![Relationship between the intake and percentage of absorbed B 12 from a dose of [ 58 Co]-cyanocobalamin (Adams et al. [34]) and [ 13 C]-cyanocobalamin (Devi et al. [35]) in n ¼ 10-12 healthy volunteers.](https://www.researchgate.net/profile/Agata-Sobczynska-Malefora/publication/351055936/figure/fig2/AS:1035071268065282@1623791629491/Relationship-between-the-intake-and-percentage-of-absorbed-B-12-from-a-dose-of-58_Q320.jpg)
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Critical Reviews in Clinical Laboratory Sciences
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ilab20
Vitamin B12 status in health and disease: a critical
review. Diagnosis of deficiency and insufficiency –
clinical and laboratory pitfalls
Agata Sobczyńska-Malefora, Edgard Delvin, Andrew McCaddon, Kourosh R.
Ahmadi & Dominic J. Harrington
To cite this article: Agata Sobczyńska-Malefora, Edgard Delvin, Andrew McCaddon, Kourosh
R. Ahmadi & Dominic J. Harrington (2021): Vitamin B12 status in health and disease: a critical
review. Diagnosis of deficiency and insufficiency – clinical and laboratory pitfalls , Critical Reviews
in Clinical Laboratory Sciences, DOI: 10.1080/10408363.2021.1885339
To link to this article: https://doi.org/10.1080/10408363.2021.1885339
© 2021 The Author(s). Published by Informa
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REVIEW ARTICLE
Vitamin B
12
status in health and disease: a critical review. Diagnosis of
deficiency and insufficiency –clinical and laboratory pitfalls
Agata Sobczy
nska-Malefora
a,b
, Edgard Delvin
c,d
, Andrew McCaddon
e
, Kourosh R. Ahmadi
f
and
Dominic J. Harrington
a,b
a
The Nutristasis Unit, Viapath, St. Thomas’Hospital, London, UK;
b
Faculty of Life Sciences and Medicine, King’s College London,
London, UK;
c
Sainte-Justine UHC Research Centre, Montreal, Canada;
d
Department of Biochemistry and Molecular Medicine, University
of Montreal, Montreal, Canada;
e
Chester Medical School, University of Chester, Chester, UK;
f
Department of Nutrition & Metabolism,
School of Biosciences and Medicine, University of Surrey, Guildford, UK
ABSTRACT
Vitamin B
12
(cobalamin) is an essential cofactor for two metabolic pathways. It is obtained princi-
pally from food of animal origin. Cobalamin becomes bioavailable through a series of steps per-
taining to its release from dietary protein, intrinsic factor-mediated absorption, haptocorrin or
transcobalamin-mediated transport, cellular uptake, and two enzymatic conversions (via methio-
nine synthase and methylmalonyl-CoA-mutase) into cofactor forms: methylcobalamin and adeno-
sylcobalamin. Vitamin B
12
deficiency can masquerade as a multitude of illnesses, presenting
different perspectives from the point of view of the hematologist, neurologist, gastroenterologist,
general physician, or dietician. Increased physician vigilance and heightened patient awareness
often account for its early presentation, and testing sometimes occurs during a phase of vitamin
B
12
insufficiency before the main onset of the disease. The chosen test often depends on its
availability rather than on the diagnostic performance and sensitivity to irrelevant factors interfer-
ing with vitamin B
12
markers. Although serum B
12
is still the most commonly used and widely
available test, diagnostics by holotranscobalamin, serum methylmalonic acid, and plasma homo-
cysteine measurements have grown in the last several years in routine practice. The lack of a
robust absorption test, coupled with compromised sensitivity and specificity of other tests (intrin-
sic factor and gastric parietal cell antibodies), hinders determination of the cause for depleted
B
12
status. This can lead to incorrect supplementation regimes and uncertainty regarding later
treatment. This review discusses currently available knowledge on vitamin B
12
, informs the reader
about the pitfalls of tests for assessing its deficiency, reviews B
12
status in various populations at
different disease stages, and provides recommendations for interpretation, treatment, and associ-
ated risks. Future directions for diagnostics of B
12
status and health interventions are
also discussed.
Abbreviations: AI: adequate intake; AD: Alzheimer’s disease; AIG: autoimmune gastritis; CSF:
cerebrospinal fluid; Cbl: cobalamin; Co: cobalt; cB
12
: combined indicator of vitamin B
12
status;
CBLA: competitive binding chemiluminescence assays; CN-Cbl: cyanocobalamin; Ado-Cbl: 5’-deox-
yadenosylcobalamin; ELISA: enzyme-linked immunosorbent assay; GWAS: genome-wide associ-
ation studies; HC: haptocorrin; holoHC: holohaptocorrin; holoTC: holotranscobalamin; OH-Cbl:
hydroxocobalamin; IGS: Imerslund-Gr€
asbeck; IM: intramuscular; IF: intrinsic factor; LC-MS/MS:
liquid chromatography-tandem mass spectrometry; MS: methionine synthase; Me-Cbl: methylco-
balamin; MMA: methylmalonic acid; MCM: methylmalonyl-CoA mutase; 5-MTHF: 5-methyltetrahy-
drofolate; MAF: minor allele frequency; MRP1: multidrug resistance protein; MS: multiple sclerosis;
NHANES: National Health and Nutrition Examination Survey; N
2
O: nitrous oxide; OC: oral contra-
ceptives; PCA: parietal cell antibodies; PA: pernicious anemia; PPIs: proton pump inhibitors; RR:
risk ratio; RYGB: Roux-en-Y gastric bypass; SAM: S-adenosylmethionine; SG: sleeve gastrectomy;
SCDC: subacute combined degeneration of the spinal cord; THF: tetrahydrofolate; tHcy: total
plasma homocysteine; TC: transcobalamin; T2D: type 2 diabetes; UK: United Kingdom; US: United
States; uMMA/C: urinary MMA/creatinine ratio; B
12
: vitamin B
12
ARTICLE HISTORY
Received 6 July 2020
Revised 10 November 2020
Accepted 1 February 2021
KEYWORDS
Vitamin B
12
; cobalamin
holotranscobalamin
methylmalonic acid
homocysteine
CONTACT Agata Sobczy
nska-Malefora agata.malefora@viapath.co.uk The Nutristasis Unit, Viapath, St. Thomas’Hospital, London, SE1 7EH, UK
This article has been corrected with minor changes. These changes do not impact the academic content of the article.
ß2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES
https://doi.org/10.1080/10408363.2021.1885339
Introduction
Historical background
As early as the nineteenth century, James Combe [1],
Thomas Addison [2], and Anthony Biermer [3]wrote
about a fatal diathesis now recognized as vitamin B
12
(B
12
, synonym cobalamin [Cbl]) deficiency caused by per-
nicious anemia (PA). William Osler and William Gardner
described “progressive pernicious anemia”(a term used
five years earlier by Biermer) in a 52-year-old Englishman
with numbness of the fingers, hands, and forearms, as
well as large red blood cells [4]. Increased marrow cellu-
larity had already been reported by Julius Cohnhein [5].
The pathophysiological consequences of PA were
extended to spinal cord lesions reported by Lichtheim
[6], with Russell et al. coining the term “subacute com-
bined degeneration of the spinal cord”(SCDC). In 1900,
Russell noted characteristics of B
12
deficiency that are
overlooked with surprising frequency today: “some of
the most typical cases presented no anemia throughout
the course, others only late in the disease, while in other
cases anemia was an obtrusive symptom …and pre-
ceded the nervous symptoms by many months”[7].
Whipple and Robscheit-Robbins discovered that
feeding liver to anemic dogs promoted the regener-
ation of hemoglobin, leading George Minot and William
Murphy to the first effective treatment for PA when
they fed raw and later cooked liver to patients [8,9]. In
1934, Whipple, Minot, and Murphy received the Nobel
Prize for their work. While searching for the remission-
inducing component, Minot used an early method for
counting reticulocytes that enabled him to address the
hematological response to different liver fractions.
Other than this “human bioassay”(i.e. testing liver frac-
tions on patients with PA) no reliable laboratory diag-
nostics were available to support the search for the
extrinsic antipernicious factor(s) over the next 20 years.
A breakthrough came in 1946 when Lester Smith
noticed that the most potent liver fractions were red-
dish. We now know that B
12
contains a system of conju-
gated double bonds, which confer the red color and
make B
12
easily detectable at high concentrations.
Several teams worked on isolating the colored material
in quick succession, providing Dorothy Hodgkin with
the opportunity to undertake her remarkable X-ray crys-
tallographic studies for which a second Nobel Prize in
the vitamin B
12
field was awarded in 1964 [10].
Vitamin B
12
structure
Vitamin B
12
is one of the largest and most structurally
complex nonpolymeric biomolecules described in
nature, with a molecular weight of 1355 daltons for its
frequently used cyano- form. The generic term cobala-
min refers to a set of structures named corrinoids, con-
sisting of one central cobalt (Co) atom coordinated
with 4 equatorial nitrogen atoms contributed by pyr-
role residues (Figure 1). The 5
th
coordination site (a)in
B
12
, below the planar structure, is occupied by a 50,60-
dimethyl benzimidazole residue linked to a a-ribosyl-
3-phosphate. The 6
th
coordination site (b), above the
corrin ring, is occupied by a methyl- (methylcobalamin
[Me-Cbl]), 50-deoxyadenosyl (50-deoxyadenosylcobala-
min [Ado-Cbl]), aquo- (hydroxocobalamin [OH-Cbl]), or
a cyano-group (cyanocobalamin [CN-Cbl]). The meta-
bolic utility of B
12
in humans is conferred by an upper
ligand consisting of either a methyl or adenosyl moi-
ety. Cyanocobalamin is the most chemically stable
form, though it has no vitamin activity per se.
Cyanocobalamin undergoes enzymatic conversions
during intracellular processing in human cells, as
described below, and it is eventually converted to the
two cofactors, Me-Cbl and Ado-Cbl [11]. Me-Cbl and
Ado-Cbl regularly undergo catalytic reductions and
oxidations and occasionally generate OH-Cbl, which
returns to the catalysis via a reactivation cycle [12].
Figure 1. The chemical structure of vitamin B12.
2 A. SOBCZYŃSKA-MALEFORA ET AL.
B
12
sources and de novo biosynthesis
Vitamin B
12
is synthesized exclusively by micro-organ-
isms, sometimes inhabiting higher plants that cannot
produce B
12
. Fermentation in the gastrointestinal tract
of herbivorous animals supports the growth of micro-
organisms that synthesize B
12
, which is then absorbed
by the animal and incorporated into its tissues [13].
Omnivores and carnivores, including humans, acquire
B
12
from animal tissues or animal products such as milk,
cheese, and eggs. Liver in particular is a very rich source
of cobalamin, followed by kidney and heart. Although
the B
12
content of various types of milk is not high,
regular intake of milk is associated with higher serum
B
12
concentration in healthy elderly adults [14]. Egg
intake does not significantly contribute to higher serum
B
12
concentration [14]. Vitamin B
12
exists in foods in
several forms; meat and fish contain mostly Ado-Cbl
and OH-Cbl, OH-Cbl predominates in milk, and Me-Cbl
with Ado-Cbl and OH-Cbl are found in nearly all dairy
products [15–17]. Animals also procure B
12
through
coprophagia and as a consequence of bacterial contam-
ination of their feed.
Biosynthesis of B
12
is restricted to certain bacterial
strains, such as Lactobacillus rossiae and Lactobacillus
reuteri [18,19], and involves one of two alternative
routes, either the aerobic or anaerobic pathway in bac-
teria and archaea, respectively. Approximately 30
enzyme-catalyzed reactions are responsible for the syn-
thesis of full Cbl. The process starts with condensation
leading to uroporphyrinogen III (the first macrocyclic
intermediate in tetrapyrrole synthesis), continues via
the transformation of uroporphyrinogen III into cobina-
mide, and ends with nucleotide loop assembly and
attachment to the corrin ring [20]. To conserve energy,
many gram-negative bacteria salvage B
12
or other corri-
noids and transport them into the cell [21].
Dietary intake and bioavailability
Dietary intake of B
12
exceeds the metabolic require-
ment for the majority of people and leads to the accu-
mulation of substantial hepatic stores of 1–5 mg. The
typical Western diet provides 4–6mg/d of B
12
, from
which 1–5mg is absorbed [22]. The bioavailability of B
12
from food is assumed to be 50% for healthy adults
with normal absorption, while the absorption of crystal-
line B
12
, present in supplements and fortified food
products, is between 55 and 74%. However, the absorp-
tion from specific foods can vary greatly, for example,
24 to 36% for egg products (B
12
dose 0.3–0.94 lg), 42%
for fish (dose 1.95–2.18 lg), and 65% for lean meat
(dose 0.95 lg) [23]. Data from the 1999–2000 National
Health and Nutrition Examination Survey (NHANES)
indicates that the median intake of B
12
for the United
States (US) population is 3.4 mg/d [24]. It should be
noted that intakes are often reported collectively for
adults from different populations [25], despite the fact
that dietary intake of B
12
varies by sex and age. For
example, the evaluation of micronutrient consumption
across United Kingdom (UK) adults in their twenties,
thirties, forties, and fifties showed the daily B
12
intake
for females to be statistically lower amongst females
aged 20–59 years when compared with males of the
same age (p<.001) [25].
