Vitamins | Diseases | Human Body and Cells

Vitamin C deficiency

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Defi ciency in humans
A defi ciency of vitamin C results in scurvy. Fully
developed scurvy is rarely seen nowadays, but clinical
signs of mild scurvy are found quite frequently in
alcoholics and drug addicts. The symptoms described
below have been observed in patients with scurvy
(Chazan & Mistilis, 1963) and in experimentally induced
scurvy (Hodges et al., 1971).
Early symptoms in adults are weakness, easy fatigue
and listlessness, followed by shortness of breath and
aching bones, joints and muscles. Progressive changes
in the skin then appear after about 4 months of complete
vitamin C deprivation. A horny material piles
up around the openings of hair follicles, and the hair
becomes fragmented and coiled. Red spots of pinpoint
to pinhead size caused by the rupture of small
blood vessels appear fi rst on the feet and ankles, and
then spread upwards. Thereafter, bruises appear over
large areas of skin, particularly on the legs. Bruising
is the manifestation of haemorrhages in subcutaneous
tissue, beneath the periosteum of bones and in
the synovia of joints. The gums become swollen and
bleeding, especially where there is advanced dental
caries. Haemorrhages are caused by rupture of capillaries,
which are fragile because of impaired ascorbic
acid-dependent synthesis of vascular basement
membrane (Priest, 1970). Wounds fail to heal and old
wounds reopen. The sufferer is visibly anaemic due in
part to haemolysis caused by peroxidative damage to
the erythrocyte plasma membrane (Goldberg, 1963).
Vitamin C defi ciency in adults may cause osteoporosis
due to a diminished production of organic matrix
in bones. The corresponding symptoms in infantile
scurvy are impaired ossifi cation and bone growth.
Kinsman & Hood (1971) studied the psychological
aspects of vitamin C defi ciency in healthy volunteers.
They measured four behavioural areas: physical fi tness
(strength, coordination and balance), mental
functions (memory, vigilance and problem solving),
psychomotor performance tasks (reaction time,
manipulative skills and hand–arm steadiness), and
personality. Three areas of change associated with
vitamin C deficiency were found: physical fitness involving
bending or twisting of the legs, psychomotor
tasks, and measures of personality. The changes in the
physical fitness could be accounted for by the pronounced
joint pain in the legs that occurred during
the deficiency period. The decrements in psychomotor
performance were attributed to a reduced motivational
level. The personality changes corresponded
to the classical ‘neurotic triad’ of the Minnesota Multiphasic
Personality Inventory, i.e. hypochondriasis,
depression and hysteria. Elevation of this triad is also
found in prolonged semi-starvation and defi ciencies
of B-complex vitamins.
In fully developed scurvy, as witnessed and recorded
at sea by James Lind in 1752, the body is covered with
spots and bruises, and the skin overlying the joints
becomes discoloured from the haemolysed blood in
and around them. There may be bleeding into the
peritoneal cavity and pericardial sac as well as into
joints. The gums become swollen, spongy and of a
livid blue-red colour. The swelling can develop to such
an extent that the gum tissue completely encases and
hides the teeth. The spongy gums bleed on the slightest
touch and become secondarily infected, leading to
loosening of the teeth and gangrene. Death preceded
by dyspnoea, cyanosis and convulsions is inevitable in
the continuing absence of vitamin C.
19.12.2 Rebound scurvy
Theoretically, the absorption of ascorbic acid could be
impaired on resumption of normal vitamin C inputs
following mega-dosing (>1 g per day), because of insuffi
cient carriers in the enterocyte cell membranes.
Based on experiments with guinea pigs, it is considered
likely that, in humans, renewed synthesis of
carriers will take place well before the onset of scurvy.
During mega-dosing, reduced ascorbate absorption is
accompanied by increased rates of ascorbate catabolism.
In adult guinea pigs, the accelerated catabolism
is not reversible after more than 2 months on subnormal
uptake of ascorbate (Sorensen et al., 1974). Guinea
pigs are thus susceptible to a systemic conditioning
effect known as rebound scurvy, caused by an induction
of ascorbic acid-metabolizing enzymes by high
dietary vitamin C. The body stores of vitamin C are
depleted more rapidly in juvenile guinea pigs than in
adults, increasing the likelihood of rebound scurvy in
juveniles. Solid evidence for the existence of rebound
scurvy in humans is tenuous (Gerster & Moser, 1988),
and reports by Schrauzer & Rhead (1973) and Siegel
et al. (1982) describe only single cases.

