Friday, June 29, 2007

Historical overview of Vitamin B1

At one time, the disease beriberi was believed to
be caused by a microorganism or toxin. The fi rst
indication of a nutritional aetiology was the virtual
elimination of beriberi in the Japanese Navy in 1885,
brought about by increasing the proportion of meat
and vegetables in the staple rice diet. In 1890, Eijkman,
a Dutch medical offi cer stationed in Java, discovered
that feeding chickens on polished rice induced a
polyneuritis closely resembling human beriberi,
which could be prevented by the addition of rice bran
to the avian diet. A few years later, Grijns extracted
a water-soluble ‘polyneuritis preventive factor’ from
rice bran and correctly concluded that beriberi is the
result of a dietary lack of an essential nutrient. By
1926, two Dutch chemists, Jansen and Donath, had
succeeded in isolating the factor (by now called vitamin
B1) in crystalline form from rice bran extracts. By
1936, Robert R. Williams had elucidated the structure
of vitamin B1, which he named ‘thiamine’, and accomplished
its synthesis. The failure of thiamin-defi cient
pigeons to metabolize pyruvate led Sir Rudolph Peters
and his colleagues in the early 1930s to establish the
essential role of thiamin in pyruvate metabolism. Lohmann
and Schuster then discovered that the active
coenzyme form of the vitamin was the diphosphate
(In this text, ‘thiamin’, rather than ‘thiamine’, is used
in accordance with the nomenclature policy of the International
Union of Nutritional Sciences Committee
on Nomenclature.)
11.2 Chemistry and biological activity
The thiamin molecule comprises substituted pyrimidine
and thiazole moieties linked by a methylene
bridge (Fig. 11.1). It is a quaternary amine, which exists
as a monovalent or divalent cation depending on
the pH of the solution. Three phosphorylated forms
of thiamin occur in nature. In living tissues the predominant
form is the diphosphate, usually referred
to as thiamin pyrophosphate (TPP) (Fig. 11.1), which
serves as a coenzyme in several metabolic pathways.
Small amounts of the monophosphate and triphosphate
esters also occur in animal tissues. Thiamin
triphosphate has no coenzyme function, but it has a
role (not yet completely understood) in nerve transmission.
Thiamin monophosphate appears to be
biologically inactive.
The name thiamin and the individual phosphates
of thiamin will be used as specifi c terms; total thiamin
means the sum of thiamin and its phosphates, and
vitamin B1 is a non-specifi c generic term.

Vitamin K deficiency

Vitamin K deficiency
10.6.1 Defi ciency in adults
In adult humans, clinical vitamin K defi ciency manifests
as occult bleeding. Abnormal blood coagulation
is more likely to arise from secondary causes such
as malabsorption syndromes or biliary obstruction
than from a dietary inadequacy of vitamin K. However,
subclinical defi ciency, manifested as decreased
urinary γ-carboxyglutamic acid excretion, has been
induced in healthy adults by dietary deprivation of
the vitamin (Ferland et al., 1993).
In a placebo-controlled study involving healthy
young and elderly adults, Binkley et al. (2000) reported
that vitamin K supplementation (1000 μg of
synthetic phylloquinone per day) resulted in a 10-
fold increase in serum phylloquinone concentration.
The mean percentage undercarboxylated osteocalcin
decreased from 7.6% to 3.4% without signifi cant
differences by age or sex. The results showed that the
usual dietary practices in the population studied did
not provide adequate vitamin K for maximal osteocalcin
carboxylation. Further research is needed to
establish whether maximal osteocalcin γ-carboxylation
is important for optimum bone mass density
and whether submaximal osteocalcin γ-carboxylation
should be used as a marker of vitamin K nutritional
10.6.2 Defi ciency in infants
Plasma concentrations of the Gla-containing bloodclotting
factors (factors II, VII, IX and X) in normal
newborns range between 30 and 60% of adult values
(Vermeer & Hamulyák, 1991). These relatively low
values are not due to vitamin K defi ciency as raising
cord blood levels of phylloquinone to the endogenous
maternal range by maternal oral supplementation
does not improve coagulation in the fetus or neonate
(Mandelbrot et al., 1988). Also, there is no detectable
difference in coagulation between breast-fed and formula-
fed infants in the fi rst month of life, despite the
marked differences in serum phylloquinone concentrations
(Pietersma-de Bruyn et al., 1990). The likely
explanation for the low neonatal concentrations of
vitamin K-dependent clotting factors is reduced
synthesis of their precursor proteins. In the mouse,
gestational factor IX mRNA levels are <5% of adult
levels up to 2 days before birth, when levels begin to
rise steeply, reaching 43% of adult levels at birth. This
is followed by a gradual increase until adult levels are
reached at about 24 days of age (Yao et al., 1991).
About 30% of full-term infants have low vitamin
K status as indicated by the presence of des-γ-carboxyprothrombin
(also known as PIVKA-II) in their
plasma during the fi rst week of life (Motohara et al.,
1985). Des-γ-carboxyprothrombin represents undercarboxylated
prothrombin and is a sensitive haemostatic
marker of subclinical vitamin K defi ciency. The
low vitamin K status, coupled with the low concentrations
of vitamin K-dependent clotting factor precursor
proteins, makes infants at birth and in early life
susceptible to a syndrome referred to nowadays as
vitamin K defi ciency bleeding (VKDB) of early infancy.
This disease, formerly known as haemorrhagic
disease of the newborn, has a reported incidence of
between 2 and 10 cases per 100 000 births (Shearer,
1995a). Three syndromes have been identifi ed according
to their time of presentation: early, classic and late
VKDB. Early VKDB presents within 24 hours of birth
and is commonly manifested as bleeding within the
gut and around the genitalia. Classic VKDB presents
1 to 7 days after birth and the bleeding is usually
gastrointestinal, dermal, nasal or from circumcision.
Late VKDB, which presents 2 to 12 weeks after birth,
is the most serious syndrome and is frequently associated
with some abnormality of liver function. It has
Vitamin K 269
a 50% incidence of intracranial haemorrhage, resulting
in death or severe and permanent brain damage
(Shearer, 1995b).
Owing to limited placental transfer of maternal
phylloquinone to the fetus, babies are born with low
liver reserves of vitamin K. After birth, it takes several
weeks before the liver stores of menaquinones attain
adult levels. The absence of an intestinal microfl ora
during the fi rst few days of life may be signifi cant in
this regard. The newborn is entirely dependent on
milk for its supply of vitamin K and hence any delay
in the establishment of lactation may be a risk factor
for classic VKDB. The vitamin K content of mature
human milk ranges from 0.85 to 9.2 μg L–1 with a
mean concentration of 2.5 μg L–1, but can be increased
by maternal intakes of pharmacological doses of the
vitamin. By comparison, cow’s milk contains 5 μg L–1
and infant formulas contain 50–100 μg L–1 (Institute
of Medicine, 2001). Two major risk factors for VKDB
are exclusive breast feeding and not giving vitamin K
prophylaxis at birth. Premature babies now routinely
receive intramuscular or (less effectively) oral doses of
vitamin K as a prophylactic measure against VKDB.

Vitamin K and atherosclerosis

Background information can be found in Section
The mRNA of matrix Gla protein (MGP) is expressed
by a wide variety of soft tissues, as well as in
developing bone (Fraser & Price, 1988). However, the
protein itself has been found only in bone and calcifi
ed cartilage (Price et al., 2000). This observation
suggests that the protein may accumulate at sites of
calcifi cation owing to its strong binding affi nity to
hydroxyapatite. Indeed, MGP, synthesized in the arterial
intima by macrophages and to a lesser extent by
vascular smooth muscle cells, accumulates in calcifi ed
atherosclerotic plaques. MGP is also synthesized by
vascular smooth muscle cells directly abutting calcifi
ed regions in the arterial media (Shanahan et al.,
Solid evidence confi rming that MGP is a potent
inhibitor of calcifi cation in vivo comes from mice
that lack MGP (Luo et al., 1997). Targeted deletion
of the MGP gene causes rapid calcifi cation of the
elastic lamellae in the tunica media of the arteries,
but not of the arterioles, capillaries or veins. The entire
media is replaced by chondrocytes, producing a
typical cartilage that starts to progressively calcify at
birth. By 3 to 6 weeks of age, calcifi cation is so extensive
that the arteries become rigid tubes and, within
8 weeks of age, death occurs due to rupture of the
thoracic or abdominal aorta. There is also inappropriate
calcifi cation of proliferating chondrocytes at
the epiphyseal plate of growing long bones, resulting
in stunted bone growth and osteopenia. The vascular
phenotype of the MGP-defi cient mouse suggests that
MGP is an essential inhibitor of arterial calcifi cation.
Furthermore, it indicates that vascular calcifi cation
occurs spontaneously if not actively inhibited. In
humans, mutations in the MGP gene are responsible
for Keutel syndrome, a rare inherited disease characterized
by multiple peripheral pulmonary stenoses,
neural hearing loss, short terminal phalanges, midfacial
hypoplasia, and abnormal calcifi cation of the
cartilage of the auricles, nose, larynx, trachea and ribs
(Munroe et al., 1999).
Contrary to expectations, Shanahan et al. (1994)
found that MGP mRNA is up-regulated in association
with vascular calcifi cation. However, this does
not necessarily mean that the protein product is
functional: function is crucially dependent on vitamin
K-dependent post-translational conversion of
Glu residues to Gla residues. Although γ-carboxylase
activity has been demonstrated in the vessel wall (de
Boer-van den Berg et al., 1986), advancing age and
environmental factors such as diet and medication
may lead to reduced levels of functional MGP. Jie et
al. (1995) reported that post-menopausal women
with calcifi ed atherosclerotic lesions had higher
levels of undercarboxylated osteocalcin and a lower
dietary vitamin K intake than women without calcifi
cations. This study demonstrated that aortic
calcifi cation is associated with a reduced vitamin K
status. Furthermore, the presence of atherosclerotic
calcifi cations was associated with a lower bone mass
(Jie et al., 1996). On the basis that MGP is produced
by the vessel wall as a defence mechanism against
calcifi cation, an insuffi ciency of vitamin K will lead
to the production of nonfunctional MGP, and hence
inappropriate calcifi cation.
Another Gla protein has been isolated from calcifi
ed human atherosclerotic plaques and partly characterized
(Gijsbers et al., 1990). This protein, named
plaque Gla protein (PGP), has a mass of 23 kDa,
268 Vitamins: their role in the human body
contains fi ve Gla residues per molecule, and is structurally
dissimilar from any of the known Gla proteins.
In vitro, PGP is extremely potent in inhibiting the
precipitation of various calcium salts, but its role in
vivo has yet to be demonstrated.
10.5.7 Possible role of vitamin K in the
nervous system
A more recently discovered Gla protein encoded by
a growth arrest-specifi c gene and known as Gas6 has
a wide tissue distribution, including the nervous system.
Gas6 is a ligand for a class of tyrosine kinase receptors
and as such is involved in cell cycle regulation
and cell–cell adhesion. In the nervous system, Gas6 is
a growth factor for Schwann cells and is implicated in
neuronal survival (Tsaioun, 1999).

