SPECIAL ARTICLE

The Many Faces of Cobalamin (Vitamin B12)

Deficiency
Bruce H.R. Wolffenbuttel, MD, PhD; Hanneke J.C.M. Wouters, BSc;
M. Rebecca Heiner-Fokkema, PhD; and Melanie M. van der Klauw, MD, PhD

Abstract

Although cobalamin (vitamin B12) deficiency was described over a century ago, it is still difficult to
establish the correct diagnosis and prescribe the right treatment. Symptoms related to vitamin B12 defi-
ciency may be diverse and vary from neurologic to psychiatric. A number of individuals with vitamin B12
deficiency may present with the classic megaloblastic anemia.
In clinical practice, many cases of vitamin B 12 deficiency are overlooked or sometimes even
misdiagnosed. In this review, we describe the heterogeneous disease spectrum of patients with
vitamin B 12 deficiency in whom the diagnosis was either based on low serum B 12 levels, elevated
biomarkers like methylmalonic acid and/or homocysteine, or the improvement of clinical
symptoms after the institution of parenteral vitamin B 12 therapy. We discuss the possible clinical
signs and symptoms of patients with B 12 deficiency and the various pitfalls of diagnosis and
treatment.
ª 2019 THE AUTHOR. Published by Elsevier Inc on behalf of Mayo Foundation for Medical Education and Research. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) n Mayo Clin Proc Inn Qual Out 2019;3(2):200-214

S
everal scientific articles and textbooks levels of methylmalonic acid (MMA) are the
have described the clinical presentation cornerstone of diagnostics, but normal levels
From the Department of of patients with cobalamin (vitamin B12) of serum B12 and MMA do not exclude symp-
Endocrinology (B.H.R.W.,
H.J.C.M.W., M.M.v.d.K.),
deficiency.1,2 After the classic presentation of tomatic B12 deficiency. In clinical practice,
Department of Haematology Addison-Biermer disease with megaloblastic many cases of B12 deficiency are overlooked
(H.J.C.M.W.), and Depart- anemia, many generations of doctors have been or sometimes even misdiagnosed because of
ment of Laboratory Medicine
(M.R.H.-F.), University of
educated with the view that vitamin B12 defi- misconceptions and misbeliefs among health
Groningen, University Medical ciency exclusively presents itself with this type care professionals. We have summarized the
Center Groningen, Gronin- of anemia. Additional cases have been reported most frequently encountered misconceptions
gen, NL-9700 RB, The
Netherlands.
in which neurologic abnormalities were the and misbeliefs regarding vitamin B12 defi-
main presenting symptom, with subacute com- ciency in Table 1.
bined degeneration of the spinal cord as one of In this review, we discuss a number of
the most feared manifestations,3 often leading typical patients who were seen at our outpa-
to permanent disability. Lindenbaum et al4 re- tient clinic, and we summarize the possible
ported a large series of 40 patients who had clinical signs and symptoms of patients with
neurologic symptoms or psychiatric disorders vitamin B12 deficiency and the pitfalls of diag-
caused by vitamin B12 deficiency but who had nosis and treatment.
no anemia or macrocytosis. Psychiatric symp-
toms may vary from depression to mania, psy- CASE DESCRIPTIONS
chosis, and occasionally suicidal thoughts
(Supplemental Table 1, available online at Patient A
http://mcpiqojournal.org).5 The reason why Patient A is a 55-year-old woman admitted to
some patients mainly present with megaloblastic the psychiatry department of our hospital
anemia and others with neurologic symptoms re- because of depression. In addition to symp-
mains unknown. toms related to her depression, she reported
Laboratory investigations with the estab- pain in the lower legs, paresthesia and numb-
lishment of low serum B12 levels and elevated ness in the feet, and difficulty walking. Her

200 Mayo Clin Proc Inn Qual Out n June 2019;3(2):200-214 n https://doi.org/10.1016/j.mayocpiqo.2019.03.002
www.mcpiqojournal.org n ª 2019 THE AUTHOR. Published by Elsevier Inc on behalf of Mayo Foundation for Medical Education and Research. This is an
open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
COBALAMIN DEFICIENCY

(reference ranges provided parenthetically)

