Vitamin B12 as a Regulator of Bone Health

Abstract

In the early 1920s, the anti-anaemic effect of liver-rich diet had been recognized. The anti-anaemic substance from the liver was isolated by 1950 and called vitamin B12 (hereafter B12). It took another 20 years to structurally define and chemically synthesize B12 in its pure form. Since then, it has been recognized that B12 modulates a variety of biological systems, from immune system to bone homeostasis. Recent clinical studies have shown that B12 deficiency is likely to be an important etiological factor in the pathogenesis of bone degeneration. In this regard, either observational studies that aimed to verify an association between low B12 level and bone mass, or clinical trials on the effect of B12 as a supplementary treatment in low bone mass patients have been presented in the emerging clinical literature. Recently, we created a mouse genetic model of B12 deficiency to elucidate its mode of action by genetic deletion of gastric intrinsic factor, a protein essential for the absorption of B12 from the gut. This has led to the identification of a novel gut-liver-bone axis that has the potential to be pharmacologically targeted for treating low bone mass diseases in humans. In this review, we revisit the history of B12 from its discovery in the early 20th century to the elucidation of its mode of action in the bone till date.

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CURRENT SCIEN CE, VOL. 114, NO. 8, 25 APRIL 201 8 1632
*e-mail: vijay@nii.ac.in
Vitamin B12 as a regulator of bone health
Vijay K. Yadav*
Metabolic Research Labora tory, Na tional Inst itute of I mmunology, Aru na Asaf Ali Mar g, New Delhi 1 10 067, India
In the early 1920s, the anti-anaemic effect of liver-rich
diet had been recognized. The anti-anaemic substance
from the liver was isolated by 1950 and called ‘vita-
min B12’ (hereafter B12). It took another 20 years to
structurally define and chemically synthesize B12 in its
pure form. Since then, it has been recognized that B12
modulates a variety of biological systems, from
immune system to bone homeostasis. Recent clinical
studies have shown that B12 deficiency is likely to be
an important etiological factor in the pathogenesis of
bone degeneration. In this regard, either observa-
tional studies that aimed to verify an association be-
tween low B12 level and bone mass, or clinical trials on
the effect of B12 as a supplementary treatment in low
bone mass patients have been presented in the emerg-
ing clinical literature. Recently, we created a mouse ge-
netic model of B12 deficiency to elucidate its mode of
action by genetic deletion of gastric intrinsic factor, a
protein essential for the absorption of B12 from the
gut. This has led to the identification of a novel gut–
liver-bone axis that has the potential to be pharmaco-
logically targeted for treating low bone mass diseases
in humans. In this review, we revisit the history of B1 2
from its discovery in the early 20th century to the elu-
cidation of its mode of action in the bone till date.
Keywords: Bone formation, endocrinology, osteo-
blasts, vitamin B12.
BONE growth during gestation and post-natally is regu-
lated by the pr ocess of bone remodelling1. Bone forma-
tion carried out by osteoblasts and resorption carried out
by osteoclasts form the two arms of this remodelling
process, which ensures that vertebrates have flexible yet
strong skeleton during most part of their adult life. A
dysregulation in either arms of the remodelling process,
formation or resorption, leads to low bone mass and
increased risk of fractures2. In the last 20 years advances
in mouse genetics and studies in clinics have identified
numerous factors that regulate bone mass. The factors
that affect bone health originate either within th e bones or
from other organs. For instan ce, serotonin that originates
from the gut acts as a hormone to regulate bone formation
by acting on osteoblasts, while bone morphogenetic pro-
teins originate within the bone marrow and affect bone
formation3. Despite the advances in mouse and human
molecular genetics, our search for an ideal therapy that
can cure low bone mass diseases continues.
Vitamins are (semi)essential nutrients required for the
proper functioning of the human-body systems4. Vitamins
perform diverse functions by acting as hormones or local
growth factors to impinge on critical biological processes
within differ ent cell types, viz. vitamin D3 regulates bone
forming activity of the osteoblasts in the bone. Multiple
vitamins have been shown to regulate bone mass and
their mechanisms of action have been well established,
viz. vitamins D, K and E5. In our quest to identify bio-
logical factors that regulate bone mass, we examined the
role of vitamin B12 (hereafter B12) in regulating bone
mass6. Before going into details of this study, it would be
appropriate to go through the historical in vestigations on
this complex vitamin that have lasted for more than a
century and led to the award of two Nobel Prizes.