Effects of menaquinone-4 on bone metabolism

Akedo et al. (1992) reported that MK-4 suppresses
the proliferation of osteoblastic cells in vitro. Warfarin
reversed this effect, implicating the γ-carboxylation
system in the modulation of proliferation. Koshihara
et al. (1996) reported that MK-4 enhanced
1,25-dihydroxyvitamin D3-induced mineralization
by human osteoblasts in vitro. This was due to enhanced
γ-carboxylation of the osteocalcin induced
by 1,25-dihydroxyvitamin D3, and accumulation of
carboxylated osteocalcin in the extracellular matrix,
causing mineralization (Koshihara & Hoshi, 1997).
Hara et al. (1993) reported that MK-4 inhibited the
bone resorption induced by interleukin-1α, prostaglandin
E2, parathyroid hormone and 1,25-dihydroxyvitamin
D3 in a dose-dependent manner in vitro.
MK-4 also inhibited the prostaglandin E2 production
stimulated by interleukin-1α. Koshihara et al. (1993)
showed that MK-4-induced inhibition of prostaglandin
synthesis in cultured human osteoblast-like
periosteal cells was reduced by cycloheximide, indicating
that newly synthesized protein participates in
the inhibitory effect.
Akiyama et al. (1994) examined the effects of MK-
4 on osteoclast-like multinucleated cell formation in
bone marrow cell cultures. MK-4 showed the most potent
inhibitory effect on cell formation when present
in cultures during the last 3 days, suggesting that the
vitamin blocks cell differentiation and/or cell fusion.
MK-4 did not infl uence 1,25-dihydroxyvitamin D3-
induced osteoclast-like cell formation when present
in the culture during the fi rst 4 days, indicating that
it does not affect proliferation of osteoclast precursor
cells. MK-4 did not affect the proliferation of many
other cell types in the bone marrow culture, suggesting
that the observed inhibitory effect of MK-4 on
osteoclast-like cells was not a result of cytotoxicity.
Hara et al. (1995) compared the effects of phylloquinone
and MK-4 on bone resorption in vitro.
Calcium concentration in the medium was used as
a parameter of bone resorption. MK-4 inhibited the
calcium release from mouse calvaria organ cultures
induced by 1,25-dihydroxyvitamin D3 or prostaglandin
E2, and it also inhibited osteoclast-like cell
formation induced by 1,25-dihydroxyvitamin D3
in co-culture of spleen cells and stromal cells at the
same concentrations. In contrast, the same doses of
phylloquinone had no effects on bone resorption and
osteoclast-like cell formation in these in vitro systems.
The inhibitory effect of MK-4 on the calcium release
from calvaria was not affected by the addition of warfarin,
suggesting that the effect of MK-4 is not due to
γ-carboxylation coupling with the vitamin K epoxide
cycle. The structures of MK-4 and phylloquinone differ
only in their side chains (see Fig. 10.1), therefore
whether the difference in their effects is related to
the differences in side chain structure was evaluated
in the co-culture system. Geranylgeraniol inhibited
osteoclast-like cell formation to almost the same degree
as MK-4, whereas the effect of phytol was weak.
Moreover, multi-isoprenyl alcohols of two to seven
units, except the four-unit geranylgeraniol, did not
affect osteoclast-like cell formation. Thus the specifi c
inhibitory effect of MK-4 is attributable to the geranylgeranyl
side chain.
Kameda et al. (1996) demonstrated that MK-4, but
not phylloquinone, inhibits bone resorption by targeting
osteoclasts to undergo programmed cell death
(apoptosis). MK-4 did not induce apoptosis in other
cell types in unfractionated bone cells. Calcitonin,
which strongly inhibits osteoclastic bone resorption
via calcitonin receptors, did not cause osteoclast apoptosis.
MK-4 might be an appropriate therapeutic
drug against bone diseases with excess bone resorp-
Vitamin K 267
tion, because of its selective and direct induction of
osteoclast apoptosis.
Clinical use of menaquinone-4 in osteoporosis
A number of Japanese studies have claimed benefi cial
results using synthetic MK-4 (menatetranone) in the
treatment of osteoporosis. The rationale includes the
possibility that MK-4 may have different effects on
bone metabolism than phylloquinone. The dosage
currently used (45 mg per day) is far in excess of daily
vitamin K requirements and any effect must be regarded
as pharmacological rather than a dietary correction
of a nutritional defi ciency. MK-4 was shown
to be effective in increasing bone mineral density of
cortical bone in osteoporotic patients (Orimo et al.,
1998) as well as preventing the occurrence of new
fractures and sustaining lumbar bone mineral density
(Shiraki et al., 2000). In the latter study, MK-4 treatment
enhanced γ-carboxylation of the osteocalcin
molecule. There were no signifi cant changes in bone
resorption markers, therefore the prevention of bone
fractures by MK-4 may not be caused entirely by inhibition
of bone resorption.

Markers of vitamin K status

Coagulation assays such as prothrombin time lack the
sensitivity to detect subclinical vitamin K defi ciency.
More sensitive tests are based on the detection in plasma
of undercarboxylated species of vitamin K-dependent
proteins that are the product of protein synthesis
when either vitamin K is in low supply or its action is
blocked by antagonists. These species are sometimes
called PIVKA (proteins induced by vitamin K absence
or antagonism). Assays to measure undercarboxylated
species in plasma have been developed for two vitamin
K-dependent proteins, prothrombin and osteocalcin,
allowing independent assessment of two different
functional roles of vitamin K (Shearer, 1995a).
Sokoll & Sadowski (1996) evaluated biochemical
markers for assessing vitamin K nutritional status in
healthy adult humans and found that undercarboxylated
serum osteocalcin is the most sensitive marker.
Both serum native osteocalcin and undercarboxylated
osteocalcin can be quantitated by radioimmunoassay
using a rabbit polyclonal antibody raised against purifi
ed bovine bone osteocalcin (Sokoll et al., 1995). The
degree of γ-carboxylation of osteocalcin can also be assessed
by determining the in vitro binding capacity of
serum osteocalcin to hydroxyapatite (Jie et al. 1992).

10.5.4 Role of vitamin K in blood
Background information can be found in Section
The liver synthesizes a group of Gla proteins that
have a regulatory function in blood coagulation: factor
II (prothrombin) and factors VII, IX and X have
a coagulant function, while proteins C and S have an
anticoagulant function.
The chick bioassay for vitamin K is based upon the
degree of lowering of elevated blood clotting times
in vitamin K-depleted chicks. Blood clotting measurements
(actually prothrombin times) are rapidly
determined following the addition of a clotting agent
(thromboplastin) and calcium chloride solution to
oxalated or citrated blood. The chick is the animal of
choice because its vitamin K requirement is fi ve-fold
that of the rat, it is readily depleted of vitamin K, and
coprophagy (faecal recycling) is easier to control. The
chick’s higher requirement for vitamin K compared
with the rat is at least partly attributable to the short
length of its colon and rapid transit time.
Matschiner & Doisy (1966) determined the molar
activities of several forms of vitamin K using the
chick bioassay. Compared to phylloquinone, which
was arbitrarily assigned an activity of 100, MK-4 had
the highest activity (156) followed by MK-7 (122) and
MK-5 (116).

Role of vitamin K in bone
Gla proteins occurring in bone
Three Gla proteins are found in bone tissue: osteocalcin
(also known as bone Gla protein), matrix Gla
protein and protein S. Osteocalcin is a relatively
small molecule (5.5 kDa) containing three Gla residues.
It is synthesized exclusively by osteoblasts and
odontoblasts and comprises about 15% to 20% of
non-collagen protein in bone. Approximately 20%
of the newly synthesized osteocalcin is not bound
to the hydroxyapatite matrix in bone, but is set free
in the bloodstream (Vermeer et al., 1995). Matrix
Gla protein is a larger molecule of 9.6 kDa containing
fi ve Gla residues. This protein is synthesized by
chondrocytes and is present in every cartilaginous
structure; it is expressed in developing bone prior
to ossifi cation. Little or nothing is known about the
precise functions of the Gla proteins. Osteocalcin has
been proposed as a specifi c regulator of the size of the
hydroxyapatite crystals in bone; it is also involved in
osteoclast recruitment (Robey & Boskey, 1996). Matrix
Gla protein inhibits inappropriate calcifi cation of
the epiphyseal (growth) plate (Olson, 1999). Protein S
has been identifi ed as a ligand of tyrosine kinase-type
receptors that modulate cell proliferation (Kohlmeier
et al., 1996). Children with inherited protein S defi -
ciency not only suffer from recurrent thrombosis, but
also have severely reduced bone mass (osteopenia)
(Pan et al., 1990).
Vitamin K status and osteoporosis
Individuals carrying the E4 allele of apoE experience
a higher incidence of bone fractures during their
lifetimes than do individuals without the E4 allele
(Kohlmeier et al., 1998). The increased risk of hip
and wrist fracture in women with the apoE4 allele
is not explained by bone density, impaired cognitive
function or falling (Cauley et al., 1999). This predisposition
toward bone fracture is consistent with
E3/E4 and E4/E4 phenotypes having lower plasma
phylloquinone levels than normal.
Vitamin K suffi ciency of the bone is related to the
degree of γ-carboxylation of osteocalcin and this in
turn is related to the plasma phylloquinone concentration.
As vitamin K intake decreases, circulating
osteocalcin seems to be the fi rst Gla protein to occur
in an undercarboxylated form (Vermeer et al., 1995).
Circulating osteocalcin is about 92% γ-carboxylated
in healthy young adults on a normal diet. Daily supplementation
with 250 μg of phylloquinone increases
osteocalcin carboxylation to 96%, while 1000-μg supplements
are required to achieve 100% carboxylation
(Binkley et al., 2002). These observations reveal that a
diet suffi cient to maintain normal clotting would not
be able to maximize γ-carboxylation of osteocalcin
and probably other vitamin K-dependent proteins.
It remains unknown whether maximal osteocalcin
carboxylation is necessary for optimal bone health.
Most studies have shown that the circulating levels
of total osteocalcin increase with ageing in normal
women, especially after the menopause. This increase
is likely to refl ect an increase in bone turnover, which
is associated with low bone mass in all skeletal regions
(Ravn et al., 1996). The γ-carboxylation of circulating
osteocalcin is signifi cantly impaired in women
over 80 years of age (Plantalech et al., 1991). Also in
elderly women, high concentrations of circulating
undercarboxylated osteocalcin is associated with low
hip bone mineral density (BMD) (Szulc et al., 1994)
and increased risk of hip fracture (Szulc et al., 1993,
1996; Vergnaud et al., 1997). Plasma levels of phylloquinone
and of the menaquinones MK-7 and MK-8
are depressed in elderly women within a few hours
of hip fracture, suggesting that vitamin K is sequestered
from the circulation for use at the fracture site
(Hodges et al., 1993). Vitamin K1 supplementation
(1000 μg per day) corrected undercarboxylation of
osteocalcin in postmenopausal women (Knapen et
al., 1989; Douglas et al., 1995) and decreased two
markers of bone resorption, urinary calcium and
hydroxyproline excretion (Knapen et al., 1989, 1993).
Booth et al. (1999) reported that 15 days of dietary
vitamin K depletion led to increased bone turnover as
measured by serum osteocalcin and urinary NTx (Ntelopeptides
of type I collagen) concentration. These
markers were subsequently normalized by 10 days of
phylloquinone repletion (200 μg per day). As elevated
bone turnover is associated with rapid bone loss,
vitamin K insuffi ciency would be expected to contribute
to the development of osteoporosis. However,
associations do not necessarily imply causation and
no direct evidence for the participation of decreased
plasma vitamin K in osteopenia in the elderly has been
In a prospective study involving 72 327 women
(Feskanich et al., 1999), dietary vitamin K intakes less
266 Vitamins: their role in the human body
than 109 μg per day were associated with an increased
risk of hip fracture. Booth et al. (2003) assessed dietary
vitamin K intake with a food-frequency questionnaire
in 1112 men and 1479 women (mean ± SD
age: 59 ± 9 years) and measured BMD of the hip and
spine. Women in the lowest quartile of vitamin K intake
(mean: 70.2 μg per day) had signifi cantly lower
BMD at the hip and spine than did those in the highest
quartile of intake (mean: 309 μg per day). No signifi -
cant association was found between dietary vitamin K
intake and BMD in men.
Tamatani et al. (1998) evaluated the possible participation
of circulating levels of testosterone, vitamin
D metabolites and vitamin K in osteopenia in elderly
men. No signifi cant correlation between plasma testosterone
and BMD was observed, despite the agerelated
decrease in plasma testosterone. However,
elderly men with decreased BMD showed signifi cant
decreases in the circulating levels of 25-hydroxyvitamin
D, phylloquinone and MK-7 compared with
elderly men with normal BMD.