TABLE 1. Frequently Reported Misconceptions and
revealed a hemoglobin level of 7.0 mmol/L
Misbeliefs Regarding Vitamin B12 Deficiency
(>7.5 mmol/L) and mean corpuscular volume
Diagnosis: You do not have vitamin B12 deficiency of 105 fl (85-98 fl). Her serum vitamin B12
because level was 51 pmol/L (145-450 pmol/L). These
d You have no anemia findings prompted the diagnosis of pernicious
d You have no macrocytic anemia
anemia caused by vitamin B12 deficiency, and
hydroxocobalamin injections were initiated
d Your serum vitamin B12 level is within the reference immediately. Within 3 to 4 weeks, the patient
range of 140 to 450 pmol/L reported that her symptoms gradually less-
ened, and her walking capacity improved.
d Your serum vitamin B12 level is only moderately
low
Additional laboratory tests revealed the pres-
ence of antibodies against parietal cells and
d Your serum vitamin B12 level may be low, but your intrinsic factor (IF). Six weeks later, the
plasma level of methylmalonic acid is completely attending psychiatric resident ordered an addi-
normal tional serum vitamin B12 measurement.
d Vitamin B12 deficiency only occurs in elderly people
Because the serum vitamin B12 level was
greater than 1476 pmol/L, hydroxocobalamin
d Vitamin B12 deficiency never occurs in children injections were stopped after consultation
with an internal medicine resident. The patient
Treatment
was discharged home 3 months later, but no
d Oral therapy is as good as or even better than pain-
follow-up appointment at our outpatient clinic
ful injections to alleviate neurologic symptoms
was made. The discharge letter did not
d You may stop treatment because your serum mention the diagnosis of pernicious anemia,
vitamin B12 level has normalized nor the treatment with hydroxocobalamin in-
jections. This exclusion went unnoticed until
d You must stop treatment because your serum
8 months later, when we realized she was
vitamin B12 level is elevated
lost to follow-up and invited her for a
d You must stop after 5 injections because vitamin B12 follow-up visit. She presented at our outpa-
can have serious adverse effects tient clinic shortly thereafter and reported a se-
vere increase of her symptoms. One month
d You must stop the injections because your symp-
earlier, her primary care physician had or-
toms have worsened
dered measurement of her serum vitamin B12
d We must measure your serum vitamin B12 level af- level, which was 251 pmol/L; he did not
ter 3 injections to see whether the treatment is restart treatment because he was unaware of
successful the earlier findings. We strongly advised the
patient to recommence hydroxocobalamin in-
d We must stop vitamin B12 injections now that you
jections, which she continued twice weekly for
are pregnant
the following 2 years. Gradually, her symp-
toms abated. Two years later, there were still
history included Graves disease, for which she bouts of pain, numbness, and paresthesia,
had undergone thyroidectomy several years but she was able to walk supported only by
earlier and was subsequently prescribed levo- a walking stick. This case is a clear example
thyroxine. We were invited for consultation of pernicious anemia in the constellation of a
because of slightly elevated free thyroxine polyglandular autoimmune syndrome. Unwar-
levels. The patient had no symptoms sugges- ranted cessation of therapy may lead to severe
tive of thyrotoxicosis. On examination, she worsening of neurologic abnormalities and
had signs of severe peripheral neuropathy of irreversible damage.
the lower legs (absent reflexes, superficial
and deep sensation) and several skin lesions Patient B
compatible with vitiligo. Laboratory studies Patient B is a 17-year-old girl who had a 2-year
(also summarized in Supplemental Table 2, history of fatigue, sleepiness, numbness in her
available online at http://mcpiqojournal.org) hands, dizziness, exertional dyspnea, and
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 MAYO CLINIC PROCEEDINGS: INNOVATIONS, QUALITY & OUTCOMES

problems with concentrating and finding the Patient C

correct words while in conversations. Table 2 Patient C is a 15-year-old girl. She had no spe-
summarizes her most important symptoms. cific symptoms. Her exercise capacity was
Because her mother had presented with symp- normal, and her school grades were fine. Because
tomatic vitamin B12 deficiency 6 months earlier, her brother was diagnosed as having symptom-
she was tested as well. Clinical examination did atic vitamin B12 deficiency at age 17 years and
not reveal major abnormalities. Laboratory ex- was subsequently successfully treated, she was
amination yielded a total serum vitamin B12 referred for evaluation. Physical examination
level of 112 pmol/L, with an intermediate level yielded no abnormalities. Laboratory investiga-
of holotranscobalamin (holoTC) of 54 pmol/L tion revealed a serum vitamin B12 level of 94
(40-125 pmol/L) and a low total haptocorrin pmol/L initially and 114 pmol/L on repeated
concentration of 162 pmol/L (203-596 pmol/ testing, MMA value of 218 nmol/L, homocyste-
L). Her MMA value was 178 nmol/L (<300 ine concentration of 78.4 mmol/L, and folate
pmol/L), homocysteine level was 11 pmol/L level of 6.7 nmol/L. Extensive evaluation showed
(<10 pmol/L), and folate concentration was no additional abnormalities, especially no signs
19 nmol/L (10-36 nmol/L). Antibodies against of celiac disease, or antibodies against parietal
parietal cells, IF, and transglutaminase/gliadin cells or IF. Whole-exome sequencing of genes
were absent. Because of our clinical suspicion related to metabolic disorders yielded only the
of vitamin B12 deficiency, she was treated with well-known homozygous c.677C>T mutation
hydroxocobalamin, 1000-mg injections twice in the gene coding for methylenetetrahydrofo-
weekly, with a beneficial effect on her symp- late reductase. It is known that people with
toms (Table 2). As can be appraised from her this mutation have increased risk of mild hyper-
symptom list, many improved considerably or homocysteinemia with vitamin B12 and/or folate
resolved completely. This case illustrates the deficiency.6 This patient had no clinical symp-
clear clinical syndrome of vitamin B12 defi- toms related to vitamin B12 deficiency and a
ciency despite intermediate holoTC and normal severely elevated homocysteine level, but the ge-
MMA levels. netic mutation is not considered to be the sole

TABLE 2. Self-Reported Symptoms of Patient B Before and 1 and 6 Months After Initiation of Hydroxocobalamin
Therapya
After treatment
Variable Before treatment 1 mo 6 mo
Numbness in hands 5 4 1
Difficulties focusing 5 4 3
Not being able to find the right words 6 4 0
Mood changes 7 6 0
Pain in mouth and tongue 6 6 4
Fatigue, weakness 9 9 0
Nausea, reduced appetite 8 6 0
Menstrual pains 9 9 8
Pain in joints 6 6 3
Dyspnea on exertion 6 8 1
Dizzy spells 7 5 0
Pale skin 5 5 4
Feeling cold 8 8 0
Muscle cramps 6 6 4
Stomach complaints, acidity 6 4 0
a
Symptoms rated on a scale of 0 to 10, in which 0 ¼ no symptoms and 10 ¼ worst symptoms.