Discovery of B12: a long dwindling road
Pernicious anaemia (PA) is a condition in which the
blood has lower than normal number of red blood cells7.
PA was fir st described by Thomas Addison in 1849, and
was generally referred to as Addison’s anaemia, a disease
with an unknown cause leading to death of affected per-
sons (hence termed pernicious). Up until 1920, there was
no cure for Addison’s anaemia. In 1923, Minot and
Murphy8 were the first to test the radical concept that die-
tary intake of raw liver might help patients with PA.
Their idea of feeding patients with liver was based on
earlier experimental work by George Hoyt Whipple with
a canine experimental model of anaemia. In this model,
Robscheit-Robbins and Whipple9 showed that amongst
the foods tested, meat products (especially liver) were
highly beneficial in reversing anaemia (http://www.
bloodjournal.org/content/bloodjournal/suppl/2006/06/12/
107.12.4970.DC1/Video1.mov). Similar to the findings in
the canine model, feeding PA patients with raw liver
cured their disease. This discovery led to Minot, Murphy
and Whipple sharing the Nobel Prize in Physiology or
Medicine in 1934. The work of Minot and Murph y led to
the conclusion that an ‘extrinsic factor’ was responsible
for this remarkable effect of liver on the health of PA pa-
tients10,11. In 1948, two teams, one directed by Karl Folk-
ers (Merck & Co., Inc.), and the other by Alexander R.
Todd (Glaxo laboratories), obtained anti-pernicious or
extrinsic factor itself and called it ‘vitamin B12’12. Up until
this point the only way one could isolate B12 was to use

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Figure 1 . Vitamin B12 physiology in anima ls. Vita min B12 is obtained from a diet rich in a nimal products, bi nds to ga stric intrinsic fa ctor (Gif) in
the duodenum and is absorbed through receptor -mediated endocytosis in the ileum. In the blood, it binds to transcobalamin II protein and is
transported to the liver, where in it is store d and recycled.
litres of cultures of B12 synthesizing bacteria or tonnes of
liver extracts and laborious chromatographic methods.
The difficulties in B12 isolation necessitated the need to
chemically synthesize it in pure form so that it is safe for
clinical use. To facilitate the elucidation of chemical
structure, Merck provided a small amount of B12 powder
to Dorothy Hodgkin13, a well-known British scientist who
described th e chemical structure of B1 2 in 1958. With the
elucidation of the structure of B1 2 by Hodgkin, chemists
now had the information to attempt synthesis of B12
(http://www.webofstories.com/play/dorothy.hodgkin/35).
Robert B. Woodward (Harvard University, USA) in close
collaboration with Albert Eschenmoser (ETH, Switzer-
land) took up the challenge to synthesize B12 (refs 14,
15). It took Woodward and Eschenmoser a decade to
chemically synthesize B12; the final announcement of
its complete synthesis came in the fall of 1972
(http://www.chem.umn.edu/groups/hoye/links/condensed
woodward.php). Thus, it took the efforts of biologists,
clinicians and the industry almost 50 years to identify,
structurally define and chemically synthesize B1 2, the
very deficiency of which leads to PA.
B12 physiology in animals
In the animal kingdom only protozoans and certain bacte-
rial species can synthesize B12, and during evolution or-
ganisms lost the ability to synthesize B12, but it
remained essential for their survival16. In the intestinal
tract, diet-derived B12 binds to gastric intrinsic factor
(Gif) produced by the parietal cells of the stomach
(Figure 1)17. The Gif–B12 complex is recognized by the
Cubilin receptor assembly in the small intestine, leading
to its endocytosis. B12 is next transported to the blood
where it binds to the protein transcobalamin II (Tcn2).