Maternal to fetal transfer of phylloquinone

The transfer of phylloquinone from the maternal to
fetal circulation is poor. Despite a 500-fold increase in
maternal plasma phylloquinone concentration following
the intravenous administration of 1 mg of phylloquinone
to pregnant women at term, the corresponding
increase in cord plasma was only about fi ve-fold. The
levels attained in cord plasma (0.10–0.14 ng mL–1) after
injection were at most near the lower end of the normal
fasting adult range (0.10–0.66 ng mL–1) (Shearer et al.,
1982). When pregnant women were given daily oral
doses of 20 mg phylloquinone for at least 3 days, cord
plasma levels of phylloquinone were boosted 30-fold at
mid-trimester and 60-fold at term. Again, these levels
were substantially lower than corresponding supplemented
maternal levels (Mandelbrot et al., 1988). The
large concentration gradient of phylloquinone between
maternal and neonatal plasma suggests that phylloquinone
does not cross the placenta readily. Alternatively,
uptake by fetal plasma is low, perhaps because of low
levels of transporting lipoproteins.
The cord plasma of premature infants increased
by an average of 2.3-fold after their mothers received
5 mg of phylloquinone intramuscularly several hours
to 35 min before delivery (Yang et al., 1989). Thus
supplemental phylloquinone given to the mother antenatally
can be transferred to premature infants, but
to a lesser degree than to term babies.
10.4.6 Storage and catabolism in the liver
The liver has a limited capacity for long-term storage
of vitamin K compared to vitamin A. Surprisingly,
phylloquinone comprises only about 10% of the total
liver stores of vitamin K. Menaquinones ranging from
MK-4 to MK-13 make up the bulk of stores with the
long-chain forms (MK-9 to MK-13), constituting
73% of total vitamin K (Usui et al., 1990). Unlike
phylloquinone, which undergoes rapid turnover, the
hepatic turnover of long-chain menaquinones is low,
presumably because of their high affi nity for membranes
(Shearer, 1992). The contrasting turnovers of
phylloquinone and menaquinones may account for
the predominance of the latter in liver. Whether the
menaquinones originate from the diet or from bacterial
synthesis, their strong retention relative to phylloquinone
would enable concentrations to gradually
build up while phylloquinone is being constantly
utilized and metabolized. In support of this concept,
hepatic stores of phylloquinone are rapidly depleted
during dietary restriction of vitamin K, but hepatic
stores of menaquinones are not (Usui et al., 1990).
Also, the common hepatic menaquinones (MK-9 to
MK-13) are not detectable in plasma, suggesting that
they are not easily mobilized. It appears, therefore,
that the large hepatic pool of menaquinones does
not contribute signifi cantly to vitamin K nutriture
but represents a very slow turnover of the extremely
lipophilic long-chain menaquinones. Further work is
needed to establish the origin of hepatic menaquinones
and their nutritional relevance.
In the liver of the human fetus, phylloquinone is detectable
as early as 10 weeks gestation, and at term the
concentration is about one-fi fth the value in adults.
Hepatic concentrations of menaquinones are usually
undetectable at birth and in the fi rst week of life. The
gradual build-up of hepatic stores of menaquinones
is consistent with the colonization of the neonatal gut
by enteric bacteria.

Absorption of dietary vitamin K

Phylloquinone, the major form of vitamin K in the
diet, is absorbed in the jejunum, and less effi ciently
in the ileum, in a process that is dependent on the
normal fl ow of bile and pancreatic juice (Shearer
et al., 1974). Both long- and short-chain menaquinones
are readily absorbed by rats after oral ingestion
(Groenen-van Dooren et al., 1995) and therefore dietary
menaquinones are likely to be incorporated into
mixed micelles through the action of bile salts and
absorbed along with phylloquinone.
The effi ciency of vitamin K absorption varies
widely depending on the source of the vitamin and
the amount of fat in the meal. Pure phylloquinone
is absorbed with an effi ciency of 80% (Shearer et al.,
1974). The phylloquinone present in cooked spinach
was only 4% as bioavailable as that from a commercial
detergent suspension of phylloquinone. Adding butter
to the spinach increased this to 13% (Gijsbers et
al., 1996). The absorption of phylloquinone was six
times higher after the ingestion of a 500-μg phylloquinone
tablet than after the ingestion of 150 g of raw
spinach containing 495 μg phylloquinone (Garber et
al., 1999). The phylloquinone from a phylloquinonefortifi
ed oil was absorbed better than that from an
equivalent amount from cooked broccoli, regardless
of adjustment to triglyceride concentrations (Booth
et al., 2002). The tight binding of phylloquinone to
the thylakoid membranes of chloroplasts explains the
poor bioavailability of the vitamin in green plants.
The free phylloquinone in vegetable oils, margarines
and dairy products is well absorbed owing to the
stimulating effect of fat.
10.4.2 Bacterially synthesized
menaquinones as a possible endogenous
source of vitamin K
The large intestine of healthy adult humans contains
a microfl ora of bacteria, many species of which synthesize
menaquinones ranging mainly from MK-6 to
MK-12. The menaquinones are incorporated into the
bacterium’s cytoplasmic membrane where they function
under reduced (anaerobic) conditions as redox
Vitamin K 259
compounds in bacterial respiration. The most prevalent
menaquinone-producing bacteria in the intestine
are Bacteroides species which synthesize MK-10,
MK-11 and MK-12. Among other prevalent species,
Escherichia coli synthesizes mainly MK-8 (Ramotar
et al., 1984).
Conly & Stein (1992a) reported quantitative and
qualitative measurements of phylloquinone and menaquinones
at different sites within the human intestinal
tract. Overall, long-chain menaquinones (MK-
9, -10 and -11) predominated. Menaquinones were
found mostly in the distal colon (10 faecal samples)
and totalled 19.85 ± 0.36 μg per g dry weight. Menaquinones
in two samples of terminal ileal contents
taken during appendectomy totalled 8.85 μg per g dry
weight. Little menaquinone was found in samples of
proximal jejunal contents collected by means of a nasojejunal
tube (total, 0.03 μg per g dry weight).
The menaquinones incorporated into membranes
of viable bacteria are not available for absorption.
However, Conly & Stein (1992b) described in vitro
experiments showing that signifi cant amounts of
biologically active menaquinones can be secreted or
liberated from bacteria. For example, when a dialysis
bag containing Staphylococcus aureus (a known producer
of menaquinones) was immersed into 100 mL
of media, 0.18% of total menaquinone was recovered
from the surrounding media, representing a concentration
of 0.6 nmol L–1. Inoculation of the media with
Bacteroides levii (a vitamin K-requiring organism)
before dialysis resulted in a luxurious growth of this
organism, but not in controls containing no Staphylococcus
Being strongly lipophilic, the bacterially synthesized
menaquinones require the presence of bile salts
and the formation of mixed micelles for absorption
to take place. Because bile salts are reabsorbed in
the distal ileum and the amounts remaining are degraded
by colonic bacteria, there is no opportunity
for absorption of menaquinones to take place in the
colon. Indeed, colonic absorption of MK-9 in rats is
extremely poor (Ichihashi et al., 1992; Groenen-van
Dooren et al., 1995). However, bearing in mind the
appreciable amounts of menaquinones found in the
terminal ileum of two subjects (Conly & Stein, 1992a),
and considering the possibility that contents from the
caecum (where large amounts of bacteria reside) may
backwash past the ileocaecal valve into the ileum,
one can envisage some degree of bile salt-mediated
absorption taking place in this region. In addition,
Hollander et al. (1977) demonstrated ileal absorption
of MK-9 in the conscious rat and showed that
the absorption rate increased with increasing bile salt
Direct evidence to support absorption of menaquinones
from the distal human intestinal tract,
where intestinal microfl ora are most prevalent, is
lacking. Indirect evidence that enteric menaquinones
are absorbed is the fact that about 90% of liver
stores of vitamin K is in the form of menaquinones
(Shearer, 1992) despite phylloquinone predominating
in the diet. Moreover, the various menaquinones
found in liver are remarkably consistent with the
menaquinone profi le of human intestinal content.
However, it has not been possible to prove that the hepatic
menaquinones do not originate from the diet.
Studies performed on human volunteers placed on
a vitamin K-defi cient diet have consistently failed to
demonstrate any signifi cant changes in prothrombin
time. However, bleeding episodes associated with a
prolonged prothrombin time have been reported in
vitamin K-deprived volunteers receiving broad-spectrum
antibiotics (Allison et al., 1987). The data from
the latter study did not support the hypothesis discussed
by Lipsky (1988) that N-methylthiotetrazolecontaining
antibiotics suppress vitamin K-dependent
clotting factor biosynthesis. Collectively, these
data imply that enteric menaquinones are absorbed
and utilized to some extent.
Conly et al. (1994) demonstrated that menaquinones
can be absorbed directly from the human
ileum and be functionally active. Their study consisted
of an experimental phase followed by a control
phase, using the same four volunteers. The volunteers
were started on a vitamin K-defi cient diet and then
given adjusted doses of warfarin to maintain a stable
elevated prothrombin time. A 1.5-mg dose of mixed
menaquinones (MK-4 to MK-9) extracted from harvested
Staphylococcus aureus was then placed directly
into the ileum by means of a nasoileal tube after an
overnight fast. Within 24 hours of menaquinone administration,
the prothrombin time decreased signifi -
cantly and the factor VII level increased signifi cantly,
indicating that the menaquinones had been absorbed
and utilized. The results of this study provide an explanation
as to why starvation or a complete lack of
dietary intake of vitamin K alone cannot induce a
clinically manifest vitamin K defi ciency.
260 Vitamins: their role in the human body
In conclusion, a report by Ferland et al. (1993) that
subclinical vitamin K defi ciency can be induced in
healthy adults by dietary deprivation of the vitamin
suggests that absorption of bacterially synthesized
menaquinones may not be suffi cient to sustain adequate
vitamin K status.