Vitamin C and cardiovascular disease

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Ascorbic acid through its numerous metabolic and
antioxidant effects may inhibit some of the steps
involved in atherosclerosis and thrombosis, thus
reducing the risk of cardiovascular disease. In a casecontrol
study, Ramirez & Flowers (1980) reported
signifi cantly lower (p < 0.001) leucocyte vitamin C
levels in 101 cases of angiographically documented
cardiovascular disease.
19.11.1 Cholesterol metabolism
Studies of animals that either synthesize (rat, rabbit)
or do not synthesize (guinea pig, monkey) vitamin
C have shown that vitamin C is intimately involved
in cholesterol metabolism. Guinea pigs subjected to
chronic vitamin C defi ciency exhibit increased cholesterol
levels in blood plasma and liver due to slower
conversion of cholesterol to bile acids (Ginter et al.,
1971; Ginter, 1973). The impaired conversion results
from a decreased activity of the rate-limiting liver enzyme
cholesterol 7α-hydroxylase (Horio et al., 1989).
When guinea pigs, rats and rabbits are rendered
hypercholesterolaemic by feeding a high-cholesterol
diet, vitamin C supplementation lowers their blood
cholesterol levels.
19.11.2 Lipoprotein profi le
Diets low in vitamin C lead to a redistribution of
cholesterol among the various plasma lipoproteins.
Vitamin C defi ciency in ODS rats (rats with an hereditary
inability to synthesize ascorbic acid) leads to
an increase in potentially pro-atherogenic LDL cholesterol
and a decrease in HDL cholesterol, resulting in
hypercholesterolaemia (Uchida et al., 1990).
19.11.3 Protection of LDL against
peroxidative modifi cation
Physiological concentrations of ascorbic acid protect
LDL against copper-catalysed peroxidative modifi cation
in vitro, maintaining the ability of LDL to be
recognized by appropriate LDL receptors and not by the scavenger receptor of macrophages (Sakuma
et al., 2001). This protective action preserves LDL’s
indigenous lipid-soluble antioxidants, except for
ubiquinol, the reduced form of coenzyme Q (Retsky
& Frei, 1995). Ascorbic acid spares, rather than
regenerates, LDL-associated α-tocopherol, i.e. prevents
α-tocopherol oxidation in the fi rst place. The
dilemma of whether ascorbate acts as a pro-oxidant or
as an antioxidant when interacting with LDL has been
addressed by Lynch et al. (1996). Ascorbate protects
native or mildly oxidized LDL against further metal
ion-dependent oxidation; only if LDL becomes extensively
oxidized does ascorbate acts as a pro-oxidant.
19.11.4 Effects on nitric oxide-mediated
arterial relaxation
Several studies have shown that an acute application
of ascorbic acid enhanced endothelium-dependent
vasodilation in patients with diabetes, coronary artery
disease, hypertension, hypercholesterolaemia, or
chronic heart failure, and in cigarette smokers (Heitzer
et al., 1996; Levine et al., 1996; Ting et al., 1996; Solzbach
et al., 1997; Ting et al., 1997; Hornig et al., 1998).
Long-term ascorbic acid treatment (500 mg per day)
produced a sustained improvement in endotheliumdependent
vasodilation in patients with coronary artery
disease (Gokce et al., 1999). Kanani et al. (1999)
demonstrated that administration of ascorbic acid
prevents induction of endothelial dysfunction by homocysteine.
These fi ndings may be attributable to the
scavenging of superoxide by ascorbate, thus preventing
the reaction between superoxide and nitric oxide
to form hydroxyl radicals and nitrogen dioxide, both
of which can initiate lipid peroxidation.
Heller et al. (1999) demonstrated that pre-incubation
of cultured endothelial cells with ascorbic acid
led to a three-fold increase of the cellular production
of nitric oxide after stimulation with ionomycin or
thrombin. Ascorbate did not induce the expression of
nitric oxide synthase and appeared to act through an
effect on the availability or affi nity of the enzyme cofactor
tetrahydrobiopterin. The fi ndings suggest that
saturation of the vascular tissue with ascorbate provides
the optimum reaction conditions for adequate
nitric oxide synthesis and that a decrease in intracellular
ascorbate leads to endothelial dysfunction.
19.11.5 Enhancement of prostacyclin
formation
The formation of prostacyclin (PGI2), a member of
the prostaglandin family which protects the arterial
wall against deposition of platelets, is inhibited by hydroperoxides
of unsaturated fatty acids. In vitro studies
have shown that physiological concentrations of
ascorbic acid enhance the formation of prostacyclin
by aortic rings by protecting the cyclooxygenase and
PGI-synthase (Beetens & Herman, 1983).
19.11.6 Effects of vitamin C
supplementation
Rifi ci & Khachadurian (1993) administered vitamin
C (1 g per day) and vitamin E (800 IU per day), both
separately and in combination, to healthy female and
male subjects and examined oxidation of lipoproteins
in vitro. Vitamin E administration alone produced a
52% inhibition and vitamin C alone a 15% inhibition
of copper-catalysed thiobarbituric acid reactive
substances production; the combination of vitamins
produced a 63% inhibition. Harats et al. (1998) reported
that in young healthy male subjects consuming
a diet high in saturated fats, supplementation with
citrus fruits containing an estimated 500 mg per day
of vitamin C reduced the in vitro susceptibility of
LDL to oxidation. Mosca et al. (1997) reported that
antioxidant supplementation with a combination of
800 IU of vitamin E, 1000 mg of vitamin C and 24 mg
of β-carotene signifi cantly reduced the susceptibility
of LDL to oxidation in patients with coronary artery
disease. The response produced by a similar combination
containing half the amounts of each antioxidant
was non-signifi cant.
19.11.7 Epidemiological studies
Current evidence from epidemiological studies on the
role of vitamin C in the prevention of cardiovascular
disease is inconclusive, with some studies showing a
very strong correlation between vitamin C intake and
incidence of cardiovascular events and other studies
showing no correlation at all (Lynch et al., 1996; Institute
of Medicine, 2000).

More in Vitamin C

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Natural killer cell activity
An in vivo effect of ascorbic acid on enhancement of
human natural killer cell activity has been reported
at a dosage of 60 mg per kg body weight (Vojdani &
Ghoneum, 1993).
19.10.8 Regulation of the complement
component C1q
When guinea pigs were fed tissue-saturating amounts
of vitamin C, plasma C1q concentrations were signifi -
cantly higher than in those animals fed only enough
ascorbate for adequate growth and for the prevention
of scurvy (Haskell & Johnston, 1991). When healthy
men and women were given 500 mg ascorbate three
times daily with meals for 4 weeks, their plasma C1q
levels were not signifi cantly altered (Johnston, 1991).
Hence, signifi cantly enhanced C1q production may
occur only during activation of the immune system,
and not in healthy, non-infected individuals.
19.10.9 Enhancement of lymphocyte blastogenesis
Using cultured spleen cells from an inbred strain of
rat that does not synthesize vitamin C, Oh & Nakano
(1988) observed that ascorbic acid enhanced
lymphocyte blastogenesis through inhibition of the
biosynthesis of immunosuppressive histamine.
19.10.10 Enhancement of interferon
synthesis
The participation of vitamin C in protection against
some viral infections may be in the enhancement of
interferon biosynthesis as demonstrated in vivo and
in vitro. The level of circulating interferon induced
in mice by inoculation with leukaemia virus was enhanced
by the addition of ascorbate to the drinking
water (Siegel, 1974). Ascorbate also enhanced the interferon
levels produced by cultured human embryo
fi broblasts in response to Newcastle Disease virus
(Dahl & Degré, 1976; Karpin´ ska et al., 1982).
19.10.11 Regulation of cytokines
Vitamin C has an indirect effect on lymphocyte proliferation
through its action on cytokines, as shown in
vitro by Cunningham-Rundles et al. (1993). Ascorbic
acid suppressed proliferation response to interleukin-
2, suggesting a basis for the vitamin’s inhibitory effect
on mitogen-induced lymphocyte proliferation. In
contrast, ascorbic acid enhanced the proliferative
response to interferon-γ, without inhibiting the
production of interferon-γ that accompanied the response
to infl uenza A (Table 19.4).
19.10.12 Clinical application to immunodeficiency diseases
Anderson (1981) administered a single oral daily dose
of 1 g sodium ascorbate to three children suffering
from chronic granulomatous disease as a supplement
to prophylactic trimethoprim–sulphamethoxazole
therapy for 2 years. In all three patients, introduction
of ascorbate to the therapeutic regimen resulted in the
correction of defective neutrophil motility and increased
activity against staphylococci. These responses
were accompanied by a decrease in the frequency of
infection and increased weight and growth rate.