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COBALAMIN DEFICIENCY

cause of this patient’s vitamin B12 deficiency and completely lost the feeling in her feet; she was
marked elevation of homocysteine. not able to walk outside her house anymore.
Even walking in her home was extremely diffi-
Patient D cult and painful. Additional laboratory exami-
Patient D is a 33-year-old woman with a history nations did not reveal any new abnormalities.
of fatigue, inertia, indolence, paresthesia in her She was admitted to the neurology department
hands and feet, difficulties concentrating, prob- in another hospital and found to have severe
lems with remembering things, and word polyneuropathy. Magnetic resonance imaging
finding disturbances. An acquaintance of the spinal cord showed symmetric bilateral
mentioned to her the possibility of vitamin B12 high signal within the dorsal columns, sugges-
deficiency. Her primary care physician ordered tive of subacute combined degeneration of the
a serum vitamin B12 measurement, which was cord; magnetic resonance imaging of the brain
190 pmol/L, and subsequently advised her to revealed minimal white matter lesions. Again,
start hydroxocobalamin injections. Her MMA her serum vitamin B12 level was normal, as
and homocysteine levels were unfortunately were thiamine, pyridoxine, and folate values.
not measured. After 5 weeks of treatment, her No signs or laboratory abnormalities consistent
symptoms had decreased considerably. Her with connective tissue disease or other causes of
physician then ordered repeated serum vitamin polyneuropathy were found. Biochemical and
B12 measurement, which revealed a value of microbiological evaluation of cerebrospinal
more than 1476 pmol/L. Subsequently, the fluid revealed no abnormalities. After 2 weeks
physician became concerned about possible of observation, her doctors decided to initiate
vitamin B12 intoxication and asked the patient hydroxocobalamin treatment. Two days later,
to stop treatment. Five to 6 weeks later, her results of additional testing were returned: her
symptoms had increased substantially. She was MMA level was 37,000 nmol/L, and her homo-
referred to our clinic for evaluation. Her main cysteine value was 165 mmol/L. These findings
question was why this successful treatment was confirmed the existence of severe vitamin B12
stopped. Hydroxocobalamin injections were deficiency. Additional testing yielded a high
restarted, in a frequency of twice weekly, which titer of antibodies against IF and parietal cells.
resulted in a gradual improvement of her symp- Because of uncertainty about the diagnosis,
toms. This case illustrates the difficulties of an she was referred to our outpatient clinic 2
incomplete diagnostic work-up and also the months later. No additional clues arose from
dangers of measuring serum vitamin B12 levels history or physical examination. Repeated ex-
during parenteral administration. Hydroxocoba- amination of the bone marrow specimen did
lamin is not toxic, and successful treatment not confirm a diagnosis of myelodysplastic syn-
should not be stopped. Usually, the period be- drome and showed only a slight increase of
tween injections can be prolonged when all erythropoiesis. The most likely diagnosis was
symptoms have disappeared. Addison-Biermer disease and interference in
the vitamin B12 assay by the IF autoantibodies.
Patient E No serum samples were left to test this hypoth-
Patient E is a 68-year-old woman who was esis. Six months after diagnosis and initiation of
treated by a hematologist in another hospital high-dose hydroxocobalamin treatment, the
for a period of 3 years because of macrocytic patient still had considerable neurologic dam-
anemia (hemoglobin, 7.5 pmol/L; mean age, loss of sensations in her feet and legs, and
corpuscular volume, 110 fl). There was no his- inability to walk without the use of a rollator
tory of alcohol abuse. Extensive evaluation walker. The anemia has resolved completely.
revealed no other abnormalities, and her serum This case is a classic example of how the assay
vitamin B12 value was 301 pmol/L. She had for serum vitamin B12 can be wrong because
already been treated for several years with levo- of interference of the assay by IF
thyroxine because of primary hypothyroidism. autoantibodies.
Myelodysplastic syndrome was diagnosed by
bone marrow aspiration, and she received Patient F
blood transfusions twice. In 2017, she experi- Patient F is a 62-year-old woman with a body
enced progressive pains in her legs and almost mass index of 36.6 kg/m2. She underwent a
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 MAYO CLINIC PROCEEDINGS: INNOVATIONS, QUALITY & OUTCOMES

Roux-en-Y gastric bypass procedure 8 months parameters. Some investigators suggest that it
earlier, which resulted in a 40-kg weight loss. is necessary to establish different reference cut-
During a routine visit to the outpatient clinic, offs according to age and the applied analytic
she reported fatigue and difficulties in concen- method.17 However, serum vitamin B12 tests
trating and executing difficult tasks. She took also may fail because many people with symp-
multivitamins that were prescribed by the toms related to cobalamin deficiency may have
physician in the bariatric surgery center. serum vitamin B12 levels above the lower refer-
Before the operation, she underwent a labora- ence level of 140 pmol/L.18,19 Although
tory evaluation that revealed the following: several factors may be of influence, in a
serum vitamin B12, 303 pmol/L; folate, 15.4 considerable number of cases this issue can
nmol/L; and vitamin D, 59 nmol/L. At reas- be caused by the earlier use of oral supplemen-
sessment, her serum vitamin B12 concentra- tation with multivitamins or high-dose oral
tion was 249 pmol/L, and her MMA level vitamin B12 preparations.20 It has been re-
was 1380 nmol/L. Because of the suspicion ported that even a dose of 10 mg/d can in-
of vitamin B12 deficiency, treatment with crease vitamin B12 levels to more than 200
hydroxocobalamin injections, 1000 mg twice pmol/L in elderly individuals (>65 years).21
weekly, was initiated, after which most of Oral supplementation may increase the serum
her symptoms disappeared within 6 to 8 vitamin B12 level but often not enough to
weeks. This case illustrates that vitamin B12 replenish the vitamin B12 levels in the tissues21
levels within the reference range do not unless very high doses (1000-2000 mg/d) are
exclude symptomatic vitamin B12 deficiency. used.