Dietary sources for vitamin K

The highest concentrations of vitamin K (in the
form of phylloquinone) are found in green leafy vegetables,
e.g. cabbage, broccoli, Brussels sprouts and
spinach. Such vegetables are the top contributors to
vitamin K intake in the American diet. Other types
of vegetables (roots, bulbs and tubers), cereal grains
and their milled products, fruits and fruit juices are
poor sources of vitamin K. Animal products (meat,
fi sh, milk products and eggs) contain low concentrations
of phylloquinone, but appreciable amounts of
menaquinones are present in liver.
Some vegetable oils, including canola (rapeseed),
soybean and olive oils, are rich sources of phylloquinone,
whereas peanut and corn (maize) are not. Soybean
oil is the most commonly consumed vegetable
oil in the American diet. The addition of phylloquinone-
rich vegetable oils in the processing and cooking
of foods that are otherwise poor sources of vitamin K
makes them potentially important dietary sources of
the vitamin. This is particularly evident, for example,
when chicken, eggs and potatoes are fried in certain
vegetable oils. Those margarines, mayonnaises and
regular-calorie salad dressings that are derived from
phylloquinone-rich vegetable oils are second to green
leafy vegetables in their phylloquinone content. The
addition of these fats and oils to mixed dishes and
desserts has an important impact on the amount of
vitamin K in the American diet.
Various menaquinones have been found in fermented
foods (Sakano et al., 1988), salmon, shellfi
sh, beef, pork, chicken, egg yolk, cheese and butter
(Hirauchi et al., 1989a) but the amounts may not
be nutritionally signifi cant in some of these foods.
Livers of ruminant species (e.g. cow) contain signifi
cant concentrations (10–20 μg per 100 g) of some
menaquinones (Hirauchi et al., 1989b), while cheese
contains signifi cant quantities of MK-8 (5–10 μg per
100 g) and MK-9 (10–20 μg per 100 g) (Shearer et al.,