Recommended intakes
The recommended intakes were set to maintain normal
hematological values and serum B
12
within normal con-
centrations. They also assume that 50% of vitamin B
12
is absorbed from the diet. Intakes of 1.5 lg/d of B
12
are
recommended by the UK government [26] and 4 lg/d
(adequate intake [AI], covers the needs of all in the
group) by the European Union for adults [27]. The US
recommends an intake of 2.4 lg/d [28]. Higher intakes
are recommended for pregnancy and lactation in the
US (2.6 and 2.8 lg/d, respectively) and lactation in the
UK (2.0 lg/d). The recommended intakes for children
vary from 0.3 lg/d for 0–6 month olds to 1.0 lg/d for
9–13 year olds in the UK and 0.4 lg/d to 1.8 lg/d for
the same age groups in the US [26,28].
It remains debatable whether the above intakes are
sufficient to achieve optimum vitamin B
12
status. Using
a factorial approach, which includes a summation of
daily losses that need to be compensated for by dietary
intake of vitamin B
12
, Doets et al. [23] estimated that
vitamin B
12
intakes ranging from 3.8 lg/d to 20.7 lg/d
are needed to prevent deficiency in apparently healthy
adults and elderly people.
In addition to the above, due to the high prevalence
of food-bound malabsorption (inability to release vita-
min B
12
from its binding proteins) in people >60 years
of age, many European countries recommend food
products fortified with crystalline vitamin B
12
, as its
absorption is not affected in this condition [29].
Vitamin B
12
absorption
There are two mechanisms whereby B
12
is absorbed: (i)
passive diffusion and (ii) a receptor-mediated process.
Only about 1–2% of an oral dose can be absorbed pas-
sively via mucous membranes and the surface of the
gastrointestinal tract [30]; hence, high doses of oral B
12
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 3
(e.g. 1 mg daily) are required to provide an “adequate”
intake of B
12
if the specific transport does not function.
Absorption via receptors is initiated after release of
cobalamin from the dietary matrix. This begins in the
stomach due to the action of gastric acid and by the
digestion of food by pepsin. The stomach contains
B
12
–specific proteins as well as intrinsic factor (IF) and
haptocorrin (HC, also known as R-binder), which are
synthesized by gastric parietal cells and the salivary
glands, respectively. HC and IF compete for the liber-
ated vitamin but, at an acidic pH, HC has a higher affin-
ity for B
12
(Figure 2). When gastric contents enter the
first part of the duodenum, with an alkaline environ-
ment, R-binders become partly digested by pancreatic
proteases, freeing up the vitamin, which subsequently
attaches to IF.
In the terminal ileum, the vitamin B
12
-IF complex
binds to cubam receptors comprised of amnionless and
cubilin protein moieties on intestinal mucosal entero-
cytes [31]. The complex is then taken up by endocytosis
into the ileal cell. This process is highly specific and
only takes place at a neutral pH in the presence of
calcium ions. The receptors only take up the vitamin
B
12
bound to IF [32]. This step is rate-limiting since ileal
receptors have a finite capacity for B
12
amounting to
1.5–2.5 mg from a single meal. Based on early studies
with isotopes, it was shown that 50% of a 1 lg oral
dose was absorbed, 20% of a 5 lg dose, and 5% of a
25 lg/d dose (Figure 3)[33,34]. A recent study by Devi
et al. [35], which used [
13
C]-cyanocobalamin, achieved
similar results (Figure 3). The receptors are recycled to
the surface and become available again within 4–6h
[36]. After internalization, IF is degraded by lysosomal
proteases, releasing the vitamin B
12
for transport by the
lysosomal transmembrane protein, LMBD1 [37]. From
the cytosol, B
12
is exported to blood circulation by the
ATP-driven exporter, multidrug resistance protein
(MRP1) [38]. In the blood, free B
12
immediately binds
two proteins: transcobalamin (TC) and HC (Figure 2).
Transcobalamin is apparently synthesized by all tissues
and circulates in a predominately unsaturated form
[39]. Most newly-absorbed B
12
binds to TC, forming hol-
otranscobalamin (holoTC). HoloTC has a much faster
turnover than the B
12
–HC complex, holohaptocorrin
Figure 2. Vitamin B
12
absorption and intracellular processing via two enzymatic pathways. In the absence of vitamin B
12
, 5-MTHF
becomes metabolically trapped in this form producing a pseudo folate-deficient state (methyl-trap) and cannot be utilized for
regeneration of THF. Cbl: cobalamin; CBS: cystathionine beta-synthase; dTMP: deoxythymidine monophosphate; dUMP: deoxyuri-
dine monophosphate; DHFR: dihydrofolate reductase; HC: haptocorrin; holoTC: holotranscobalamin; HO-Cbl: hydroxocobalamin; IF:
intrinsic factor; MS: methionine synthase; Me-Cbl: methylcobalamin; MTHFR: methylene tetrahydrofolate reductase; MMA: methyl-
malonic acid; MCM: methylmalonyl-CoA mutase; 5-MTHF: 5-methyltetrahydrofolate; SAH: S-Adenosyl homocysteine; SAM: S-
Adenosyl methionine; THF: tetrahydrofolate; TS: thymidylate synthase; TC: transcobalamin.
4 A. SOBCZYŃSKA-MALEFORA ET AL.
(holoHC), but controversies exist over its rate of clear-
ance, which is likely to be attributed to methodologies
used for this estimation. This is best exemplified by
Hom et al., where authors first estimated half-life (t1/2)
¼1.5 h of labeled B
12
bound to TC [40], and 15 h [41]in
their later study, based on the assumption that radio-
active B
12
does not reappear due to reutilization once
cleared from plasma. This latter approach calculated
the plasma disappearance half-life obtained from the
final slope of the plasma curve, which probably
included both clearance and reappearance of B
12
[41].
A half-life of 6 min was reported by others and, more
recently, 7 min for clearance of a holoTC-conjugate in
patients with malignancies [42].
Only 10% of total TC is normally bound to B
12
and
this fraction is responsible for the transport of 4 nmol/
dofB
12
into cells [41]. If B
12
is no longer absorbed, cir-
culating holoTC is not formed. Because the half-life of
holoTC is much shorter than that of holoHC (10 days)
[41], low holoTC is believed to be the earliest indicator
that a patient is no longer absorbing vitamin B
12
from
food [43].
Conversely, HC in blood is almost fully saturated
with B
12
and inactive B
12
analogs, and although this
protein carries the majority of the vitamin in the circula-
tion (80%), only few sites are available for absorption
of HC-B
12
. Haptocorrin attaches to cell surface receptors
on the liver, with a turnover of 0.1 nmol/d of B
12
[44].
There is no evidence that HC has any role in the cellular
uptake of vitamin B
12
, however, its role is still poorly
understood. One suggestion for its function is the trans-
portation of potentially harmful cobalamin analogs
(other corrinoids) to the liver for secretion into the bile,
thus ensuring that only holoTC is available for cellular
uptake [45].
Vitamin B
12
excreted in the bile is effectively reab-
sorbed through enterohepatic circulation. The amount
of B
12
excreted in bile varies from 1 to 10 lg/d. Biliary
B
12
is reabsorbed across the ileal enterocytes and
requires IF, in the absence of which nearly all cobalamin
is excreted with the feces. People with a low dietary
intake of B
12
increase their efficiency of reabsorption of
B
12
. Efficient reabsorption is the reason why it can take
some time for deficiency to develop, even in those peo-
ple whose diets are very low in B
12
. In their systematic
review, Doets et al. [23] estimated that 0.13% of the
total body store is lost daily, while the mean store val-
ues were between 1.1 and 3.9 mg. Based on these val-
ues, total losses of B
12
would range from 1.4 to 5.1 lg/
d. Assuming a normal initial store (1.1–3.9 mg) and a
loss of 0.13% of B
12
per day, we can expect that 10% of
the whole store remains after approximately 692 days.
In his study, Paul Golding [46] experimentally addressed
the time scale for development of B
12
deficiency and
assessed it as 500–700 days based on vitamin B
12
markers being in deficiency ranges following a period
of lower or nil B
12
intake. There was a 71% decrease in
serum B
12
after 728 days of B
12
intake depletion and a
70% (609 days) and 81% (658 days) increase in plasma
homocysteine and MMA concentrations, respectively.
Figure 3. Relationship between the intake and percentage of absorbed B
12
from a dose of [
58
Co]-cyanocobalamin (Adams et al.
[34]) and [
13
C]-cyanocobalamin (Devi et al. [35]) in n¼10–12 healthy volunteers.
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 5
The subject in this study was diagnosed with B
12
defi-
ciency in the past, was B
12
replete at baseline, and
gradually decreased his B
12
intake to 0 mg on day 371
of this experiment; he then took 100 mg of vitamin B
12
for 4 weeks, but from day 497 to 751 his B
12
intake was
0mg[46]. Despite the variations in B
12
intake, the results
produced in this work suggest that the liver’s stores
may not maintain adequate vitamin B
12
status for a
long time. This was also recently suggested by
Kornerup et al. [47] in their study on patients following
bariatric surgery, where increased methylmalonic acid
and decreased holoTC were found as early as 2 months
after surgery.
For factors that can affect B
12
absorption, the reader
is referred to the malabsorption section.
Metabolic processing and function
There are only two intracellular enzymes for which vita-
min B
12
is required, namely, methionine synthase (MS)
in the cytosol and methylmalonyl-CoA mutase (MCM) in
the mitochondria. Cellular processing leading to forma-
tion of methylcobalamin and adenosylcobalamin, the
active vitamin forms supporting enzyme activity, starts
with the transport of holoTC into cells. There are recep-
tors for holoTC on the surface of all DNA-synthesizing
cells. Following endocytosis of the holoTC complex by
the CD320 receptor [48], the complex is degraded in
lysosomes and B
12
is released into the cytoplasm via
two proteins, LMBD1 (cblF) [37] and ABCD4 (cblJ) [49].
In the cytosol, the cblC protein dealkylates methylcoba-
lamin and adenosylcobalamin as well as decyanates
cyanocobalamin to cob(I)alamin, which, under aerobic
conditions, is oxidized to cob(II)alamin and
hydroxycob(III)alamin (cob(III)alamin) [12,50,51].
Kolhouse et al. [52] provided evidence that
cob(II)alamin was the form most active in binding to
apo-methionine synthase (cblG). CblC combines with
the cytosolic form of cblD to direct B
12
, probably as
cob(II)alamin, to methionine synthase (cblG) for the for-
mation of methylcobalamin (Me-Cbl) in the presence of
methionine synthase reductase (cblE) [53–55]. The mito-
chondrial form of cblD in combination with cblC directs
B
12
to mitochondria where it is converted to adenosyl-
cobalamin (Ado-Cbl) in the presence of the gene prod-
ucts of cblA, cblB, and methylmalonyl-CoA mutase
(MCM) (Figure 2)[11].
Methylcobalamin is essential for the remethylation
of homocysteine to methionine by MS; 5-methyltetrahy-
drofolate (5-MTHF) supplies the methyl group and is
converted to tetrahydrofolate (Figure 2). Methionine is
subsequently adenosylated to S-adenosylmethionine
(SAM) to supply methyl groups that are critical for the
methylation of proteins, phospholipids, neurotransmit-
ters, RNA, and DNA.
Conversely, Ado-Cbl is a cofactor for MCM, catalyzing
the conversion of methylmalonyl-CoA to succinyl-CoA.
Methylmalonyl-CoA is a degradation product of propi-
onate. In most mammals, propionate arises from the
utilization of some amino acids (isoleucine, valine,
methionine, threonine, thymine) or cholesterol as well
as after b-oxidation of odd-chain fatty acids.
Inborn errors of vitamin B
12
absorption and
intracellular metabolism
A number of genes that encode for proteins involved in
B
12
absorption and transport harbor highly penetrant
genetic mutations that cause inborn errors of vitamin
B
12
absorption and metabolism. All of these diseases
are characterized by a deficiency of the coenzyme
forms of B
12
. They can be successfully diagnosed and
treated with regular high doses of B
12
injections, B
12
supplements, and other therapies [56].
Inborn errors of B
12
absorption
Congenital PA is caused by mutations in a gene (GIF,
CBLIF) and defects in IF synthesis. The condition usually
presents during the first five years of life, but it can
manifest later if a partial defect is present [57]. This dis-
ease exhibits many features resembling non-dietary
adult PA, an autoimmune disorder associated with
autoantibodies to gastric parietal cells or gastric IF, as
well as Immerslund-Gr€
asbeck (IGS) disease [58].
IGS is a rare, potentially life-threatening, autosomal
recessive disorder caused by mutations in either the
CUBN or AMN gene, responsible for the synthesis of the
cubam receptors. This disorder is clinically highly het-
erogeneous. It is accompanied by a mild proteinuria in
50% of cases, owing to cubam receptor expression
also in kidney cells. In terms of treatment, life-long B
12
injections lead to resolution of PA and IGS-
anemia [58,59].
