominent in vitamin B12 deficiency (cognitive slowing and
neuropathy).

These were seen more commonly in the early 20th century when deficiencies were often quite
advanced upon presentation. These findings may be present, but often more subtle
presentations are seen in healthy individuals living in resource-rich countries. Routine

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supplementation of grains with folic acid and routine CBC testing have made dietary
deficiencies less common and have led to earlier diagnosis in many individuals.

Macrocytic anemia — The presence of symptoms attributable to anemia depends on the rate
that deficiency has developed, the severity of the deficiency, the hemoglobin level, and the
person's overall health [23]. Many individuals with vitamin B12 or folate deficiency have vague
or nonspecific symptoms (fatigue, irritability, cognitive decline), which are likely to be due at
least in part to anemia. This is because red blood cell (RBC) precursors in the bone marrow
divide rapidly and thus are sensitive to the lack of nucleic acids that occurs when these vitamins
are deficient. (See "Causes and pathophysiology of vitamin B12 and folate deficiencies".)

In the era of frequent CBC testing, many deficiencies come to medical attention as an incidental
finding of anemia and/or macrocytosis. Individuals with vitamin B12 deficiency can also develop
pancytopenia. (See 'CBC and blood smear' below.)

Symptoms attributable to tissue hypoxia and organ ischemia (eg, chest pain, shortness of
breath) may occur if the anemia is severe and/or the individual has underlying ischemic heart
disease. However, the anemia typically develops gradually in vitamin B12 deficiency, and
compensatory mechanisms may mitigate symptoms related to tissue hypoxia, such as
palpitations, light-headedness, and shortness of breath [23]. Individuals with more severe
anemia may have skin pallor. When combined with mild jaundice due to hemolysis, this may
cause the skin to be a "peculiar lemon-yellow color" [23].

The laboratory characteristics of the anemia are discussed below. (See 'CBC and blood smear'
below.)

Gastrointestinal symptoms — Vitamin B12 deficiency can cause glossitis (including pain,
swelling, tenderness, and loss of papillae and/or hyperpigmentation of the tongue), and folate
deficiency can cause oral ulcers [24]. This occurs because cells in the gastrointestinal tract divide
rapidly and are thus sensitive to the lack of nucleic acids that occurs when these vitamins are
deficient.

Other individuals may have gastrointestinal symptoms related to the underlying condition that
caused the deficiency, such as pain or diarrhea related to inflammatory bowel disease, celiac
disease, or other malabsorptive states. The mechanisms by which the deficiencies cause
gastrointestinal symptoms and the underlying conditions cause the deficiencies are presented
separately. (See "Causes and pathophysiology of vitamin B12 and folate deficiencies".)

Neuropsychiatric changes — Neuropsychiatric manifestations may be present in both vitamin

B12 and folate deficiencies. Although these findings are most commonly ascribed to vitamin

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B12 deficiency, neurocognitive and other changes have been reported with folate deficiency as
well [2,25-29]. The mechanism is discussed separately. (See "Causes and pathophysiology of
vitamin B12 and folate deficiencies" and "Evaluation of cognitive impairment and dementia".)

The most common neurologic findings in vitamin B12 deficiency are symmetric paresthesias or
numbness and gait problems [20,30]. The neuropathy is typically symmetric and affects the legs
more than arms. The classic neurologic finding in vitamin B12 deficiency is subacute combined
degeneration of the dorsal (posterior) and lateral columns (white matter) of the spinal cord due
to demyelination. It is associated with progressive weakness, ataxia, and paresthesias that may
progress to spasticity and paraplegia. These findings are helpful diagnostically if present but
may not occur in all cases, especially if diagnosed earlier in the course of the deficiency. The
neurologic features of spinal cord disorders are discussed in more detail separately. (See
"Disorders affecting the spinal cord".)

Other findings may include one or more of the following [2,20,31-34]:

● Depression or mood impairment

● Irritability
● Insomnia
● Cognitive slowing
● Forgetfulness
● Dementia
● Psychosis
● Visual disturbances, which may be associated with optic atrophy
● Peripheral sensory deficits
● Weakness, which may progress to paraplegia and incontinence if severe
● Impaired position sense
● Impaired vibration sense
● Lhermitte sign, a shock-like sensation that radiates to the feet during neck flexion
● Ataxia or positive Romberg test
● Abnormal deep tendon reflexes
● Extrapyramidal signs (eg, dystonia, dysarthria, rigidity)
● Restless legs syndrome

Nonspecific fatigue may be a presenting symptom even in the absence of anemia, but fatigue is
usually associated with some of the findings above, particularly depression/mood impairment
or cognitive slowing. Although evaluation for fatigue might include evaluation of vitamin B12
level, we do not recommend supplementation unless vitamin B12 levels are low. (See
"Treatment of vitamin B12 and folate deficiencies", section on 'Vitamin B12'.)
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In individuals with vitamin B12 deficiency, neuropsychiatric symptoms can be present even in
the absence of anemia or macrocytosis, and the lack of these hematologic changes cannot be
used to exclude vitamin B12 deficiency as a cause of neuropsychiatric symptoms. This was
illustrated in a study that assessed hematologic findings in 141 consecutive patients with
neuropsychiatric symptoms caused by vitamin B12 deficiency [26]. Of these, 40 (28 percent) had
a normal hematocrit, MCV, or both. Vitamin B12 replacement led to a clinical response in all
evaluable patients.

Retrospective studies have suggested a correlation between vitamin B12 levels and declining
cognitive function over time, but a causal relationship has not been demonstrated [35]. (See
"Evaluation of cognitive impairment and dementia", section on 'Laboratory testing' and
"Prevention of dementia", section on 'Vitamins B6, B12, and folate'.)

Neuroimaging is generally not performed, but if done as part of the evaluation for other
conditions, magnetic resonance imaging (MRI) of the spinal cord in an individual with vitamin
B12 deficiency may show classic changes described as "an inverted V-shaped pattern" in the
cervical and thoracic spinal cord [20,33,36]. The localization of some of the specific neurologic
manifestations (eg, Lhermitte sign, related to changes in the cervical spinal cord) is discussed in
more detail separately. (See "Anatomy and localization of spinal cord disorders".)

Subtle neurologic, cognitive, or psychiatric changes are among of the most commonly
encountered symptoms in primary care practice, particularly in older individuals. A full
discussion of the complex issues related to these findings and their possible causes is
presented in detail in separate topic reviews. (See "Evaluation of cognitive impairment and
dementia" and "Unipolar depression in adults: Assessment and diagnosis" and "Approach to the
patient with sensory loss" and "Overview of cerebellar ataxia in adults".)

Infants and maternal vitamin B12 deficiency — Vitamin B12 is transferred to the fetus during
gestation and via breast milk after birth. Thus, an infant born to a mother with vitamin B12
deficiency can be deficient at birth and at risk for further deficiency if exclusively breast-fed.

Vitamin B12 deficiency in an infant may present with pancytopenia and/or macrocytosis; there
may be associated developmental delay or regression, feeding difficulties, hypotonia, irritability,
tremors, or convulsions [20,37].

Newborn screening may be able to identify infants with vitamin B12 deficiency at birth. (See
'Epidemiology' above.)

Other rare features — Although rarely the presenting complaint, the following have also been
associated with vitamin B12 and/or folate deficiency:

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● Vitamin B12 – Other findings in vitamin B12 deficiency:

• Skin – Skin hyperpigmentation and hypopigmentation can occur [20,31,38]. Case

reports have described hyperpigmentation on the hands ( picture 2) and feet
( picture 3) that resolved after vitamin B12 repletion [38]. Reports have also
described maculopapular skin lesions that respond to vitamin B12 supplementation
[39].

• Bones – Increased risk of osteoporosis and fractures of the hip or spine (mechanism
unknown, possibly due to suppression of osteoclast activity) [40-46]. These data are
observational, and some studies have not observed the association [47].

• Cancer – Increased risk of gastric cancer in individuals with pernicious anemia. (See
'Determining the underlying cause of vitamin B12 deficiency' below.)

● Folate – Other findings in folate deficiency:

• Cancer – Possible increased risk of certain malignancies (eg, colon cancer), but the
evidence is not strong, and there is possible increased risk with excess folic acid
supplementation. (See "Colorectal cancer: Epidemiology, risk factors, and protective
factors", section on 'Folic acid and folate'.)

• Neural tube defects – Folate deficiency during embryogenesis is known to increase

the risk of neural tube defects, as discussed in detail separately. (See "Neural tube
defects: Overview of prenatal screening, evaluation, and pregnancy management",
section on 'Folate deficiency' and "Preconception and prenatal folic acid
supplementation".)

Distinguishing vitamin B12 from folate deficiency — There are no presenting features that
can definitively distinguish between vitamin B12 and folate deficiencies. However, the typical
clinical presentations differ as follows:

● Diet – Vitamin B12 deficiency may be seen in strict vegans or vegetarians who do not take
supplemental vitamin B12. Individuals living in resource-rich settings who are able to take
in a normal diet are extremely unlikely to become folate deficient. Folate deficiency may be
seen in individuals with a history of markedly decreased dietary intake or excessive alcohol
use, even without other signs of malnutrition [48].

● Associated clinical findings – The classical clinical picture of subacute combined

degeneration of the cord (eg, progressive weakness, ataxia, and paresthesias that can
advance to spastic paraplegia) is confined to patients with vitamin B12 deficiency. A variety
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of neuropsychiatric changes have been ascribed to both vitamin B12 and folate deficiency
as discussed above; however, because of the widespread prevalence of neuropsychiatric
disorders in the general population and the lower frequency of symptomatic vitamin B12
and folate deficiencies, an informed approach to evaluation of neuropsychiatric changes is
warranted. (See 'Neuropsychiatric changes' above and 'Other rare features' above.)

● Time course – The time course of developing the deficiency may be helpful, if known.