Vitamin E deficiency

Vitamin E defi ciency
9.8.1 Defi ciency in animals
Vitamin E defi ciency in animals is readily demonstrable
and results in a variety of pathological conditions
that affect the muscular, cardiovascular, reproductive
and central nervous systems as well as the liver, kidney
and erythrocytes. There is a marked difference between
animal species in their susceptibility to different
defi ciency disorders. A complex biochemical interrelationship
exists between vitamin E and the trace element
selenium. Unsaturated fat, sulphur-containing
amino acids and synthetic fat-soluble antioxidants are
also implicated in some disorders. Consequently, in
order to experimentally induce a particular defi ciency
syndrome in a given species, it is usually necessary to
adjust the balance of these nutrients in the diet. The
most extensively studied defi ciency syndromes are
listed in Table 9.3.
Fetal resorption
In female rats deprived of vitamin E all reproductive
events are normal up to implantation of the fertilized
ova. Several days later, however, the developing fetus
shows abnormalities followed by intra-uterine death,
rapid autolysis and resorption. A defect in the fetal
blood vessels may be the primary event leading to
death of the fetus (Nelson, 1980). This disease can
be prevented by administering an adequate dose of
vitamin E as late as the tenth day of pregnancy. The
synthetic antioxidant DPPD is at least as effective
as α-tocopherol in preventing fetal resorption, but
ethoxyquin, which readily prevents encephalomalacia
in chicks, is inactive (Draper et al., 1964). Selenium
compounds have no effect on fetal resorption in rats.
Erythrocyte haemolysis
Erythrocyte plasma membranes are particularly vulnerable
to lipid peroxidation because of their direct
exposure to molecular oxygen and the presence of
haemoproteins which are catalysts of peroxidation.
Erythrocytes isolated from blood samples of vitamin
E-depleted rats exhibit spontaneous haemolysis when
added to dilute solutions of dialuric acid, whereas
erythrocytes of rats receiving vitamin E are resistant
to this haemolysis. This early manifestation of vitamin
E defi ciency can be prevented by certain synthetic
antioxidants administered to the animal or added to
the cell suspension in vitro as well as by vitamin E.
Selenium compounds have no effect on erythrocyte
This nutritional disorder occurs in growing chicks
fed vitamin E-defi cient diets containing adequate
amounts of selenium for the prevention of exudative
diathesis and suffi cient methionine or cystine for the
prevention of necrotizing myopathy. Encephalomalacia
is manifested by lesions of the cerebellum, the part
of the brain concerned with coordination of movement.
The cerebellum is softened, swollen and oedematous
with minute haemorrhages on the surface and
greenish-yellow necrotic areas. The necrosis may be
the result of thrombosis in the capillaries. Once established,
the lesions are irreversible. The main symptoms
are ataxia of gait and stance, backward or downward
retraction of the head, tremors, spasms of the
limb muscles, and eventually prostration, stupor and
death within a few hours. The incidence and severity
of the disease are markedly increased with increasing
levels of linoleic acid in the diet. Low concentrations
of synthetic antioxidants such as DPPD and ethoxyquin
in the diet readily prevent encephalomalacia, but
selenium has no effect.
Exudative diathesis
This is a vascular disease of chicks which develops
as a result of feeding diets that are low in both vitamin
E and selenium. The disease can be induced
for experimental purposes by feeding diets based on
Torula yeast, which is low in both micronutrients and
contains substantial amounts of unsaturated fatty
acids. The most obvious manifestation is a massive
accumulation of a greenish fl uid under the skin of the
breast and abdomen. Internally, the oedema extends
to the muscles and many organs, including the heart
and lungs. The oedema is the result of a leakage of
plasma from the capillaries caused by an increased
permeability of the capillary walls. The disease can
be prevented by administration of either vitamin E
or selenium, provided that the selenium defi ciency is
not too severe (Thompson & Scott, 1969). A severe
defi ciency of selenium causes degeneration of the
exocrine component of the pancreas and consequent
impairment of dietary lipid absorption, which will
affect the absorption of vitamin E (Thompson &
Scott, 1970). In this event, extremely high doses of
vitamin E are required to prevent exudative diathesis.
Some synthetic antioxidants, including DPPD and
ethoxyquin, are also effective, but only at concentrations
distinctly greater than those required to prevent
Liver necrosis
Necrotic liver degeneration develops in weanling rats
after commencement of a diet based on Torula yeast,
which is defi cient in both vitamin E and selenium and
low in sulphur-containing amino acids. Necrosis is
preceded by degeneration of the sinusoidal cellular
plasma membrane and lipid peroxidation has been
detected late in the progress of the disease. The onset
of necrosis is delayed by cystine, which appears to have
a sparing action on the amount of vitamin E or selenium
required to prevent the disease.
Testicular atrophy
In male rats depleted of vitamin E from early life there
is no testicular injury until the onset of sexual maturity,
when a progressive degeneration of the germinal
epithelium of the seminiferous tubules occurs and the
testes atrophy. The resultant sterility does not respond
to vitamin E and is truly permanent.
Necrotizing myopathy
This disease is manifested as a progressive muscular
weakness which affects the skeletal muscles of many
vertebrate species. It was originally called nutritional
muscular dystrophy, but this term suggests an aetiological
relationship between the myopathy of vitamin
E defi ciency and human muscular dystrophy. Although
many of the pathological lesions are similar
in these two diseases, human muscular dystrophy is
genetically determined and does not respond to vitamin
E treatment.
Necrotizing myopathy is characterized histologically
by marked variation in the cross-sectional
diameter of the muscle fi bres, segmental fragmentation
with interstitial oedema and necrosis and, in the
later stages, extensive replacement of muscle tissue by
connective tissue. The disease can be detected in its
early stages by an increased excretion of creatine in
the urine (creatinuria), which is the result of a loss of
creatine from the affected muscles. Creatine excretion
is often expressed as the creatine:creatinine ratio, the
excretion of creatinine being relatively constant on a
body weight basis.
Necrotizing myopathy in rabbits, guinea pigs, rats
and monkeys responds primarily to vitamin E. Selenium
is not capable of completely replacing vitamin
E in these species, although it does reduce the vitamin
requirement. The myopathy, as studied in the chick,
does not respond to dietary synthetic antioxidants at
levels several times those needed to prevent encephalomalacia.
The disease is induced in the chick when
the dietary vitamin E is accompanied by a defi ciency
in the sulphur-containing amino acids, methionine
and cystine. Approximately 0.5% of dietary linoleic
acid (but not linolenic acid) is necessary to produce
myopathy. Concentrations above 0.5% do not increase
the amount of vitamin E required for preven-
Vitamin E 249
tion. The chick appears to be unique in that the myopathy
can be prevented in the absence of vitamin E
by supplementing the diet with cystine or methionine.
Cystine is about twice as effective as methionine on an
equal sulphur basis (Scott, 1970).
9.8.2 Defi ciency in humans
Apart from haemolytic anaemia in premature infants,
vitamin E, in the context of human nutrition,
has long been considered ‘a vitamin looking for a
disease’. It is now recognized that vitamin E is responsible
for the neurological abnormalities that had
been described in patients with long-term disorders
of fat absorption.
Haemolytic anaemia
Newborn infants generally have low serum vitamin
E levels because of the vitamin’s limited transfer
through the placenta. A haemolytic anaemia associated
with vitamin E defi ciency in premature infants
6 to 10 weeks after birth was fi rst reported by Oski &
Barness (1967). This defi ciency syndrome was further
investigated in infants fed commercial milk formulas
that were high in PUFA and relatively low in vitamin
E (Hassan et al., 1966; Ritchie et al., 1968). Control infants
were fed identical formulas supplemented with
vitamin E. The syndrome consisted of haemolytic
anaemia, oedema and skin lesions. The erythrocytes
lysed when treated in vitro with dilute hydrogen peroxide
(i.e. the cell contents leaked out of the damaged
cell membrane) and the blood fi lm showed abnormal
red cell morphology, such as spiky and fragmented
cells. Erythrocyte survival was shortened and an
increase in the number of reticulocytes (erythrocyte
precursors newly arrived in the blood from the bone
marrow) indicated a response to increased erythrocyte
destruction. Erythroid hyperplasia was observed
in the bone marrow and an increased platelet count
was indicative of a general increase in bone-marrow
activity. The infants were restless, breathing was noisy
and there was a watery nasal discharge. Oedema appeared
and slowly progressed until it involved the
entire face, lower limbs and genitalia. The oedema is
analogous to the exudative diathesis observed in vitamin
E-defi cient chicks. The skin lesions began on the
sides of the face extending into the neck and adjacent
parts of the scalp. All of the symptoms were associated
with low serum vitamin E levels; the symptoms
were not observed in the controls, which had higher
serum vitamin E levels. The symptoms disappeared
in response to oral vitamin E therapy; there was no
response to iron or vitamin B12. The lengthening of
erythrocyte survival coincident with the rise in serum
vitamin E was direct in vivo evidence that vitamin E
prevented haemolytic anaemia. The therapeutic effect
of vitamin E in these experiments is presumably
attributable to its ability to protect the vital phospholipids
in cell membranes from peroxidative degeneration.
Nowadays, infant milk formulas contain
added vitamin E and an adequate ratio of vitamin E to
PUFA; this has almost completely eradicated haemolytic
It is well documented that a diet rich in polyunsaturated
fat, but which does not contain a correspondingly
high amount of vitamin E, induces defi ciency
signs in animals. This also applies to humans as shown
by the above experiments with premature infants. In
a long-term human study (the Elgin project), adult
male volunteers received a diet in which about half of
the fat content was composed of vitamin E-stripped
lard. After 30 months this fraction of the fat content
was replaced by stripped corn oil and 9 months later
the amount of stripped corn oil was doubled. No
manifestations of anaemia were observed and it was
not until the 72nd month that a well-controlled study
of erythrocyte survival was performed. The data obtained
showed that the erythrocytes of the vitamin
E-depleted subjects were being destroyed at a rate
about 8–10% faster than in the subjects in the control
groups. The experiment was terminated soon after
these observations, but it is logical to assume that if
the diet had been made more defi cient, the pathology
would have been more severe (Horwitt, 1976).
Fat malabsorption
Because of the intimate association between intestinal
absorption of dietary fat and vitamin E, any condition
causing the prolonged malabsorption of fat (steatorrhoea)
will lead to a secondary defi ciency of vitamin
E. Thus, patients with a variety of chronic fat malabsorption
conditions exhibit low plasma vitamin E
concentrations. The major nongenetic causes of
steatorrhoea associated with a symptomatic vitamin
E defi ciency state are chronic cholestatic hepatobiliary
disorders, cystic fi brosis and short bowel syndrome.
Abetalipoproteinaemia and homozygous hypobetalipoproteinaemia
are genetic causes of steatorrhoea.
250 Vitamins: their role in the human body
Chronic cholestatic hepatobiliary disorders
These disorders include diseases of the liver and of
the intrahepatic and extrahepatic bile ducts. The
impaired bile fl ow leads to an insuffi cient concentration
of bile constituents in the intestinal lumen and
a consequent failure to produce micelles. The result
is malabsorption of dietary fat-soluble substances.
Because of their low vitamin E body stores, infants
with cholestatic liver disease show symptoms of
neuropathy as early as the second year of life, the
neurological damage becoming irreversible if the
vitamin E defi ciency is not corrected. Correction of
the defi ciency by oral administration requires very
high doses of vitamin E (100–200 IU per kg per day)
or the use of a water-soluble form (α-tocopheryl polyethylene
glycol-1000 succinate) which forms micelles.
Alternatively, vitamin E can be administered by intramuscular
Cystic fi brosis
In cystic fi brosis, increased viscosity of pancreatic
secretions causes obstruction of pancreatic ducts
leading ultimately to destruction and fi brosis of the
exocrine pancreas. The resultant failure to secrete
pancreatic digestive enzymes causes steatorrhoea and
vitamin E defi ciency. Despite the common observation
of neuroaxonal lesions in the posterior column
of the spinal cord at autopsy, overt neurological dysfunction
is rare in vitamin E-defi cient cystic fi brosis
patients. Most patients who do exhibit neurological
dysfunction also have fi brotic livers.
Short bowel syndrome
Short bowel syndrome is a collection of signs and
symptoms used to describe the nutritional consequences
of major surgical resections of the small
intestine. Resections are carried out for treatment of
Crohn’s disease and mesenteric vascular thrombosis,
among other disorders. The causes of vitamin E defi
ciency in these conditions are a reduced intestinal
absorptive surface area and excessive faecal bile acid
losses. Although low plasma vitamin E concentrations
may be present within several years of surgical resection,
10 to 20 years of severe malabsorption are generally
required before the manifestation of neurological
symptoms. This is because of the prior accumulation
of vitamin E in most tissues and its relatively slow release
from nervous tissues.
Chylomicrons contain apoB-48, among other apoproteins,
while VLDL and LDL contain apoB-100.
These two apoB proteins are encoded by the same
gene, apoB-48 being synthesized in the intestinal
mucosa and apoB-100 in the liver. Abetalipoproteinaemia
is a rare inborn error of lipoprotein production
and transport characterized by undetectable or very
small amounts of apoB-containing lipoproteins (chylomicrons,
VLDL and LDL) in the circulation. The
underlying genetic defect in abetalipoproteinaemia
is a mutation in the gene coding for the microsomal
triglyceride-transfer protein. This protein is essential
for lipoprotein assembly in the Golgi apparatus; without
it the lipoproteins are not secreted by the intestine
or liver. Abetalipoproteinaemia patients become
vitamin E defi cient because the steatorrhoea caused
by the absence of chylomicrons severely impairs
absorption of the vitamin. Furthermore, the lack of
VLDL secretion by the liver means that no LDL can
be formed, and so any vitamin E that might have been
absorbed cannot be transported in the usual manner.
The treatment of abetalipoproteinaemic patients
with massive oral doses of vitamin E (100 IU per kg
per day) allows a small proportion to be absorbed,
resulting in detectable plasma levels and correction of
in vitro erythrocyte haemolysis (Traber et al., 1993).
Normal plasma levels are rarely, if ever, attained. Interestingly,
the enterocytes of abetalipoproteinaemic
patients are able to synthesize HDL, which do not
require apoB for their formation (Deckelbaum et al.,
1982). It is possible that this abnormally produced
enteric HDL facilitates the intestinal secretion and
plasma transport of vitamin E in the absence of the
apoB-containing lipoproteins. The principal clinical
features of abetalipoproteinaemia are steatorrhoea
and spiky erythrocytes (both congenital), pigmented
retinopathy and a chronic progressive neurological
disorder. The characteristic neurological and retinal
symptoms manifest in the fi rst decade of life, evolving
into a crippling ataxia with visual impairment by the
second or third decades (Sokol, 1989).
Homozygous hypobetalipoproteinaemia
Patients with this condition have a defect in the
apoB gene and secrete lipoproteins containing truncated
forms of apoB. These defective lipoproteins can
transport minor amounts of vitamin E but they have
Vitamin E 251
a rapid turnover and constitute only a tiny fraction of
the circulating lipoproteins in these patients.
Ataxia with vitamin E defi ciency (AVED)
Ataxia with vitamin E defi ciency (AVED) uniquely
represents a primary vitamin E-defi cient state.
Originally called ‘isolated vitamin E defi ciency syndrome’,
and later ‘familial isolated vitamin E’ (FIVE)
defi ciency, AVED is the result of a mutation in the
gene for α-tocopherol transfer protein (α-TTP) on
chromosome 8. Infants born with this syndrome have
normal gastrointestinal function and yet their plasma
vitamin E levels are only 1% of normal. There is either
a complete absence of α-TTP or a defect in the
α-tocopherol-binding region of the protein (Traber,
1994); in either case, there is impaired hepatic secretion
of α-tocopherol in VLDL. The dramatic fall in the
plasma level of vitamin E is due to the rapid removal of
α-tocopherol from the plasma to the liver and excretion
in the bile, with no α-TTP to salvage it. The ataxia
and other neurological symptoms appear between the
ages of 4 and 18 years. They are manifestations of the
neurological damage that arises from the impaired
delivery of vitamin E to the nervous tissues, which are
especially sensitive to variations in plasma vitamin E.
When given vitamin E supplements (about 1 g per
day), patients maintain normal plasma α-tocopherol
concentrations and progression of the neurological
damage is halted. If patients stop taking the supplements,
their plasma concentrations fall to defi ciency
levels within days and the damage progresses.
Clinical features and histopathology of vitamin E
defi ciency
Sokol (1988) compared the clinical features found in
abetalipoproteinaemia, chronic childhood cholestasis,
other fat malabsorption disorders and isolated
vitamin E defi ciency (now known as AVED). The
most common fi ndings include loss of deep tendon
refl exes, truncal and limb ataxia, loss of positional and
vibratory sensation, muscle weakness and dysarthria.
Ophthalmoplegia (impairment of eye movements)
and pigmented retinopathy are common features in
abetalipoproteinaemia, cholestasis and other fat malabsorption
disorders, but they are not seen in AVED. A
possible explanation is the fact that the fi rst three disorders
represent secondary defi ciency states, malabsorption
being the primary cause. In contrast, AVED,
with no evidence of fat malabsorption or of other
nutritional defi ciencies, represents a primary defi -
ciency state. A concomitant defi ciency of vitamin A
is probably required to produce the ocular symptoms
present in cases of secondary vitamin E defi ciency, the
two vitamins acting synergistically.
Sokol (1988) also described the histopathology of
vitamin E defi ciency in humans. Axonal degeneration
and demyelination of large-calibre neurons are
universal in both primary and secondary advanced
defi ciency. Disturbance in function of the posterior
columns of the spinal cord, sensory nerves and
spinocerebellar tracts account for the loss of vibratory
and positional sensation and truncal and limb ataxia.
The nerve degeneration presumably originates from
peroxidation of constituent phospholipids. Peroxidative
injury and the formation of lipopigments is the
cause of pigmented retinopathy commonly seen in
older patients with abetalipoproteinaemia.