Discussion BIOCHEMISTRY AND DIAGNOSIS

We have described the disease spectrum of a Vitamin B12 deficiency may be easily overlooked
number of patients who attended our outpa- in patients when only total serum vitamin B12 is
tient clinic with possible vitamin B12 defi- used as a status marker.18,22e25 Many investiga-
ciency in whom the diagnosis was either tors therefore advocate measuring one or both
based on low serum vitamin B12 levels, of the additional functional biomarkers MMA
elevated biomarkers like MMA and/or homo- and homocysteine to establish vitamin B12 defi-
cysteine, or the improvement of clinical symp- ciency. Especially in people with so-called
toms after the institution of parenteral vitamin borderline vitamin B12 levels, ie, those between
B12 therapy. Altogether, these cases show the 140 and 300 pmol/L, elevated MMA and/or ho-
considerable heterogeneity of vitamin B12 defi- mocysteine values may aid in establishing a
ciency. Not all cases were easy to recognize or possible diagnosis of deficiency.18,26,27 The
diagnose. The most prevalent symptoms of mechanism by which these biomarkers may
vitamin B12 deficiency are neurologic, such demonstrate cobalamin deficiency can be derived
as paresthesia in hands and feet, muscle from the role of vitamin B12 in our body. Vitamin
cramps, dizziness, cognitive disturbances, B12 is a pivotal cofactor in 2 enzymatic reactions.
ataxia, and erectile dysfunction, as well as fa- Its deficiency will impair the proper function of
tigue, psychiatric symptoms like depression, these enzymes and lead to accumulation of the
and macrocytic anemia.1,2,7e10 However, substrate. Methylmalonylecoenzyme A (CoA)
there is evidence that in the Western world mutase, which catalyzes the isomerization of
as well as China, less than 20% of people methylmalonyl-CoA to succinyl-CoA, is one of
with demonstrable low serum vitamin B12 the vitamin B12edependent enzymes, and
levels have macrocytic anemia.11e14 Although impaired function will cause elevations of
the demonstration of low serum vitamin B12 methylmalonyl-CoA and thereby MMA after
levels is considered diagnostic, there is a cleavage of CoA. The other vitamin
poor correlation between these levels and B12edependent enzyme is methionine synthase,
symptoms,8,9,15 and even people with vitamin which regenerates methionine from homocyste-
B12 levels below 140 pmol/L may not have ine, and impaired activity as the consequence of
symptoms.16 This factor sheds a different light vitamin B12 deficiency will lead to accumulation
on the discussion regarding appropriate cutoff of homocysteine.28,29 From this process, it can
levels for serum vitamin B12 and related be understood how low tissue levels of cobalamin
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COBALAMIN DEFICIENCY

will increase MMA and homocysteine, and a clear indeterminate levels,27 and the reference
relationship between serum vitamin B12, MMA, values strongly depend on the assay method
and homocysteine has been described in the Na- used.39 It has been suggested that
tional Health and Nutrition Examination Survey patients with holoTC levels between 23 and
population.29 Nevertheless, the sensitivity and 75 pmol/L require further testing of MMA
specificity of elevated MMA and/or homocysteine levels to document, or rule out, true vitamin
levels in patients with symptoms associated with B12 deficiency.41 Also, approximately 63% of
vitamin B12 deficiency are unknown. Also, it has people with low holoTc levels (<27 pmol/L,
been documented that MMA levels are elevated in indicative of true deficiency) have normal
people with severely impaired renal function.30 levels of MMA, while 9% of patients with hol-
Similarly, elevated homocysteine values can also oTC values above 63 pmol/L have elevated
be the consequence of folate or vitamin B6 defi- MMA levels (defined as >300 nmol/L). This
ciency, as well as impaired renal function, hypo- issue raises questions about whether holoTC
thyroidism, and certain medications.6 In a measurement is really superior to measure-
separate study, we calculated from the National ment of total serum vitamin B12 plus MMA
Health and Nutrition Examination Survey and for determining vitamin B12 deficiency41 but
Lifelines epidemiological studies that MMA and/ also indicates that MMA is a poor indicator
or homocysteine levels are elevated above current of vitamin B12 deficiency. Indeed, in another
reference values (>300 nmol/L and 10 mmol/L, study, both serum vitamin B12 and holoTC
respectively) in only 73% of people with low levels were weak predictors of abnormal
serum vitamin B12 levels of less than 140 pmol/ MMA levels.23
L and in 28% of patients with serum vitamin A more mathematical approach toward
B12 levels between 140 and 300 pmol/L establishing vitamin B12 deficiency was pro-
(B.H.R.W., H.J.C.M.W., J.E. Kootstra-Ros, et al, posed by Fedosov et al,42,43 who calculated a
unpublished data, 2019). Other studies have single combined indicator of vitamin B12 sta-
confirmed that normal levels of MMA may be tus in which levels of total serum vitamin
measured even in situations of very low vitamin B12, holoTC, MMA, and homocysteine are
B12 levels.31 In addition, there are isolated reports taken into account. This is an elegant
that serum vitamin B12, homocysteine, and MMA approach to making a proper diagnosis, but
levels are unreliable predictors of vitamin its validation in the context of both functional
B12eresponsive neurologic disorders.32 Genetic vitamin B12 deficiency and systematically eval-
studies have found that in addition to mutations uated response to vitamin B12 supplementa-
in the MMUT gene (for expansion of gene sym- tion therapy (see subsequent discussion) is
bols, use search tool at www.genenames.org) required.
associated with methylmalonylaciduria, single-
nucleotide polymorphisms in HIBCH and ASSAY INTERFERENCE
ACSF3 have been associated with MMA Several published case reports have shown
levels.33,34 Similarly, serum vitamin B12 levels functional vitamin B12 deficiency in patients
may also be influenced by specific polymor- with apparently normal serum vitamin B12
phisms or mutations.35e37 One of these factors levels. In some of these patients, interference
is the gene FUT2, in which the FUT2 secretor of serum vitamin B12 assays by IF antibodies
variant genotype AA is associated with a 10% to has been demonstrated,44e47 and it has been
25% higher total and haptocorrine-bound reported that assays fail to measure low total
vitamin B12 level but not holoTC/active vitamin vitamin B12 concentrations in some samples
B12.38 because of an unknown artifact.48 Carmel
During recent years, the clinical usefulness and Agrawal48 reported that in 25% of pa-
of measuring holoTC as a screening for tients with pernicious anemia, the assay may
vitamin B12 status has received attention. Hol- have produced false-normal values. We
oTc is the biologically active form of vitamin strongly believe that our patient E fits the
B12 in plasma. Some studies have suggested description of interfering anti-IF antibodies.
that holoTC has a better diagnostic accuracy She had overt macrocytic anemia, initially
than total serum vitamin B12 level.39,40 How- diagnosed as myelodysplastic syndrome.
ever, holoTc also has a large window with Repeated serum vitamin B12 measurements
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 MAYO CLINIC PROCEEDINGS: INNOVATIONS, QUALITY & OUTCOMES