• Vitamin B12 deficiency typically develops over the course of years, as total body stores
are large (approximately 3 to 5 mg, which in many cases is sufficient to provide
adequate levels of vitamin B12 for 5 to 10 years) [49]. However, in infants born to
vitamin B12-deficient mothers, there may not be any stores established, and the infant
may be born deficient. Another exception is exposure to nitrous oxide, which causes
rapid depletion of vitamin B12 [50,51].

• Folate deficiency can develop rapidly (weeks to months, depending on baseline stores)
as body stores are limited (approximately 5 to 10 mg) and become rapidly depleted
during normal cell division.

DIAGNOSTIC EVALUATION

Overview of evaluation — In countries in which dietary deficiency is less of a concern, vitamin

B12 and/or folate deficiency may be suspected in a patient with one or more of the following
( algorithm 1):

● Unexplained anemia, macrocytosis (mean corpuscular volume [MCV] >100 fL)

( picture 4), pancytopenia, or hypersegmented neutrophils ( picture 5).

● Unexplained neurologic or psychiatric symptoms.

● Strict vegan diet or conditions that may interfere with absorption. (See 'Associated
conditions' above.)

● Certain autoimmune disorders such as thyroiditis or vitiligo, or those taking chronic

metformin therapy.

● Gastrointestinal symptoms such as sore tongue, anorexia, or diarrhea [22].

Periodic monitoring of vitamin B12 levels may be appropriate in individuals with intestinal
disorders that might affect absorption of vitamin B12, including celiac disease, inflammatory

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bowel disease, small intestinal bacterial overgrowth, ileal resection, radiation enteritis, and
chronic metformin therapy. Information on which tests to order and the frequency of
monitoring is discussed in topic reviews on these disorders.

Medications associated with lower vitamin B12 levels including metformin and proton pump
inhibitors should be assessed. (See 'Associated conditions' above.)

For an individual with suspected vitamin B12 or folate deficiency, the history should include
questions about previously diagnosed associated conditions, particularly celiac disease or
inflammatory bowel disease; bariatric, gastric, or intestinal surgery; reduced dietary intake (eg,
vegan or vegetarian diet, lack of fresh vegetables); alcohol use (as an independent cause of
macrocytic anemia and as a possible predictor of reduced dietary intake); and any symptoms,
including subtle neurologic or psychiatric symptoms, such as those described above [20,34].
(See 'Clinical presentation' above and "Causes and pathophysiology of vitamin B12 and folate
deficiencies".)

The physical examination should focus on gastrointestinal and dermatologic findings, as well as
hepatosplenomegaly and lymphadenopathy, which may suggest other causes of macrocytic
anemia ( table 5). A thorough neurologic examination should be conducted for signs of
altered affect or mentation and/or findings associated with central or peripheral neuropathy
(eg, impaired sense of vibration, proprioception, or light touch, ataxia, weakness) [20]. These
other causes are discussed below. (See 'Differential diagnosis' below.)

We test for both deficiencies when evaluating individuals with gastrointestinal conditions
known to affect absorption of both vitamins ( algorithm 1). In the absence of these
gastrointestinal abnormalities in individuals who are eating a healthy diet that includes food
fortified with folic acid, and in the absence of other reasons to suspect folate deficiency, we only
test for vitamin B12 deficiency. The specific tests required (and their sequence) depend on the
clinical picture, findings on the complete blood count (CBC), and the results of initial testing for
the vitamin B12 (and folate level if appropriate), as described in the following sections. (See
'Laboratory testing' below.)

Our approach is consistent with a 2014 guideline from the British Committee for Standards in
Haematology [24]. Links to this and other guidelines are presented separately. (See 'Society
guideline links' below.)

Routine population screening for vitamin B12 deficiency has been debated, but there is no
evidence that this improves outcomes or is cost effective, and we do not screen otherwise
healthy individuals without clinical findings or risk factors for deficiency. However, we suggest a
lower threshold for testing populations at increased risk of deficiency due to the clinical factors
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discussed above, as well as older adults in general if they develop signs or symptoms indicative
of deficiency or as part of the evaluation of cognitive decline. (See 'Associated conditions' above
and 'Epidemiology' above.)

Laboratory testing

CBC and blood smear — Deficiencies of vitamin B12 and folate can both cause megaloblastic
anemia. The findings on the CBC and the blood cell morphology on the peripheral blood smear
are identical with both deficiencies and may include one or more of the following:

● Anemia
● Macrocytic red blood cells (RBCs; eg, mean corpuscular volume [MCV] >100 fL) or macro-
ovalocytosis ( picture 4)
● Mild leukopenia and/or thrombocytopenia
● Low reticulocyte count
● Hypersegmented neutrophils ( picture 5) on the peripheral blood smear (ie, >5 percent
of neutrophils with ≥5 lobes or ≥1 percent of neutrophils with ≥6 lobes)

Macrocytosis and hypersegmentation of neutrophils precede the development of anemia and

may be present as isolated findings (without anemia). The mechanisms by which RBCs become
macrocytic and neutrophils become hypersegmented (both features of megaloblastic anemia)
are discussed separately. (See "Macrocytosis/Macrocytic anemia", section on 'Megaloblastic
anemia' and "Causes and pathophysiology of vitamin B12 and folate deficiencies", section on
'Hematopoiesis'.)

On rare occasions, severe acquired vitamin B12 deficiency or heritable gene variants affecting
cobalamin metabolism may be associated with schistocytes and may resemble a thrombotic
microangiopathy [52]. (See "Diagnostic approach to suspected TTP, HUS, or other thrombotic
microangiopathy (TMA)", section on 'Exclude systemic disorders associated with MAHA and
thrombocytopenia' and "Diagnostic approach to suspected TTP, HUS, or other thrombotic
microangiopathy (TMA)", section on 'Overview of primary TMA syndromes'.)

An MCV value >115 fL is more specific to vitamin B12 or folate deficiency than other conditions
in the differential diagnosis such as hypothyroidism or myelodysplastic syndrome. However, a
normal MCV does not exclude vitamin B12 or folate deficiency. In a 1994 series of 100
consecutive patients with a confirmed diagnosis of vitamin B12 deficiency based on the
presence of autoantibodies to intrinsic factor (IF), a positive Schilling test, or both, only 29
percent had anemia and only 36 percent had an MCV >100 fL [53]. In a cohort of healthy older
adults (age 67 or older) who participated in the Framingham heart study, most of the

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individuals who were found to have laboratory values of low vitamin B12 or folate on
population screening had a normal MCV [5].

The MCV does not help distinguish vitamin B12 deficiency from folate deficiency. Further, if a
patient has concomitant microcytosis due to iron deficiency or thalassemia, the macrocytosis
may be masked on a CBC because the MCV reflects the average volume of all RBCs. However, in
this instance (combined macrocytosis and microcytosis), the red cell distribution width (RDW)
may be increased, and a blood smear may reveal both small and large red blood cells [54].
Other forms of anemia may also blunt the macrocytosis caused by vitamin B12 or folate
deficiency.

In addition to affecting the morphology of mature RBCs and neutrophils, megaloblastic anemia
can cause premature destruction of developing RBCs in the bone marrow (ineffective
erythropoiesis leading to intramedullary hemolysis) and the peripheral circulation (hemolysis).
(See "Causes and pathophysiology of vitamin B12 and folate deficiencies".)

Laboratory findings associated with hemolysis may be present, including increased lactate
dehydrogenase (LDH; increases may be dramatic with extremely high LDH levels), increased
indirect bilirubin, and low haptoglobin. Unlike other forms of hemolytic anemia, the reticulocyte
count in vitamin B12 and folate deficiency is also low (or normal), reflecting reduced blood cell
production.

Serum vitamin B12 and folate levels — For individuals with typical findings on the CBC and a
low reticulocyte count, the only initial testing needed is a serum vitamin B12 and, in some cases,
a folate level [20,24]. We often omit the folate level if the individual is consuming a varied diet
containing folate-supplemented grains and has normal gastrointestinal anatomy and function,
as folate deficiency in these individuals is rare ( algorithm 1). We test both vitamin B12 and
folate levels in individuals with gastrointestinal conditions, excess alcohol use, or dietary
patterns known to cause both deficiencies. (See 'Epidemiology' above.)

Most individuals can be determined to have normal or deficient levels using laboratory-specific
cutoff values provided by the testing laboratory with the patient's results. For those with a
clearly normal or deficient level, additional testing to diagnose deficiency is unlikely to be
required [20,24,55]. For those with borderline levels, additional testing can be used to
determine the clinical significance. (See 'Metabolite testing (MMA and homocysteine)' below.)

Additional postdiagnostic testing to determine the underlying cause of the deficiency and/or
other associated medical conditions is discussed below. (See 'Post-diagnostic testing' below.)

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Vitamin B12 normal ranges — The following reflects typical serum vitamin B12 levels and
their interpretation [55,56]:

● Above 300 pg/mL (above 221 pmol/L) – Normal; deficiency unlikely (sensitivity of
approximately 90 percent; however, the assay may not be as sensitive in individuals with
anti-intrinsic factor [IF] antibodies) [20].

● 200 to 300 pg/mL (148 to 221 pmol/L) – Borderline; deficiency is possible and additional
testing is useful. (See 'Additional testing for selected individuals' below.)

● Below 200 pg/mL (below 148 pmol/L) – Low; consistent with deficiency. Additional testing
may be appropriate to determine the accuracy of the diagnosis and possibly its cause (see
'Determining the underlying cause of vitamin B12 deficiency' below). Treatment is
discussed separately. (See "Treatment of vitamin B12 and folate deficiencies", section on
'Vitamin B12'.)

If the level is clearly normal and another cause of macrocytosis, anemia, and/or other clinical
features has been identified (see 'Differential diagnosis' below), we do not perform further
testing. For patients with fatigue or other neuropsychiatric symptoms and either normal
vitamin B12 levels or low/borderline B12 levels with normal results on metabolite testing, there
is no evidence that vitamin B12 deficiency is responsible or that treatment corrects fatigue.
Patients may request injections based on material they access in the lay press; if this happens,
we counsel patients that there is no medical rationale for that approach [57,58]. (See
"Treatment of vitamin B12 and folate deficiencies", section on 'Individuals without documented
vitamin B12 deficiency'.)