Inhibition of vascular smooth muscle cell proliferation

Inhibition of vascular smooth muscle
cell proliferation
Vascular smooth muscle cell proliferation represents
a signifi cant central event in the formation of the fi -
brous atherosclerotic plaque. α-Tocopherol at physiological
concentrations specifi cally inhibits mitogeninduced
proliferation of vascular smooth muscle cells
and certain other cell types in a parallel manner to
the inhibition of PKC activity (Boscoboinik et al.,
1991a,b; Chatelain et al, 1993). The degree of inhibition
of cell proliferation depends on the mitogen
responsible for stimulating growth. Proliferation induced
by platelet-derived growth factor (PDGF), endothelin
and unmodifi ed LDL was almost completely
inhibited by α-tocopherol, whereas proliferation
produced by other mitogens, such as bombesin and
lysophosphatidic acid, was only moderately or slightly
inhibited (Azzi et al., 1993). In cultured smooth mus muscle
cells, α-tocopherol activated the cellular release of
the growth-inhibiting transforming growth factor-β
(TGF-β) (Özer et al., 1995). Calphostin C, a specifi c
PKC inhibitor, also inhibits smooth muscle cell proliferation,
supporting the notion that the antiproliferative
effect of α-tocopherol is mediated through the
inhibition of PKC activity (Tasinato et al., 1995).

9.7.6 Protection of prostacyclin generation
in arteries
Prostacyclin (PGI2), a member of the prostaglandin
family, is a product of arachidonic acid metabolism
(see Fig. 5.3). Prostacyclin is a potent stimulator of
adenylyl cyclase, the enzyme which converts ATP to
cyclic AMP. Because platelet aggregation is inhibited
by cyclic AMP, prostacyclin acts as a platelet anti-aggregating
agent. This effect upon platelets is opposed
by another product of arachidonic metabolism, thromboxane A2, which inhibits adenylyl cyclase.
Prostacyclin is also a strong vasodilator; moreover,
it inhibits polymorphonuclear leucocyte adhesion to
endothelial cells in vitro (Boxer et al., 1980).
In experimental atherosclerosis the capacity of
arterial endothelial cells to generate prostacyclin at
the site of plaque formation is considerably impaired
(Gryglewski et al., 1978). This decrease in prostacyclin
production may be caused by oxidative stress.
Prostacyclin synthesis in cultured aortic endothelial
cells was depressed by the presence of high glucose
concentration in the medium and synthesis could be
restored by the simultaneous addition of vitamin E
(Kunisaki et al., 1992). Vitamin E probably acted as
an antioxidant by preventing the build-up of lipid
hydroperoxides which are inhibitory to prostacyclin
synthesis. The results suggested that vitamin E may
restore depressed prostacyclin production by the vascular
wall in hyperglycaemic conditions such as those
seen in patients with diabetes mellitus. Szczeklik et
al. (1985) reported that feeding an atherogenic diet
to rabbits for a week resulted in elevation of plasma
lipid hydroperoxides and a 90% decrease in arterial
generation of prostacyclin. Enrichment of the atherogenic
diet with 100 mg of vitamin E daily prevented
the increase in lipid hydroperoxides and protected the
prostacyclin generating system in arteries.
Chan & Leith (1981) showed that prostacyclin
synthesis in dissected rabbit aorta is decreased when
the tissue is depleted of vitamin E. Enrichment of cultured
human endothelial cells with vitamin E caused
an increase in arachidonic acid release and spontaneous
prostacyclin synthesis (Tran & Chan, 1988, 1990).
A potentiating effect of vitamin E on arachidonic acid
release in megakaryocytes was attributed to an upregulation
of phospholipase A2 (Chan et al., 1998).
The above fi ndings suggest that a defect in the local
production of prostacyclin may be an underlying factor
in the pathogenesis of atherosclerosis in terms of
platelet aggregation and monocyte adhesion to endothelial
cells. An increased dietary intake of vitamin
E can overcome this defect.
9.7.7 Preservation of nitric oxide-mediated
arterial relaxation
Background information can be found in Section 4.5.9.
Nitric oxide, an important relaxant of arterial
smooth muscle, is released from vascular endothelial
cells in response to acetylcholine (Furchgott &
Zawadzki, 1980) and other agents. Exposure of the
vascular wall to oxidized LDL (oxLDL) inhibits the
release of nitric oxide, thereby preventing relaxation
(Kugiyama et al., 1990). This dysfunction has, in part,
been attributed to PKC stimulation (Ohgushi et al.,
Keaney et al. (1996) fed rabbits diets defi cient in or
supplemented with α-tocopherol and examined the
effects of the vitamin on oxLDL-induced endothelial
dysfunction. Exposure of thoracic aorta segments
from vitamin-defi cient animals to ox-LDL produced
dose-dependent inhibition of acetylcholine-mediated
relaxation, while similarly treated segments from
animals consuming α-tocopherol showed no such inhibition.
Vessel resistance to endothelial dysfunction
in the vitamin-replete animals was strongly correlated
with the vascular content of α-tocopherol. Incorporation
of α-tocopherol into the vasculature limited
endothelial dysfunction induced by phorbol ester, a
direct activator of PKC. Using cultured human aortic
endothelial cells, Keaney et al. (1996) confi rmed that
oxLDL stimulates endothelial PKC and that cellular
incorporation of α-tocopherol inhibits this stimulation.
Desrumaux et al. (1999) showed that plasma
phospholipid transfer protein, by supplying vascular
endothelial cells with α-tocopherol, plays a distinct
role in the prevention of endothelial dysfunction.
9.7.8 Inhibition of platelet aggregation
α-Tocopherol has long been known to inhibit platelet
aggregation in vitro, an effect that was initially attributed
to the inhibition of lipid peroxidation. However,
α-tocopheryl quinone (which is not an antioxidant)
also inhibits platelet aggregation (Cox et al., 1980),
making this theory unlikely. Freedman et al. (1996)
found that inhibition of platelet aggregation by α-tocopherol
was closely linked to its incorporation into
platelets. Incorporation of α-tocopherol inhibited
phorbol ester-induced stimulation of platelet PKC,
thus implicating this enzyme in the inhibition of aggregation.
Platelet-rich plasma from healthy individuals receiving
varying doses of supplemental vitamin E showed a
marked decrease in platelet adhesion compared to presupplementation
adhesiveness. A daily supplement of
400 IU was near the optimum to reduce platelet adhesivity
(Jandak et al., 1988). Vitamin E supplementation
246 Vitamins: their role in the human body
(727 mg per day) of healthy humans with low antioxidant
status over a 5-week period showed no effects on
the capacity of platelets to aggregate in vitro (Stampfer
et al., 1988). Over a 5-month period, supplementation
of human diets with a combination of 300 mg vitamin
E, 600 mg ascorbic acid, 27 mg β-carotene and 75 μg
selenium in yeast signifi cantly reduced platelet aggregability
(Salonen et al., 1991).
9.7.9 Protection against oxLDL-mediated
Marchant et al. (1995) incubated LDL with copper
ions for varying periods in the presence or absence of
α-tocopherol. They then incubated these LDL preparations
with human monocyte-macrophages and
measured the toxicity produced in the cells. Toxicity
increased with increasing duration of copper-catalysed
oxidation in the absence of α-tocopherol. The
presence of α-tocopherol protected the LDL from
oxidation and the cells from toxicity. Martin et al.
(1998) reported that enrichment of human aortic
endothelial cells with α-tocopherol in vitro dosedependently
increased their resistance to cytotoxic
injury from oxLDL.
9.7.10 Effects of vitamin E supplementation
on experimentally induced atherosclerosis
Verlangieri & Bush (1992) studied the effects of
α-tocopherol supplementation on experimentally
induced atherosclerosis in monkeys using duplex
ultrasound scanning and B-mode imaging to monitor
lesion formation and progression. The animals
were randomly assigned to one of four groups and
atherosclerosis was monitored over a 36-month period.
One group was fed a basal diet, while three other
groups consumed an atherogenic diet (basal diet plus
0.4% cholesterol, w/w). Two of the latter groups also
received α-tocopherol, one at the onset of the study
(prevention group) and the other after atherosclerosis
was established (regression group). The dosage
of α-tocopherol was approximately eight times the
daily requirement for primates. A steady initial rise
in percent stenosis was detected in the three groups
fed an atherogenic diet. However, stenosis in the
unsupplemented animals progressed more rapidly
and to greater extent than stenosis in the prevention
group. In the regression group, stenosis reached a
plateau at 4 months post-supplementation, while
that of the unsupplemented group continued to rise.
The data indicated that, while α-tocopherol does not
totally prevent atherosclerosis, it appears to lessen the
severity and reduce the rate of disease progression.
Moreover, supplemental vitamin E may regress wellestablished
9.7.11 Epidemiological studies and clinical
Epidemiological studies have shown increased protection
against coronary artery disease in subjects
consuming vitamin E in daily doses >100 mg taken
for more than 2 years (Rimm et al., 1993; Stampfer et
al., 1993).
The CHAOS clinical trial (Stephens et al., 1996)
was designed to study the effects of α-tocopherol at
doses of 400 IU or 800 IU daily on the risk of cardiovascular
death and nonfatal myocardial infarction in
patients with established coronary atherosclerosis at
recruitment. The results showed that α-tocopherol,
compared to placebo, reduced the risk of non-fatal
myocardial infarction by 77%; there was no difference
in deaths due to cardiovascular causes. The results of
more recent clinical trials have not agreed with the
results of the CHAOS trial. HOPE Study Investigators
(2000) reported that 400 IU of vitamin E administered
daily for 4 to 6 years had no benefi cial effects on
cardiovascular outcomes in a high-risk population of
patients who were 55 years or older. In an Italian trial
(GISSI-Prevenzione Investigators, 1999), the number
of patients with nonfatal myocardial infarction was
slightly higher in patients receiving 300 IU of vitamin
E per day than those receiving placebo, and the
number of deaths from coronary heart disease was
slightly smaller. Neither difference was statistically
signifi cant.
The lack of agreement between CHAOS and GSSI
has been discussed by Pryor (2000) in his extensive review
of the effects of vitamin E on heart disease, Pryor
concluded that in view of the diffi culty in obtaining
more than about 30 IU per day from a balanced diet,
vitamin E supplementation (100 to 400 IU per day)
should be part of a general programme of hearthealthy
behaviour that includes a fruit- and vegetable-
rich diet and regular exercise.