Immune function of Vitamin C

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There is a large body of evidence that vitamin C plays
an important role in the biochemistry of the human
immune system, particularly in the stimulation of
phagocytosis. Leucocytes accumulate ascorbic acid
after uptake from the plasma by active transport
(Moser, 1987), suggesting an involvement of the vitamin
in the normal function of these cells. The concentration
of ascorbate in monocytes, for example, is over
80 times higher than that in plasma (Evans et al., 1982)
and macrophages contain about twice as much ascorbate
as neutrophils and monocytes (Schmidt & Moser,
1985). Vitamin C accumulation in activated human
neutrophils is increased as much as ten-fold above the
concentrations present in resting neutrophils as a result
of a novel vitamin recycling mechanism. Extracellular
ascorbate is oxidized to dehydroascorbic acid by
oxidants generated by the activated neutrophil. The
dehydroascorbic acid is preferentially taken up by the
neutrophil and reduced intracellularly to ascorbate
within minutes (Washko et al., 1993). Ascorbate, as an
antioxidant, protects phagocytes from self-destruction
by reactive oxidants (Muggli, 1993). It also neutralizes
reactive oxidants released extracellularly by
activated phagocytes, thereby preventing damage to
surrounding host tissue (Anderson & Lukey, 1987).

Antioxidant role of Vitamin C

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Ascorbate is an effective scavenger of all aggressive
reactive oxygen species within the aqueous environment
of the cytosol and extracellular fl uids. These
species include hydroxyl, superoxide anion and nonlipid
peroxyl radicals together with the non-radicals
singlet oxygen and hydrogen peroxide (Sies & Stahl,
1995). Ascorbate reacts with free radicals to produce
the ascorbyl radical and detoxifi ed product through
a single-electron transfer. Fig. 19.8 shows a possible
scheme in which ascorbate can be recycled during the
scavenging process.
Ascorbate is not the only antioxidant in aqueous
systems: other water-soluble antioxidants such as protein thiols and urate are also present. However,
ascorbate is the only endogenous antioxidant that effectively
protects the lipids in blood plasma (and also
low-density lipoprotein) against oxidative damage
initiated by non-lipid peroxyl radicals generated in
the aqueous phase. This is observed as a complete cessation
of lipid peroxidation when ascorbate is added
to plasma; other endogenous antioxidants, including
α-tocopherol, do not have this effect (Frei, 1991). Apparently,
ascorbate traps virtually all peroxyl radicals
generated in the aqueous phase before they can diffuse
into the lipid phase. Thus, ascorbate acts as the fi rst
and major line of antioxidant defence in the protection
of lipoidal plasma constituents and low-density
lipoprotein. In this action, ascorbate spares vitamin
E, the chain-breaking antioxidant in the lipid phase
(Doba et al., 1985).
In its role as a lipid-soluble, chain-breaking antioxidant
in biomembranes and lipoproteins (see Section
9.5), vitamin E (tocopherol, T-OH) scavenges
lipid peroxyl free radicals and itself is converted to
the tocopheroxyl radical (T-O•). Lipid peroxyl radicals,
because of their location in lipid environments,
cannot be scavenged by ascorbate anion. However, in
vitro studies using phospholipid liposomes as model
biomembranes have shown that ascorbate (AH–)
restores the antioxidant activity of vitamin E by converting
the tocopheroxyl radical back to the phenolic
tocopherol (reaction 19.7). Ascorbate works at the
lipid–water interface of membranes, very close to the
polar head groups of tocopherol.
T-O• + AH– → T-OH + A–• (19.7)
Whether vitamin C regenerates vitamin E in vivo is
debatable. Burton et al. (1990) found no evidence for
an interaction between the two vitamins in guinea
pigs and concluded that any such interaction must be
negligible in comparison with the normal turnover
of vitamin E.
As discussed above, ascorbate is an excellent antioxidant
but, paradoxically, it can also behave as a pro-oxidant
at lower concentrations (Buettner & Jurkiewicz,
1996). This crossover effect from pro-oxidant to
antioxidant is dependent on the ability of transition
metals in their reduced forms (e.g. Fe2+ and Cu+) to
catalyse the generation of free radicals. Ascorbate,
being a powerful reducing agent, maintains transition
metals in their catalytic reduced forms. At a high concentration
of ascorbate, the length of free radical chain
reactions will be small owing to ascorbate’s free radical
scavenging action. As the concentration of ascorbate is
lowered, there will come a point where its antioxidant
action is negligible but its capacity to reduce catalytic
metals is still suffi cient. At this crossover point ascorbate
switches from being an antioxidant to a prooxidant.
The antioxidant/pro-oxidant behaviour of
ascorbate has implications in the protection of plasma
LDL from oxidative modifi cation (Section 19.11.3).
The antioxidant action of vitamin C has a wide variety
of protective roles in the body. For example:
• the DNA in human sperm is protected from free
radical damage (Fraga et al., 1991);
• lung tissue is protected from free radical damage
resulting from inhalation of tobacco smoke, pollutants
and ozone;
• ocular tissue is protected from photo-oxidative
damage that can ultimately result in cataract formation;
• the high concentrations of ascorbate in neutrophils
and macrophages and its release on stimulation
protect these phagocytes and host tissue during the
respiratory burst in which reactive oxygen species
are produced to kill phagocytosed pathogens.