If the level is clearly deficient, we confirm and treat the deficiency and determine the underlying
cause, which has implications for the route of replacement and the duration of therapy, as
discussed below. (See 'Basis for confirmed diagnosis' below and 'Determining the underlying
cause of vitamin B12 deficiency' below.)

In most laboratories, serum vitamin B12 levels are measured using a competitive
chemiluminescence assay that has an estimated sensitivity of approximately 95 percent in
symptomatic patients (a specificity of approximately 80 percent or less) [59]. The assay uses
binding to intrinsic factor (IF) following dissociation from binding proteins, with a readout based
on the amount of unbound IF remaining.

There are several important caveats regarding the performance of automated tests, and assays
by different laboratories using different methods often show poor agreement. Additional
testing is warranted if levels are in the borderline range or if the results of laboratory testing are

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discordant with the clinical picture (eg, patient with chronic or worsening macrocytic anemia for
which all other testing is unrevealing). This testing is discussed below. (See 'Additional testing
for selected individuals' below.)

Examples of the caveats related to vitamin B12 testing include [20,34,56,59-61]:

● Normal ranges can vary based on the characteristics of the assay used for testing. In a
retrospective record review of 456 ambulatory individuals, vitamin B12 levels fluctuated by
a median of 23 percent over a period of weeks [62]. One-fifth of the individuals had
variations greater than 100 pg/mL.

● Tissue stores of vitamin B12 are apparently only moderately correlated with serum vitamin
B12 levels, possibly due to variations in serum vitamin B12 binding proteins.

Causes of spuriously low vitamin B12 — Vitamin B12 circulates in plasma bound to
haptocorrin (TCI; also called R-factor) and transcobalamin II. Approximately 80 percent of
vitamin B12 is bound to haptocorrin and is functionally inactive. Vitamin B12 bound to
transcobalamin II (holotranscobalamin) is the active form absorbed by cells.

Decreased production of vitamin B12 binding proteins has been proposed to be responsible for
the spuriously low values, as levels of the active holotranscobalamin were unchanged in a series
of individuals with decreased vitamin B12 levels [63]. The effect of vitamin B12 binding proteins
on levels of functional vitamin B12 is illustrated in the figure ( figure 4).

Certain conditions or medications may be associated with spuriously low serum vitamin B12
levels and thus might cause the appearance of vitamin B12 deficiency when the patient is not
deficient. Examples include:

● Multiple myeloma
● HIV infection
● Pregnancy
● Oral contraceptives
● Diphenylhydantoin

An example of spuriously low vitamin B12 level in pregnancy was demonstrated in a series of 50
pregnant individuals with low vitamin B12 levels (45 to 199 pg/mL), in whom metabolite testing
for methylmalonic acid (MMA) and homocysteine showed no correlation with vitamin B12 level.
(See 'Metabolite testing (MMA and homocysteine)' below.)

Causes of spuriously high vitamin B12 — Certain conditions may be associated with
spuriously increased vitamin B12 levels and thus might cause the appearance of normal vitamin
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B12 levels when the patient is deficient. Examples include [23]:

• Occult malignancy
• Myeloproliferative neoplasm
• Alcoholic liver disease
• Kidney disease
• Certain inborn errors of metabolism
• Nitrous oxide exposure (which can also cause vitamin B12 deficiency, as evidenced by
clinical symptoms and high methylmalonic acid [MMA] levels) [64]

In addition, autoantibodies to IF in individuals with pernicious anemia may compete with IF in

the chemiluminescence assay and result in spuriously normal vitamin B12 levels [65-67]. If the
vitamin B12 level is very high (eg, 800 pg/mL), we do not worry about this effect in the absence
of clinical features suggesting vitamin B12 deficiency; however, if the vitamin B12 level is
borderline or low normal and/or other clinical features suggest vitamin B12 deficiency (see
'Clinical presentation' above), it is prudent to obtain other testing such as MMA and
homocysteine levels. (See 'Additional testing for selected individuals' below.)

Folate normal ranges — When indicated, routine testing for folate deficiency is usually
done using the serum folate level.

The following reflects typical serum folate levels and their interpretation [55,56]:

● Above 4 ng/mL (above 9.1 nmol/L) – Normal. Suggests folate is not deficient, unless the
individual has recently consumed a folate-containing meal or supplement. In such cases,
RBC folate can be obtained (see 'RBC folate' below), although we generally prefer
metabolite testing (see 'Metabolite testing (MMA and homocysteine)' below). The RBC
folate level is not needed in routine testing because it does not provide additional
information and is more costly to obtain.

● From 2 to 4 ng/mL (from 4.5 to 9.1 nmol/L) – Borderline. Additional testing may be
indicated depending on the clinical circumstances and the degree of suspicion for folate
deficiency. (See 'Additional testing for selected individuals' below.)

● Below 2 ng/mL (below 4.5 nmol/L) – Low. Consistent with folate deficiency. (See 'Basis for
confirmed diagnosis' below.)

Values may be slightly higher in the first six months of life. For hospitalized patients, blood
samples should be obtained immediately on admission, before any meals have been consumed

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and before any blood transfusions have been given, as even a single meal or transfusion may
normalize serum folate levels for several weeks even if deficiency is present.

Additional testing for selected individuals

Metabolite testing (MMA and homocysteine) — Additional testing for intermediates in

vitamin B12 and folate metabolism (methylmalonic acid [MMA] and homocysteine) can be
reserved for cases in which initial test results for vitamin B12 and/or folate levels are borderline
(near the lower limit of normal) or inconclusive, or if clinical findings are discordant with initial
testing values (eg, low-normal vitamin B12 level in an individual with unexplained macrocytic
anemia or unexplained neurologic findings) ( algorithm 1).

Because of concerns regarding the accuracy of vitamin B12 testing, in most cases of suspected
deficiency, we often confirm the diagnosis by testing for the intermediates methylmalonic acid
[MMA] and homocysteine [68,69]. Ideally, at least two biochemical abnormalities should be
sought to make the diagnosis in individuals who are asymptomatic since an isolated
abnormality, such as elevated MMA, may be spurious [49].

The normal ranges for MMA and homocysteine are laboratory-dependent; laboratory-specific
and assay-specific cutoffs should be used. Examples of typical normal ranges are: MMA 70 to
270 nmol/L; homocysteine 5 to 15 micromol/L. For MMA, the American Board of Internal
Medicine uses 0 to 400 nmol/L (0 to 0.40 micromol/L). (See "Laboratory test reference ranges in
adults", section on 'Methylmalonic acid (MMA), serum'.)

Interpretation is as follows:

● MMA and homocysteine normal – No deficiency of vitamin B12 or folate.

● MMA and homocysteine elevated – Deficiency of vitamin B12 (does not eliminate the
possibility of folate deficiency).

● MMA normal, homocysteine elevated – No deficiency of vitamin B12. Consistent with

deficiency of folate.

MMA is elevated in vitamin B12 deficiency but not in folate deficiency. This is because vitamin
B12 is a cofactor in conversion of methylmalonyl-CoA to succinyl-CoA, a reaction that occurs in
mitochondria and is catalyzed by methylmalonyl-CoA mutase [59]. In the absence of vitamin
B12, this reaction cannot proceed normally, and MMA accumulates ( figure 3).

Urinary MMA can also be measured, although this is not done in routine practice [7].

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MMA can be elevated in the absence of vitamin B12 deficiency in individuals with chronic kidney
disease. Other alternatives for testing in this setting include measuring autoantibodies to
intrinsic factor and examining the clinical response to administration of parenteral vitamin B12.
For individuals with a low serum vitamin B12 level and a high MMA, we test for autoantibodies
to intrinsic factor to establish whether pernicious anemia (PA) is the underlying cause. (See
'Autoantibodies to intrinsic factor' below and 'Response to vitamin replacement' below.)

The sensitivity of MMA and homocysteine for vitamin B12 deficiency was addressed in a 1994
study that measured these metabolites in a series of 406 individuals diagnosed with vitamin
B12 deficiency based on a low vitamin B12 level (<200 pg/mL) plus a clinical finding (diagnostic
bone marrow, blood smear, and response to vitamin B12 administration) [70]. Most of the
patients had PA. Of these 406 individuals, 94.5 percent had elevations of both metabolites, and
all but one had an increase in at least one metabolite, with a sensitivity of 99.8 percent for the
diagnosis of vitamin B12 deficiency.

These findings were very useful for characterizing the metabolite patterns in individuals with
obvious clinical deficiency, and they suggest that metabolite testing has a high sensitivity in
people with PA who have overtly low serum vitamin B12 levels and associated clinical features.
The sensitivity and specificity of this testing in individuals with borderline vitamin B12 levels
and/or the absence of clinical features, where metabolite testing would be most useful, is not as
clear.

Homocysteine is elevated in both vitamin B12 and folate deficiencies. This is because both
vitamins are required for the metabolism of homocysteine to methionine ( figure 3). In the
absence of either vitamin, this process cannot occur normally, and homocysteine accumulates.
Thus, a normal MMA and an increased homocysteine level is consistent with folate deficiency. In
the 1994 study that measured metabolites in individuals with clinically obvious deficiencies,
homocysteine was increased in 91 percent of those with folate deficiency (based on a low folate
level [<4 ng/mL], normal vitamin B12 level [>300 pg/mL], and presence of an underlying
disorder associated with folate deficiency) [70].

There may be substantial fluctuations in the measured MMA and homocysteine levels, and
unexpectedly normal or abnormal levels should be repeated [62,71]. Conditions other than
vitamin B12 and folate deficiency that have the potential to alter the MMA include the following:

● Chronic kidney disease (increased MMA levels).

● Methylmalonic acidemia/aciduria (increased MMA levels). (See "Methylmalonic acidemia".)
● Hereditary homocysteinemia (increased homocysteine levels). (See "Overview of
homocysteine".)