Genetically predetermined haptocorrin deficiency
leads to a benign condition not requiring treatment
[45]. Interestingly, some animals such as rodents do not
produce HC and survive with just TC and IF [60].
Diagnosis is often suspected following a low serum B
12
concentration in the absence of any symptoms related
to deficiency. Conversely, genes that cause transcobala-
min deficiency will often result in a severe phenotype
[61] if not treated with high doses of vitamin B
12
from
an early age.
6 A. SOBCZYŃSKA-MALEFORA ET AL.
Inborn errors of intracellular metabolism
To rationalize the etiological heterogeneity of these dis-
orders, they have been catalogued into a total of eight
defects of Cbl metabolism [57], designated cblA-cblG
and mut, defined by means of in vitro somatic comple-
mentation analysis. Defects in genes encoding for cbl
proteins result in a failure to utilize B
12
by the target
cells irrespective of its normal supply. In the cblC, cblD,
cblF, and cblJ group of disorders, the synthesis of both
Ado-Cbl and Me-Cbl are affected, leading to combined
methylmalonic aciduria and homocystinuria. In cblA
and cblB disorders, the synthesis of Ado-Cbl is impaired,
resulting in methylmalonic aciduria but normal homo-
cysteine concentrations, whilst cblE and cblG disorders
result in homocystinuria without methylmalonic acidu-
ria [57]. Finally, the mut defect describes mutations
(>200 identified) in the MUT gene (encodes for MCM)
that partially (class mut
) or totally (class mut
0
) abolish
MCM-mediated conversion of methylmalonyl-CoA to
succinyl-CoA and so perturb substrate (odd-chain fatty
acids, branched-chain amino acids, and cholesterol) util-
ization and energy metabolism in the mitochon-
dria [62].
Of the eight defects, the cblC defect is the most
common, although still rare with an incidence 1/
200,000 births. Clinical manifestations often appear in
infancy but can also occur later in life. Typical symp-
toms associated with early-onset disease are feeding
difficulties, failure to thrive, hypotonia, seizures, pig-
mentary retinopathy, developmental delay, and macro-
cytic anemia. Late-onset disease is frequently
accompanied by neurological dysfunction, including
cognitive decline, confusion, psychosis, or dementia. In
comparison with early-onset patients, late-onset
patients have better survival and response to therapy
[57,63]. For a detailed description of other intracellular
disorders and response to treatment the reader is
referred to detailed reviews on this subject [57,62,64].
Genetic polymorphisms associated with poor
B
12
status
Developments in genomic technology have allowed us
to move beyond single-gene disorders and to study the
polygenic basis of vitamin B
12
status. This has led to the
discovery of a myriad of new genes/pathways that har-
bor genetic polymorphisms associated with variable B
12
status among different population groups. The most
extreme consequences of genetic variation have
already been described in the case of rare inborn errors
of B
12
metabolism. More common effects of genetic
variation in B
12
levels can be described by its heritability
(h
2
) in different populations as well as associations
between specific polymorphisms in genes and the vari-
ous indicators of B
12
status. Dib et al. [65] have pro-
vided robust h
2
estimates for all markers of vitamin B
12
status for UK-based Caucasian populations that range
from 15% in the case of MMA right up to 80% for
holoTC, highlighting that B
12
status is a complex and
multifactorial trait, whereby several polymorphisms in
multiple genes interact with the environment to cause
variable B
12
status. So far, candidate gene and genome-
wide association studies (GWAS) have identified 59
SNPs from 19 genes involved in cobalamin metabolism
and various indicators of its status [66]. We refer the
reader to Surendran et al. [66], who provide a very use-
ful guide for each gene and polymorphism associated
with B
12
status, but would like to highlight that most, if
not all, of the findings thus far refer to “associations”
identified mainly through observational studies with
currently unclear clinical and pathophysiological signifi-
cance. Therefore, “causal”genetic determinants of vita-
min B
12
status, functional consequences, and context
dependency remain poorly understood [65,67].
Corrinoids and microbiome
As stated before, cobalamin belongs to a family of com-
pounds referred to as corrinoids. Corrinoids with a
cobalt ion in the corrin ring are called cobamides.
Vitamin B
12
is differentiated from other cobamides (ana-
logs), which usually cannot provide cofactor activity in
mammalian cells, by having a 50,60-dimethyl benzimida-
zole group attached to the lower nucleotide loop.
There are also other corrinoids (analogs) that differ in
the tetrapyrrole ring or have a central atom other than
cobalt, such as nickel, copper, or zinc. Corrinoids are
synthesized exclusively by bacteria and archaea, and
are abundant in the large intestine due to the activity
of the gut microbiota [68]. Corrinoids play key roles in
shaping the gut microbial community composition
and diversity [69]. A majority of human gut microbial
species either directly require access to the host’s diet-
ary-derived B
12
or are dependent on analogs [70] pro-
duced by other bacteria [71]. In humans, bacterial
synthesis of vitamin B
12
and analogs takes place only in
the large intestine and the cecum, and there is
evidence that humans cannot utilize bacterial B
12
/ana-
logs [32] as part of the orchestrated steps that ensure
adequate absorption and reabsorption of dietary-only
Cbl. However, there is some evidence to show that cer-
tain diseases or age-related changes to the gastrointes-
tinal tract can lead to protein-bound vitamin B
12
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 7
malabsorption and that bacterial-derived analogs may
play a role [72–74].
Analogs act as cofactors for corrinoid-dependent
enzymes for gut microbes, hence the constituents of the
microbiome compete with the host, as well as other bac-
teria, for B
12
or corrinoid analogs, in order to ensure via-
bility and stability of the microbiome ecology. Previous
studies have predicted that 86% of human gut bacteria
species are dependent on corrinoids as cofactors but
<25% have the capacity to synthesize them de novo
[75]. Although many bacteria cannot synthesize B
12
, they
can remodel it by removing the original nucleotide and
adding a new one. About 50% of total dietary Cbl is
thought to be converted to other corrinoids by the gut
bacteria [76]. Therefore, a majority of gut bacteria either
rely on Cbl-uptake mechanisms from either the host’s
diet or are dependent on related corrinoids [77] pro-
duced by other bacteria [78]. This is likely associated
with the individual variability in dietary Cbl exposure as
well as the specificity of the individual gut microbial
community composition. For example, Visconti et al. [79]
have shown that only 43% of bacterial species are
shared by any two randomly selected individuals from
the population. Estimates suggest high dietary deficiency
rates in the population, especially in an exponentially
increasing number of individuals who, for ethical and
health reasons and also encouraged by scientific obser-
vations, have been shifting toward a higher consumption
of plant-based food [80]. Although Cbl deficiency is a
modifiable risk factor, which itself is associated with a
number of rare/common non-communicable (neuro-
logical/vascular) diseases [81], evidence of the specific
role of Cbl and other corrinoids, in shaping the gut bac-
terial community and as mediators of human-micro-
biome symbiosis, and their impact on human health,
remains largely unknown [76,82,83]. Analogs were found
to delay growth in chicks [84] or induce severe demyeli-
nating disease in baboons [85]. Mechanisms that prevent
absorption and tissue dissemination of analogs have
evolved, including the poor affinity for IF. Of those that
are bound by IF, most appear to be retained by the
ileum. Analogs that do reach plasma are largely bound
to haptocorrin and are transported to the liver and
excreted via the bile and feces. Analogs may also be
bound by transcobalamin and transported to tissue [83],
but the amount absorbed might be insignificant [86].
Diagnosis of deficiency and insufficiency –
clinical and laboratory pitfalls
One might suppose that diagnosing B
12
deficiency
should be relatively simple. The laboratory provides a
diagnostic “cutoff”point, the patient is proclaimed to
be deficient, and the vitamin should be replenished.
The truth can be far more complicated, especially if
insufficiency of B
12
(subclinical deficiency) is also con-
sidered. Clinical vitamin B
12
deficiency is often the
result of prolonged and severe malabsorption and
patients are symptomatic, while in insufficiency, symp-
toms associated with B
12
deficiency are not well
expressed but biochemical markers, most notably total
plasma homocysteine (tHcy) and methylmalonic acid
(MMA), are elevated [87]. In such cases, serum B
12
con-
centration is often normal. However, relatively high
holoTC, low serum B
12
, and high MMA may also be
seen in some patient cohorts with B
12
insufficiency [88].
Therefore, in the course of B
12
status assessment, the
physician can encounter pitfalls at each step of the pro-
cess including: (A) consideration of B
12
deficiency as a
major cause for presenting illness; (B) testing for B
12
deficiency/insufficiency and interpreting results; (C)
investigating the potential causes of deficiency/insuffi-
ciency; (D) treatment; and (E) considering the associ-
ated risks.
Consideration of B
12
deficiency as a major cause
of presenting illness
B
12
deficiency can masquerade as a multitude of ill-
nesses; therefore, the physician requires considerable
clinical acumen. The disease presents different perspec-
tives from the point of view of the hematologist, neur-
ologist, gastroenterologist, general physician, dietician,
and psychiatrist, and is a prime example of
“elephanomics”[89]. A high index of suspicion is
required. Importantly, the absence of anemia does not
preclude a positive diagnosis, a trap easily fallen into
since many physicians erroneously equate B
12
defi-
ciency with PA, though the latter is only one potential
cause of the former [90].
Classical features of deficiency include those relating
to anemia (weakness, tiredness, dyspnea on exertion),
gastrointestinal symptoms (appetite loss, sore tongue
and mouth, epigastric discomfort, nausea, vomiting,
heartburn), neurological symptoms (numbness of
extremities, pins and needles, impaired fine finger
movements, gait ataxia, positive Romberg’s sign,
impaired vibration and position sense, orthostatic dizzi-
ness, loss of taste or smell), and those relating to psy-
chological and psychiatric disturbances (irritability,
personality change, memory and intellectual impair-
ment, disorientation, depression, psychomotor slowing,
delirium, dementia). The deficiency predominantly
affects hematological and neurological parameters, but
8 A. SOBCZYŃSKA-MALEFORA ET AL.
neuropsychiatric symptoms are often the first clinical
manifestation [91]. Gynecological and urological symp-
tomatology (infertility, cystitis, and pyelonephritis),
though rare, should also be considered. The full spec-
trum of clinical features associated with deficiency is
perhaps broader. In a survey of individuals with PA,
symptomatology also included brittle nails, flushing,
fever, hair loss and graying, and nominal aphasia [92].
Occasionally, the deficiency is entirely asymptomatic, its
discovery arising from the alert physician pursuing
abnormal results from a routine full blood count.
Megaloblastic anemia describes the morphological
features of hematopoietic tissue caused by a deficiency
of B
12
(and/or folate). It includes ineffective erythropoi-
esis, moderate hemolysis, and inefficient leukopoiesis
and thrombopoiesis [93]. Abnormal morphology of
blood elements include normochromic and macrocytic
erythrocytes, lower reticulocyte count, hyper seg-
mented neutrophils, abnormal morphology of bone
marrow cells, and megaloblastic changes in granulo-
cytes and platelet precursors [93]. Megaloblastic cells
are in a state of unbalanced growth with impairment of
DNA synthesis and an increase in the RNA/DNA ratio.
Morphological mimics include myelodysplastic syn-
dromes and sideroblastic anemia [94]. Conversely, B
12
deficiency can co-exist with iron deficiency or thalas-
semia, thus masking macrocytosis.
It was generally believed that hematological features
preceded any neuropsychiatric abnormality, but in the
last few decades it has been noted that these manifes-
tations might be considered as two major, and some-
times entirely separate (or even excluding each other),
clinical syndromes [95,96]. It remains unclear why some
B
12
-deficient patients present with neurological disor-
ders in the absence of hematological changes and why
some develop a predominantly cerebral picture, while
others a spinal or peripheral nerve disorder. This may
be partially related to folate status, as folate deficiency
is primarily responsible for hematological changes due
to defective DNA synthesis. A good supply of folates
may delay the hematological symptoms of B
12
defi-
ciency, even though tetrahydrofolate (THF) is not
regenerated from 5-MTHF (Figure 2). In the absence of
vitamin B
12
, 5-MTHF becomes metabolically trapped in
this form, producing a pseudo folate-deficient state
(methyl-trap) [97]. The methyl-trap hypothesis explains
why vitamin B
12
deficiency produces apparently identi-
cal megaloblastic anemia to that seen in folate defi-
ciency. In both cases (folate and vitamin B
12
deficiency),
the cells would be deficient in folate cofactors required
for DNA and RNA synthesis. It also explains the clinical
observation that treating a patient with megaloblastic
anemia caused by vitamin B
12
deficiency with a
pharmacological dose of folic acid allows the cells of
the bone marrow to start to divide again. It is thought
that the new folate molecules would carry out several
such cycles before they are trapped as 5-MTHF (Figure
2). If folic acid continued to be administered to a
patient with megaloblastic anemia, which would occur
if the wrong clinical diagnosis had been made, hemato-
logical remission would be maintained [97].