Biosynthesis of collagen

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Collagen is the major macromolecule of most connective
tissues. It is composed of three α chain
sub units that are wound together to form a triple
helix. Cross-linking gives the molecule a rigid and
inextensible structure. There are over 25 different α
chains that associate to yield 15 different types of collagen.
Type I collagen, which is found in large quantities
in skin and bone, comprises two α1(I)-chains and
one α2(I)-chain. The amino acid composition of collagen
is unusual among animal proteins in that it has
an abundance of proline and 4-hydroxyproline and a
few residues of 3-hydroxyproline and hydroxylysine.
The hydroxyproline residues are necessary for proper
structural conformation and stability; hydroxylysine
residues take part in cross-linking and facilitate subsequent
glycosylation and phosphorylation.
Collagen α chains are synthesized in a precursor
form known as proα chains, which have additional
non-collagenous amino acid sequences (propeptides)
at both amino and carboxyl termini. The presence of
hydroxyproline and hydroxylysine arises through the
post-translational hydroxylation of particular proline
and lysine residues in the polypeptide chain. Within
the cisternae of the rough endoplasmic reticulum,
the newly synthesized proα chains encounter three
hydroxylating enzymes. Two of these enzymes, prolyl-
4-hydroxylase and prolyl-3-hydroxylase, convert
proline residues to 4-hydroxyproline or 3-hydroxyproline
respectively, and the third, lysyl hydroxylase,
converts lysine residues to hydroxylysine. Following
amino acid modifi cation, the propeptides at the carboxyl
termini of two proα1 and one proα2 chains associate
and bond through disulphide bridges. Triple
helix formation then takes place as the protein passes
through the endoplasmic reticulum. Following attachment
of carbohydrate moieties to the carboxy
terminal propeptides, the procollagen molecules are
transported to the cell surface within secretory granules.
Enzymatic removal of the propeptides during
the process of extrusion allows the collagen molecules
to spontaneously assemble into fi brils. These are then
cross-linked by a series of covalent bonds and deposited
into the extracellular matrix.
Ascorbic acid stimulates collagen synthesis through
increased transcription of procollagen genes (Hitomi
& Tsukagoshi, 1996). Also, ascorbic acid is an essential
cofactor for the post-translational hydroxylation of
proline and lysine residues in the polypeptide chain.
Each of the enzymes concerned contains an iron ion
(maintained in the ferrous state by ascorbate) and
requires molecular oxygen and α-ketoglutarate as
co-substrates (Prockop et al., 1979) (Fig. 19.4). The
absence of wound healing is one of the features of
scurvy that can be attributed to impaired collagen
synthesis arising from lack of vitamin C.
The pathway of collagen synthesis is tightly coupled
through feedback regulation (Schwarz et al., 1987).
Proline hydroxylation stabilizes the triple helical conformation of the procollagen. This conformation
increases the secretion rates by six-fold and this in
turn leads to an increase in translational effi ciency.
Therefore ascorbate levels, solely by controlling the
activity of the proline hydroxylation step, can control
the chain of events through the whole pathway.

Renal reabsorption of Vitamin C

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General principles
The kidney actively reabsorbs ascorbate present in the
glomerular fi ltrate, thereby maximizing vitamin C
conservation in the body and helping the intestine to
maintain the circulating vitamin in its useful, reduced
state. The kidneys of all mammals handle vitamin C in
a similar manner. Renal reabsorption of vitamin C is
an essential process for humans as, without it, urinary
loss would far exceed the average daily intake of the
vitamin. Although species that have the ability to synthesize
ascorbic acid might be able to replace that lost
in the urine, the metabolic costs would be high.
Transport mechanisms
Ascorbic acid
Renal uptake of the L-ascorbate anion at the brushborder
membrane of the absorptive cell of the proximal
convoluted tubule is, like intestinal uptake in the
human or guinea pig, a sodium-coupled, secondary
active transport system (Rose, 1986; Bowers-Komro
& McCormick, 1991). Unlike the corresponding intestinal
transport system, however, the renal system
is electrogenic, indicating a Na+/ascorbate– coupling
ratio of 2:1 (Toggenburger et al., 1981). As the loaded
carrier bears a net positive charge, its transport is accelerated
by the negative membrane potential. Rapid
renal reabsorption of ascorbate is essential considering
that the transit time in the proximal tubule is only
about 10 s. Ascorbate is transported across the basolateral
membrane by sodium-independent facilitated
diffusion (Bianchi & Rose, 1985a).
Dehydroascorbic acid
The mechanism of dehydroascorbic acid transport in
renal brush-border (Bianchi & Rose, 1985b) and basolateral
(Bianchi & Rose, 1985a) membrane vesicles
appears to be facilitated diffusion. A favourable gradient
for continued renal reabsorption is maintained
dehydroascorbic acid (Rose, 1989). Dehydroascorbic
acid is taken up also from the blood across the basolateral
cell membrane and subsequently reduced to
ascorbate, which is then returned to the circulation
(Rose, 1989). The kidney participates with the intestine
and blood components in promoting reduction
of dehydroascorbic acid derived from the blood.

Intestinal absorption of Vitamin C

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General principles
Approximately 80–90% of the vitamin C content of
a given foodstuff exists in the reduced form, ascorbic
acid; the remainder is in the oxidized form, dehydroascorbic
acid. Ascorbic acid and dehydroascorbic
acid are absorbed by separate transport mechanisms
in animal species that depend upon dietary vitamin
C (Fig. 19.3). Inside the absorptive cell (enterocyte)
of the intestinal epithelium, dehydroascorbic acid is
enzymatically reduced and the accumulated ascorbic
acid is transported across the basolateral membrane
to the bloodstream. In addition to uptake at the
brush-border membrane, dehydroascorbic acid from
the bloodstream can be taken up at the basolateral
membrane, reduced within the cell, and returned to
the circulation in the form of the useful and non-toxic
ascorbic acid. The serosal uptake of dehydroascorbic
acid from the bloodstream and intracellular reduction
to ascorbic acid take place in animal species which do
not have a dietary vitamin C requirement as well as
those species that do. The ability of the enterocyte to
absorb dehydroascorbic acid effi ciently is important
because, apart from the indigenous dehydroascorbic
acid content of the diet, additional oxidation of
ascorbic acid occurs in the gastrointestinal tract as
the vitamin functions to maintain other nutrients
such as iron in the reduced state. The intestinal uptake
and reduction of dehydroascorbic acid explains
why this compound, orally administered, maintains
plasma concentrations of ascorbic acid and prevents
scurvy. The overall system of intestinal transport and
metabolism is designed to maximize the conservation
of vitamin C and also to maintain the vitamin in its
non-toxic reduced state, whether it is derived from the
diet .