Following this, Tcn2–B12 complex is transported to th e
liver, where B12 is released. This pool of B12 serves as a
store to provide a continuous supply of the vitamin or its
derivatives to other parts of the body16 –19. Within the
cells, B12 is converted into two known cofactors, methyl-
B12 and 5-adenosyl-B12 (Figure 2)1 6–19. Methyl-B12 is
essential for the functioning of methionine synthase, an
enzyme required for the production of methionine in the
animal cells. Methionine in turn is responsible for the
generation of S-adenosyl methionine or is incorporated
into proteins. 5-adenosyl-B1 2 on the other hand, is essen-
tial for the functioning of the enzyme methyl malonyl
CoA mutase that generates succinate. However, methion-
ine can be derived from the diet and succinate can be
generated as a by-product of th e Krebs cycle. Studies are
needed to identify other factors that are produced by
B12-dependent reactions to understand the importance of
B12 in the cells.
B12 deficiency: genetic and induced causes
The historical perspective provided above should not give
the view that B12 deficiency is only associated with PA.
Anaemia is only one of the symptoms of B12 deficiency,
which has multiple causes, can happen at any stage of a
person’s life from the time he/she is growing in his/her
mother’s womb to adult life, and can lead to multiple or-
gan dysfunctions20–2 3. In clinical practice, diagnosis of
B12 deficiency is typically established through the meas-
urement of serum cobalamin (Cbl) levels, and a person is
defined as deficient when B12 serum levels fall below
200 pg/ml. However, given that most of the B12 is stored
in the liver and is not circulating fun ctional B12 defi-
ciency can occur at any serum level, with or without
anaemia and/or macrocytosis20. B12 deficiency can be re-
flected in elevated methylmalonic acid (MMA) and
homocysteine (Hcy) levels, but these two molecules are
not routinely tested unless the initial B12 levels are equi-
vocal, because MMA and Hcy can be elevated in other
conditions as well that are independent of B12 levels. B12
deficiency is common in all age groups and is sometimes
diagnosed late due to the lack of a reliable assay and its
complex etiology19,20 .

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Figure 2. B12 -dependent enzy me reactions and associated metabolic processes. Vitamin B12 is taken up by the cells through t he process of endo-
cytosi s and then through a series o f enzymatic reactions is conv erted into two cofa ctors, methyl B12 and 5 adensyl B 12 . The B 12 derived cofact ors
are essential for two known enzymat ic reactions, methioni ne synthase and methylmalonyl CoA mutase, and through their downstream product s
affect a variety of biological processes.
During gestation, placental transfer of B12 from th e
mother provides adequate source to the foetus that lasts
up to 1 year after birth21. A deficiency of B12 in the
mother due to inadequate B12 source in the food (pure
vegetarian diet), therefore leads to various abnormalities in
the offspring during gestation or after birth. In adults, B1 2
deficiency is generally a result of certain conditions that
affect stomach Gif synthesis, such as atrophic gastritis, in
which the person’s stomach lining has undergone thin-
ning; gastric bypass surgery that r emoves part of the
stomach or small intestine, including weight loss surgery;
conditions that affect the small intestine where B12 is
absorbed, such as Crohn’s disease, celiac disease, bacte-
rial gr owth, or a parasite; heavy alcohol consumption,
and immune system disorders, such as Graves’ disease.
B12 deficiency is more common in persons who do not eat
any animal products (including meat, milk, cheese and
eggs), or persons who do not eat enough eggs or dairy
products to meet B12 needs24 .
Changes in B1 2 levels and bone health in humans
In humans, changes in serum B12 levels have been re-
ported to affect growth, bone mineral density and bone
fracture risk24–28 . The effect of B12 deficiency or supple-
mentation on bone is site-specific and dependent on a
variety of factors, in cluding age, gender and duration of
deficiency/treatment.
B12 deficiency has been shown to lead to a decrease or
no change in bone mineral density (BMD). Framingham
Osteoporosis Study identified that in both men and women
B12 deficiency was associated with lower BMD27 . In men,
significant effect was observed at th e hip, while in
women at the spine. Dhonukshe-Rutten29 examined 1267
subjects of the Amsterdam Longitudinal Aging Study
(subjects were 615 men and 652 women with a mean age
of 76  6.6 years) for changes in B12 levels, bone bio-
markers and fracture risk. This study showed that low B12
levels (<200 pM) were significantly associated with
markers of bone turnover, and an increased risk of frac-
tures. Cagnacci et al.30 studied 117 healthy postmeno-
pausal women, at two time-points five years apart who
volunteered for a cross-sectional evaluation of BMD and
levels of serum folates, h omocysteine and B12. Their goal
was to determine whether in postmenopausal women, levels
of folates, homocysteine or B12 could predict changes in
vertebral BMD. Their study could not find any significant
corelation in vertebral BMD and homocysteine or B12 in
healthy women immediately after menopause. Morris et
al.31 studied vitamin status indicators and bone health on
data collected from older (i.e. aged >55 years) men and
women who underwent DEXA scans of the hip as partici-
pants in the US National Health and Nutrition Examination
Survey (n = 1550). This analysis showed that in older
Americans serum B1 2 levels were positively associated
with BMD in a dose-dependent fashion up to ~200 pmol/l.