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● Antibiotic treatment (lower MMA levels in two case reports, possibly due to alterations in
intestinal flora that led to a lack of the MMA precursor propionic acid) [69].

In such cases, it may be necessary to test for autoantibodies associated with PA or to determine
the hematologic response to vitamin administration. (See 'Autoantibodies to intrinsic factor'
below and 'Response to vitamin replacement' below.)

Autoantibodies to intrinsic factor — Testing for autoantibodies to intrinsic factor (IF) is

used to identify pernicious anemia (PA), an autoimmune condition that leads to severely
impaired vitamin B12 absorption. We test for anti-IF antibodies in individuals who have
biochemical evidence of vitamin B12 deficiency (low serum vitamin B12 level and/or high MMA
level) without another obvious cause. The testing is typically done with an immunoassay. The
sensitivity of IF autoantibodies is relatively low. As an example, in a 1992 study of 324 patients
with documented PA, autoantibodies to IF were present in 228 (70 percent) [72]. The specificity
of autoantibodies to IF is high; if present, anti-IF autoantibodies are considered confirmatory for
a diagnosis of PA [23].

Many patients with PA have autoantibodies to parietal cells; however, antiparietal cell antibodies
are also seen in some patients with gastritis and are not diagnostic of PA [23].

If PA is strongly suspected and testing for autoantibodies to IF is negative, we may make the
diagnosis based on other findings such as clinical manifestations and laboratory findings
consistent with vitamin B12 deficiency (high MCV, hypersegmented neutrophils), especially if
these findings resolve with vitamin B12 administration. Such findings would be sufficient to
continue lifelong treatment. (See "Treatment of vitamin B12 and folate deficiencies", section on
'Treatment of vitamin B12 deficiency'.)

In selected cases, it may be possible to measure serum gastrin levels or pepsinogen I and II
levels, although elevated gastrin and low ratio of pepsinogen I to II are also not highly specific
for PA [73,74].

RBC folate — RBC folate is a surrogate for tissue folate levels. RBC folate provides
information about folate status over the lifetime of RBCs, similar to hemoglobin A1C for blood
glucose levels.

We no longer use this test; rather, if the serum folate is borderline and there is clinical
suspicion, we use metabolite testing and/or response to folate repletion (See 'Metabolite testing
(MMA and homocysteine)' above and 'Response to vitamin replacement' below.)

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Some clinicians consider it to be helpful to test for RBC folate if results of serum folate testing
are normal or borderline low in an individual with a strong clinical suspicion for folate deficiency
(see 'Folate normal ranges' above). This especially applies if they have just eaten a folate-
containing food, such as a hospital snack or meal. However, studies comparing serum folate
with RBC folate have found that addition of RBC folate to serum folate testing does not provide
substantially more clinical information [75,76].

Variation in the use of the RBC folate assay was illustrated in a 2003 benchmarking survey of
hematology laboratories in the United Kingdom, which found that 42 percent of laboratories
used serum folate, 45 percent used RBC folate, and 13 percent used both [75]. This review
suggested that approximately 5 percent of people with normal serum folate levels may have
evidence of folate deficiency using RBC folate testing.

An RBC folate level below 150 ng/mL (<150 mcg/L; <340 nmol/L) is consistent with folate
deficiency as long as there is not concomitant vitamin B12 deficiency (RBC folate is lower in
individuals with vitamin B12 deficiency) [24].

Response to vitamin replacement — Another option for individuals who have borderline
laboratory values, conditions that interfere with measurement (eg, kidney disease), or
discordance between laboratory testing and clinical picture is to provide replacement therapy
for the vitamin suspected to be deficient (eg, parenteral vitamin B12 1000 mcg weekly for three
weeks, or oral or parenteral folic acid 1 mg daily for one to two weeks).

The hematologic response can be assessed by measuring the reticulocyte count and CBC before
and approximately two weeks after supplementation. Both the reticulocyte count and
hemoglobin level are expected to increase as hematopoiesis improves. There will no longer be
hypersegmented neutrophils on the blood smear, and the MCV will eventually return to normal,
although it may remain high initially (during reticulocytosis) since reticulocytes are larger than
mature RBCs.

Measuring hematologic parameters is likely to be more accurate than general well-being or

neurologic symptoms since many individuals report the phenomena of more energy following a
vitamin B12 shot even if they were not deficient.

Vitamin B12 administration is extremely safe and inexpensive. However, there are potential
problems with using the response to vitamin B12 administration as a diagnostic test. (See 'Basis
for confirmed diagnosis' below.)

Alternatively, there may be comorbidities such as anemia of chronic disease/anemia of

inflammation (ACD/AI), that prevent a full reticulocyte response. However, the resolution of

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hypersegmented neutrophils on the peripheral blood smear is correlated to correction of the

deficiency.

Folic acid administration is also nontoxic and inexpensive, but administration of folic acid alone
in an individual with concurrent vitamin B12 deficiency may be associated with progression or
worsening of neurologic abnormalities. Thus, it is prudent to check the vitamin B12 level, as
discussed below. (See 'Other contributing factors to anemia' below.)

Schilling test (historical interest) — The Schilling test is primarily of historical interest;
this test is no longer routinely available.

Prior to the availability of other testing for pernicious anemia, the Schilling test was used to
determine if an individual with vitamin B12 deficiency was able to absorb vitamin B12 via the
oral route. The test involved an oral dose of radiolabeled vitamin B12, with or without another
substance to promote absorption, such as intrinsic factor, and a large parenteral dose of
unlabeled vitamin B12 to saturate the vitamin B12 binding proteins and lead to massive
excretion of the excess vitamin B12. If radiolabeled vitamin B12 was absorbed, it would be
excreted in urine.

The Schilling test may be falsely normal in patients with achlorhydria who are unable to extract
vitamin B12 from food but may be able to absorb it from a pill [77].

Lack of access to the radiolabeled vitamin B12 used in the test, declining expertise, and
increased availability of other testing with equal or better accuracy has made the Schilling test
obsolete. Alternative tests for vitamin B12 absorption are under investigation [23].

Bone marrow (generally not indicated) — A bone marrow aspirate and biopsy is not
used to evaluate for vitamin B12 or folate deficiencies, and the findings on bone marrow
morphology typically do not help to distinguish these deficiencies from other hematologic
disorders or from each other. However, if a bone marrow evaluation is performed in an
individual with vitamin B12 or folate deficiency (eg, for another suspected condition, such as
myelodysplastic syndrome [MDS]), it is likely to reveal a markedly hypercellular marrow with
megaloblastic erythroid hyperplasia, giant metamyelocytes, and frequent mitoses ( picture 6
and picture 1). (See 'Differential diagnosis' below.)

Bone marrow abnormalities due to vitamin B12 or folate deficiency resolve rapidly after
treatment is initiated.

Basis for confirmed diagnosis — We consider the diagnosis of vitamin B12 or folate deficiency
to be confirmed in the following scenarios:

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● Serum vitamin level(s) in the "low" range, especially if there are clinical findings consistent
with deficiency.

● Elevated serum MMA and/or homocysteine levels consistent with deficiency and serum
vitamin levels in the intermediate range.

● Hematologic response to administration of the deficient vitamin, as long as the response

cannot be explained by another intervention. Based on concerns outlined above, this is
generally used as supportive evidence and typically is not the sole basis for confirming the
diagnosis. (See 'Response to vitamin replacement' above.)

We do not interpret lack of a response to administration of vitamin B12 or folate to indicate lack
of deficiency, because there may be other causes of the clinical findings and/or reasons for lack
of hematologic response. In such cases, we provide parenteral replacement if the oral route was
used initially, and we continue to evaluate for other causes of anemia, such as ACD/AI, as well
as for other conditions, such as malabsorption. (See "Treatment of vitamin B12 and folate
deficiencies" and "Diagnostic approach to anemia in adults".)

Specialist referral — Specialist referral is not generally required for those with suspected
vitamin B12 or folate deficiency as the diagnosis is straightforward in the vast majority of cases.
However, there may be instances in which specialist referral is indicated, such as the following:

● Concern about other conditions in the differential diagnosis, such as myelodysplastic

syndrome, which may require bone marrow evaluation. (See "Diagnostic approach to
anemia in adults" and "Approach to the adult with pancytopenia".)

● Concern about gastrointestinal conditions that may require additional evaluations and/or
treatments. (See 'Determining the underlying cause of vitamin B12 deficiency' below.)

● Lack of the expected response to therapy. (See "Treatment of vitamin B12 and folate
deficiencies".)

DIFFERENTIAL DIAGNOSIS

The differential diagnosis of vitamin B12 and folate deficiencies includes other causes of
macrocytic anemia (or macrocytic RBCs without anemia), other causes of pancytopenia or
multilobed neutrophils, and other causes of neurologic findings. Many of these findings are
quite common in primary care practice. Their full evaluation is discussed in more detail in the
linked topic reviews listed for each finding.

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● Other causes of macrocytosis and/or anemia with low reticulocyte count – There are
numerous other causes of macrocytosis and/or anemia with an impaired reticulocyte
response ( table 5). Like vitamin B12 and folate deficiency, these other conditions have
varied presentations; they often present as an incidental finding or during the evaluation
for relatively nonspecific symptoms, such as fatigue. Unlike vitamin B12 and folate
deficiency, these conditions have abnormal laboratory findings other than those described
above. (See "Macrocytosis/Macrocytic anemia", section on 'Causes of
macrocytosis/macrocytic anemia' and "Anemia of chronic disease/anemia of
inflammation".)

● Other causes of pancytopenia – Other causes of pancytopenia include a number of bone

marrow disorders and autoimmune conditions, some of which can be life-threatening. Like
vitamin B12 and folate deficiency, patients with these conditions may present with
nonspecific symptoms of cytopenias. Unlike vitamin B12 and folate deficiency, the
cytopenias in many of these other conditions can be severe, and there may be a variety of
other associated findings. Also unlike vitamin B12 and folate deficiency, these conditions
have abnormal laboratory findings other than those described above and diagnosis often
requires bone marrow evaluation. (See "Approach to the adult with pancytopenia".)