Effi ciency of ascorbate absorption in humans
The usual dietary intake of vitamin C ranges from 30–
180 mg per day and over this range the effi ciency of
absorption is 70–90% (Institute of Medicine, 2000).
Brush-border uptake by the sodium-coupled, secondary
active transport mechanism reaches its maximum
rate at a relatively low luminal concentration. Beyond
physiological intakes, absorption becomes progressively
less effi cient, falling from 75% of a single 1-g
dose to 16% of a single 12-g dose (Table 19.2). This
fall-off in effi ciency occurs because absorption of
high luminal concentrations of vitamin C takes place
mainly by simple diffusion, and this passive movement
proceeds at a very low rate.
The ingestion of eight 0.125-g doses of ascorbate
spaced throughout the day produced a 72% increase
in absorption compared to a single 1-g dose (Yung et
al., 1981). The absorption effi ciency of a single dose
can be improved if the ascorbate is ingested in the
form of a sustained-release capsule (Sacharin et al.,
1976). The ingestion of 1 g of ascorbate immediately
after a fatty meal produced a 69% increase in absorption
compared to the same dose given on an empty
stomach (Yung et al., 1981). The divided dose effect
is consistent with a saturable absorption mechanism,
while the after-meal effect indicates a slowing of gastric
emptying.
Adaptive regulation of ascorbate absorption in
guinea pigs
In the guinea pig, intestinal absorption of ascorbate
is adaptively regulated in a transient and reversible
manner by the level of dietary ascorbate (Karasov et
al., 1991). The mechanism of regulation is an increase
or decrease in the number of carriers at both brushborder
and basolateral membranes of enterocytes in
response to low or high concentrations of ascorbate
in the blood. The rationale for adaptive regulation
is that carriers are most needed at low dietary ascorbate
levels; at excessive levels the required amount of
ascorbate can be absorbed by fewer carriers, aided by
passive diffusion. As ascorbate does not provide metabolizable
energy, there is nothing to gain from the
cost of synthesizing and maintaining carriers when
the vitamin supply is in excess. The issue of adaptive
regulation has not been examined in humans.

Vitamin C

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The structure of vitamin C, designated as a hex uronic
acid, was established in 1933 at the University of
Birmingham in England by Walter Haworth and
his associates, who also accomplished its synthesis.
Szent-Györgyi and Haworth renamed hexuronic acid
‘L-ascorbic acid’ to convey its antiscorbutic properties;
the new name was offi cially accepted in 1965.
Both Szent-Györgyi and Haworth were to be awarded
the Nobel Prize in 1937, the former for Physiology
and Medicine and the latter for Chemistry. Synthetic
ascorbic acid proved to have identical physicochemical
and biological properties to the vitamin C isolated
from plant or animal tissues, and there was no difference
in biological potency between the synthetic and
natural products. In 1934, Reichstein and Grüssner
in Switzerland worked out a chemical route for synthesizing
ascorbic acid commercially, starting from
glucose.

The term ‘vitamin C’ refers to both ascorbic acid
and dehydroascorbic acid, since the latter oxidation product is reduced back to ascorbic acid in the body.
The principal natural compound with vitamin C activity
is L-ascorbic acid. There are two enantiomeric
pairs (mirror images) of the 2-hexenono-1,4-lactone
structure; namely, L- and D-ascorbic acid and L- and
D-isoascorbic acid (Fig. 19.1). D-Ascorbic acid and
L-isoascorbic acid are devoid of vitamin C activ- ity and do not occur in nature. D-Isoascorbic acid
(commonly known as erythorbic acid) is an epimer
of L-ascorbic acid, the structural difference being the
orientation of the hydrogen and hydroxyl group at
the fi fth carbon atom. D-Isoascorbic acid is also not
found in natural products, apart from its occurrence
in certain microorganisms. It possesses similar reductive
properties to L-ascorbic acid, but exhibits only
5% of the antiscorbutic activity of L-ascorbic acid in
guinea pigs (Pelletier & Godin, 1969).
At around neutral pH, ascorbic acid exists as the
ascorbate anion due to facile ionization of the hydroxyl
group on C-3. Ascorbate is easily and reversibly
oxidized to dehydro-L-ascorbic acid, forming the
ascorbyl radical (also known as semidehydroascorbate)
as an intermediate (Fig. 19.2). The delocalized
nature of the unpaired electron in the ascorbyl radical
makes it a relatively unreactive free radical and two
ascorbyl radicals can react together non-enzymatically
to produce ascorbate and dehydroascorbic acid. In
the body, enzymes are available to reduce the ascorbyl
radical and dehydroascorbic acid back to ascorbate.
Dehydroascorbic acid is not a true organic acid as
it contains no readily ionizable protons. In aqueous
solution, dehydroascorbic acid exists not as the
2,3-diketo compound, but as the bicyclic hemiketal
hydrate. In buffered solution at neutral or alkaline
pH, dehydroascorbic acid undergoes a non-reversible
oxidation in which the two rings open to give 2,3-
diketogulonic acid in a straight-chain structure.

Dietary sources of Vitamin C
Fresh fruits (especially citrus fruits and blackcurrants)
and green vegetables constitute rich sources
of vitamin C. Potatoes contain moderate amounts
but, because of their high consumption, represent the
most important source of the vitamin in the British
diet. Liver (containing 10–40 mg per 100 g), kidney
and heart are good sources, but muscle meats and cereal
grains do not contain the vitamin in measurable
amounts. Human milk provides enough ascorbic acid
to prevent scurvy in breast-fed infants, but preparations
of cow’s milk are a poor source owing to oxidative
losses incurred during processing.

Causes of vitamin B12 malabsorption

2007-07-03T20:19:00.000-07:00

Vitamin B12 deficiency results from a failure of intestinal
absorption or subsequent transport to the tissues;
it is rarely, if ever, caused by a lack of B12 in the diet.
Disorders of vitamin B12 absorption and transport
have been discussed by Kapadia & Donaldson (1985)
and just a few examples of malabsorption are mentioned
here.
• Elderly people are prone to atrophic gastritis, a
condition in which the gastric oxyntic mucosa
atrophies to such an extent that virtually no hydrochloric
acid or intrinsic factor is secreted.
• In patients with diverticula, strictures and fi stulas
of the small intestine, stagnant regions of the lumen
may become contaminated with colonic bacteria
which can take up much of the dietary vitamin B12
passing by. Bacteria can take up vitamin B12 bound
to intrinsic factor, although not as avidly as they can
take up the free vitamin. Intrinsic factor and bacteria
have a similar affi nity for B12, so it is possible
that bacterial uptake could take place following the
vitamin’s release from haptocorrin but before its
transfer to intrinsic factor.
• The fish tapeworm Diphyllobothrium latum competes
with the host for B12, making it less available
for absorption.
• A common inherited disorder is an auto-immune reaction
with formation of antibodies against intrinsic
factor. Such cases involve two types of antibody: type
I prevents intrinsic factor from binding to cobalamin
and type II blocks the binding of the intrinsic factor–
cobalamin complex to the ileal receptor.