Vitamin E and atherosclerosis

Background information can be found in Section
Vitamin E counteracts many of the atherogenic
effects of oxidized LDL (listed in Section 4.5.3) by
mechanisms that may be independent of its antioxidant
properties. As discussed in this section, some
mechanisms are attributable to an inhibitory effect of
vitamin E on protein kinase C (PKC) activity.
9.7.1 Inhibition of protein kinase C activity
Physiological concentrations of α-tocopherol markedly
inhibit PKC activity in vascular smooth muscle
cells (Azzi & Stocker, 2000). Inhibition is obtained
only at the cellular level; addition of α-tocopherol to
recombinant PKC in vitro does not result in inhibition.
β-Tocopherol, which possesses 89% of the antioxidant
potency of α-tocopherol, is not inhibitory; however, it
is able to reverse the inhibitory effect of α-tocopherol.
Other tocochromanols (γ- and δ-tocopherols and α-
and γ-tocotrienols) are also not inhibitory (Chatelain
et al., 1993). Thus the inhibitory effect of vitamin E on
PKC activity is specifi c to α-tocopherol and is apparently
unrelated to its antioxidant activity. Although
various isoforms of PKC (α, β, δ, ε, ζ and μ) have been
shown to be present in rat aortic smooth muscle cells,
only PKCα is inhibited by α-tocopherol (Ricciarelli et
al., 1998). The inhibition is indirect and not attributable
to a decreased synthesis of the enzyme. There is
evidence that α-tocopherol induces the activity of a
type 2A phosphatase (Ricciarelli et al., 1998), an enzyme
which desensitizes the PKC signalling pathway
242 Vitamins: their role in the human body
by dephosphorylating PKCα (Hansra et al., 1996).
Whether or not the type 2A phosphatase is the only
target of α-tocopherol is under investigation.
9.7.2 Protection of low-density lipoprotein
from oxidation
Several reports indicate protection of LDL from
oxidation following supplementation of human diets
with α-tocopherol (Dieber-Rotheneder et al., 1991;
Jialal & Grundy, 1992; Princen et al., 1992; Reaven
et al., 1993; Jialal et al., 1995). Supplementation can
increase the vitamin E content of LDL to about four
times its basal level (Esterbauer et al., 1990). In one
report (Reaven et al., 1993), dietary supplementation
with 1600 mg all-rac-α-tocopherol per day (1760 IU
per day) for 5 months resulted in a 2.5-fold increase in
LDL vitamin E levels and a 50% decrease in LDL susceptibility
to oxidation as measured by in vitro assays.
Jialal et al. (1995) showed that the minimum dose of
α-tocopherol needed to signifi cantly decrease the susceptibility
of LDL to oxidation was 400 IU per day.
The release of superoxide by phagocytic monocytes
during the respiratory burst (Section 5.2.3) induces
oxidation of LDL and renders it toxic to proliferating
cells (Cathcart et al., 1989). Monocytes from healthy
human subjects taking oral vitamin E supplements
(1200 IU per day) showed lower superoxide production
and a reduced capacity to oxidize LDL (Devaraj
et al., 1996). This effect of vitamin E appeared to be
mediated via inhibition of PKC. Vitamin E may therefore
protect circulating LDL from oxidation induced
by activated phagocytes. The enzyme responsible for
superoxide production in phagocytes is NADPH-oxidase.
Activation of this enzyme, elicited by appropriate
stimulation of the phagocytic cell, requires translocation
of several cytosolic enzyme components to
the membrane. PKC is involved in the activation of
NADPH-oxidase and can phosphorylate one of its
cytosolic components, p47phox. Cachia et al. (1998a)
studied the effect of vitamin E on NADPH-oxidase
activation elicited by phorbol myristate acetate in
human monocytes. They found that α-tocopherol
inhibited translocation and phosphorylation of
p47phox. The results suggested that the attenuating effect
of α-tocopherol on the respiratory burst is due to
inhibition of PKC activity.
The lysolecithin that accumulates in oxidized LDL
increases production of superoxide anion in the
walls of blood vessels, which may further enhance
LDL oxidation (Ohara et al., 1994). When human
monocytes were stimulated by phorbol ester to produce
superoxide in vitro, the addition of native LDL
inhibited superoxide production in a manner highly
sensitive to the increasing α-tocopherol content; the
free form of α-tocopherol produced lower inhibition
compared with the lipoprotein-associated form
(Cachia et al., 1998b). It was suggested that a vitamin
E-induced decrease in monocyte superoxide production
could lead to a decrease in lysolecithin production
in LDL. Lysolecithin is responsible for many of
the atherogenic properties of oxidized LDL and any
means of reducing its production would promote an
anti-atherogenic status of vessels.
9.7.3 Prevention of monocyte
Incubation of co-cultures of human aortic endothelial
and smooth muscle cells with LDL in the presence
of human serum resulted in an increased synthesis of
monocyte chemotactic protein 1 (MCP-1) mRNA
and protein. This was accompanied by an increase in
the adhesion of monocytes (but not neutrophil-like
cells) to the endothelial monolayer and an increased
transmigration of monocytes into the subendothelial
space. The increase in monocyte migration was most
likely due to the increased levels of MCP-1, since it was
completely blocked by a specifi c antibody to MCP-1.
Pre-treatment of the co-cultures with α-tocopherol
before the addition of LDL prevented the LDL-induced
monocyte transmigration (Navab et al., 1991).
9.7.4 Inhibition of monocyte–endothelial
cell adhesion
The induced expression of the endothelial adhesion
molecules, ICAM-1, VCAM-1 and E-selectin, is a
key event in the pathogenesis of atherosclerosis. The
genetic expression of protein molecules is regulated
by transcription factors which, when activated, bind
to specifi c regulatory elements on the DNA of target
genes where they mediate gene transcription and
synthesis of the encoded protein. Expression of genes
involved in early defence reactions, such as the genes
for cytokines and cytokine receptors, endothelial and
leucocyte adhesion molecules, and some growth and
differentiation factors, depends upon a particular
Vitamin E 243
transcription factor, nuclear factor-κB (NF-κB).
NF-κB is found in many different cell types and tissues,
but has been characterized best in cells of the
immune system, such as lymphocytes, monocytes and
In the absence of a stimulus, NF-κB resides in the
cytoplasm as an inactive complex composed of three
subunits – two DNA-binding subunits (p65 and p50)
and an inhibitory subunit called 1κB. Various extracellular
activators cause an alteration in 1κB, allowing
it to be released from the complex. The NF-κB dimer
then migrates to the nucleus where it binds to the
DNA recognition site.
The cytoplasmic NF-κB–1κB complex is activated
by a great variety of agents. These include the cytokines
IL-1 and TNF-α, viruses, double-stranded
RNA, bacterial lipopolysaccharide (LPS), endotoxins,
T-cell mitogens, phorbol 12-myristate 13-acetate
(PMA), protein synthesis inhibitors (e.g. cycloheximide)
and UV radiation (Schreck et al., 1992).
Schreck et al. (1991) reported that treatment of
T lymphocytes with micromolar concentrations of
hydrogen peroxide activated NF-κB; that is, hydrogen
peroxide induced the nuclear appearance and DNAbinding
of the transcription factor. Hydrogen peroxide
also induced the expression of the HIV-1 provirus,
whose gene is controlled by NF-κB. The activation of
NF-κB by hydrogen peroxide was inhibited by the
antioxidant and free radical scavenger N-acetyl-Lcysteine
(NAC). These experiments strongly supported
the preconceived idea that oxygen free radicals
were involved in the activation process. After its passive
diffusion through the cell plasma membrane, the
relatively innocuous hydrogen peroxide can be converted
into the highly reactive hydroxyl radical (Section
4.3.1). Activation of NF-κB by cycloheximide,
double-stranded RNA, IL-1 and LPS (Schreck et al.,
1991) and TNF-α and PMA (Staal et al., 1990) was
also inhibited by NAC.
Every type of cell produces oxygen radicals constitutively.
It is well established that different cell types
are stimulated to enhance the production of oxygen
radicals by the binding of extracellular cytokines such
as TNF-α and IL-1 to their respective cell surface receptors.
Since these cytokines and other agents, and
also hydrogen peroxide (a free radical precursor),
are able to activate NF-κB, and all of these activators
can be inhibited by a radical-scavenging antioxidant,
Schreck & Baeuerle (1991) postulated that oxygen
radicals act as second messengers in relaying extracellular
signals to the cytosolic NF-κB–1κB complex.
Oxygen radicals are well suited for this purpose; they
are small, diffusible and ubiquitous, and can be synthesized
and destroyed rapidly. The oxygen radicals
somehow activate NF-κB, which then migrates to the
nucleus and binds to its transcription site on the DNA
(Fig. 9.3).
Faruqi et al. (1994) observed that agonist-induced
adhesion of monocytes to cultured human umbilical
vein endothelial cells was inhibited by prior treatment
with α-tocopherol. The inhibition correlated with a
decrease in steady-state levels of E-selectin mRNA
and cell surface expression of E-selectin. Probucol and
NAC were also inhibitory, whereas other antioxidants
had no signifi cant effect. PKC did not appear to play
a role in the α-tocopherol effect since no suppression
of phosphorylation of PKC substrates was observed.
Cominacini et al. (1997) showed that expression of
ICAM-1 and VCAM-1 induced by oxidized LDL
could be reduced by pre-treatment of either the
oxLDL or the endothelial cells with vitamin E. Martin
et al. (1997) demonstrated an inhibitory effect of
α-tocopherol upon LDL-induced adhesion of monocytes
to human aortic endothelial cells and an accompanying
decrease in the release of ICAM-1. Devaraj
et al. (1996) reported that monocytes isolated from
healthy human subjects supplemented with 1200 IU
per day of α-tocopherol over 8 weeks were less able,
when activated, to adhere to activated endothelial cells
compared with monocytes isolated from placebo controls.
The vitamin E-enriched monocytes also showed
a 90% decrease in the release of interleukin 1β (IL-1β)
when activated. IL-1β is a proatherogenic, proinfl ammatory
cytokine that promotes monocyte–endothelial
cell adhesion; it also augments smooth muscle cell
proliferation via induction of platelet-derived growth
factor. Enrichment of monocytes with α-tocopherol
resulted in a reduced expression of the monocyte
adhesion molecules MAC-1 and VLA-4 (Islam et al.,
1998). Furthermore, pre-treatment of monocytes
with α-tocopherol signifi cantly decreased the LPSinduced
activation of NF-κB. The results of these and
other experiments suggest that α-tocopherol inhibits
transcription of adhesion molecule genes by preventing
the activation of NF-κB by oxygen radicals generated
within the cell.