● Other causes of hypersegmented neutrophils – As noted above, hypersegmented

neutrophils are one of the typical findings of megaloblastic anemias, such as vitamin B12
and folate deficiency. Other conditions that can produce hypersegmented neutrophils
include other causes of megaloblastic anemia and heat stroke. Like vitamin B12 and folate
deficiency, these conditions may be associated with gastrointestinal symptoms and
anemia with a low reticulocyte count. Unlike vitamin B12 and folate deficiency, these
conditions will have other findings on the examination and laboratory testing.
Hypersegmented neutrophils may be seen in individuals treated with hydroxyurea (eg, for
sickle cell disease, essential thrombocythemia, or polycythemia vera). (See
"Macrocytosis/Macrocytic anemia", section on 'Megaloblastic anemia' and "Evaluation of
the peripheral blood smear", section on 'Lobulation'.)

● Other causes of neuropsychiatric findings – Other causes of neurologic findings are

numerous. Like vitamin B12 and folate deficiency, some of these conditions are also
associated with hematologic abnormalities (eg, copper deficiency, systemic lupus
erythematosus, hypothyroidism, hepatic or uremic encephalopathy, infection,
medications). Also like vitamin B12 and folate deficiency, findings may be nonspecific,
especially in older adults. Unlike vitamin B12 and folate deficiency, these conditions have
other abnormal findings on laboratory testing and/or neuroimaging. (See "Evaluation of

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cognitive impairment and dementia" and "Unipolar depression in adults: Assessment and
diagnosis" and "Approach to the patient with sensory loss" and "Overview of cerebellar
ataxia in adults" and "Copper deficiency myeloneuropathy".)

● Other causes of fatigue – Fatigue is a nonspecific symptom with numerous causes. Like
vitamin B12 and folate deficiency, individuals may have subtle neurologic or
neuropsychiatric symptoms. Unlike vitamin B12 and folate deficiency, with these other
causes of fatigue, the vitamin B12 and folate levels are normal. Causes and assessment
are presented separately. (See "Approach to the adult patient with fatigue".)

POST-DIAGNOSTIC TESTING

Once the diagnosis of vitamin B12 and/or folate deficiency is made, additional historical
information and/or testing is used to determine the underlying cause. This is important to
prevent future deficiency, to choose the appropriate route and duration of therapy, and to
identify other conditions that may require treatment (eg, celiac disease, inflammatory bowel
disease [IBD]). Common causes are listed in the tables on causes of vitamin B12 ( table 2) and
folate ( table 3) deficiencies and discussed in more detail separately. (See "Causes and
pathophysiology of vitamin B12 and folate deficiencies".)

Other contributing factors to anemia — The diagnosis of vitamin B12 deficiency does not
eliminate the possibility of folate deficiency. This is an important consideration in those with
folate deficiency, who may have an apparent response to folic acid supplementation with
normalization of the hematologic findings and even apparent normalization of the serum
vitamin B12 level. Nor does diagnosis of vitamin B12 or folate deficiency eliminate the
possibility of other causes of anemia. As an example, many of the conditions that interfere with
absorption of vitamin B12 and/or folate also impair the absorption of iron, leading to iron
deficiency, and cause inflammation, leading to a component of anemia of chronic
disease/anemia of inflammation. (See "Causes and diagnosis of iron deficiency and iron
deficiency anemia in adults" and "Anemia of chronic disease/anemia of inflammation".)

Likewise, the diagnosis of folate deficiency does not eliminate the possibility of vitamin B12
deficiency. Individuals with folate deficiency and concomitant vitamin B12 deficiency may have
an apparent response to folic acid supplementation with normalization of the hematologic
findings and even apparent normalization of the serum vitamin B12 level. However, folic acid
cannot correct the neurologic deficits caused by vitamin B12 deficiency; these deficits may
become progressive and irreversible if not treated with supplemental vitamin B12. Thus, it is

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prudent to check the vitamin B12 level in an individual diagnosed with folate deficiency. (See
'Neuropsychiatric changes' above.)

It is also important to assess that treatment has been effective in correcting the deficiency, as
discussed separately. (See 'Response to vitamin replacement' above and "Treatment of vitamin
B12 and folate deficiencies".)

Determining the underlying cause of vitamin B12 deficiency — The major consideration in
an individual with vitamin B12 deficiency involves determining the underlying cause, which in
turn determines the route and duration of therapy [21].

As examples, individuals with pernicious anemia (PA; vitamin B12 deficiency due to
autoantibodies against intrinsic factor or gastric parietal cells) are often treated with indefinite
parenteral vitamin B12 supplementation. However, high-dose oral vitamin B12 (1000 to 2000
mcg daily) is an option if there are no active neurologic complications and the individual is
expected to be adherent.

Strict vegans should take an oral supplement, and individuals with celiac disease or IBD may
require only temporary oral supplementation until their gastrointestinal disorder is
treated/controlled. Treatment will not interfere with the results of this testing and should not be
withheld while deciding which testing to pursue.

In a 1994 study, PA was found to account for approximately three-fourths of vitamin B12
deficiency, with treatable intestinal diseases accounting for another 14 percent [70]. It is not
clear whether this distribution has shifted along with increasing use of bariatric surgery, use of
proton pump inhibitors and metformin, and evolving dietary practices.

Testing for the following may be appropriate, depending on the patient's clinical status,
symptoms, and available interventions should an abnormality be found:

● Celiac disease – (See "Diagnosis of celiac disease in children" and "Diagnosis of celiac
disease in adults".)

● IBD – (See "Clinical presentation and diagnosis of inflammatory bowel disease in children"
and "Endoscopic diagnosis of inflammatory bowel disease in adults".)

● Pancreatic insufficiency – (See "Chronic pancreatitis: Clinical manifestations and diagnosis

in adults" and "Exocrine pancreatic insufficiency".)

● Other causes of malabsorption – (See "Chronic complications of short bowel syndrome in

children", section on 'Nutritional complications' and "Approach to the adult patient with

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suspected malabsorption".)

● Hypergastrinemia or gastritis – (See "Physiology of gastrin", section on 'Hypergastrinemia'

and "Metaplastic (chronic) atrophic gastritis".)

● Autoimmune thyroiditis – (See "Pathogenesis of Hashimoto's thyroiditis (chronic

autoimmune thyroiditis)".)

However, patients should be aware that there are not good data to support this testing.

H. pylori infection has been proposed to be associated with vitamin B12 deficiency, but there is
limited evidence of a causal relationship.

In a prospective cohort of 138 patients with anemia and vitamin B12 deficiency who underwent
upper gastrointestinal endoscopy, H. pylori was detected in 77 (56 percent) [78]. Of these 77, 31
(40 percent) had improvement in vitamin B12 levels after H. pylori was eradicated. However, as
an editorialist noted, the patients were not tested for vitamin B12 absorption or for
autoantibodies to IF or gastric parietal cells, and administration of antibiotics might have
improved vitamin B12 absorption by treating another cause of deficiency, such as small
intestinal bacterial overgrowth [79].

We do not test for H. pylori infection unless the patient has other indications for testing. These
are outlined in a separate topic review. (See "Indications and diagnostic tests for Helicobacter
pylori infection in adults".)

Additional testing in pernicious anemia — Additional considerations such as screening for

gastrointestinal malignancy and the risk of other autoimmune disorders are discussed
separately. (See "Treatment of vitamin B12 and folate deficiencies", section on 'Additional
considerations for pernicious anemia'.)

SOCIETY GUIDELINE LINKS

Links to society and government-sponsored guidelines from selected countries and regions
around the world are provided separately. (See "Society guideline links: Anemia in adults".)

INFORMATION FOR PATIENTS

UpToDate offers two types of patient education materials, "The Basics" and "Beyond the Basics."
The Basics patient education pieces are written in plain language, at the 5th to 6th grade reading

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level, and they answer the four or five key questions a patient might have about a given
condition. These articles are best for patients who want a general overview and who prefer
short, easy-to-read materials. Beyond the Basics patient education pieces are longer, more
sophisticated, and more detailed. These articles are written at the 10th to 12th grade reading
level and are best for patients who want in-depth information and are comfortable with some
medical jargon.

Here are the patient education articles that are relevant to this topic. We encourage you to print
or e-mail these topics to your patients. (You can also locate patient education articles on a
variety of subjects by searching on "patient info" and the keyword(s) of interest.)

● Basics topics (see "Patient education: Vitamin B12 deficiency and folate deficiency (The
Basics)" and "Patient education: Pernicious anemia (The Basics)")

SUMMARY AND RECOMMENDATIONS

● Prevalence – Vitamin B12 deficiency affects 1 to 2 percent of the general population and
10 to 15 percent of older adults and hospitalized patients. Most cases of vitamin B12
deficiency in resource-rich settings are due to malabsorption rather than dietary
deficiency. Folate deficiency is rare in individuals consuming a varied diet with folic acid-
supplemented food, as long as they have normal gastrointestinal anatomy and function.
(See 'Epidemiology' above.)

● When to suspect – The tables summarize medical conditions and dietary practices
associated with vitamin B12 deficiency ( table 2) and folate deficiency ( table 3).
History of using metformin or a proton pump inhibitor, symptoms related to anemia or
pancytopenia, gastrointestinal features, neuropsychiatric changes (including irritability or
cognitive decline), and neuropathy are also suggestive. In the era of frequent complete
blood counts (CBCs), many individuals come to medical attention due to the incidental
finding of anemia or macrocytosis. (See 'Clinical presentation' above and 'CBC and blood
smear' above.)

● Diagnostic evaluation – The evaluation includes a history for associated conditions and
symptoms; an examination for gastrointestinal, dermatologic, neurologic, and other
findings; and laboratory testing, including CBC and vitamin B12 and folate levels. We often
omit the folate level in those with a folate-enriched diet without gastrointestinal disorders.