yielded normal results, and in the following 3 constituents can be demonstrated in the
years, she had development of symptoms blood.50e53 Table 3 summarizes the most
compatible with severe polyneuropathy and prevalent disorders, varying from autoimmune
nerve damage, signs of subacute combined thyroid disease (Hashimoto or Graves disease)
degeneration of the spinal cord. After both to type 1 diabetes, Addison disease, and viti-
MMA and homocysteine levels were found to ligo. In the situation of vitamin B12 deficiency,
be grossly elevated, it was realized that she both antibodies against IF and antibodies
had severe vitamin B12 deficiency. An addi- against parietal cells can be found, although
tional factor that may have added to her pro- it must be realized that the sensitivity of these
tracted course was that she had been treated measurements is low. One study found that
with high doses of folate. It is well known only 55% of people of Western European
that folate therapy may mask anemia, and descent with documented pernicious anemia
not treating with cobalamin may accelerate had anti-IF antibodies.54 However, this was
neurologic damage in people with vitamin not a very recent study, and methodology to
B12 deficiency.49 demonstrate antibodies may have changed.
Studies that assessed different assays to mea-
POSSIBLE CAUSES OF VITAMIN B12 sure anti-IF antibodies have yielded discrepant
DEFICIENCY results.54e56 Conversely, some investigators
The polyglandular autoimmune syndrome is have argued that testing for gastric parietal
easily recognizable as a cause of deficiency in cell antibodies is an appropriate screening
patients A and E. Polyglandular autoimmune test for pernicious anemia.57
syndromes are characterized by a number of Other causes that can lead to vitamin B12
(endocrine) diseases in which autoantibodies deficiency are summarized in Supplemental
directed against certain organs or cell Table 3 (available online at http://

TABLE 3. Spectrum of Polyglandular Autoimmune Syndrome

Organ Disease Antigen
Thyroid Hashimoto disease Thyroid peroxidase
Graves disease Thyrotropin receptor, thyroid-stimulating immunoglobulins
Pancreas Type 1 diabetes Glutamic acid decarboxylase 65, Islet antigen 2, zinc
transporter 8
Adrenal glands Addison disease 21-Hydroxylase
Gonads Autoimmune oophoritis 17a-Hydroxylase, side-chain cleavage enzyme
Premature menopause Aldehyde (retinal) dehydrogenases, selenium-binding
protein 1
Autoimmune orchitis Various antigens of sperm cell or testicular basement
membrane
Pituitary gland Lymphocytic hypophysitis Various antigens suggested
Parathyroid glands Hypoparathyroidisma NACHT leucine-rich repeat protein 5
Intestine Celiac disease Gluten
Stomach Atrophic gastritis Parietal cells, intrinsic factor
Liver Autoimmune hepatitis Cytochrome P450 1A2, 2A6
Sclerosing cholangitis Unknown
Primary biliary cirrhosis Mitochondrial antigens, like subunits (E2) of the pyruvate
dehydrogenase complex
Blood Thrombocytopenia Platelet surface glycoproteins
Hemolytic anemia Various antigens
Skin Vitiligo Tyrosinase
Hair follicles Baldness/alopecia Tyrosinase
a
Often in combination with mucocutaneous candidiasis.

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COBALAMIN DEFICIENCY

mcpiqojournal.org). One important cause is Imerslund-Gräsbeck syndrome is mentioned