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In contrast to the negative effects of B12 deficiency on
bone density, data on B12 supplementation have not been
equivocal. The effect of B1 2 supplementation is dependent
on age and other factors. Rejnamrk et al.32 examined
whether dietary intake of B12 (as assessed by food re-
cords) affects BMD and fracture risk. They utilized a
population-based cohort, including 1869 peri-menopausal
women from the Danish Osteoporosis Prevention Study,
and examined any possible associations between B12 intake
and BMD at baseline and after five years of follow-up.
They did not find any association between changes in die-
tary intake of B12 and BMD. Although supplementation
with B12 alone has not been found to affect fracture risk,
but in combination with other molecules B12 has shown
either a positive or n o effect. Herrmann et al.33 tested the
effect of supplementation of vitamin D3 in combination
with B12 but could not find any further improvement in
bone biomarkers in these subjects (>54 years). In con-
trast, combinatorial supplementation of healthy subjects
with B12 and folate showed a positive effect. Sato et al.34
tested whether treatment with folate and B12 reduced the
incidence of hip fractures in patients (>65 years) with
hemiplegia following stroke. The study showed that in
this Japanese population with a high baseline fracture
risk, combined treatment with folate and B12 was effec-
tive in reducing the risk of a hip fracture.
Overall, a large set of clinical trials have shown a
negative effect of B1 2 deficiency, while the effect of B1 2
supplementation is dependent on a variety of other con-
founding factors such as age, sex and genetics. Despite
these associations of B12 and bon e in clinic, the mecha-
nisms through which B12 deficiency mediates these skele-
tal effects in humans are poorly understood.
Animal models of short-term dietary
B12 deficiency and bone health
To understand how B1 2 deficiency leads to bone abnor-
malities in humans, studies were carried out on rodents
with diets deficient in B12, alone or in combination with
other molecules. Herrmann et al.35 developed a rat model
of sh ort-term B12 and folate deficiency, and showed
that it did not affect parameters of bone quality. Similar
findings have been reported in a mouse model with
short-term deficiency in B12 and/or folic acid. Holstein et
al.36 using a mouse model of dietary B12 deficiency
showed that short-term B12 deficiency did not alter frac-
ture healing. Consistent with these in vivo findings,
decreasing B12 or folate levels in vitro do not affect
human osteoblast activity. Together, these studies have
shown that short-term dietary B12 deficiency does not
affect bone quality or fracture healing in mice. These ani-
mal or cell culture studies therefore have not been able to
illustrate how B1 2 deficiency leads to skeletal abnormali-
ties in humans.