Our “preconception”of B
12
deficiency still associates it
with anemia, and its diagnosis usually proceeds in three
steps: the recognition of anemia, the observation that it
is macrocytic, and finally, the confirmation of an underly-
ing deficiency. However, if suspicion of the deficiency
were based on the presence of macrocytic anemia alone,
a substantial proportion of B
12
-deficient individuals would
escape detection [90,98], a point elegantly expressed by
Carmel, who notes that “The proscription that vitamin
B
12
deficiency should not be diagnosed unless megalo-
blastic changes are found is akin to requiring the pres-
ence of jaundice to diagnose liver disease”[99].
Early presentation, due to increased physician vigi-
lance and heightened patient awareness, can result in
tests during a phase of the illness when laboratory find-
ings of a “full-blown”deficiency are not yet apparent.
Before patients become clinically deficient, they tra-
verse earlier stages, beginning with insufficiency. The
concept of B
12
deficiency as a gradually progressive dis-
order [99] is an important development over the last
30 years [100]. The five stages of vitamin B
12
status/defi-
ciency described by Victor Herbert in 1987 [100] are
likely to be applicable to the majority of cases today:
stage I –normal vitamin B
12
status; stage II –negative
vitamin B
12
balance; stage III –vitamin B
12
depletion
accompanied by possible clinical signs and symptoms
such as fatigue, dizziness, nausea or poor appetite, diar-
rhea, palpitations and rapid heartbeat, bleeding gums,
and mouth sores; stage IV –deficient erythropoiesis
with potentially reversible neurological symptoms such
as numbness and tingling in the hands and feet, ataxia,
muscle weakness, depression and psychosis; and stage
V–related anemia with possible irreversible neuro-
logical symptoms.
Testing for B
12
deficiency/insufficiency and
interpretation of laboratory results
“We sanguinely hope that the laboratory will answer our
questions with a dogmatic ‘yes’or ‘no’; its usual answer
is perhaps”(Anon).
There are four biological markers of B
12
deficiency: total
serum B
12
, holoTC (also known as “Active B
12
”), and
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 9
measures of the related metabolites tHcy and MMA.
Each has its limitations, which the physician should be
alert to.
Serum B
12
test
Serum B
12
is the most frequently used laboratory
marker of B
12
status, measuring the circulatory concen-
tration of B
12
bound to both vitamin binding proteins,
TC, and HC. As is the case for a number of other
vitamins, serum B
12
was initially measured by microbio-
logical methods, for example, using Lactobacillus leich-
mannii, for which cobalamin is an obligate nutrient
[101]. Microbiological assays for B
12
suffered from inter-
laboratory variability, interference by antibiotics, and an
extended turnaround time inherent to the assay prin-
ciple. The main disadvantage however is its low specifi-
city when compared to IF-based assays [102]. These
assays were challenged by nonspecific R-protein-bind-
ing assays due to availability of R-proteins from saliva,
for example. However, the values obtained with these
were markedly higher than those obtained with the
microbiological assays, as they measured a broad var-
iety of cobalamin analogs that are not necessarily bio-
logically active [83].
Since the 1990s, clinical laboratories have opted for
high throughput automated competitive binding
chemiluminescence assays (CBLA), using purified IF as a
reagent, to measure total cobalamin after its release
from endogenous binding proteins [103]. Caution
should however be observed when interpreting results,
since some of these assays could be influenced by the
presence of interfering anti-IF antibodies, particularly in
patients with PA, thereby providing spuriously elevated
serum cobalamin concentrations in otherwise B
12
-defi-
cient patients [104–106].
Serum B
12
can also be determined using B
12
-anti-
bodies and B
12
enzyme-linked immunosorbent assay
(ELISA) kits. Various kits are available. Although these
could potentially provide an alternative to IF-based
assays, they have not been completely veri-
fied [107–109].
Functional indicators of B
12
status, tHcy and MMA,
are based on the rationale that deficiency leads to the
deactivation of two key enzymes of the one-carbon
cycle, namely MS and mitochondrial MCM, causing the
accumulation of tHcy and MMA, respectively [110].
While providing a better insight into the B
12
status of
different groups of individuals, the lack of consensus in
choosing cutoff values for each of the biomarkers
remains problematic in diagnosing B
12
deficiency.
Reported cutoffs for B
12
vary from as low as 100 pmol/L
(Immulite 2000) when associated with plasma MMA
concentrations 260 nmol/L [111], to as high as
258 pmol/L (IF radioassay, Quantaphase) on the basis of
increased MMA [112]. These two reports exemplify the
uncertainties in the reported cutoffs for individual bio-
markers of B
12
status. Shelub et al. [113], in a study
based on NHANES III 1991–1994 and NHANES
1999–2002 involving 5000 middle-aged participants
in both cohorts, used <148 pmol/L as the conventional
cutoff for B
12
deficiency. Using this cutoff, they reported
a prevalence of 1.6 to 2.2% of the deficiency in NHANES
III and NHANES, respectively, and showed significant
differences in circulating tHcy and MMA between sub-
jects with B
12
deficiency vs normal status. In a 2001 epi-
demiological study based on combined NHAHES
1999–2000, 2001–2002 and 2003–2004 data totaling
12,612 middle-aged adult participants, Bailey et al.
[114] reported a steady increase in the prevalence of
B
12
deficiency, from 2.9% to 25.7% by moving decision
points from <148 pmol/L to >296 pmol/L of total
serum B
12
. They observed a similar trend but of much
less importance (2.3 ± 0.2% to 5.8 ± 0.3%) when the cut-
off point for MMA was decreased from >376 to
>271 nmol/L. Concerned by the impact of different B
12
and MMA cutoffs in the diagnosis of clinical outcomes
and using data collected from the 1999–2004 surveys
and different statistical models, Bailey et al. [115]
attempted to identify a single change point at which
the relation between plasma MMA and serum B
12
changes slope to differentiate between inadequate and
adequate B
12
status, but the results were not as simple
as expected. They reported three slopes resulting in
two change points for three subgroups. The first sub-
group had serum B
12
<126 pmol/L and the highest
median plasma MMA of 281 nmol/L. This group, repre-
senting 1.2% of the population studied, was considered
at high risk for severe deficiency because of the fre-
quently accompanying elevation of MMA and tHcy. The
second group, representing 67.6% of the population
studied, in which serum B
12
was >287 pmol/L, likely
had adequate B
12
status with a median MMA concen-
tration of 120 nmol/L. The third group (33%) was classi-
fied as indeterminate and difficult to interpret because
of intermediate serum concentrations of B
12
(126–287 pmol/L). Although none of these studies pro-
vide a clear cutoff, they all call for the measurement of
associated metabolites when assessing B
12
status.
Holotranscobalamin testing
HoloTC, the bioactive form of B
12
, is also routinely used
for evaluating B
12
status, often in combination with an
MMA test [116,117]. It remains debatable whether
holoTC “alone”serves any better than the measurement
10 A. SOBCZYŃSKA-MALEFORA ET AL.
of serum B
12
. It has particularly been criticized by
Golding [118], who provides an extensive review and
argumentation concluding that “the holoTC immuno-
assay cannot be used to measure B
12
status any more
reliably than total B
12
, or to predict the onset of a meta-
bolic deficiency, because it is based on an erroneous
hypothesis and a flawed model for the staged develop-
ment of B
12
deficiency.”This conclusion is challenged
by Kornerup et al. [47], who provide evidence that
holoTC is superior to total B
12
in detecting early
changes in the vitamin’s status following bariatric sur-
gery. Jarquin Campos et al. [119] also demonstrated
from a cohort study of 11,833 samples that holoTC had
the highest diagnostic accuracy for recognizing B
12
insufficiency, with significantly higher diagnostic accur-
acy than B
12
and tHcy. The sensitivity of holoTC was
found to be between 0.55–0.87 vs 0.33–0.76 for total
B
12
, whilst the specificity ranged from 0.50–0.96 and
0.41–0.98 for holoTC and B
12
, respectively, in various
studies summarized by Golding [118]. The variations in
sensitivity and specificity values largely depended on
the MMA cutoffs used, which served as the proxy gold
standard marker representative of metabolic vitamin
B
12
deficiency in these studies. Most of these studies
demonstrated only a slightly higher sensitivity and spe-
cificity for holoTC than total B
12
. Conversely, the correl-
ation coefficient (r), which reflects the relationship
between holoTC and B
12
, ranged from 0.42 to 0.882 in
twenty-two studies summarized by Golding in his
review [118]. A high correlation over a wide range of
values may imply that holoTC could not detect the
onset of B
12
deficiency earlier than total B
12
[118].
However, separate r values for low and high B
12
were
not provided in most of the studies. Herrmann and
Obeid [120] additionally reported r¼0.524 for low total
B
12
<300 pmol/L and 0.403 for values above
300 pmol/L. The authors concluded that total B
12
was
a poor predictor of holoTC, and low holoTC occurred at
higher total vitamin B
12
concentrations [120]. The r
value (Spearman’s) based on randomly selected, con-
secutive diagnostic results from the authors’laboratory
(AS-M and DJH) was 0.741 (N¼298) across the whole
holoTC and B
12
range (unpublished data). Separate cor-
relation analysis of holoTC <25 pmol/L (deficiency cut-
off) with total B
12
gave r¼0.404 (N¼175) and for
holoTC 25 pmol/L, r¼0.627 (N¼123) (Figures 4 and
5). Assuming B
12
deficiency based on holoTC
<25 pmol/L, 56.0% of patients would have been classi-
fied as sufficient if the serum total B
12
test with a cutoff
of 138 pmol/L had been used (Figure 3).
In addition, holoTC has been shown to be unaffected
by assay interference from high-titer IF antibody levels
[121]. Furthermore, holoTC is not subject to the 50%
fall as seen for total B
12
in pregnancy [122]. The diag-
nostic utility of holoTC in other cohorts requires further
study and evaluation.
The holoTC assay is useful when transcobalamin and
haptocorrin deficiencies are suspected (Table 1). In hap-
tocorrin deficiency, holoTC concentration is normal/
high while total serum B
12
(the sum of holohaptocorrin
and holoTC) can indicate severe deficiency. In haptocor-
rin deficiency, the serum B
12
test measures the holoTC
fraction only. Without the availability of holoTC testing,
a clinician may wrongly diagnose a patient and
Figure 4. The correlation between holoTC and serum B
12
in the low holoTC range, <25 pmol/L. The horizontal line on the y-axis
represents the serum B
12
deficiency cutoff of 138 pmol/L used in the authors’laboratory (AS-M and DJH).
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 11
incorrectly prescribe B
12
replacement for an otherwise
benign condition. In transcobalamin deficiency how-
ever, early treatment can lead to a good clinical out-
come. In this condition, holoTC is unmeasurable since
deficiency of the transcobalamin protein does not allow
for B
12
binding and the formation of holoTC. Ultimately,
genetics analysis can confirm the diagnosis of
both conditions.
Conversely, unmeasurable holoTC may rarely imply
variants in the transcobalamin gene that interfere with
assays such as “Active B
12
”[123,124]. For instance, the
minor allele rs35838082 (p.R215W), which is rare in
Caucasians with a minor allele frequency (MAF) of
<0.01 but more common in South Asians (MAF 0.02)
and those of African origin (MAF 0.25), was found to
interfere with the “Active B
12
”test [124]. HoloTC results
in these patients are erroneously low (<5 pmol/L), des-
pite all other B
12
laboratory markers being normal and
an absence of clinical symptoms [124].
Transcobalamin receptor (TCblR/CD320) polymor-
phisms may also have an impact on holoTC concentra-
tions. In an elderly population, 5% were found to have
a heterozygous codon 88 GAG deletion [125]. This is
associated with proportionately more serum B
12
bound
to holoTC. Hence, holoTC might not be a marker of
“true”intracellular B
12
status in some individuals; an ele-
vated holoTC may reflect decreased cellular uptake due
to the GAG deletion.
Figure 5. The correlation between holoTC and serum B
12
in the holoTC range 25 pmol/L. The horizontal line on the y-axis rep-
resents the serum B
12
deficiency cutoff of 138 pmol/L and the vertical line represents the holoTC cutoff of 70 pmol/L for B
12
replete patients as used in authors’laboratory (AS-M and DJH). Samples with holoTC results between 25–70 pmol/L are referred
for additional testing.
Table 1. The commonly seen vitamin B
12
marker patterns in selected clinical scenarios.
Serum holoTC Serum total B12 Plasma MMA Plasma tHcy Possible diagnosis
NN""Suboptimal B
12
status
##Nor""Mild B
12
deficiency, on antibiotics
NN"N Bacterial overgrowth, B
12
replete
### ### "" "" Pernicious anemia
Nor##Nor"N Pregnancy, B
12
replete
###"Nor"Pregnancy, B
12
deficiency
NNN"-""" Mild to severe folate deficiency, B
12
replete
Nor"##N N Haptocorrin deficiency
### Nor#or """ ""Transcobalamin deficiency
NN""" """ CblC, D, F, J disorder
NN""" N CblA, B disorder
NNN""" CblE, G disorder
Nor#Nor#Nor" """ Nitrous oxide abuse
"" "" N N On vitamin B
12
injections
""" """ N N Chronic myeloid leukemia
Cbl: cobalamin; holoTC: holotranscobalamin; MMA: methylmalonic acid; tHcy: total plasma homocysteine. ###: very low/undetectable concentration; ##:
low; #: decreased; N: within reference range; ": elevated; "": highly elevated; """: very highly elevated.