The CBC may show macrocytosis, anemia, pancytopenia (typically mild), and/or
hypersegmented neutrophils. Laboratory-specific and assay-specific normal values for
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vitamin B12 and folate should be used if possible ( algorithm 1). Bone marrow
evaluation is not required. (See 'Overview of evaluation' above.)

• Additional testing (vitamin B12) – Methylmalonic acid (MMA), homocysteine, and/or

autoantibodies to intrinsic factor (IF) or gastric parietal cells may be appropriate if the
vitamin B12 level is borderline low or discordant with the clinical picture. (See
'Metabolite testing (MMA and homocysteine)' above and 'Autoantibodies to intrinsic
factor' above.)

• Additional testing (folate) – MMA, homocysteine, or red blood cell (RBC) folate may be
appropriate if the serum folate is borderline or discordant with the clinical picture,
especially if the patient has just consumed a folate-fortified meal. (See 'Metabolite
testing (MMA and homocysteine)' above and 'RBC folate' above.)

• Response to treatment – Response to administration of the vitamin (especially

hematologic response) is supportive. In some cases this may be the sole basis for the
diagnosis, although we prefer laboratory testing. (See 'Response to vitamin
replacement' above.)

• General approach to evaluating macrocytic anemia – (See "Macrocytosis/Macrocytic

anemia".)

● Diagnostic confirmation – A low serum vitamin B12 or folate level is confirmatory, as are
increased MMA and homocysteine for vitamin B12 deficiency and homocysteine for folate
deficiency ( figure 3), with caveats discussed above. Response to vitamin repletion can
also be used for diagnostic confirmation. Specialist referral is not required unless there is
concern for another hematologic or gastrointestinal condition that should be addressed.
(See 'Basis for confirmed diagnosis' above and 'Specialist referral' above.)

● Differential diagnosis – The differential diagnosis includes other causes of macrocytosis,

anemia, pancytopenia, hypersegmented neutrophils, neuropsychiatric findings, and
fatigue. Many of these findings are common in primary care practice. (See 'Differential
diagnosis' above.)

● Determining the cause – Other testing may be appropriate to determine the cause, and
route and duration of therapy. Individuals with pernicious anemia may require increased
monitoring for gastrointestinal malignancy, those with celiac disease may require
additional attention to bone health and other nutritional deficiencies, and most individuals
with folate deficiency require testing of vitamin B12 levels before folic acid is administered.

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(See 'Post-diagnostic testing' above and "Causes and pathophysiology of vitamin B12 and
folate deficiencies".)

● Management – (See "Treatment of vitamin B12 and folate deficiencies".)

ACKNOWLEDGMENTS

UpToDate gratefully acknowledges Stanley L Schrier, MD (deceased), who contributed as Section

Editor on earlier versions of this topic and was a founding Editor-in-Chief for UpToDate in
Hematology.

The UpToDate editorial staff also acknowledges the extensive contributions of William C
Mentzer, MD, to earlier versions of this and many other topic reviews.

Use of UpToDate is subject to the Terms of Use.

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74. Alonso N, Granada ML, Salinas I, et al. Serum pepsinogen I: an early marker of pernicious
anemia in patients with type 1 diabetes. J Clin Endocrinol Metab 2005; 90:5254.

75. Galloway M, Rushworth L. Red cell or serum folate? Results from the National Pathology
Alliance benchmarking review. J Clin Pathol 2003; 56:924.
76. MacMillan TE, Gudgeon P, Yip PM, Cavalcanti RB. Reduction in Unnecessary Red Blood Cell
Folate Testing by Restricting Computerized Physician Order Entry in the Electronic Health
Record. Am J Med 2018; 131:939.
77. Mazokopakis EE. The old Schilling test as a necessary criterion at present for the diagnosis
of food-cobalmin malabsorption (FCM) syndrome. Hell J Nucl Med 2012; 15:262.
78. Kaptan K, Beyan C, Ural AU, et al. Helicobacter pylori--is it a novel causative agent in
Vitamin B12 deficiency? Arch Intern Med 2000; 160:1349.
79. Stopeck A. Links between Helicobacter pylori infection, cobalamin deficiency, and
pernicious anemia. Arch Intern Med 2000; 160:1229.
Topic 7155 Version 78.0

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12/26/23, 12:13 AM Clinical manifestations and diagnosis of vitamin B12 and folate deficiency - UpToDate

GRAPHICS

Clinical symptoms of selected vitamin deficiencies

Vitamin Deficiency syndrome

Water-soluble vitamins

Vitamin B1 Beriberi – Congestive heart failure (wet beriberi), aphonia, peripheral neuropathy,
(thiamine) Wernicke encephalopathy (nystagmus, ophthalmoplegia, ataxia), confusion, or coma

Vitamin B2 Nonspecific symptoms including edema of mucous membranes, angular stomatitis,

(riboflavin) glossitis, and seborrheic dermatitis (eg, nose, scrotum)

Niacin Pellagra – Dermatitis on areas exposed to sunlight; diarrhea with vomiting,

(nicotinic acid) dysphagia, mouth inflammation (glossitis, angular stomatitis, cheilitis); headache,
dementia, peripheral neuropathy, loss of memory, psychosis, delirium, catatonia

Vitamin B6 Anemia, weakness, insomnia, difficulty walking, peripheral neuropathy, nasolabial

(pyridoxine, seborrheic dermatitis, cheilosis, stomatitis
pyridoxal)

Vitamin B12 Megaloblastic anemia (pernicious anemia), peripheral neuropathy with impaired
(cobalamin) proprioception; may include axonal or optic neuropathy and slowed mentation

Folate Megaloblastic anemia; may include sensory predominant neuropathy

Biotin Nonspecific symptoms including altered mental status, myalgia, dysesthesias,

anorexia, maculosquamous dermatitis

Pantothenate Nonspecific symptoms including paresthesias, dysesthesias ("burning feet"), anemia

gastrointestinal symptoms

Vitamin C Scurvy – Fatigue, petechiae, ecchymoses, bleeding gums, depression, dry skin,
(ascorbate) impaired wound healing

Fat-soluble vitamins

Vitamin A Night blindness, xerophthalmia, keratomalacia, Bitot spot, follicular hyperkeratosis

(retinol, retinal,
retinoic acid)

Vitamin D Rickets, osteomalacia, craniotabes, rachitic rosary

(cholecalciferol,
ergocalciferol)

Vitamin E Sensory and motor neuropathy, ataxia, retinal degeneration, hemolytic anemia
(tocopherols)

Vitamin K Hemorrhagic disease

(phylloquinone,

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menaquinone,
menadione)

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12/26/23, 12:13 AM Clinical manifestations and diagnosis of vitamin B12 and folate deficiency - UpToDate

Roles of vitamin B12 and folate in the methionine cycle, folate cycle, and DNA
synthesis showing the methyl folate "trap"

The roles of vitamin B12 and folate in the methionine cycle, folate cycle, and DNA synthesis are shown.
The key intersection of vitamin B12 and folate occurs at the methionine synthase (MS) reaction (shown at
the left edge between the yellow and blue panels) in which the one-carbon methyl group of methyl-
tetrahydrofolate (methylTHF) is transferred to homocysteine to form methionine. Vitamin B12 (as
methylcobalamin) acts as a cofactor for this reaction. Tetrahydrofolate regains a one-carbon methylene
group through the serine-hydroxymethyltransferase (SHMT) reaction, and the resulting
methylenetetrahydrofolate (methyleneTHF) is essential for conversion of deoxyuridine to thymidine in
the thymidylate synthase (TS) reaction, which is rate-limiting for DNA synthesis. In vitamin B12 deficiency,
folate becomes trapped as methylTHF (the methyl folate "trap"). Administration of folic acid can
temporarily overcome this block through formation of tetrahydrofolate (THF). The other product of the
MS reaction is the essential amino acid methionine, which becomes adenosylated and serves as a
universal methyl donor in numerous methyltransferase reactions. The product S-adenosyl-
homocysteine (SAH) undergoes reversible hydrolysis, completing the methionine (or remethylation) cycle.
The alternative pathway for Hcy disposal, which involves transsulfuration and requires vitamin B6, is not
shown.

SAM: S-adenosyl-methionine; ATP: adenosine triphosphate; SAH: S-adenosyl-homocysteine; NADP + : NAD

phosphate; NADPH + : reduced NAD phosphate; H + : proton; DHFR: dihydrofolate reductase; AHCY
hydrolase: adenosyl-homocysteine hydrolase; MS: methionine synthase; B12: Vitamin B12; THF:
tetrahydrofolate; SHMT: serine hydroxymethyltransferase; TS: thymidylate synthase; MethylTHF:
methylene THF; MTHFR: methylene tetrahydrofolate reductase.

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12/26/23, 12:13 AM Clinical manifestations and diagnosis of vitamin B12 and folate deficiency - UpToDate

Reprinted from: Green R. Vitamin B12 deficiency from the perspective of a practicing hematologist. Blood 2017; 129(19):2603, with
permission from American Society of Hematology. Copyright © 2017. https://www.journals.elsevier.com/blood.

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12/26/23, 12:13 AM Clinical manifestations and diagnosis of vitamin B12 and folate deficiency - UpToDate

Pathways of homocysteine metabolism

Homocysteine is metabolized by one of two divergent pathways: transsulfuration and remethylation. The
transsulfuration of homocysteine to cysteine is catalyzed by CBS, a process that requires pyridoxal
phosphate (vitamin B6) as a cofactor. Remethylation of homocysteine produces methionine. This reaction
is catalyzed either by methionine synthase or by betainehomocysteine methyltransferase. Vitamin B12
(cobalamin) is the precursor of methylcobalamin, which is cofactor for methionine synthase.

MS: methionine synthase; MTHFR: methylene tetrahydrofolate reductase; CBS: cystathionine-beta

synthase; CL: cystathionine-gamma-lyase; BHMT: betaine-homocysteine methyltransferase; DMG:
dimethylglycine; B2: riboflavin; THF: tetrahydrofolate; SAM: S-adenosyl methionine; SAH: S-adenosyl
homocysteine.