Vitamin B12

2007-07-03T20:18:00.000-07:00

A type of anaemia attributed to a digestive disorder
was reported by Combe in 1822 and later recognized
as pernicious anaemia by Addison in 1849. It was not
until 1926 that Minot and Murphy started to cure patients
suffering from pernicious anaemia by feeding
them with large amounts of raw liver. The idea for this treatment originated from the discovery by Whipple
that dietary liver improved haemoglobin production
in iron-defi cient dogs. In 1929, Castle showed that the
intestinal absorption of the ‘antipernicious anaemia
principal’ required prior binding to a specifi c protein
(intrinsic factor) secreted by the stomach.
Research into isolating the active principal from
liver was hampered by the inability to induce pernicious anaemia in animals. For many years, the
only known bioassay was the haemopoietic response
of patients with the disease. Eventually, in 1948, a red
crystalline substance having the clinical activity of
liver and designated as vitamin B12 was isolated by
several independent scientifi c groups. The success of
one group, headed by Folkers (Merck and Co., USA),
was largely attributable to a microbiological assay developed
by Shorb in 1947. The complicated structure
of vitamin B12 was established by Hodgkin using X-ray
crystallography in 1955. Its complete chemical synthesis
was achieved in 1973, but because of the large
number of stages required (over 70) the procedure is
of no commercial interest.

In accordance with the literature on nutrition and
pharmacology, the term vitamin B12 is used in this
text as the generic descriptor for all cobalamins that
exhibit antipernicious anaemia activity. Individual
cobalamins will be referred to by their specifi c names
(e.g. cyanocobalamin).
The cobalamin molecule depicted in Fig. 18.1 contains
a corrin ring system and a cobalt atom, which
may assume an oxidation state of (I), (II) or (III).
There are two vitamin B12 coenzymes with known
metabolic activity in humans, namely methylcobalamin
and 5´-deoxyadenosylcobalamin (frequently
abbreviated to adenosylcobalamin and also known
as coenzyme B12). The methyl or adenosyl ligands of
the coenzymes occupy the X position in the corrin
structure. The coenzymes are bound intracellularly
to their protein apoenzymes through a covalent peptide
link, or in milk and plasma to specifi c transport
proteins. The enzyme-bound cobalamins exist as
cob(I)alamins.
Cyanocobalamin is the most stable of the vitamin
B12-active cobalamins and is the one mostly used in
pharmaceutical preparations and food supplementation.
Aqueous solutions of cyanocobalamin are stable
in air at room temperature if protected from light. On
exposure to light, the cyano group dissociates from
cyanocobalamin and hydroxocobalamin is formed.
In neutral and acid solution hydroxocobalamin exists
in the form of aquocobalamin (Gräsbeck & Salonen,
1976). This photolytic reaction does not cause a loss
of activity.

Naturally occurring vitamin B12 originates solely
from synthesis by bacteria and other microorganisms
growing in soil or water, in sewage, and in the
rumen and intestinal tract of animals. Any traces of
the vitamin that may be detected in plants are due to
microbial contamination from the soil or manure or,
in the case of certain legumes, to bacterial synthesis in
the root nodules.
Vitamin B12 is ubiquitous in foods of animal origin
and is derived from the animal’s ingestion of cobalamin-
containing animal tissue or microbiologically
contaminated plant material, in addition to vitamin absorbed from the animal’s own digestive tract. Liver
is the outstanding dietary source of the vitamin, followed
by kidney and heart. Muscle meats, fi sh, eggs,
cheese and milk are other important food sources.
Vitamin B12 activity has been reported in yeast, but
this has since been attributed to the presence of noncobalamin
corrinoids or vitamin B12 originating
from the enriching medium (Herbert, 1988). About
5 to 30% of the reported vitamin B12 in foods may be
microbiologically active non-cobalamin corrinoids
rather than true B12 (National Research Council,
1989).
Vitamin B12 in foods exists in several forms as reported
by Farquharson & Adams (1976). Meat and fi sh
contain mostly adenosyl- and hydroxocobalamins;
these compounds, accompanied by methylcobalamin,
also occur in dairy products, with hydroxocobalamin
predominating in milk. Sulphitocobalamin is found
in canned meats and fi sh. Cyancobalamin was only
detected in small amounts in egg white, cheeses and
boiled haddock.