12 A. SOBCZYŃSKA-MALEFORA ET AL.
Functional markers of B
12
status
Plasma tHcy and serum MMA are very sensitive markers,
especially for B
12
insufficiency, but are also influenced
by other conditions independently of B
12
status. Most
notably, renal impairment and hypothyroidism lead to
elevations of tHcy and MMA, making these tests unreli-
able. MMA begins to accumulate from the early stages
of renal impairment, leading to a decreased specificity
Table 2. Selected serum/plasma results from longitudinal studies of uncomplicated pregnancies: pre-conception, pregnancy,
labor, post-delivery and cord samples.
Geo
region Variable (unit) Methodology
Sampling time
Pre-conception/weeks
pregnancy/labor/cord/
weeks postD (n)
Concentration
(range)
Concentration/
range definition Reference
Germany Vitamin B
12
(pmol/L)
FP IF
Ligand assay
9–12 weeks (31) 257 (226–292) Geometric Mean
(95% CI)
Koebnick et al.
2002 [111]20–22 weeks (39) 239 (212–268)
36–38 weeks (38) 178 (161–198)
tHcy (mmol/L) HPLC 9–12 weeks (31) 6.9 (6.2–7.6) Mean (95
th
CI)
20–22 weeks (39) 5.9 (5.4–6.3)
36–38 weeks (38) 6.6 (5.9–7.2)
Spain Vitamin B
12
(pmol/L)
Microbiological assay
L. leichmannii
Pre-conception (89) 293 (155–535) Geometric Mean
(10
th
-90
th
percentiles)
Murphy et al.
2007 [109]8 weeks (88) 267 (144–449)
20 weeks (90) 230 (123–432)
32 weeks (90) 198 (107–339)
Labor (84) 224 (117–444)
Cord (82) 325 (146–641)
holoTC
(pmol/L)
MEIA Pre-conception (56) 63 (38–98)
8 weeks (58) 47 (31–74)
20 weeks (61) 48 (34–78)
32 weeks (60) 45 (26–82)
Labor (49) 40 (23–79)
Cord (23) 92 (45–214)
MMA
(nmol/L)
GC-MS Pre-conception (88) 120 (90–170)
8 weeks (87) 110 (90–170)
20 weeks (89) 110 (80–150)
32 weeks (90) 140 (90–200)
Labor (83) 140 (90–210)
Cord (72) 240 (130–400)
Denmark Vitamin B
12
(pmol/L)
MEIA 18 weeks (441) 216 (96–484) Mean (±1.96SD) Milman et al.
2007 [112]32 weeks (310) 169 (73–388)
39 weeks (266) 154 (71–333)
8 weeks PostD (170) 315 (148–672)
MMA
(nmol/L)
SID-MS 18 weeks (413) 110 (40–290)
32 weeks (390) 130 (50–340)
39 weeks (250) 150 (60–360)
8 weeks PostD (160) 160 (80–350)
Hcy
(mmol/L)
GC-MS 18 weeks (416) 6.1 (3.4–11.0)
32 weeks (291) 6.6 (3.9–11.1)
39 weeks (252) 7.8 (4.7–12.8)
8 weeks PostD (158) 11.0 (5.8–20.6)
Canada Vitamin B
12
(pmol/L)
Competitive-binding
immunoenzymatic assay
12–16 weeks (188–342) 219 (210–229) Mean (95
th
CI) Visentin et al.
2016 [113]Labor (188–342) 169 (162–176)
Cord (188–342) 321 (300–344)
MMA
(nmol/L)
LC-MS/MS 12–16 weeks (188–342) 109 (105–114)
Labor (188–342) 136 (129–142)
Cord (188–342) 313 (297–331)
tHcy
(mmol/L)
Enzymatic assay 12–16 weeks (188–342) 5.0 (4.9–5.1)
Labor (188–342) 6.0 (5.8–6.2)
Cord (188–342) 4.9 (4.7–5.1)
USA Vitamin B
12
(pmol/L)
Electrochemiluminescent
immunoassay
12–30 weeks (124) 343 (238–401) Median (IQR) Finkelstein et al.
2019 [114]Labor (75) 216 (173–312)
Cord (58) 569 (479–844)
Canada Vitamin B
12
(pmol/L)
Binding Immuno
Enzymatic Assay
8.3–13.9 weeks (596) 216 (89.9,519) G-Mean (lower and
upper limits [90%
CI] of central
95% RI)
Schroder et al.
2019 [115]14.9–20.9 weeks (640) 198 (84.0, 473)
holoTC
(pmol/L)
MEIA 8.3–13.9 weeks (587) 83.6 (29.5, NA
a
)
14.9–20.9 weeks (567) 76.5 (26.0, NA
a
)
MMA
(nmol/L)
LC-MS/MS 8.3–13.9 weeks (586) 130 (69.6, 371)
14.9–20.9 weeks (601) 120 (51.0, 374)
CI: confidence interval; GC: gas chromatography; FP: fluorescence polarization; HPLC: high performance liquid chromatography; holoTC: holotranscobala-
min; IQR: interquartile range; IF: intrinsic factor; LC-MS/MS: liquid chromatography-tandem mass spectrometry; MS: mass spectrometry; MMA: methylma-
lonic acid; MEIA: microparticle enzyme immunoassay; NA: not applicable; postD: post-delivery; SID: stable isotope dilution; n: study size; tHcy: total plasma
homocysteine.
a
The upper limit for holoTC is undefined.
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 13
for B
12
deficiency. Urinary MMA/creatinine ratio (uMMA/
C) may have diagnostic utility, due to the stable urinary
excretion of MMA before severe renal failure [126]. With
a threshold of 1.45 mmol/mmol, the uMMA/C ratio has
been associated with good diagnostic performance for
B
12
deficiency as a second line assay [127].
High tHcy is also a strong indicator of folate defi-
ciency, therefore concomitant assessment of both vita-
mins is required if tHcy is being utilized as a marker of
B
12
status. Postprandial and orthostatic variations, as
well as the prompt separation of plasma following
blood collection, are preanalytical factors that need to
be addressed before a sample is collected for tHcy
measurement [128]. Usage of special tubes may be con-
sidered if separation of plasma within 1 h of blood col-
lection is not viable [128]. Bacterial overgrowth and
hemoconcentration may additionally influence MMA
concentrations. Both markers are age-dependent, and
tHcy is higher in males.
All the above factors need to be considered when
interpreting B
12
results (Table 1). A combination of tests
for the assessment of B
12
status is preferable, but tests
need to be carefully chosen while considering factors
affecting B
12
markers independently of status. An
understanding of the laboratory reference ranges, and
using them as a guidance only rather than an indicator
of “firm”B
12
status, will also help to avoid misdiagnos-
ing patients [129].
Combined indicator of B
12
status
If markers of B
12
status do not show a “clear-cut”defi-
ciency, the absence of a definitive diagnostic test can
cause difficulty. Fedosov et al. [130]proposedasolu-
tion to this diagnostic dilemma using a “combined”
indicator of B
12
status (cB
12
or 4cB
12
). This is a rigor-
ously derived mathematical model combining all four
markers (total B
12
, holoTC, tHcy, and MMA), which
makes adjustments for age and folate status. It yields
one of five diagnoses: elevated B
12
,adequateB
12
,
decreased B
12
,possiblyB
12
-deficient, and probably
B
12
-deficient [131]. The interpretive comments from
individual B
12
markerscombinedvstheoutcomefrom
4cB
12
calculations in n¼44 patient samples analyzed
in the authors’laboratory (AS-M and DJH) showed
100% agreement in B
12
status assessment (unpub-
lished data).
The main drawbacks of 4cB
12
are cost and the rela-
tive lack of availability of all tests in routine practice. A
refined model now provides “missing markers”[131],
and although additional clinical research is required,
this approach should be more widely appreciated. Our
calculations using the 2cB
12
formula (holoTC and MMA
values only) for n¼3290 patients with holoTC 25-
70 pmol/L showed that 1% of patients within this group
were classified as possibly or probably B
12
-deficient
(“need immediate intervention with intramuscular (IM)
injections”), while 14% were classified as having
decreased B
12
(“start B
12
supplements”). Using the MMA
test as a confirmatory marker for this holoTC range in
the same cohort yielded 24% of patients with elevated
MMA [116].
The cB
12
model has currently not been validated for
use in pregnancy, however, our unpublished results
suggest its potential use [132].
Considering groups at risk and potential causes
for B
12
deficiency/insufficiency
Once a diagnosis of B
12
deficiency/insufficiency is
made, it is incumbent on the physician to determine
potential causes of deficiency/insufficiency, an import-
ant but often overlooked stage. Dietary and family his-
tory, pregnancy, age, concomitant nutrient deficiencies
(folate and iron in particular), malabsorption, surgery,
bacterial overgrowth, or pharmaceutical interactions
should all be considered or investigated, as appropriate.
This will determine the appropriate type, and duration,
of treatment.
Restricted dietary intake (veganism/vegetarianism)
Vegetarian and vegan diets have become very popular
in the last few decades, especially in developed coun-
tries, in view of their potential benefits to prevent cor-
onary heart disease, cancer, and type 2 diabetes, as well
as due to ethical and environmental issues [133].
However, these diets have restricted the intake of cer-
tain nutrients, including vitamin B
12
. Also, non-vegeta-
rians in the developing world can have low vitamin B
12
intake due to the high cost of meat in these areas
[134]. In addition, in countries such as India or
Bangladesh, vegetarianism has been practiced for cen-
turies for religious and cultural reasons [135] and it is
well documented that the prevalence of vitamin B
12
deficiency is high in these regions. Both vegans/vegeta-
rians are unlikely to achieve recommended dietary
allowances, since plant derived foods have no, or trace,
B
12
contents [136,137]. A certain amount of B
12
may be
provided by plants contaminated with B
12
-producing
bacteria through fertilization with manure [135], since
feces are a good source of vitamin B
12
[76]. It was also
observed that in largely vegetarian populations in pla-
ces where hygiene standards might be low, poor peo-
ple had a better B
12
status than the urban middle-class.
This is possibly due to microbial vitamin B
12
from
14 A. SOBCZYŃSKA-MALEFORA ET AL.
ingestion of contaminated food and water. Using
150 pmol/L as threshold, 81% of urban middle-class
men had low vitamin B
12
-concentration compared to
51% of slum residents [138].
In a European study of 29 vegans, 66 lacto-vegeta-
rians or lacto-ovo-vegetarians, and 79 omnivores who
were not taking supplements, vegans had the lowest
B
12
status; 92% had holoTC <35 pmol/L and 83% had
MMA >271 nmol/L [139]. The prevalence of low holoTC
and high MMA was also high in lacto-vegetarians and
lacto-ovo-vegetarians (77% and 68%, respectively) com-
pared to omnivores in this study (11% and 5%, respect-
ively) [139]. Therefore, dietary supplements are
essential to vegans and should be considered by those
with poor intake of milk and milk products.
The choice of supplement should also be carefully
considered, since the B
12
content of dietary supple-
ments can vary greatly. When the B
12
content was
determined in 19 dried Chlorella cells, which are used
in dietary supplements, it varied from <0.1 mg/100 g to
415 mg/100 g of dried weight [140]; as Chlorella does
not synthesize B
12
and is instead dependent on symbi-
otic microorganisms, their content is greatly variable
[141]. Chlorella represents a group of eukaryotic green
microalgae. Further analysis by liquid chromatography-
tandem mass spectrometry (LC-MS/MS) showed the
presence of inactive corrinoid compounds, a cobalt-free
corrinoid, and 5-methoxybenzimidazolyl cyanocoba-
mide in four and three high B
12
-containing Chlorella
tablets, respectively. In four Chlorella tablet types with
high and moderate B
12
content, Ado-Cbl (32%) and
Me-Cbl (8%) were present, whereas the unnaturally
occurring corrinoid cyanocobalamin was present at the
lowest concentrations. Chlorella sorokiniana is com-
monly used in dietary supplements and does not have
a requirement of B
12
for growth despite B
12
uptake
from the medium being observed. In further experi-
ments, B
12
-dependent MCM and MS activities were
detected in cell homogenates. These results suggest
that B
12
contents of Chlorella tablets reflect the pres-
ence of B
12
-generating organic ingredients in the
medium or the concomitant growth of B
12
-synthesizing
bacteria under open culture conditions [140].
Pregnancy state
Balanced nutrition during pregnancy is essential for the
mother’s and offspring’s health status. Most import-
antly, pregnant women are at high risk of developing
deficiency as the requirements for vitamin B
12
are
exceptionally high during pregnancy as a result of
increased metabolic rate and fetal demands. Studies
conducted on maternal-fetal dyads demonstrate higher
cord blood B
12
concentrations than those of the
respective mothers and a direct relationship between
the two pools [142–145].