Analysis of the effect of long-term B12 deficiency
on bone using a mouse genetic model
Gastrointestinal absorption of B1 2 requires an essential
protein, Gif, produced by the stomach parietal cells. To
generate a mouse genetic model of B12 deficiency, we
recently inactivated Gif in the mouse through homolo-
gous recombination6. Recombination analysis showed
that we had successfully inactivated this protein in the
mouse. To generate Gif–/– animals that are unable to ab-
sorb B12, we crossed Gif+/– male with Gif+/– female
generating Gif+/+, +/– and –/– offspring. Oral gavage
showed that Gif–/– offspring were indeed unable to a b-
sorb B12 from their diet. These Gif–/– offspring contained
20-fold less B12 in their blood; yet they showed signifi-
cant amount of B12 in their serum. This level of B12, al-
though extremely low, was sufficient to support offspring
growth and bone mass up to one year of age; at this point
these mutants showed osteoporosis6. Serum B1 2 in th e
first generation of Gif–/– offspring was derived from the
placental transfer from their Gif+/– mothers during gesta-
tion. With th e r easoning that further depletion of this B12
store will lead to more severe and early onset of osteopo-
rosis, we n ext used F1 Gif–/– females as mothers. This
genetic cross further lowered serum B12 in the F2 Gif–/–
offspring and led to growth retardation and early onset
osteoporosis as early as four weeks of age. These mouse
genetic crosses using Gif+/– and Gif–/– mothers produc-
ing first (F1) and second (F2) generation of Gif–/– mice
indicated that Gif–/– (F2) and not Gif–/– (F1) animals
show consequences of B1 2 deficiency during early postna-
tal growth, suggesting that maternally derived B12 is a
powerful determinant of growth and bone mass in the off-
spring. To further confirm the importance of maternally
derived B12 in the regulation of bone mass, we gave a
single injection of B12 to the Gif–/– mother during gesta-
tion day 14.5. This was sufficient to cure growth retarda-
tion and osteoporosis in the Gif–/– (F2) mice, confirming
the importance of B12 derived from the mother in this
process. These studies using F1 and F2 Gif–/– are consis-
tent with earlier studies that have shown no effect of
short-term B12 deficiency in animal models, and provided
a model in which effect of B1 2 deficiency on skeletal and
other abnormalities could be further studied.
B12 deficiency specifically affects osteoblast
numbers and bone formation
Bone histology and histomorphometry analysis revealed
that B12 deficiency profoundly affected osteoblast num-
bers and bone formation, but had no effect on bone
resorption parameters6. These results suggested that B12
deficiency may dir ectly affect osteoblast cells. Depletion
of B12 in the culture medium for 24 h from 10,000 to
0 ng/ml did not lead to any significant negative effect on

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Figure 3. Mecha nistic model of B12 regulati on of bone mass. Vitamin B12 is either derived
from the moth er during ea rly childhood or is a bsorb ed from a diet rich in animal produ cts. B12 is
then stored in the liver a nd recycled. In the liver B12 affect s the synthesis of ta urine which posi-
tively regu lates bone mas s through GH– IGF1 axis.
osteoblast proliferation or function. This could easily be
anticipated given high stability of B12 in animal cells.
Moreover, animal cells in their life are never exposed to
this severe depletion of B12 in the extracellular fluid,
including in humans, where serum B12 levels below
200 pM are classified into B12 deficiency. Given these
surprising negative effects of B1 2 deficiency on os-
teoblasts in vivo but not in vitro, we examined the organs
that might be affected by B12 deficiency and could ex-
plain low osteoblasts numbers in these mutant mice.
B12 deficiency causes growth hormone resistance
Given that B12-deficient animals showed growth retarda-
tion during early postnatal life, we examined the levels of
growth hormone (GH), a hormone that regulates growth
profoundly during peripubertal period. GH regulates
various biological pr ocesses by either directly acting on
the target cell types or by indirectly regulating production
of insulin-like growth factors 1 (IGF1) from the liver37.
For instance, GH regulates bone mass indir ectly through
production of liver IGF1 that in turn acts on osteoblasts
to increase bone mass. Analysis of GH–IGF1 axis in B12-
deficient animals revealed that these mutants had two- to
three-fold high levels of GH in their serum but very low
levels of IGF1, suggesting a state of GH resistance in
B12-deficient animals. These results suggest a model in
which B12 is an essential molecule that regulates liver GH
responsiveness to produce IGF1, which in turn acts on the
osteoblasts to regulate their proliferation and bone mass.
We reasoned that if we normalize the low levels of IGF1
in B12 deficient animals, it may rescue growth retardation
and low bone mass observed in them. To address this, we
next administered twice-daily recombinant IGF1 that has
been shown earlier to rescue growth retardation in the
state of GH resistance6. Surprisingly, however, IGF1 ad-
ministration in B12-deficient animals led to their death
within a few days following the start of the treatment.
Further investigation into the cause of death of B12-
deficient animals following IGF1 administration led to the
discovery that GH resistance was associated with hypo-
glycaemia in B12-deficient animals and IGF1 injections
further decreased their glycaemic state likely leading to
the death of these animals. These results t ogether suggest
that absence of at least one other molecule in B1 2-
deficient animals besides IGF1 is responsible for their
growth retardation and low bone mass.