celiac disease, in which gluten hypersensitivity as an example of a genetic cause of vitamin
will cause atrophy of the intestinal villi and B12 deficiency due to selective malabsorption,
consequently malabsorption, not only for but its prevalence is very low. Other causes
vitamin B12 but also for a variety of other vita- may be mutations in the gene for intrinsic fac-
mins. Celiac disease is one of the disorders in tor,70 genes encoding the vitamin B12 trans-
which vitamin B12 absorption may be restored porting transcobalamins,71,72 and genes
after dietary adjustment with complete avoid- involving intracellular vitamin B12 meta-
ance of gluten. Other factors that may bolism. The list of single-nucleotide polymor-
contribute to vitamin B12 malabsorption, phisms associated with serum vitamin B12
hence deficiency, are certain medications like concentration is gradually increasing, and a
metformin and proton pump inhibitors,58,59 recent review reported 59 gene polymor-
H2 receptor blockers, and antacids. Metformin phisms found in a wide variety of populations,
use was associated with an increase in MMA although the majority were reported in people
and worsening neuropathy score in patients of Western European descent.37 Recent
with type 2 diabetes.60 In addition, vitamin studies have found an association between ho-
B12 malabsorption may be more prominent mozygosity of the TT methylenetetrahydrofo-
in older individuals, rendering these people late reductase C677T genotype and vitamin
at increased risk of symptomatic vitamin B12 B12 deficiency.73e75 In the near future, dedi-
deficiency.61,62 cated evaluation of genetic mutations in genes
Inadequate dietary intake is another impor- that are associated with vitamin B12 meta-
tant cause of vitamin B12 deficiency. Because bolism, for instance with whole-exome
vitamin B12 is mainly present in food products sequencing, may shed light on familial cases.
from animal origin, vegetarians and vegans are
at especially high risk for development of CONSEQUENCES OF MATERNAL AND IN-
vitamin B12 deficiency.63 Finally, a new group FANT LOW B12 STATUS
of vitamin B12edeficient individuals is rapidly Maternal vitamin B12 deficiency during preg-
developing as the number of people undergoing nancy may be associated with an increased
bariatric surgery worldwide is increasing.64,65 incidence of neural tube defects and brain
Bariatric surgery has become one of the major development retardation,76 as well as preterm
treatments for (morbid) obesity, and reduced birth and low birth weight.77 Wnt signaling
production of both IF and gastric acid, as well has been reported to be disrupted in the devel-
as reduced intake of vitamin B12econtaining oping cerebellum of the offspring of vitamin
foods, contribute to the development of vitamin B12e and folate-deficient female rats78 and is
B12 deficiency (patient F). A recent study from associated with long-term disabilities of
The Netherlands reported a 60% incidence of behavior and memory.79 Ethnic differences
postebariatric surgery vitamin B12 deficiency, in vitamin B12 levels have been observed in
defined as a serum MMA level greater than pregnancy, possibly related to differences in
300 pmol/L.66 Interestingly, these investigators intake of animal-derived foods.80 Children
also reported that positive results of parenteral born to mothers with normal to high folate
vitamin B12 administration were reported by levels and low serum vitamin B12 values have
patients without functional vitamin B12 defi- higher truncal adiposity and insulin resis-
ciency (MMA <300 nmol/L), suggesting that tance,81 which may influence long-term risk
supplementation itself, regardless of the actual of development of type 2 diabetes and cardio-
vitamin B12 status, improves clinical symp- vascular disease.82,83 Insufficient vitamin B12
toms.66 Finally, severe cases of vitamin B12 defi- status has an important negative influence on
ciency have been reported in young individuals children’s development and cognitive func-
using nitrous oxide as a recreational drug.67e69 tioning.84,85 Nutritional vitamin B12 deficiency
in children, and a late diagnosis of this condi-
FAMILIAL/GENETIC CAUSES OF VITAMIN tion, may lead to irreversible neurologic dam-
B12 DEFICIENCY age such as growth and motor retardation and
The literature on familial cases of vitamin B12 even convulsions,86 and recent studies indi-
deficiency is limited. Classically, the cate a relationship between maternal vitamin
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 MAYO CLINIC PROCEEDINGS: INNOVATIONS, QUALITY & OUTCOMES

B12 status and bone mass in the offspring.87 in this study, a mixture of nutrients was
Disturbed vitamin B12 status as well as used, including multivitamins and vitamin
(maternal) genetic factors may influence B12 orally. Another study reported that paren-
DNA methylation patterns in the newborn teral vitamin B12 administration was more
and thereby predispose individuals to specific effective than nortriptyline in the treatment
disease later in life.88 of painful diabetic neuropathy.93 A 2005
meta-analysis94 reported that a high-dose
NATURAL COURSE OF VITAMIN B12 oral cyanocobalamin or methylcobalamin had
DEFICIENCY beneficial effects on symptoms of diabetic neu-
Importantly, the natural course of vitamin B12 ropathy, such as pain and paresthesia. In 3
deficiency is not clearly understood. In typical studies, methylcobalamin therapy also
patients, in whom clear signs and symptoms improved autonomic symptoms. Effects on vi-
associated with (poly)neuropathy and/or bration perception and electrophysiologic
megaloblastic anemia are explained by the un- measures were not consistent. However, one
equivocal finding of low serum vitamin B12 review did not find any evidence that the use
levels, like in patient A, it is not particularly of oral vitamin B12 supplements is associated
difficult to make a diagnosis and institute with improvement in the clinical symptoms
treatment. However, more widespread of diabetic neuropathy.95 Low central nervous
screening for vitamin B12 deficiency may result system cobalamin levels may play a role in the
in situations in which low serum vitamin B12 development of multiple sclerosis.96,97 Similar
levels are found but without clear symptom- observations, and even reports on beneficial
atology.10 Parenteral vitamin B12 administra- effects of vitamin B12 injection therapy, have
tion is also useddaccording to the package been reported in patients with myalgic
insertdfor the prevention of vitamin encephalomyelitis, with and without fibromy-
B12eassociated problems. In some individuals algia.98,99 Also, there are clues that low
without symptoms, low levels of serum vitamin B12 status is related to the develop-
vitamin B12 may be associated with reductions ment of chemotherapy-induced peripheral
in the binding protein haptocorrin.89 Howev- neuropathy.100 Prospective studies to assess
er, this condition may be difficult to discrimi- whether vitamin B12 supplementation may
nate from true vitamin B12 deficiency in the prevent chemotherapy-induced peripheral
presence of symptoms, as is evidenced by pa- neuropathy are planned or ongoing.101
tient B. A recent intriguing study comes from US in-
vestigators, who demonstrated low vitamin B12
JUST A VITAMIN, OR MORE? levels in the brain tissue of aged individuals,
Some of the cases reported in this article, as especially those older than 60 years, and of peo-
well as the study by Smelt et al66 mentioned ple with autism and schizophrenia.102 They
previously, suggest that supplementation of speculate that on one hand vitamin B12 status
vitamin B12 itself, regardless of the actual in the brain compartment is distinctly regulated
vitamin B12 status, improves clinical symp- during aging from the rest of the body, while on
toms. This hypothesis may put our thinking the other hand may contribute to impaired
on the pathophysiology of vitamin B12 defi- brain function and in the etiology of neurologic
ciency in a different perspective. Maybe we disorders. These observations are supported by
should not regard vitamin B12 as a vitamin a recent study that reported improvement of
that needs to be restored to normal levels to symptoms in children with autism spectrum
ensure proper functioning of metabolic path- disorder treated with frequent hydroxocobala-
ways but rather as a general nerve-protecting min injections.103 Finally, in a prospective
and nerve-regenerating compound. There are study, Brito et al104 found that one injection of
some clues in the medical literature that sup- high-dose (10 mg) cyanocobalamin, pyridox-
port this concept. Cobalamin may regulate ine, and thiamine increased several metabolic
the balance between neurotoxic and neurotro- markers of mitochondrial function oxidative
phic agents.90,91 Micronutrients including stress, nerve function, and myelin integrity
vitamin B12 might improve the neuropathy such as acylcarnitines, plasmalogens, phospho-
score in patients with type 2 diabetes,92 but lipids, and sphingomyelins.
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COBALAMIN DEFICIENCY