Moreover, maternal nutrition before and during
pregnancy is the main determinant of the nutritional
status of the offspring. Layden et al. [146] demonstrated
that not only the mean cord blood B
12
concentration
correlated to that of the mother at delivery, but it was
almost tripled (maternal: 230.9 pmol/L, cord blood:
662.1 pmol/L, p<.0001), confirming fetal dependency
on good maternal B
12
status. Low cobalamin intake
prior to and during pregnancy has been related to
increased risk of preterm delivery, low birth weight
[147,148], and lower colostrum and early milk B
12
con-
tents [149,150]. Importantly, the deficient stores, result-
ing from inadequate B
12
intake during pregnancy, have
been shown to reflect on the newborn’s hepatic B
12
stores [17]. Vitamin B
12
deficiency has also been sug-
gested as a cause of recurrent spontaneous pregnancy
loss [151].
Pregnancy is probably the most challenging state for
the diagnosis of B
12
deficiency, since all B
12
markers are
affected due to physiological and hormonal changes
during pregnancy while B
12
status is also declining.
Decades ago, observational studies of apparently
healthy pregnant women with a normal hematological
pattern and megaloblastic anemia showed a fall in
serum B
12
concentration during pregnancy but that
pre-pregnancy concentrations resumed soon after
delivery [152–154]. In one study in the early 1960s, 33%
of women had B
12
concentrations below 147 pmol/L
compared with only 17% of non-pregnant controls
[153]. In another study conducted five decades ago and
involving 518 patients, 70% of pregnant women had
serum B
12
concentrations below the normal value for
non-pregnant subjects (110 pmol/L), including 20% of
women with exceptionally low concentrations
<37 pmol/L (L. Leichmannii assay was used) [152]. The
question was raised whether this fall in circulating B
12
was the result of physiological processes or reflected
true deficiency. To answer this question, Metz et al.
[154] compared serum tHcy and MMA in pregnant
women who had low B
12
(<150 pmol/L) vs women who
had B
12
concentrations within the reference range for
non-pregnant women. Interestingly, they found no cor-
relation between these biomarkers and serum B
12
, but
they observed elevated MMA (>376 nmol/L) in one-
third of women and elevated tHcy (>13.8 mmol/L) in
only two women (3%). The authors concluded that
there was a lack of evidence concerning the association
of functional deficiency with low serum B
12
status. The
same publication suggested homocysteine as a
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 15
valuable marker in assessment of B
12
status during
pregnancy. Pardo et al. [155] later confirmed the above
observations and conclusions. In short, these early stud-
ies indicated that using references ranges for non-preg-
nant women during pregnancy is specious and
warrants the use of separate reference ranges. We now
know that serum B
12
concentrations decrease during
pregnancy because of hemodilution and decreased syn-
thesis of haptocorrin, making this test unreliable [122].
HoloTC is considered a superior diagnostic tool in preg-
nancy as it is less impacted by pregnancy-related
changes [122,143]. Hemodilution and hormonal
changes have likewise been proposed as modulators of
MMA and tHcy concentrations during pregnancy [143].
In addition, folic acid supplementation, usually taken in
the first trimester, influences tHcy concentration, mak-
ing this marker less specific for B
12
deficiency [156].
Table 2 lists the values for B
12
markers measured in lon-
gitudinal studies in pregnant women, which were con-
ducted more recently [142–144,157–159]. Although
absolute values may differ, these studies unanimously
show changes in B
12
markers during pregnancy, which
are likely to be a result of both pregnancy-related fac-
tors and declining B
12
status. In addition, Milman et al.
[158] and Schroder and Tan et al. [159] established
pregnancy related reference intervals for some B
12
markers for all trimesters and the first two trimesters,
respectively. Serum B
12
and MMA reference intervals
were in good agreement between these studies.
Schroeder and Tan et al. [159] also showed differences
between women of European and South-Asian descent,
reporting change points based on MMA in pregnancy
and interestingly finding that these were not ethnicity-
dependent. Accordingly, total B
12
<186 and
<180 pmol/L and holoTC concentration <62.2 and
<67.5 pmol/L in the 1
st
and 2
nd
trimesters, respectively,
would indicate an increased risk of deficient B
12
status.
Newborns and infants
The mechanisms that determine B
12
status in newborns
are not well defined, particularly during the early
breastfeeding period. During the immediate postnatal
period, the gastrointestinal tract undergoes profound
colostrum-dependent growth, morphological changes,
and functional maturation, including the capacity of
acid secretion [160]. From binding studies of IF and of
purified human milk HC using the immortalized entero-
cyte cell line Caco-2, and IF gastric secretion measured
in terms of breastfed infant fecal extracts, Adkins et al.
[161] have shown IF-dependent B
12
absorption mecha-
nisms in breast-fed newborns. However, with IF concen-
trations appearing seemingly too low in this period of
life to participate effectively in B
12
absorption, they pro-
posed that HC could take control until maturation of
the IF component.
Importantly, the deficient stores resulting from inad-
equate B
12
intake during pregnancy have been shown
to reflect on the newborn’s hepatic B
12
stores [17].
Furthermore, exclusively breastfed infants are at risk of
developing symptoms of deficiency, including anemia,
irritability, failure to thrive, and developmental delays,
often after five months of life if maternal B
12
stores, and
hence breast milk, remain low [129,162,163]. Lower
serum B
12
and holoTC were found in breastfed infants
of mothers with poor vitamin B
12
status compared with
breastfed infants receiving solid foods or solid foods/
milk substitutes [164]. Chandyo et al. [165] reported
that 11% and 24% of 6–11 month old breastfed infants
(n¼316) were B
12
-deficient (<148 pmol/L) or margin-
ally deficient (148–221 pmol/L), respectively.
Interestingly, raised tHcy (>10 mmol/L) and MMA
(>280 nmol/L) concentrations were observed in 53%
and 75% of toddlers, respectively. They attributed these
high frequencies to sub-optimal maternal nutritional
status and the largely vegetarian additional feeding.
Intervention clinical trials with 400 mg IM cobalamin
showed a marked improvement in B
12
markers (serum
B
12
, tHcy, and MMA) of breastfed infants compared to
controls, as well as an improvement in motor function,
providing evidence that biochemical abnormalities
seen in infants reflect vitamin B
12
status in addition to
immature metabolism and that long-term exclusive
breastfeeding does not provide adequate B
12
to the
growing infant [166–168]. A plasma homocysteine
value of 6.5 mmol/L was suggested as the cutoff for
defining impaired B
12
function in infants [168].
Therefore, infants suspected of poor development and
having feeding difficulties should be screened for vita-
min B
12
deficiency before tests of the inborn errors of
metabolism are carried out. Exclusive breastfeeding
beyond 4–5 months of age should be another pointer
for B
12
assessment.
Malabsorption
Malabsorption leading to B
12
deficiency is not only
prevalent in the elderly; it can result from gastritis,
intestinal diseases and infections, surgical resections,
drugs, or it can have a genetic origin as described ear-
lier. In B
12
-deficient patients, for whom no “obvious”
cause is apparent and who are IF antibody negative,
the capacity to absorb the vitamin should be estab-
lished. Unfortunately, the traditional Schilling tests used
for this purpose have been discontinued due to the
safety factors related to use of radioactive B
12
. One
16 A. SOBCZYŃSKA-MALEFORA ET AL.
option is the CobaSorb test based on changes in circu-
lating holoTC before and after administrating oral B
12
[122]. Importantly, this test cannot be used once the
patient is treated with B
12
[169]. Although the
CobaSorb test has a potential diagnostic use, poor
availability of holoTC measurements in many clinical
settings, its dynamic design, as well as a lack of inter-
pretative knowledge hinder its wider use.
Ageing. Malabsorption due to autoimmune disease or
gastritis is the main cause of B
12
deficiency in the eld-
erly, followed by inadequate dietary intake. For
example, in a French study of 172 elderly patients with
B
12
deficiency, 2% of the cases were a consequence of
inadequate intake [170]. Atrophic gastritis is the main
cause of dietary cobalamin malabsorption in the elderly
and its prevalence increases with age [170,171] from as
much as 24% in those aged 60–69 years to 37% in
those aged >80 years [172].
The incidence of B
12
deficiency increases overall with
age. In one longitudinal study, it was estimated that the
mean annual decline in serum B
12
is 3.4 pmol/L for men
and 3.2 pmol/L for women [173]. Estimates of B
12
defi-
ciency of 1.6% were found in subjects over 51 years
[174]. Five percent of deficiency was estimated in peo-
ple 65–74 years and more than 10% in people 75 years
or older (based on serum B
12
<150 pmol/L or B
12
<150 pmol/L and tHcy >20 mmol/L) [175]. In the same
population-based cross-sectional study of people living
in the UK, it was found that folate deficiency also
increased with age, but only about 10% of people with
low B
12
concentrations also had low folate concentra-
tions [175]. This is relevant in view of the findings that
elderly people who have high folate concentrations
(>45.3 nmol/L) accompanied by low B
12
(<148 pmol/L)
have higher metabolic evidence of B
12
deficiency than
when folate is normal [176]. This has a potential impli-
cation for vitamin B
12
status, especially in the countries
where mandatory folic acid has been implemented but
not B
12
fortification.
The prevalence of vitamin B
12
deficiency in the eld-
erly may be even higher in other countries. Using a
serum B
12
cutoff of 220 pmol/L, an Iranian study found
that 56% and 93% of people aged 65–74 and
75 years, respectively, were at high risk of B
12
defi-
ciency [177].
The mechanisms behind decreasing B
12
status in the
elderly are not known. However, decreases of B
12
with
age were also found in total brain levels, particularly
decreases in Me-Cbl that were observed in the frontal
cortex [178]. The authors attributed these to changes in
activity to one or more transport processes across the
lifespan, such as transport across the choroid plexus via
megalin. Equally, cognitive deficits have been observed
in elderly people who are well-nourished but have low
B
12
status [179]. For more information on cognition, the
reader is referred to the vitamin B
12
, cognition, and
dementia section below.
Autoimmune disease and gastritis. Some cases of
pernicious anemia (PA) can be described as the end
stage of autoimmune gastritis (AIG), in which parietal
cell antibodies (PCAs) lead to the destruction of IF-pro-
ducing parietal cells in the stomach, leading to B
12
malabsorption and, consequently, deficiency [180].
The prevalence of PA in the general population is
0.1%, increasing to 1.9% in those over the age of 60
[59,181]. Clinical features often include anemia,
thrombocytopenia, neutropenia, glossitis, cardiomyop-
athy, jaundice, weight loss, and neurological symp-
toms [180]. PCAs are detected in more than 90% of
patients with PA. However, these antibodies are not
only specific to PA and are found in other autoimmune
conditions that often occur concomitantly with PA,
such as Graves’disease, hypothyroidism, thyroiditis,
and Addison’sdisease[180]. Two types of antibodies
related to IF have been detected in the sera of PA
patients: type I blocks the binding of B
12
to IF (found
in 70% of PA patients); type II prevents the attachment
of IF or the IF-B
12
complex to ileal receptors (in 40%
of PA). Antibodies to vitamin B
12
binding proteins
have also been reported [182]. These antibodies lead
to high serum B
12
and holoTC concentrations, and
they have not only been seen in cases of B
12
injections
but also in patients not receiving B
12
supplements
[183]. The presence of these antibodies does not
appear to interfere with the binding of B
12
to TC, but
they may impair the ability of cellular delivery. One
study has shown that the ability to deliver B
12
to cells
in vitro was impaired and clearance of B
12
in vivo of
B
12
was abnormal in the presence of these TC anti-
body complexes [183]. Precipitation of these com-
plexes with polyethylene glycol resulted in the
normalization of serum B
12
concentrations in many
patients with autoimmune diseases, hematological dis-
orders, and plasma cell neoplasms [182]. Although the
presence of these complexes is thought not to be
pathological, one needs to be aware of them, espe-
cially in patients with normal/high B
12
concentrations
and clinical signs of deficiency [180,182]. Development
of diagnostic methods for the presence of immune
complexes would help with the assessment of vitamin
B
12
status.
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 17
Intestinal diseases and infections. Hypochlorhydria
associated with atrophic gastritis hinders the release of
B
12
from food and additionally causes intestinal bacter-
ial overgrowth. The bacteria might compete for the
available B
12
, contributing further to B
12
defi-
ciency [184,185].
Additionally, Helicobacter pylori infection in the intes-
tines, which is highly prevalent in the general popula-
tion, predisposes individuals to PA through inducing
autoantibodies to antigens in the gastric mucosa. This
effect is observed in half of infected patients, suggesting
that gastric atrophy may be caused by a Helicobacter
pylori driven autoimmune process [180,186].
Some patients develop B
12
deficiency as a result of
conditions that slow the movement of food through
the intestine, allowing intestinal bacteria to multiply
and overgrow in the upper part of the small intestine.
These bacteria utilize vitamin B
12
for their own metab-
olism, rather than allowing it to be absorbed by the
body. Parasites, most notably the fish tapeworm, also
lead to B
12
malabsorption since they consume B
12
for
their own needs [187].
Patients with ileal Crohn’s and Celiac disease will
develop vitamin B
12
deficiency because of reduced
absorption as a result of villous atrophy and mucosal
impairment, respectively. One study has found B
12
defi-
ciency in 33% of patients with Crohn’s disease com-
pared to 16% of patients with ulcerative colitis [188].