Taurine deficiency underlies growth hormone
resistance and low bone mass in B12 deficient
animals
Unbiased metabolomics analysis of B12-deficient mice
liver led to the identification that these animals have a
major reduction in a variety of metabolites6. Furth er
downstream analysis of the metabolomics data, which in-
cluded variable importance in projection analysis, showed
that taurine was the most highly dysregulated molecule in
B12-deficient livers and that it could separate wild-type
(WT) animals from B12-deficient animals with th e highest
accuracy. Taurine is an atypical amino acid that contains
sulphonyl group in place of carboxyl group, and therefore

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is not incorporated into proteins. Taurine is synthesized
in the animal livers, but can also be obtained in diet. De-
spite the fact that taurine is present in abundant amounts
in the animal body, its function is poorly understood. Fur-
ther in vitro investigations using a liver cell line (HepG2)
revealed that GH positively regulated taurin e synthesis in
hepatocytes and that B12 deficiency abrogated GH-
dependent taurine synthesis. Further addition of taurine in
the culture medium of B12-deficient hepatocytes rescued
their GH resistance and restored IGF1 synthesis in these
cells. Given these promising in vitro results, we tested the
model in which taurine was an essential factor down-
stream of B12 that regulated growth and bone mass in vivo
through the regulation of the GH–IGF1 axis. To test this
hypothesis, we gavage-fed B12-deficient animals with
taurine daily from day-16 post-birth befor e the onset of
growth retardation. Taurine-fed Gif–/– (F2) animals grew
as well as WT mice, demonstrating that taurine is an
essential factor that can rescue B12 deficiency-induced
growth retardation and low bone mass. Taurine does so
by increasing IGF1 synthesis from the liver and promot-
ing IGF1 action on the osteoblasts. In the osteoblasts
taurine promotes IGF1R signalling by increasing the phos-
phorylation of IGF1R and other downstream signalling
proteins. Together, these studies revealed that one
mechanism through which B12 regulates growth and bone
mass is through the regulation of IGF1 and taurine pro-
duction from th e liver downstream of GH (Figur e 3)3 8.
B12–taurine–bone axis is operational in humans
The demonstration of taurine as a factor downstream of
B12 that regulates osteoblasts functions in a mouse model
of B12 deficiency begged the question whether this axis is
operational in humans. This was addressed in a small
cohort of patients that had B12 deficiency either due to
maternal insufficiency of B12 or due to age-related
decline in B12 absorption. Correlation analysis between se-
rum levels of B12, taurine and osteocalcin showed that like
the B12-deficient mouse model, patients had a significant
positive correlation between these parameters. These
clinical studies provided evidence that B12–taurine–bone
axis is operational in humans as it is in mice6.
Summary
The study on the regulation of growth and bone disorders
through long-term B12 deficiency has led to the discovery
of an important role played by the liver in this process.
These findings have led to the identification of taurine as
an important amino acid produced by liver, whose func-
tion in the whole-body homeostasis is only now begin-
ning to be understood. Multiple evidences point towards
the fact that the manipulation of this gut–liver bone axis
has the potential to increase bone mass in low bone mass
disorders. First, B12 and taurine specifically regulate bone
formation and therefore have the potential to increase
bone formation in diseases that specifically affect this
process. Second, both these pr oducts ar e naturally de-
rived and therefore use of these molecules will likely
pose no safety issues. For instance, taurine is produced in
the body and B12 is stored in the body in high amounts in
the liver. However, as in oth er discoveries of novel path-
ways, we have a long way to go before the true potential
of B12–taurine axis in the treatment of bone and other
metabolic disorders will be realized.
Conflict of interest: The author does not have any con-
flict of interest with regard to this manuscript.
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ACKNOWLEDGEMENTS. This work was su pported by Ra mali n-
gaswamy Fellowshi p from the Depar tment of Biotechnology, Govern-
ment of India. I tha nk Kiran Mahendroo for constru ctive comments on
the ma nuscr ipt.
Receiv ed 6 January 2017; revised accepted 14 November 201 7
doi: 10.18 520/cs/v114/i0 8/163 2-1638