HOW SHOULD TREATMENT BE GIVEN? (n¼18) compared to the IM group (n¼15).

Traditionally, vitamin B12 is administered by Similar numbers of patients reported improve-
intramuscular (IM) injection because of the ment (n¼2) or clearance of symptoms (n¼2)
low degree of resorption after oral administra- in both groups. Typically in patients using
tion as a consequence of the underlying dis- vitamin B12 injections, serum vitamin B12
ease. For parenteral therapy in cases of levels exceed the upper limit of normal
megaloblastic anemia, it has been advised to (1476 pmol/L), and this finding suggests that
administer 1000 mg hydroxocobalamin IM there may be underdosing in the injection
twice weekly for a period of 5 weeks, and group. Patients were randomized to receive
the dose is reduced to 1000 mg IM every 2 2000 mg of oral cyanocobalamin administered
months thereafter. In the United States, often with breakfast daily for 4 months or 1000 mg
cyanocobalamin is used.10,15 In case of neuro- of cyanocobalamin administered IM with
logic symptoms or abnormalities, it is sug- increasing intervals. Evaluation was at 4
gested to administer hydroxocobalamin, months, when the oral group was still using
1000 mg once or twice weekly for a period supplementation but the IM group had
of up to 2 years, and the package insert for received the most recent injection 1 month
hydroxocobalamin has included these partic- earlier. In addition, parenteral administration
ular instructions for several decades. However, consisted of cyanocobalamin, not hydroxoco-
it is ill defined which neurologic symptoms or balamin, and it has been reported that hydrox-
abnormalities require such intensive treat- ocobalamin has a longer retention in plasma
ment. Clinical practice has shown that in a and a lower excretion in urine than an equiv-
substantial number of patients seen in a ter- alent dose of cyanocobalamin.109 A second
tiary care setting, injection frequency cannot study performed in Turkey110 included 70 pa-
be reduced after the initial loading regimen. tients with megaloblastic anemia. In 8 of 24
This topic has gained much interest recently, participants in the oral group, vitamin B12
and studies evaluating this phenomenon are deficiency was caused by poor nutrition. Ten
ongoing.105 patients were excluded because they did not
Since the 1990s, interest in the possibilities appear for follow-up, and it should be noted
of oral instead of parenteral administration has that these patients were mainly randomized
emerged. In 2014, a Dutch Viewpoint on to the oral supplementation group. Research
Vitamin B12 Treatment was presented by the into the etiology of vitamin B12 deficiency
Dutch Organization of General Practi- was rather poor. Only 19 patients had
tioners.106 Motivation to publish this View- antieparietal cell antibodies assessed, which
point was the observed increase in serum was due to costs as stated by the authors.
vitamin B12 testing and findings of abnormal Finally, the prevalence of neurologic symp-
test results. In this Viewpoint, it was stated toms was low. Only 7 of 60 participants re-
that oral vitamin B12 administration was ported altered cognitive function. Taken
preferred over parenteral supplementation. together, the articles provide very weak sup-
This advice has been based on a very limited port for oral rather than parenteral vitamin
number of clinical trials. A 2005 Cochrane re- B12 administration, and the large number of
view evaluated 2 randomized controlled trials participants in the study by Bolaman et al110
comprising a total of 108 participants and sug- with an etiology of poor nutrition (and not
gested that oral vitamin B12 may be as effective malabsorption) strongly biases these results
as IM administration in obtaining short-term in favor of simple oral vitamin B12 administra-
hematologic and neurologic responses in tion, which is the default in any nutritional
vitamin B12edeficient patients.107 The first insufficiency of vitamin B12 including vegetar-
study by Kuzminski et al108 was performed ianism. However, dosage is important in this
in 33 vitamin B12edeficient patients, the ma- respect. A study by Hill et al21 found that sup-
jority with atrophic gastritic or pernicious ane- plementation with 500 mg of cyanocobalamin
mia, and reported that serum vitamin B12 did not normalize moderately elevated MMA
levels were significantly higher and MMA levels in healthy elderly people (aged 65 years
levels significantly lower in the oral group or older). A recent Cochrane review confirmed