Chromosome damage: implications for cancer

2007-07-03T20:17:00.000-07:00

Division of cells with unrepaired or misrepaired DNA
damage leads to mutations. If these relate to critical
genes, such as proto-oncogenes or tumour suppressor
genes, cancer may result. Folate is essential for
DNA synthesis and repair through its role in purine
and pyrimidine synthesis. Its role in the synthesis of
S-adenosylmethionine (SAM) is also relevant to cancer.
SAM donates its methyl group to DNA, among
other acceptors, and a defi ciency of folate can lead to
hypomethylation of DNA. As DNA methylation is a
mechanism for silencing transcription (Ng & Bird,
1999), hypomethylation of DNA has the potential
to alter the normal control of gene expression. Hypomethylation
also alters chromatin conformation,
thereby allowing access of DNA-damaging agents and
endonucleases, which destabilize the DNA and make
it more susceptible to strand breaks (Kim et al., 1997).
Imbalanced DNA methylation is a common occurrence
in carcinogenesis (Laird & Jaenisch, 1994).
Low cytosolic levels of 5,10-methylene-THF associated
with folate defi ciency result in decreased synthesis
of deoxythymidine monophosphate (dTMP) and
the accumulation of deoxyuridine monophosphate
(dUMP). This leads to DNA polymerase-mediated
incorporation of dUMP into the DNA molecule in
place of dTMP. Normal DNA repair processes remove
the misincorporated dUMP, forming transient singlestrand
breaks (nicks) that could result in a doublestrand
break if two opposing nicks are formed. Kim
et al. (1997) showed that, in rats, dietary folate depletion
is capable of producing DNA strand breaks and
hypomethylation within a highly conserved region of
the p53 tumour suppressor gene. The p53 gene was
chosen for study because alterations in it have been
implicated in >50% of human cancers. On a genomewide
basis such alterations either did not occur or
were delayed, indicating some selectivity for the exons
examined within the p53 gene.
In epidemiological studies, dietary folate deficiency
is associated with an increased risk of several specific malignancies, notably cancer of the cervix, lung,
colorectum and brain (Glynn & Albanes, 1994). The
presence of micronucleated erythrocytes in marginal
folate defi ciency is indicative of chromosomal damage
(Everson et al., 1988). Both high DNA dUMP
levels and elevated erythrocyte/reticulocyte micronucleus
frequency are reversed by folate administration
(Blount et al., 1997). Duthie & Hawdon (1998)
showed that a dietary intake of folate adequate for the
prevention of clinical defi ciency may not be suffi cient
to maintain DNA stability.
In a study of American male physicians, Ma et al.
(1997) showed that the C677T polymorphism in the
MTHFR gene reduces the risk of colorectal cancer.
Subjects with the homozygous mutation (15% in controls)
had half the risk of colorectal cancer (odds ratio
0.49; 95% confi dence interval 0.27 to 0.87) compared
with the homozygous normal or heterozygous genotypes.
The protection due to the polymorphism was
absent in subjects with folate defi ciency and reduced
in those with high alcohol consumption. It can be
reasoned that, provided folate status is adequate, the
reduced activity of the thermolabile MTHFR enzyme
variant would lead to an increased level of intracellular
5,10-methylene-THF and this would reduce the
likelihood of dUMP misincorporation into DNA.

Megaloblastic anaemia

2007-07-03T20:12:00.000-07:00

A defi ciency of folate gives rise to a type of anaemia
known as megaloblastic anaemia. A clinically indistinguishable
anaemia is also produced by vitamin
B12 deficiency but, because it is accompanied by
neurological damage, it is referred to as pernicious
anaemia. Both types of anaemia are the result of abnormal
nuclear maturation caused by impaired DNA
synthesis. The impaired DNA synthesis is presumed
to be attributable to reduced intracellular levels of
polyglutamyl 5,10-methylene-THF. As shown in
reaction 17.7, this important folate is involved in
the formation of deoxythymidine monophosphate,
one of the two pyrimidine nucleotide constituents of
DNA. The defect in DNA synthesis leads to a variety of
secondary disturbances which result in the premature
death of many haemopoietic cells in the bone marrow,
possibly without ever completing the S phase of cell
replication.

Megaloblastic anaemia caused by folate defi ciency
manifests as megaloblastosis of the bone marrow
and macrocytosis of the circulating erythrocytes.
Examination of the bone marrow is of great diagnostic
importance. The erythrocyte precursor cells
(erythroblasts) in the bone marrow fail to proliferate
rapidly and exist as gigantic cells called megaloblasts
at all stages of maturation. It is the existence of such
cells that gives rise to the term megaloblastic anaemia.
The increased size of megaloblasts is apparent both in
the cytoplasm and in the nucleus. The nuclei contain
smaller quantities of condensed chromatin than the
nuclei of normoblasts of similar maturity and thus
have an open, sieve-like appearance.
The circulating erythrocytes which are derived from
mature megaloblasts are also abnormally large and are
referred to as macrocytes. The mean corpuscular volume
of macrocytes ranges from 100 to 160 μm3 compared
with 90 to 95 μm3 for normal erythrocytes. The
macrocytes are generally oval in shape (erythrocytes
are biconcave discs) but some fragmented and irregularly
shaped cells are also present. There is a reduction
in red cell count and sometimes incredibly low values
are found. The haemoglobin content of individual macrocytes is increased owing to their larger size, but
there is little change in the haemoglobin concentration
of whole blood.
White cells and platelets are also produced in the
bone marrow. In the differentiating granulocyte series
of white blood cells (neutrophils, eosinophils and
basophils) giant, abnormally shaped metamyelocytes
are found. Megakaryocytes (precursors of platelets)
may also be larger than usual. In advanced folate/B12
defi ciency, the total white cell count and platelet count
may be low. Circulating neutrophils are characterized
by an increased number of nuclear segments. The
presence of hypersegmented neutrophils is a valuable
clue in diagnosing folate/B12 deficiency when red cell
changes are masked by a coexistent iron defi ciency or
the anaemia of chronic disease.
As in all cases of anaemia, the body adjusts its
cardiopulmonary system to compensate for the diminished
oxygen-carrying capacity of the blood, so
in mild anaemia the subject may not be aware of any
problems. Eventually, however, the progressing anaemia
leads to symptoms of weakness, fatigue, shortness
of breath and palpitations. The sufferer may also
experience headache, irritability and an inability to
concentrate. Visible signs of megaloblastic anaemia
in white-skinned people are a marked pallor and a
slight jaundice, giving the skin a distinctive lemonyellow
tinge.
Megaloblastosis is not confi ned to developing cells
in the bone marrow – all other rapidly dividing cell
types will be affected, including epithelial cells lining
the gastrointestinal, respiratory, and urinogenital
tracts. A notable feature is glossitis where the tongue
is sore at the edges, bright red in colour and smooth in
texture. Gastrointestinal disturbances caused by defective
gut epithelia have adverse consequences upon
overall nutritional status. Male infertility results from
impaired spermatogenesis.
Folate defi ciency can result, in the absence of disease,
from reduced ingestion or absorption, or from
increased utilization. Dietary folate defi ciency is common
among people who, for various reasons, eat little
fruit or fresh vegetables. Absorption is impaired in
alcoholics. There is an increased utilization of folate
during pregnancy owing to the need to transfer an
extra 100–300 μg of folate per day to the fetus (Beatty
& Wickramasinghe, 1993). Experimental folate
defi ciency is diffi cult to produce under normal circumstances,
but the study of patients suffering from
malabsorption problems such as tropical sprue or the
use of folate antagonists has yielded much clinical
information.