The study also showed that the serum B
12
test was
insensitive during B
12
deficiency detection in these
patients, as only 5% of the cases were deficient using
this marker, compared to 32% using holoTC/MMA in 89
patients who underwent paired testing [188].
Patients suffering from chronic pancreatitis also have
affected vitamin B
12
absorption since pancreatic
enzymes are required for the release of cobalamin from
R-binders [189]. Other intestinal disorders affecting vita-
min B
12
include tropical sprue, intestinal lymphoma,
amyloidosis, and short bowel syndrome [32].
In HIV infected patients, B
12
deficiency is also quite
common. The causes of this deficiency can be ileal dys-
function, diminished IF secretion, or IF antibodies [190].
Surgery. Surgery involving any part of the gastrointes-
tinal tract will lead to diminished absorption of vitamin
B
12
and, consequently, B
12
deficiency in some patients.
About 30% of patients following a partial gastrectomy
will develop vitamin B
12
insufficiency [32]. Vitamin B
12
deficiency was found in 48% and 65% of patients with
ileal resections of 20 cm and >20 cm, respectively [188].
B
12
deficiency is also common in patients under-
going bariatric surgery, causing poor absorption of
food-bound B
12
postoperatively. The prevalence of defi-
ciency following bariatric surgery varies between stud-
ies but may depend on the type of surgery. The highest
prevalence of B
12
deficiency, ranging from 35% to
75%, was reported following Roux-en-Y gastric bypass
(RYGB), as no gastric acid remains in the gastric pouch
[191], compared to post-sleeve gastrectomy (SG) [47]. It
was also recently found that MMA increased as early as
two months following surgery, and holoTC decreased in
patients following RYGB and SG, indicating a negative
B
12
homeostasis immediately following surgery [47].
However, changes in serum B
12
were observed after
6 months in this study indicating that holoTC and MMA
may be more appropriate for monitoring B
12
status
post-bariatric surgery.
Surgical anastomoses, which create gastrointestinal
cul de sacs, can lead to severe bacterial overgrowth
[192] and, consequently, B
12
inadequacy.
Pharmaceutical interactions.
Metformin. Metformin is a medication for the treatment
of type 2 diabetes (T2D), prediabetes, and polycystic
ovary syndrome. It is a drug known to affect mitochon-
drial function and glucose metabolism [193]. A range of
evidence since 2003, including observational, experi-
mental, and meta-analyses, suggests that the use of
this oral hypoglycemic medication interacts negatively
with and leads to a marked reduction in B
12
status
[194,195]. However, the associations between B
12
levels
and metformin appear not to be linked to the duration
of therapy and dosage [196]. Not many metformin-
related studies have used functional markers of B
12
sta-
tus. In those that have, elevations in tHcy and MMA
were found in addition to low serum B
12
concentrations
[197]. In other studies, no difference or lower MMA was
found in T2D patients using metformin [198,199].
Although it is widely accepted that metformin indu-
ces malabsorption of B
12
via inhibition of the Ca
þ
-
dependent binding of the IF-Cbl complex to cubam
receptors in the ileum (an effect that can be reversed
by increasing calcium intake [200]), other mechanisms
affecting B
12
status are also possible. Notably, Liu et al.
[201] showed that adaptations in mitochondrial sub-
strate utilization underlie metformin sensitivity/resist-
ance and can result in perturbations in the tricarboxylic
acid cycle and ATP production, generation of reactive
oxygen species, and alterations to nucleotide/lipid syn-
thesis. It was also shown that genetic variation in
HIBCH, MUT genes, as well as genes encoding for pro-
teins/enzymes in the branched chain amino acids path-
way, affected the mitochondria as a direct consequence
of metformin exposure [201]. We suggest that current
18 A. SOBCZYŃSKA-MALEFORA ET AL.
data support the view that patients on long-term met-
formin treatment should have their functional B
12
status
checked at regular intervals.
Protein pump inhibitors and H
2
-receptor antagonists.
Proton pump inhibitors (PPIs), such as omeprazole, lan-
soprazole, and esomeprazole, and H
2
-receptor antago-
nists, such as ranitidine, cimetidine, famotidine, and
nizatidine, are used to reduce gastric acid secretion for
the treatment of gastroesophageal reflux disease and
peptic ulcers [194]. Similar to metformin, associations of
these drugs with lower serum B
12
have been found.
However, none of these studies were a randomized
controlled trial, nor did they assess changes in B
12
markers or the onset of clinical indications of deficiency
[194]. A higher incidence of B
12
deficiency has been
found in older adults using PPIs for more than
12 months in case-control studies of [202,203]. The
same was not concluded in a study of elderly patients
taking PPIs for more than three years [204]; however,
the cross-sectional design of this work, as well as a
slightly higher proportion of controls positive for
Helicobacter pylori infection, albeit not significant
(p¼.09), may have accounted for the lack of difference
in B
12
status between PPI users and controls. The use of
omeprazole in patients with duodenal ulcers with con-
current Helicobacter pylori infections augments the pH-
increasing effect of PPIs [205].
Genetic polymorphisms that diminish the microsomal
cytochrome P450 driven catabolism of omeprazole have
been shown to have an impact on serum B
12
concentra-
tion [206,207]. Those with a heterozygous mutation in
the CYP2C19 polymorphism with a prevalence of 47% in
Orientals and 30% in Caucasians had much lower B
12
concentrations compared to homozygotes (wt/wt) after
>1 year therapy with 20 mg omeprazole daily [206].
Drinking acidic fruit juice concurrently with B
12
may
improve B
12
absorption in PPIs users [208].
Oral contraceptives. Oral contraceptives (OCs) are
among the most commonly used drugs in developing
countries. They frequently contain both estrogen and
progestin or progestin-only pills. The serum concentra-
tion of B
12
has consistently been reported as lower in
OC users when compared to nonusers [209]; however,
the values were still within normal limits in most
women [210]. The findings were the same for users of
the combined pills [209,211] as well as progestin-only
pills [210]. Lower vitamin binding capacity and lower
haptocorrin levels have been suggested as potential
causes for lower B
12
in OC users [209]. It has been
hypothesized that hormones used in OCs may affect
the synthesis of haptocorrin, since the same is observed
in pregnancy as well [209]. Some studies also measured
holoTC in addition to total B
12
; in one cross-sectional
study of 264 females, holoTC was found to be 25%
lower in OC users than in controls [211]. The women in
this study were most frequently on combination tablets
containing the synthetic estrogen ethinylestradiol and
the progestogens, levonorgestrel or drospirenon [211].
The proportional decreases in both cobalamin markers
in this study indicates that cobalamin bound to HC and
to TC was equally affected in OC users. However, the
same group has previously shown that total TC is not
significantly lower in OC users [212]. Hence, the mech-
anism for the observed decrease in plasma holoTC is
not clear and warrants further investigation.
Consequently, it seems reasonable to take OC usage
into consideration when interpreting B
12
markers, espe-
cially total B
12
and holoTC. Nonetheless, this fall in
abundance does not appear to reflect a functional defi-
cient state, as no differences in tHcy or MMA have been
observed [211]. Therefore, redistribution of B
12
, rather
than depletion of intracellular cobalamin, has been pro-
posed in OC users [211].
Figure 6. Empty nitrous oxide canisters in a London residen-
tial street, February 2020.
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 19
Nitrous oxide. Nitrous oxide (N
2
O) is commonly used
for sedation and pain relief, but it is also abused. The
gas is also a food additive when used as a propellant
for whipped cream, hence its low price and easy access
[213]. The gas is inhaled, typically by discharging N
2
O
cartridges into another object, such as a balloon, or dir-
ectly into the mouth. Empty silver canisters of N
2
O have
become a common sight not only outside night clubs
but also on the streets in residential areas in the UK
(Figure 6).
Nitrous oxide oxidizes the active cobalt atom of
cob(I)alamin from the 1þto the 3þvalence state at a
high rate, outperforming the reductive recovery system
via cblC and MS-reductase [214,215]. This leads to a sta-
ble shift toward cob(III)alamin, followed by dissociation
of Cbl from MS and inactivation of apo-MS (Figure 2)
[216]. Inactivation of the MS enzyme will lead to the
impairment of homocysteine remethylation to methio-
nine and, subsequently, all methylation reactions
become affected due to a low level of SAM. In contrast,
the second Cbl-dependent enzyme, MCM, is unaffected
by N
2
O from the start, because MCM does not generate
cob(I)alamin but instead generates cob(II)alamin, which
is insensitive to N
2
O. However, long-term abuse of N
2
O
leads to repeated reductions and oxidations of Cbl, giv-
ing off reactive oxygen species upon oxidation (e.g.
H
2
O
2
[214]). These reactive molecules can convert Cbl
to inactive “stable yellow corrinoids”with a broken pat-
tern of the conjugated bonds [217]. Such degradation
of Cbl causes gradual inactivation of MCM as well.
Therefore, biochemically, highly elevated tHcy is seen
first in patients who abuse N
2
O, followed by a slow
decrease in serum B
12
and holoTC, as well as slight ele-
vations in MMA. Serum B
12
is often normal at presenta-
tion [218,219], which may lead to an incorrect
differential diagnosis. It has been also suggested that
inactive Me-Cbl is converted to Ado-Cbl [216].
Some of the short-term effects of N
2
O include
euphoria, numbness, sedation, giddiness, uncontrolled
laughter, uncoordinated movements, blurred vision,
confusion, dizziness and/or lightheadedness, sweating,
and tiredness. If taken in large doses, N
2
O can cause
loss of blood pressure, heart attack, or death due to
hypoxia. Long-term effects lead not only to severe B
12
deficiency but also memory loss, myelopathy, incontin-
ence, demyelinating polyneuropathy, subacute com-
bined degeneration, limb spasms, weakened immune
system, disruption to reproductive systems, depression,
and psychosis [213,218]. In the authors’hospital, which
is located in central London, N
2
O poisoning cases are
seen regularly. Most patients are in their early twenties
and often present to the A & E department with lower
leg weakness and sensory loss, urinary incontinence,
tingling in the fingertips, and difficulty walking. Their
plasma tHcy is frequently >100 mmol/L. Some patients
reported ordering canisters off the internet in bulk and
taking 100 canisters when at a party or an event. A
marked improvement in neurological symptoms is
often seen following IM hydroxycobalamin injections
and homocysteine normalization.
A single use of N
2
O for anesthetic purposes should
not pose a health risk; however, more work may be
required to confirm this since some studies suggested
possible adverse health effects. In a study by Zanardo
et al. [220] both plasma homocysteine of mothers and
cord blood of the respective offspring were higher in
25 women undergoing an elective cesarean section
under nitrous oxide general anesthesia than in 25
undergoing vaginal delivery. Moreover, maternal homo-
cysteine levels significantly correlated with cord levels
of cesarean and vaginally delivered neonates (r¼0.57;
p<.01 and r¼0.66; p<.001, respectively) [220].
In a different study involving 394 patients, postoper-
ative hyperhomocysteinemia was associated with an
increased risk of major complications with a risk ratio
(RR) of 2.8 (95% CI: 1.4–5.4, p¼.002) and cardiovascular
events, with an RR of 5.1 (95% CI: 3.1–8.5, p<.0005) in
in the N
2
O group [221].
Data on occupational exposure to N
2
Oisscarce.One
study reported increased oxidative DNA damage in med-
ical personnel of operating theaters. The extent of genetic
injury was especially evident among nurses and anes-
thesiologists exposed to N
2
O in concentrations exceeding
occupational exposure limits (180 mg/m
3
)[222].
Treatment and prevention regimens
The treatment choice for clinical deficiency depends on
whether there is neurological involvement; a specialist
should manage such patients. If a specialist is not
immediately available, 1 mg of hydroxocobalamin
should be given intramuscularly on alternate days until
there is no further improvement, then intramuscularly
every 2 months [223]. For those without neurological
involvement, 1 mg hydroxocobalamin should be admin-
istered intramuscularly three times a week for 2 weeks
to replenish stores. This should result in retention of up
to 3–4mg of B
12
, sufficient to bring the body’s stores to
within the normal range [33]. On-going maintenance
dosage depends on whether the deficiency is diet-
related. For patients with deficiency that is not of diet-
ary or drug-related origins, but caused by life-long mal-
absorption, one should continue with 1 mg
intramuscularly every 2 months for life [223].
20 A. SOBCZYŃSKA-MALEFORA ET AL.
Patients who have undergone bariatric surgery are
recommended oral, sublingual, or liquid B
12
with
350–1000 mg daily, as well as nasal sprays as directed
by manufacturer or parenteral (IM or subcutaneous)
with 1000 mg monthly prevent deficiency [224]. A
1000 mg/d of B
12
to achieve normal levels is recom-
mended for bariatric patients with deficiency [224]. An
oral vitamin B
12
with 350 lg deemed appropriate to
correct low B
12
concentrations in many patients follow-
ing bariatric surgery [225].