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 MAYO CLINIC PROCEEDINGS: INNOVATIONS, QUALITY & OUTCOMES

that there is only very low-quality evidence benefit of continued frequent (twice weekly)
that oral vitamin B12 appears as safe as IM hydroxocobalamin injections vs oral
vitamin B12.111 The authors concluded that supplementation.
further trials should conduct better randomi-
zation and blinding procedures, recruit more
CONCLUSION
participants, provide adequate reporting, and
The spectrum of symptoms and signs sugges-
measure important outcomes such as the clin-
tive of vitamin B12 deficiency is reasonably
ical signs and symptoms of vitamin B12 defi-
well defined. Nevertheless, many of the symp-
ciency, health-related quality of life,
toms are nonspecific and may occur as a
socioeconomic effects, and adverse events
consequence of other diseases. Currently, no
adequately.111 Serious adverse effects, even
research has documented the positive and
with high doses of hydroxocobalamin, have
negative predictive values of specific symp-
never been reported.112,113 However, some
toms or symptom scores for the presence of
patients do report acneiform eruptions or
vitamin B12 deficiency.
rosacea.114
Patients with low serum vitamin B12 levels
may have no symptoms (yet). Nevertheless,
POSSIBLE PLACEBO EFFECTS UNLIKELY they are at high risk for development of symp-
In discussions on the effect of hydroxocobala- toms. There is a tendency among physicians
min treatment, often the suggestion is made to consider a serum vitamin B12 level higher
that part of the effect is that of a placebo. than 140 pmol/L as normal, but many symp-
Indeed, there is evidence that placebo treat- tomatic patients may present with such levels,
ments can have large and sustained effects for instance because of taking oral vitamin sup-
on clinical outcomes in multiple disorders, as plementation. This does not mean that their tis-
reviewed by Ashar et al,115 who suggested sue vitamin B12 levels are normal as well.
that this effect may be particularly prominent Methylmalonic acid and homocysteine are not
in disorders in which emotion and motivation very sensitive biomarkers, but there is currently
play a central role. In vitamin B12 deficiency, no good alternative, although systematic evalu-
there is no evidence that recovery of megalo- ation of more advanced metabolic factors may
blastic anemia or normalization of elevated lead to the application of better biomarkers.
MMA levels as a consequence of hydroxocoba- When serum total vitamin B12 levels are low
lamin injections can be regarded as a placebo or questionable, the combination of total
effect. Yet, when patients’ neurologic or cogni- vitamin B12, active vitamin B12, MMA, and ho-
tive symptoms improve by injection therapy, it mocysteine may be the best strategy, but the
is often ascribed to a placebo effect. Several validity of this combined biomarker approach43
studies have assessed the effects of oral vitamin needs to be validated in larger prospective
B12 supplementation in a variety of disorders, studies and especially validated against objec-
usually in people with normal serum vitamin tive markers of treatment response. In case of
B12 levels,116,117 with a surprising benefit in, doubt, when results of biomarker measure-
for example, aphthous stomatitis.118 We ments are equivocal, a trial with parenteral
have not found many well-designed studies hydroxocobalamin injections may be consid-
of hydroxocobalamin injections in patients ered, as was done in patients B and D. Because
with neurologic symptoms caused by vitamin symptom improvement in long-standing (sub-
B12 deficiency. One study reported favorable clinical) vitamin B12 deficiency may take some
effects of hydroxocobalamin injections in pa- time, we usually advise a treatment regimen of
tients with mechanical or irritative twice weekly hydroxocobalamin injections for
lumbago,119 whereas another well-designed 3 months, after which a thorough reevaluation
study reported improvement of symptoms in is performed with systematic evaluation of
children with autism spectrum disorder.103 symptom score as demonstrated in patient B
Clearly, there is a need for well-conducted (Table 2). There is no proof in large prospective,
double-blind, randomized studies in patients double-blind studies that oral supplementation
with vitamin B12 deficiency who report the is as effective in reducing symptoms associated

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COBALAMIN DEFICIENCY

with vitamin B12 deficiency as parenteral dwelling elderly in Ankara, Turkey. Arch Gerontol Geriatr.
2015;60(2):344-348.
treatment. 14. Wong CW, Ip CY, Leung CP, Leung CS, Cheng JN, Siu CY.
Vitamin B12 deficiency in the institutionalized elderly: a
SUPPLEMENTAL ONLINE MATERIAL regional study. Exp Gerontol. 2015;69:221-225.
15. Solomon LR. Low cobalamin levels as predictors of cobalamin defi-
Supplemental material can be found online at ciency: importance of comorbidities associated with increased
http://mcpiqojournal.org. Supplemental mate- oxidative stress. Am J Med. 2016;129(1):115.e9-115.e16.
rial attached to journal articles has not been 16. Martin SS, Daya N, Lutsey PL, et al. Thyroid function, cardio-
vascular risk factors, and incident atherosclerotic cardiovascu-
edited, and the authors take responsibility lar disease: the Atherosclerosis Risk in Communities (ARIC)
for the accuracy of all data. Study. J Clin Endocrinol Metab. 2017;102(9):3306-3315.
17. Aparicio-Ugarriza R, Palacios G, Alder M, Gonzaléz-Gross M.
A review of the cut-off points for the diagnosis of vitamin B12
Abbreviations and Acronyms: CoA = coenzyme A; hol- deficiency in the general population. Clin Chem Lab Med.
oTC = holotranscobalamin; IF = intrinsic factor; IM = 2015;53(8):1149-1159.
intramuscularly; MMA = methylmalonic acid 18. Solomon LR. Cobalamin-responsive disorders in the ambula-
tory care setting: unreliability of cobalamin, methylmalonic
acid, and homocysteine testing. Blood. 2005;105(3):978-985.
Potential Competing Interests: The authors report no 19. Roessler FC, Wolff S. Rapid healing of a patient with dramatic
conflicts of interest. subacute combined degeneration of spinal cord: a case report.
BMC Res Notes. 2017;10(1):18.
Correspondence: Address to Bruce H. R. Wolffenbuttel, 20. Dobson R, Alvares D. The difficulties with vitamin B12. Pract
Neurol. 2016;16(4):308-311.
MD, PhD, Department of Endocrinology, University of Gro-
21. Hill MH, Flatley JE, Barker ME, et al. A vitamin B-12 supple-
ningen, University Medical Center Groningen, HPC AA31 ment of 500 mg/d for eight weeks does not normalize urinary
9700 RB Groningen, The Netherlands ([email protected]). methylmalonic acid or other biomarkers of vitamin B-12 sta-
tus in elderly people with moderately poor vitamin B-12 sta-
tus. J Nutr. 2013;143(2):142-147.
22. Vugteveen I, Hoeksma M, Monsen AL, et al. Serum vitamin
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