Effects of vitamin D on insulin secretion

1α,25-Dihydroxyvitamin D3 is considered to be a
modulator of insulin secretion because vitamin D
defi ciency in rats is associated with marked impairment
of insulin secretion (Chertow et al., 1983) and
the insulin-secreting β-cells of the pancreas contain
the vitamin D-regulated protein calbindin-D28k
(Buffa et al., 1989) as well as the VDR (Clark et al.,
1980). Lee et al. (1994) speculated that 1α,25(OH)2D3
may primarily affect intracellular calcium mobilization,
resulting in an inhibition or stimulation of insulin
secretion depending on the vitamin D status and
other biochemical variables.
8.9 Vitamin D-related diseases
8.9.1 Rickets and osteomalacia
Rickets, a word from the Anglo-Saxon wrikken (to
twist) is the classic vitamin D defi ciency disease in
children. The disease is characterized by bow legs or
knock knees, curvature of the spine, and pelvic and
thoracic bone deformities. These deformities result
from the mechanical stresses of body weight and muscular
activity applied to the soft uncalcifi ed bone.
Without vitamin D, the cartilaginous growth plate
of the growing child fails to calcify. With this defect,
the cartilage cannot be replaced by bone on the diaphyseal
side and the growth plate becomes progressively
thicker. This results in enlargement of the joints
in the knees, wrists and ankles.
The prevalence of rickets among city-dwelling
children during the industrial revolution was attributable
to a limited exposure to sunlight and a lack of
suffi cient vitamin D in the diet. The narrow streets
and alleys in which the children lived and the smokepolluted
atmosphere were responsible for the lack of
sunlight. During the 1930s, the practice of adding
provitamin D to milk followed by UV irradiation
drastically reduced the incidence of rickets in the
222 Vitamins: their role in the human body
United States and some European countries. Later,
the commercial production of crystalline vitamin D2
led to its use in the fortifi cation of foods. Nowadays,
rickets is a rare disease among the indigenous populations
of the United States and Europe, but it is still evident
among the children of immigrants, particularly
Asians in Europe.
In adults, when the skeleton is fully developed, vitamin
D is still necessary for the continuous remodelling
of bone. During prolonged vitamin D defi ciency,
the newly formed, uncalcifi ed bone tissue gradually
takes the place of the older bone tissue and the weakened
bone structure is easily prone to fracture. This
condition, osteomalacia, should not be confused with
osteoporosis in which the ratio of mineral to osteoid
is unchanged. In osteomalacia, the epiphyses do not
swell, as they do in rickets, because the epiphyseal
growth plates no longer exist. Patients with osteomalacia
frequently suffer from muscle weakness and
bone tenderness or pain in the spine, shoulder, ribs or
pelvis. Pelvic deformation can occur causing potential
problems with childbirth. Women with low vitamin
D status may develop osteomalacia after several pregnancies
because they are unable to replace the calcium
lost from their bone reserves to the fetus in utero and
in lactation.
8.9.2 Vitamin D-dependent rickets
Vitamin D-dependent rickets is a rare inherited disorder
in which clinical and biochemical features of rickets
are evident despite an adequate intake of vitamin D.
This disorder is classifi ed into type I and type II disease
states, both of which appear to follow an autosomal
recessive pattern of inheritance (Brown et al., 2000).
Type I
Type I vitamin D-dependent rickets arises from impaired
renal synthesis of 1α,25(OH)2D3, which may
be due to a mutation in the gene encoding 25(OH)D-
1α-hydroxylase. The disease is diagnosed by normal
blood levels of 25(OH)D and profoundly decreased
levels of 1α,25(OH)2D3. At birth, affected children
appear healthy, but during the fi rst year or two of life
severe hypocalcaemia with tetany becomes evident.
The hypocalcaemia leads to secondary hyperparathyroidism
with elevated PTH levels and hypophosphataemia.
The calcium and phosphate defi ciencies
result in impaired mineralization of newly forming
bone, producing the classical symptoms of rickets.
The treatment of type I vitamin D-dependent rickets
is long-term administration of physiological doses of
Type II
Type II vitamin D-dependent rickets, now more
commonly called hereditary vitamin D-resistant
rickets (HVDRR), arises from a lack of responsiveness
of target tissues to 1α,25(OH)2D3 and in almost
all cases is due to a mutation in the gene encoding
the VDR. Some mutations lead to defective ligand
binding, while others lead to defective binding of the
hormone–receptor complex to the DNA. Hewison et
al. (1993) described an exceptional case attributable
not to a mutation of the VDR gene, but to a defect in
VDR translocation to the nucleus. Whereas the type
I disease state is characterized by depressed levels
of 1α,25(OH)2D3, this metabolite is elevated in the
type II state. Impaired hormonal function at the intestine
and bone causes defi ciencies in calcium and
phosphate, leading to rickets within months of birth.
Affl icted children are often growth retarded and suffer
convulsions due to tetany. Some children have total
scalp and body alopecia, including eyebrows and, in
some cases, eyelashes. The treatment of type II vitamin
D-defi cient rickets is supra-physiological doses
of 1α,25(OH)2D3 (Malloy et al., 1999).
8.9.3 Vitamin D-resistant rickets
Vitamin D-resistant rickets is a group of hereditable
abnormalities of renal phosphate transport, the most
common of which is X-linked hypophosphataemia.
The rickets cannot be explained solely by the severe
hypophosphataemia that is present and the undefi ned
pathological mechanism may involve both abnormal
phosphate transport and renal 1-hydroxylase function.
Treatment entails a combination of oral phosphate
and 1α,25(OH)2D3 (Brown et al., 2000).
8.10 Therapeutic applications of vitamin
D analogues
There have been several trials to assess the effi cacy of
vitamin D compounds in the treatment of postmeno-
Vitamin D 223
pausal osteoporosis. The most critical parameter for
successful treatment, a decrease in fracture rate, was
observed in some, but not all, studies (Brown et al.,
2000). In one Japanese study (Shiraki et al., 1996),
new fracture occurrence in the group treated with
1α(OH)D3 was around one-third of that in the placebo
group. The ideal vitamin D analogue would be one
which promotes bone formation and slow resorption,
yet has less tendency than 1α,25(OH)2D3 to produce
When cultured human epidermal keratinocytes
are exposed to physiological concentrations of
1α,25(OH)2D3, the cells cease to proliferate and start
to differentiate (Smith et al., 1986). The inhibition of
proliferation has been utilized in the treatment of hyperproliferative
diseases of the skin. Psoriasis, for example,
can be effectively treated by topical application
of the vitamin D analogue calcipotriol, which is about
200 times less potent than 1α,25(OH)2D3 in its effects
on calcium metabolism, although similar in receptor
affi nity (Kragballe, 1992). Non-toxic derivatives of
1α,25(OH)2D3 also have potential for the treatment
of some cancers and a variety of autoimmune disorders
(Holick, 1995a).
8.11 Toxicity
An excessive chronic intake of vitamin D can result
in toxicity with a fatal outcome. As in vitamin A
toxicity, hypervitaminosis D results from the excessive
consumption of vitamin D supplements, and
not from the consumption of usual diets. Toxic
concentrations of vitamin D have not resulted from
unlimited exposure to sunshine. Vitamin D intoxication
can be a concern in patients with specifi c diseases
being treated with unusual amounts of vitamin D or
analogues of the vitamin. In Great Britain, during
the 1940s and early 1950s, an epidemic of ‘idiopathic
hypercalcaemia’ broke out in newborn infants, who
failed to thrive and exhibited symptoms of toxicity.
This epidemic was eventually traced to over-supplementation
of commercial infant milk formulas
with vitamin D. The government policy was to supplement
milk with up to 2000 IU (50 μg) of vitamin
D to compensate for nutritional deprivation that
British children had suffered during World War II. To
allow for anticipated degradation of vitamin D during
processing and storage, some manufacturers put
1.5 to 2 times the correct amount of vitamin D into
the pre-processed milk.
Vitamin D toxicity is due primarily to the hypercalcaemia
caused by the increased intestinal absorption
of calcium, together with increased resorption of
bone. The cause of the hypercalcaemia is therefore
a drastic exaggeration of the normal physiological
action of vitamin D. The increased serum calcium
level can lead to a variety of non-specifi c symptoms,
such as anorexia, nausea, vomiting, muscle weakness
and constipation. Polyuria and polydipsia result from
the failure of the kidney to concentrate the urine.
The hypercalciuria that accompanies hypercalcaemia
encourages the formation of kidney stones in
the renal tubules. Chronic hypercalcaemia results in
irreversible calcifi cation of the kidneys (nephrocalcinosis),
causing permanent damage to the glomeruli
and renal tubules. Calcium salts may be deposited in
other extra-skeletal tissues as well, such as the heart,
blood vessels and lungs. The renal damage results in
a decrease in the glomerular fi ltration rate and severe
hypertension. In long-term hypervitaminosis D, the
excessive bone resorption results in part of the bone
being replaced by fi brous tissue. Where hypervitaminosis
D is fatal, the usual cause of death is renal
insuffi ciency.
High amounts of 25(OH)D3 can promote calcium
translocation in intestine and bone in vitro, suggesting
that overwhelming concentrations of 25(OH)D3
can displace 1α,25(OH)2D3 from the VDR and
directly elicit the biological responses. Brumbaugh
& Haussler (1973) predicted from their data that
25(OH)D3 must be present in 150 times the concentration
of 1α,25(OH)2D3 to displace the physiological
hormone. Hypervitaminosis D patients typically
exhibit a 15-fold increase in plasma 25(OH)D concentrations
compared to normal individuals, but
their 1α,25(OH)2D levels are not substantially altered
(Hughes et al., 1976). These observations have led to
the general conclusion that 25(OH)D, rather than
1α,25(OH)2D, is responsible for vitamin D toxicity.
An alternative hypothesis, presented by Vieth
(1990), is that 1α,25(OH)2D is, in fact, the agent
causing toxicity. This hypothesis is based on the differential
binding affi nities of the various vitamin D
metabolites for the DBP in the plasma. The 25(OH)D
metabolite binds much more tightly to the DBP than
does 1α,25(OH)2D. Therefore, when the plasma concentration
of 25(OH)D increases many-fold, a certain
224 Vitamins: their role in the human body
fraction of the circulating 1α,25(OH)2D will be displaced
from the DBP by 25(OH)D, thereby increasing
the concentration of free 1α,25(OH)2D. The liberated
hormone is now able to interact with a greater than
normal number of VDRs in target cells and elicit an
exaggerated response.
Hypercalcaemia resulting from excessive intake of
the parent vitamin D can persist for weeks or months
after intake has ceased, because of the accumulation of
this vitamin in adipose tissue and its gradual release
into the circulation. Treatment must therefore be
continued for a long time to counteract the hypercalcaemic
response. The treatment includes drugs to
enhance urinary excretion of calcium and drugs to
diminish the calcium effl ux from bone and absorption
of calcium from the intestine. The duration of
the patient’s toxic episode is brief if the administered
agent is 1α,25(OH)2D, because the half-life of this
hormone is only 4–6 h.