Those whose deficiency is likely diet-related can con-
sider oral supplementation with 50–150 mgB
12
daily or
have a twice-yearly 1 mg hydroxocobalamin injection
[223]. In severe cases, one could also consider com-
mencing treatment with several injections to replenish
B
12
stores. In vegans, treatment may need to be life-
long. In others, replacement can be stopped once B
12
levels are corrected and the diet has improved; it is
important to give dietary advice about foods rich in B
12
such as meat, fish, eggs and dairy products.
If there are doubts regarding the formal diagnosis of
deficiency, there is little to be lost, and much to be
gained, by instigating a trial of replacement. Alternative
administration routes (see further) can be considered
instead of injections to keep costs low [226].
Dosage, formulation, and schedule of administration
vary between countries, probably reflecting a paucity of
randomized research fulfilling the strict criteria of evi-
dence-based medicine [59]. Cyanocobalamin and
hydroxocobalamin are the most commonly used formu-
lations, but methylcobalamin and adenosylcobalamin
are also available, especially via the internet. In some
countries, the market has been flooded with the meth-
ylcobalamin form of vitamin B
12
, promulgating it as the
preferred formulation for treatment [227]. However,
using unstable photo-sensitive methylcobalamin and
adenosylcobalamin may not be advantageous over
cyanocobalamin and hydroxocobalamin, as they are
likely to be converted to hydroxocobalamin in vivo dur-
ing intracellular processing. The newly internalized
alkylcobalamins (i.e. methylcobalamin and adenosylco-
balamin) probably undergo dealkylation by the enzyme
MMACHC (cblC) [50]. It is therefore likely that these
forms of B
12
are no superior to cyanocobalamin in daily
oral therapy regimes [228].
Moreover, the solvent vehicle the vitamin is dis-
solved in can have an impact on treatment frequency.
It was shown that cyanocobalamin in an oily suspen-
sion (Betolvex) had a longer treatment response than
cyanocobalamin in aqueous solution (Cytobion) [229].
Cyanocobalamin and hydroxocobalamin have differ-
ing pharmacokinetic properties. Hydroxocobalamin is
retained far more efficiently than cyanocobalamin. It
disperses more slowly from the injection site, binds
more strongly to plasma, and has a slower diffusion
rate than cyanocobalamin [230–232]. Hence, cyano-
cobalamin is administered at 1000 mg once a month,
while the same dose of hydroxocobalamin can be
administered every two to three months. Treatment for
PA in France involves daily administration of 1 mg of
cyanocobalamin for one week, followed by 1 mg per
week for 1 month, and then by monthly 1 mg injections
for life [59]. In some other countries, such as Sweden
and Canada, a high oral dose is the treatment of choice
[233]. Oral doses 500 mg per day of cyanocobalamin
have been found effective at treating B
12
deficiency, as
1% of this quantity is absorbed via passive diffusion
[30]. A recent pragmatic randomized multi-center trial
of the effectiveness of oral vs intramuscular B
12
in eld-
erly deficient patients showed that 1 mg oral adminis-
tration was no less effective than IM administration at
8 weeks, although small differences were found
between administration routes after 1 year (73.6%
achieved normal B
12
in the oral arm vs 80.4% in the IM
arm [234]. This study confirms the findings of other ear-
lier studies, which found that oral cobalamin treatment
with large vitamin B
12
doses (1–2 mg daily) was as
effective as IM cobalamin treatment [235,236].
Vitamin B
12
administered sublingually and intrana-
sally has also been shown to be effective at treating B
12
deficiency [237,238]. A sublingual dose of 50 mg/d
(350 mg/week) of cobalamin, instead of the commonly
used 2000 mg/week provided in a single dose was sug-
gested by Del Bo et al. [239] to reach a state of nutri-
tional adequacy of B
12
in vegetarians and vegans.
A recent survey found that one in ten of Pernicious
Anemia Society members resort to unlicensed formula-
tions to “supplement”their prescribed treatment regime
[92]. These include nasal-sprays, sublingual drops and
sprays, transdermal patches, suppositories, self-adminis-
tered subcutaneous injections, and even intravenous
infusions. None can be deemed to be any superior, as
there is very little evidence base for these preparations.
It is noteworthy that early publications concerning
parenteral B
12
refer to IM and subcutaneous routes as
modes of administration. Self-administered B
12
via sub-
cutaneous injection should perhaps be explored as an
alternative to current treatment regimes. This would
significantly reduce costs and undoubtedly benefit
developing countries where deficiency is highly preva-
lent but nursing care is scarce. However, there is an
inadequate research base differentiating between “IM”
and “subcutaneous”routes, and further work is required
to fully evaluate their relative efficacies.
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 21
Many people choose to supplement with vitamin B
12
as a conscious health decision, and supplementation of
vitamin B
12
has been shown to be well-tolerated with-
out significant adverse effects when consumed by
healthy adults or B
12
-deficient patients [240]. The eld-
erly, vegans, and vegetarians may likely benefit from
voluntary supplementation. Vitamin B
12
supplementa-
tion is also highly recommended during pregnancy;
however, the recommendations for the dose and the
duration have not been fully validated. Doses of 50 mg
and 250 mg daily have been effectively used in clinical
trials in countries with a high prevalence of vitamin B
12
deficiency [149,150], but whether a physiological dose
of B
12
will be protective against deficiency during preg-
nancy remains to be established [241]. In their study in
Indian lactovegetarians with low vitamin B
12
, Naik et al.
[242] have shown that the physiological dose of B
12
(2 2.8 mg/d) for eight weeks cannot provide a fast
replenishment of cobalamin stores and restoration of a
normal metabolic status.
Considering associated risks
It is beyond the scope and size of this review to address
all the potential risks associated with low B
12
status
thus far. Here we can count (i) hyperhomocysteinemia,
which results from vitamin B
12
deficiency among other
causes; (ii) its impact on disease risk [243] and; (iii)
cobalamin interactions with folate [244].
Importantly, in a hospital setting, there are twice as
many patients with high serum B
12
than with its low
concentrations [116]. Whilst many of these patients are
on B
12
supplements, some cases of high B
12
can be
indicative of disease state/risk [245,246].
Only the selected health risk/diseases will be dis-
cussed in the following section.
Vitamin B12, PA, and cancer risk
Pernicious anemia is considered to be a premalignant
condition. Some tumors in PA patients arise as a conse-
quence of gastric atrophy, and subsequently, the pro-
longed elevated gastrin levels have a trophic effect on
cells that gastric tumors originate from [247]. Patients
with PA have a significantly increased risk of gastric car-
cinoid tumors, adenocarcinomas, tonsillar cancer, hypo-
pharyngeal cancer, esophageal squamous cell
carcinoma, small intestinal cancer, liver, myeloma, acute
myeloid leukemia, and myelodysplastic syndrome, but
a lower risk of rectal cancer [247]. Based on their study
in PA patients, Kokkola et al. [248] confirmed a high risk
for gastric carcinoids in this patient group, though dis-
crepancies exist regarding advice about regular
gastroscopic follow-up of PA patients [247]. However, it
would be prudent to consider thrice-yearly endoscopic
surveillance of those PA patients with a family history
of gastric cancer, or advanced age (70 years), pending
further prospective cohort studies and cost effective-
ness analyses [249].
It is unknown if vitamin B
12
deficiency contributes to
cancer risk by affecting DNA synthesis and methylation
potential (via the remethylation pathway), or if an
excess of cobalamin is associated with cancer risk.
Similar mechanisms were purported in folate deficiency
and excess in relation to cancer risk [244]. The associa-
tions of high B
12
with cancer or liver diseases have
been explained by a release of HC from proliferating
leukocytes/malignant tissues/damaged hepatocytes, as
well as by an increased synthesis or decreased clear-
ance of TC and HC proteins [245,246], hence high B
12
concentrations are accumulated in blood. A screening
strategy for addressing high serum B
12
values was pro-
posed [246]; however, this strategy did not suggest
using cobalamin as a diagnostic marker for cancer, but
only suggested following it, if unexplainable high con-
centrations were encountered. A value of 1000 pmol/L
has been suggested as a cutoff for high serum/plasma
B
12
concentrations if the elevated level did not origin-
ate from regular intakes of high vitamin doses [246].
Importantly, Arendt et al. [250] showed that high
plasma B
12
increased the risk of subsequently diag-
nosed cancer, mostly within the first year of follow-up.
Moreover, a recent large study, which used pre-diag-
nostic biomarker data from 5183 case-control pairs
nested within 20 prospective cohorts and genetic data
from 29,266 cases and 56,450 controls, found that
higher vitamin B
12
concentrations were positively asso-
ciated with overall lung cancer risk, with the authors
concluding that high vitamin B
12
status increases the
risk of lung cancer [251]. Of note is the fact that
patients with values of B
12
>850 pmol/L were excluded
from analysis in this study.
Vitamin B12, cognition, and dementia
B
12
deficiency can present as “cognitive impairment”in
some individuals and is a recognized cause of reversible
dementia [252]. Its relative contribution to the etiology
of specific dementias, including Alzheimer’s disease
(AD), is of high interest given the dearth of current
prophylaxis for dementias. Associations between B
12
deficiency and AD emerged in the 1980s [253,254], and
it was unclear whether the two were definitely related.
The discovery of low B
12
concentrations in a family with
genetically determined AD confirmed a genuine associ-
ation [255]. The advent of homocysteine as a marker for
22 A. SOBCZYŃSKA-MALEFORA ET AL.
B
12
deficiency resulted in an avalanche of literature
describing its association with AD, vascular dementia,
and other neurological disorders [243,256]. Lowering
homocysteine with high dose B vitamins, including B
12
,
has proven effective in slowing cognitive decline and
brain atrophy [257,258]. Credible mechanisms underlie
the association, which fulfills Bradford-Hill’s criteria sug-
gesting causality [259]. Further trials are needed, but
the physician should be especially alert to middle-aged
and elderly patients with insufficiency of B
12
, in order to
potentially slow cognitive decline and perhaps avert or
delay a devastating dementia.
Vitamin B12 and multiple sclerosis
Multiple sclerosis (MS) is an autoimmune neurodege-
nerative disease of the central nervous system leading
to axonal loss and demyelination. Cobalamin deficiency
is prevalent in MS patients, although not all studies,
which used serum B
12
as a deficiency marker, have
demonstrated this, thereby questioning the role of B
12
in MS [260,261]. Significantly higher unsaturated R-
binder capacities were also found in MS, compared to
patients with other neurological impairments or normal
controls [262]. It is possible that B
12
deficiency, what-
ever its cause might be, could render the patient more
vulnerable to the putative viral and/or immunologic
mechanisms, which have been suggested as the patho-
genesis of this disease [260]. Conversely, chronic
immune reactions or recurrent myelin repair processes
in MS may increase the demand for vitamin B
12
[262].
More importantly, Nijst et al. [263] showed significantly
lower B
12
in cerebrospinal fluid (CSF) in MS patients
than in reference patients, but the same was not seen
for serum B
12
. Serum and CSF B
12
correlated positively
in 293 neurological patients, including 58 with MS in
this study, but in individual patients, CSF B
12
concentra-
tions varied considerably for a given serum concentra-
tion in some patients. Of note is the fact that CSF B
12
was very low, while serum B
12
was normal, suggesting
a blood-brain barrier transport defect [263]. A resem-
blance of MS to PA was also suggested (despite age of
presentation differences), because the sex, racial, and
geographical distribution of MS and PA are simi-
lar [264].
The studies using B
12
supplementation in MS are
scarce and the benefits of regular B
12
supplementation
to MS patients are yet to be demonstrated. For
example, high methylcobalamin supplementation
improved visual and brainstem auditory evoked poten-
tials in one study [265], while adenosylcobalamin injec-
tions restored memory and speech as well as improved
balance and mobility in another study, though only one
patient was involved [266].
Conclusions and future directions
Despite significant advances in elucidating mechanisms
of vitamin B
12
absorption and intracellular processing,
challenges remain in the diagnosis and prognosis of
vitamin B
12
deficiency/insufficiency, as well as the fre-
quency and the type of treatment being administered.
We recommend that robust assessment of B
12
status
should include at least two markers, especially in
instances where discrepancies between values and clin-
ical presentation exist, and the combined index cB
12
may also be calculated. Finding the cause of deficiency
before commencing treatment is detrimental to the
future management of patients. In the event where
treatment should be initiated promptly due to severe
presentation, blood samples should be taken and saved
for future analysis as appropriate.
The elucidation of the mechanism of B
12
crossing
the blood-brain barrier, a better understanding of the
role of the microbiome and corrinoids on vitamin B
12
status and their impact on markers of B
12
, validation of
the CobaSorb or development of new absorption tests
for use in diagnostic settings, as well as a better under-
standing of the impact of long term vitamin B
12
injec-
tions on B
12
status/markers as well as cobalamin’s role
in cancer, MS, and dementia are needed to improve B
12
diagnosis and health interventions.
Acknowledgments
Renata Gorska of the Nutristasis Unit and Karol Piera of the
Free University of Berlin/ETH Z€
urich are thanked for their art-
istic skills in drawing the figures.
Disclosure statement
No potential conflict of interest was reported by
the author(s).
ORCID
Agata Sobczy
nska-Malefora http://orcid.org/0000-0001-
7349-9517
Dominic J. Harrington http://orcid.org/0000-0003-
4786-9240
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