Reprinted by permission from: Springer: Cardiovascular drugs and therapy. Kang SS, Rosenson RS. Analytic approaches for the
treatment of hyperhomocysteinemia and its impact on vascular disease. Cardiovasc Drugs Ther 2018; 32:233. Copyright © 2018.

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https://link.springer.com/journal/10557.

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Roles of vitamin B12 and folate in metabolic pathways

Both folate and the vitamin B12 derivative methylcobalamin are used in methyl transfer to convert
homocysteine to methionine. This is why deficiency of vitamin B12 or folate causes increased
homocysteine.

Only vitamin B12 is involved in hydrogen transfer to convert methylmalonic acid (MMA) to succinyl-CoA.
This is why only vitamin B12 deficiency causes increased MMA.

Refer to UpToDate for additional details of these pathways.

MMA: methyl-malonyl-CoA.

Courtesy of Clifford M Takemoto, MD.

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Bone marrow aspirate in a patient with vitamin B12 deficiency due to

pernicious anemia

Typical megaloblastic changes consistent with nuclear-cytoplasmic dyssynchrony are shown.

These include abnormal nuclear maturation in erythroid precursors, with variegated, finely granular
chromatin (salt and pepper appearance, rather than the normal ground glass appearance) in a
proerythroblast in (A) and a giant granulocyte precursor in (B). Original magnification ×100; Wright-
Giemsa stain.

Reprinted from: Green R. Vitamin B12 deficiency from the perspective of a practicing hematologist. Blood 2017; 129(19):2603, with
permission from American Society of Hematology. Copyright © 2017. https://www.journals.elsevier.com/blood.

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12/26/23, 12:13 AM Clinical manifestations and diagnosis of vitamin B12 and folate deficiency - UpToDate

Causes of vitamin B12 deficiency

Gastric abnormalities

Autoantibodies to intrinsic factor or gastric parietal cells (ie, pernicious anemia)

Gastrectomy/bariatric surgery

Gastritis

Autoimmune metaplastic atrophic gastritis

Small bowel disease

Malabsorption syndrome

Ileal resection or bypass

Inflammatory bowel disease (eg, Crohn disease)

Celiac disease

Bacterial overgrowth

Blind loop

Dibothriocephalus latus (fish tapeworm)

Pancreatitis

Pancreatic insufficiency

Diet
Breastfed infant of a mother with vitamin B12 deficiency

Vegan diet

Strict vegetarian diet

Limited intake of animal protein during pregnancy or lactation

Agents that block or impair absorption or metabolism

Neomycin

Biguanides (eg, metformin)

Proton pump inhibitors (eg, omeprazole)

Histamine 2 receptor antagonists (eg, cimetidine)

Nitrous oxide (N2O) gas, used for anesthesia or recreationally

Inherited transcobalamin II deficiency

Refer to UpToDate topics on vitamin B12 and folate deficiency for further information on evaluation and

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management.

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12/26/23, 12:13 AM Clinical manifestations and diagnosis of vitamin B12 and folate deficiency - UpToDate

Causes of folate deficiency

Nutritional deficiency

Substance use disorders that lead to inadequate dietary intake

Chronic excessive alcohol use, which may be associated with malnutrition and increased metabolic
demands

Inadequate dietary intake due to severe malnutrition or restrictive diets

Consumption of overcooked foods (cooking inactivates folates)

Reduced intake of green leafy vegetables if residing in a country where cereals and grains are not
supplemented with folic acid

Malabsorption

Celiac disease (sprue)

Inflammatory bowel disease

Infiltrative bowel disease

Short bowel syndrome

Bariatric surgery

Drugs (various mechanisms)

Methotrexate

Trimethoprim

Ethanol

Phenytoin

Increased requirements

Pregnancy, lactation

Chronic hemolysis

Exfoliative dermatitis or severe eczema

Other conditions with high cellular turnover

Several countries (eg, United States, Canada, Costa Rica, Chile, South Africa) have required manufacturers
to enrich cereals and grain products with folic acid to reduce the risk of neural tube defects. Thus, folate
deficiency has become uncommon in individuals in these countries that provide routine fortification.
However, individuals with the above conditions may be at increased risk of developing folate deficiency.
Refer to UpToDate for additional details of mechanisms and other causes.

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12/26/23, 12:13 AM Clinical manifestations and diagnosis of vitamin B12 and folate deficiency - UpToDate

Micronutrient management after bariatric surgery [1]

Preoperative Postoperative Symptoms of

RDA [2] Suppleme
prevalence prevalence deficiency

Vitamin Up to 17% [3] 8 to 11% after Early signs: Men: 900 LAGB: 5000 I
A [1,3] RYGB Night blindness mcg (3000
RYGB or SG:
Bitot's spots IU)
70% after 10,000 IU da
BPD/DS Hyperkeratinization Women:
BPD/DS: 10,0
of skin 700 mcg
daily
Loss of taste (2300 IU)

Advanced signs:
Corneal damage
Blindness

Vitamin D 25 to 68% 25 to 80% Hypocalcemia, tetany, General: 3000 IU D3 d

tingling, cramping, 600 IU from all sour
metabolic bone disease, maintain a 2
Pregnancy,
muscle pain level of >30 n
lactation,
or over 71
years of
age: 800
IU

Vitamin E 2.2% Uncommon Neuromuscular General: Adults and

disorders and hemolysis 15 mg adolescents
(22.4 IU) older: 15 mg
IU) daily
Lactation:
19 mg Lactation: 19
(28.4 IU) (28.4 IU) dail

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Vitamin K Uncommon Uncommon Impaired coagulation 90 to 120 LAGB, RYGB,

mcg 90 to 120 mc

BPD/DS: 300
daily

Vitamin 16 to 29% 1 to 49% Numbness, tingling in 1.5 mg >12 mg daily

B1 extremities, gait ataxia, preferably 50
(Thiamine) edema, vomiting, mg daily from
confusion complex sup

Wernicke-Korsakoff With IV hydr

syndrome: 100 mg of th
Encephalopathy should be ad
Ataxia the solution
not contain
Oculomotor
if Wernicke
dysfunction
encephalopa
Confabulation
suspected)
Impaired memory
Impaired learning

Beriberi:
Neuropathy
Pain
Paresthesia
Loss of reflexes

Vitamin 0 to 18% 33% after RYGB; Macrocytic 2.4 mcg Oral dose of
B12 4 to 20% after SG (megaloblastic) anemia, 1000 mcg da
mild pancytopenia, 1000 mcg IM
neuropsychiatric monthly, or b
findings (eg, depression, spray
neuropathy)

Folate 0 to 54% Up to 65% after Macrocytic 400 mcg General: 400

RYGB; 18% after (megaloblastic) anemia, mcg daily fro
SG mild pancytopenia, multivitamin
neural tube defects
Women of
childbearing

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12/26/23, 12:13 AM Clinical manifestations and diagnosis of vitamin B12 and folate deficiency - UpToDate

800 to 1000
daily

Should not e
mg per day

Iron 0 to 58% LAGB 14%, SG Anemia Men ages Males, post-

<18%, RYGB 20 19 and menopausal
Pica
to 55%, BPD 13 older and and patients
to 62%, DS 8 to Impaired learning women history of an
50% ages 51 mg of iron fr
and older: multivitamin
8 mg per
Menstruatin
day
women and
Women women who
between undergone R
the ages SG, or BPD/D
of 19 to to 60 mg of
50: 18 mg elemental iro
per day from all sour

Zinc 24 to 28% 70% after Growth retardation, Women: 8 BPD/DS: 16 t

overall; 9 to BPD/DS, 40% delayed sexual maturity, mg (200% RDA)
74% seeking after RYGB, 19% impotence, impaired
Men: 11 RYGB: 8 to 22
BPD/DS after SG, 34% immune function
mg (100 to 200%
after LAGB
SG or LAGB:
mg (100% RD

Maintain a ra
to 15 mg of z
1 mg of copp

Copper 68% in women 90% after Anemia, neutropenia, 900 mcg BPD/DS or R
seeking BPD BPD/DS, 10 to ataxia mg daily (20
20% after RYGB
SG or LAGB:
daily (100% R

Maintain a ra
to 15 mg of z
1 mg of copp

Selenium 2% 14 to 22% after Skeletal muscle 55 mcg Unknown bu

RYGB and dysfunction and higher than
BPD/DS cardiomyopathy, mood mcg/day [4]

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disorder, impaired
immune function,
macrocytosis

Calcium 1 to 10% [6] 3.6% after Bone disease, secondary 1000 to RYGB, SG, or
bariatric surgery hyperparathyroidism 1200 mg 1200 to 1500
(1.9% after RYGB, daily in divid
9.3% after SG,
BPD/DS: 180
and 10% after
2400 mg dai
BPD/DS)
divided dose

RDA: Recommended Daily Allowance; RYGB: Roux-en-Y gastric bypass; BPD/DS: biliopancreatic diversion
with duodenal switch; IU: international unit; LAGB: laparoscopic adjustable gastric band; SG: sleeve
gastrectomy; IM: intramuscular; IV: intravenous; SQ: subcutaneous.

References:
1. Mechanick JI, Apovian C, Brethauer S, et al. Clinical practice guidelines for the perioperative nutrition, metabolic, and
nonsurgical support of patients undergoing bariatric procedures - 2019 update: Cosponsored by American Association of
Clinical Endocrinologists/American College of Endocrinology, The Obesity Society, American Society for Metabolic &
Bariatric Surgery, Obesity Medicine Association, and American Society of Anesthesiologists. Surg Obes Relat Dis 2020;
16:175.
2. U.S. Department of Health and Human Services and U.S. Department of Agriculture. 2015–2020 Dietary Guidelines for
Americans, 8th Edition, December 2015. Available at: https://health.gov/our-work/food-nutrition/previous-dietary-
guidelines/2015 (Accessed on April 7, 2021).
3. Stein J, Stier C, Raab H, Weiner R. Review article: The nutritional and pharmacological consequences of obesity surgery.
Aliment Pharmacol Ther 2014; 40:582.
4. Institute of Medicine (U.S.). Panel on Dietary Antioxidants and Related Compounds. Dietary Reference Intakes for Vitamin C,
Vitamin E, Selenium, and Carotenoids, National Academy Press, Washington DC 2000.
5. Al-Matary A, Hussain M, Ali J. Selenium: a brief review and a case report of selenium responsive cardiomyopathy. BMC
Pediatr 2013; 13:39.
6. Shah M, Sharma A, Wermers RA, et al. Hypocalcemia after bariatric surgery: Prevalence and associated risk factors. Obes
Surg 2017.

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Hyperpigmentation in vitamin B12 deficiency

A 59-year-old man presented to the endocrinology clinic with a 2-month history of fatigue and a 1-year
history of progressively darkening skin on his palms and soles. He worked as a tile layer and had initially
attributed his symptoms to occupational exposures. On physical examination, his tongue was smooth
with patchy areas of mucosal darkening (A). There was also hyperpigmentation on the palms (B) and
soles. Laboratory studies showed a hemoglobin level of 9.4 g per deciliter (reference range, 14 to 18), a
mean corpuscular volume of 117 fl (reference range, 80 to 94), and mild leukopenia and
thrombocytopenia. The vitamin B12 level was 40 pg per milliliter (30 pmol per liter; reference range, 200
to 1100 pg per milliliter [150 to 810 pmol per liter]), and the serum level of intrinsic factor antibody was
greater than 200 reference units per milliliter (reference value, <18), findings consistent with a diagnosis
of vitamin B12 deficiency due to autoimmune gastritis. Skin hyperpigmentation occurs in persons with
vitamin B12 deficiency owing to increased melanin synthesis and is seen more frequently in persons with
darker skin. The hyperpigmentation typically reverses with treatment. The patient received treatment

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with parenteral vitamin B12, and at follow-up 4 months after presentation, the hyperpigmentation had
resolved (C,D), as had his fatigue.

From: Maharajh S, Teelucksingh S. Hyperpigmentation in Vitamin B12 Deficiency. N Engl J Med 2022; 386:172. Copyright © 2022
Massachusetts Medical Society. Reprinted with permission from Massachusetts Medical Society.

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Cutaneous lesions and vitamin B12 deficiency

The picture shows hyperpigmentation on both feet. A 34-year-old woman complained of skin lesions that
had developed on both her feet over the past 1.5 months. She was a nonvegetarian. She was under the
care of an endocrinologist for hyperthyroidism and was using a 7.5-mg dose of carbimazole daily. On
examination, she had non-itchy, hyperpigmented macular lesions predominantly on the dorsum of the
middle phalanges of the toes of both feet. There was no rash or dermatitis preceding the onset of
hyperpigmentation. This was her only physical sign. Clinical examination was otherwise unremarkable.
There were no clinical features suggestive of autoimmune disorders. The vitamin B12 level was 113
pmol/L (normal range 132 to 857 pmol/L). The full blood count and mean corpuscular volume were
within normal limits (normal range 80 to 97 fL). As there was no other cause for the hyperpigmentation
except for a low serum vitamin B12 level, she was started on intramuscular injections of 1000 mcg of
vitamin B12 followed by a vitamin B complex (vitamins B1, B6, and B12) tablet daily. The vitamin B12 level
after treatment was 300 pmol/L. The hyperpigmented lesions improved within 2 weeks of starting
treatment.

Reproduced with permission from: Kannan R, Ng MJ. Cutaneous lesions and vitamin B12 deficiency: an often-forgotten link. Can
Fam Physician 2008; 54:529. Copyright © 2008 The College of Family Physicians of Canada.

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12/26/23, 12:13 AM Clinical manifestations and diagnosis of vitamin B12 and folate deficiency - UpToDate

Diagnostic testing for suspected vitamin B12 or folate deficiency

Some clinicians may choose an alternate testing algorithm depending on patient factors. UpToDate
topics on vitamin B12 and folate deficiency discuss the presenting findings, diagnostic approach,
differential diagnosis, and treatment in more detail, as well as additional post-diagnostic testing for the
underlying causes of these deficiencies. Refer to laboratory-specific lower limits of normal.
Typical values for vitamin B12 are as follows:
Deficient: <200 pg/mL
Borderline: 200 to 300 pg/mL
Normal: >300 pg/mL
Typical values for folate are as follows:
Deficient: <2 ng/mL
Borderline: 2 to 4 ng/mL
Normal: >4 ng/mL

MMA: methylmalonic acid; RBC: red blood cell.

​ Folate testing may be omitted if diet and gastrointestinal anatomy and function are normal. If dietary
*
folate deficiency is suspected in a patient who has recently received a normal meal, RBC folate should be
measured instead of serum folate. If one level is deficient and the other is borderline, then it may be
necessary to follow more than one diagnostic path (eg, if folate is deficient and vitamin B12 is borderline,
then folate deficiency may be confirmed but MMA and homocysteine testing may be required to
determine vitamin B12 status). Another alternative in this setting would be to administer both vitamins.

¶ Additional testing may be appropriate. Examples include testing of MMA and homocysteine levels to
further support a diagnosis of vitamin B12 deficiency; testing for autoantibodies to intrinsic factor if
there is vitamin B12 deficiency not attributable to a known gastrointestinal condition; or screening
endoscopy for malignancy in individuals with newly diagnosed pernicious anemia.

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Macro-ovalocytes in vitamin B12 deficiency

Peripheral smear shows marked macro-ovalocytosis in a patient with vitamin B12 deficiency. In this case,
teardrop cells are an advanced form of macro-ovalocytes.

Courtesy of Stanley L Schrier, MD.

Graphic 74901 Version 6.0

Normal peripheral blood smear

High-power view of a normal peripheral blood smear. Several platelets (arrowheads) and a normal
lymphocyte (arrow) can also be seen. The red cells are of relatively uniform size and shape. The diameter
of the normal red cell should approximate that of the nucleus of the small lymphocyte; central pallor
(dashed arrow) should equal one-third of its diameter.

Courtesy of Carola von Kapff, SH (ASCP).

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Graphic 59683 Version 5.0

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Peripheral blood smear showing megaloblastic changes

Peripheral blood smear showing a hypersegmented neutrophil (seven lobes) and macroovalocytes, a
pattern that can be seen with vitamin B12 (cobalamin) or folate deficiency.

Courtesy of Stanley L Schrier, MD.

Graphic 58820 Version 5.0

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12/26/23, 12:13 AM Clinical manifestations and diagnosis of vitamin B12 and folate deficiency - UpToDate

Causes and mechanisms of macrocytosis

Abnormalities of DNA metabolism

Vitamin B12 (cobalamin) deficiency

Folate deficiency

Drugs

Antiretroviral therapies for HIV infection (eg, zidovudine)

Azathioprine or 6-mercaptopurine

Capecitabine

Cladribine

Cytosine arabinoside

Hydroxyurea

Imatinib, sunitinib

Methotrexate

Shift to immature or stressed red cells

Reticulocytosis

Action of erythropoietin - skip macrocytes, stress erythrocytosis

Aplastic anemia/Fanconi anemia

Pure red cell aplasia

Primary bone marrow disorders

Myelodysplastic syndromes

Congenital dyserythropoietic anemias

Some sideroblastic anemias

Large granular lymphocyte (LGL) leukemia

Lipid abnormalities

Liver disease

Hypothyroidism

Mechanism unknown
Alcohol abuse

Multiple myeloma and other plasma cell disorders

Graphic 66772 Version 3.0

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Total versus functional vitamin B12 are affected by levels of vitamin B12
binding proteins

Levels of vitamin B12 binding proteins alter the amount of total and functional vitamin B12. The majority
of vitamin B12 is complexed with haptocorrin (HC, light blue boxes) and is not functional. In contrast,
vitamin B12 bound to transcobalamin II (TC, yellow boxes) is functional; it can be transported into cells as
the holotranscobalamin complex.

(A) Normal levels of vitamin B12 binding proteins HC and TC II, with normal total and functional vitamin
B12.

(B) Reduction in HC results in low total vitamin B12, but TC II levels and levels of functional vitamin B12
are normal.

(C) Increased HC results in normal total vitamin B12, but decreased TC II results in low functional vitamin
B12.

B12: vitamin B12; HC: haptocorrin; TC: transcobalamin II.

Courtesy of Clifford M Takemoto, MD.

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Megaloblastic erythropoiesis

Comparison of normal and megaloblastic erythropoiesis with respect to erythroid precursors in the bone
marrow.

(Left panel) Normal erythropoiesis.

(Right panel) Megaloblastic erythropoiesis.

Courtesy of Stanley L Schrier, MD.

Graphic 68005 Version 2.0

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Contributor Disclosures
Robert T Means, Jr, MD, MACP Consultant/Advisory Boards: Affinergy [Iron-related diagnostic tests];
Pharmacosmos Therapeutics Inc. [Iron deficiency in pregnancy]. All of the relevant financial relationships
listed have been mitigated. Kathleen M Fairfield, MD, DrPH No relevant financial relationship(s) with
ineligible companies to disclose. Clifford M Takemoto, MD Grant/Research/Clinical Trial Support: Daiichi
Sankyo [Thrombosis]; Forma Therapeutics [Sickle cell disease]; Global Blood Therapeutics [Sickle cell
disease]. Consultant/Advisory Boards: Genentech [Hemophilia]; Merck [Anticoagulants]; Novartis [DSMB –
Aplastic anemia]. All of the relevant financial relationships listed have been mitigated. Jennifer S Tirnauer,
MD No relevant financial relationship(s) with ineligible companies to disclose. Jane Givens, MD, MSCE No
relevant financial relationship(s) with ineligible companies to disclose.

Contributor disclosures are reviewed for conflicts of interest by the editorial group. When found, these are
addressed by vetting through a multi-level review process, and through requirements for references to be
provided to support the content. Appropriately referenced content is required of all authors and must
conform to UpToDate standards of evidence.

Conflict of interest policy

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