Hyperhomocysteinaemia

2007-07-03T20:11:00.000-07:00

In plasma, 70–80% of homocysteine is bound to
plasma proteins, chiefl y albumin; only about 1% circulates
as free homocysteine. The remaining 20–30%
circulates as homocysteine disulphide (homocystine)
or as the mixed disulphide, homocysteine–cysteine.
Plasma homocysteine assays measure total homocysteine,
which is the sum of the homocysteine moieties
present in all of the above forms. Because variable
changes in plasma homocysteine concentration have
been observed post-prandially, it is customary to obtain
measurements in the fasting state. Normal levels
of fasting plasma homocysteine are considered to be
between 5 and 15 μmol L–1. Higher fasting values are
classifi ed arbitrarily as moderate (16–30), intermediate
(31–100) and severe (>100 μmol L–1) hyperhomocysteinaemia.
The methionine loading test has been
used to accentuate abnormalities of the homocysteine
metabolic pathways. The test measures fasting plasma
homocysteine before and 2 hours after an oral dose
of methionine (100 mg per kg body weight). An elevated
post-loading homocysteine level indicates an
abnormality.
Hyperhomocysteinaemia can result from inherited
defects in enzymes necessary for either trans-sulphuration
or remethylation and from acquired defi ciencies
in vitamin coenzymes. Renal insuffi ciency can
also lead to hyperhomocysteinaemia. Subclinical
folate defi ciency is commonly associated with hyperhomocysteinaemia,
presumably because of decreased
remethylation of homocysteine. Kang et al. (1987)
found elevated total homocysteine levels in 84% of
subjects with subnormal folate levels. The mean homocysteine
level in the low-folate subjects was about
four-fold greater than the mean level in the control
subjects.
An association between mild hyperhomocysteinaemia
and increased risk of occlusive vascular disease in
the coronary, cerebral and peripheral arteries has been
demonstrated in case-control (Selhub et al., 1995; European
Concerted Action Project, 1997) and prospective
(Stampfer et al., 1992; Arneson et al., 1995; Perry
et al., 1995) studies. Plasma homocysteine concentration
is a strong predictor of mortality in patients with
angiographically confi rmed coronary artery disease
(Nygard et al., 1997). Whether hyperhomocysteinaemia
is a causal risk factor for the disease or simply
a marker of another prothrombotic risk factor(s) is
debatable (Kuller & Evans, 1998).
Up to 30% of patients with coronary artery disease
had homocysteine elevations that were 10–50%
greater than the level observed among normal subjects
(Clarke et al., 1991). Subjects with hyperhomocysteinaemia
have a two-fold to three-fold increase in
risk of developing cardiovascular disease or venous
thrombosis (den Heijer et al., 1998). In vitro studies
have shown that high concentrations of homocysteine
can promote a prothrombotic state at the luminal surface
of the blood vessel (Lentz, 1998). An association
between impaired endothelium-dependent vasodilation
and hyperhomocysteinaemia was demonstrated
in children with homozygous homocystinuria (Celermajer
et al., 1993), in monkeys fed a methionine-enriched
diet (Lentz et al., 1996), in methionine-loaded
healthy humans (Bellamy et al., 1998; Chambers et
al., 1998) and in non-induced hyperhomocysteinaemic
healthy middle-aged (Woo et al., 1997) and
elderly (Tawakol et al., 1997) humans. In healthy
human subjects, even physiological increments in
plasma homocysteine following oral administration
of methionine or an animal protein meal impaired
endothelium-dependent vasodilatation (Chambers et
al., 1999a). Plasma homocysteine concentration can
be decreased by dietary supplementation with folic
acid, which suggests that hyperhomocysteinaemia
may be a treatable risk factor for vascular disease.

Folate homeostasis

2007-07-03T20:10:00.002-07:00

The majority of 5-methyl-THF arriving at the liver
from the intestine and taken up is not demethylated
and converted to polyglutamate; instead it is quickly
released for distribution to extrahepatic tissues. The
initial route for this distribution is the enterohepatic
circulation, whereby the folate is discharged into the
bile and subsequently reabsorbed by the small intestine
before re-entering the systemic circulation.
Accompanying 5-methyl-THF in the bile are larger
amounts of non-methylated tetrahydrofolates which
represent folates salvaged from dying cells such as
senescent erythrocytes and hepatocytes (Shin et al.,
1995). Any folic acid that might have been absorbed
and released into the portal circulation without
modifi cation is exclusively taken up by the liver and
either converted into one-carbon derivatives of THF
prior to rapid release into bile or polyglutamated and
incorporated into the hepatic folate pool (Steinberg,
1984). Hepatic reduction and derivatization of folic
acid provides another source of non-methylated tetrahydrofolates
present in bile (Shin et al., 1995).
The recycling of folate via the enterohepatic pathway
may account for as much as 50% of the folate
that ultimately reaches the extrahepatic tissues. Disruption
of the enterohepatic cycle by bile drainage
results in a fall of the serum folate level to 30–40% of
normal within 6 hours – a much more dramatic drop
than that seen with a folate-defi cient diet. Eventually,
the serum folate level stabilizes, despite continuing
losses in the bile. This suggests a net fl ux of folate into
the plasma compartment from tissue pools. Release
of stored folate from cells of any tissue requires hydrolysis
of the polyglutamates to monoglutamates by
intracellular conjugase.
The maintenance of a normal level of plasma folate
depends on regular increments of exogenous folate
from the diet. The enterohepatic circulation of folate
evens out the intermittent intake of dietary folate. The
liver plays a major role in maintaining folate homeostasis
because of its capacity to store about 50% of the
total body folate, its relatively rapid folate turnover,
and the large folate fl ux through the enterohepatic
circulation (Steinberg, 1984). In situations of dietary
folate defi ciency, the liver does not respond by releasing
its folate stores. Rather, the non-proliferating,
less metabolically active tissues mobilize their folate
stores and return monoglutamyl folate to the liver.
This folate is released by the liver via the enterohepatic
cycle and distributed to the tissues that most require
it – in particular, those with actively proliferating
cells. Preferential uptake of folate by certain tissues
(e.g. placenta and choroid plexus) is made possible
by the presence of the folate receptor on their cellular
surfaces. The kidney plays its part in conserving
body folate by actively reabsorbing folate from the
glomerular fi ltrate. In addition, a pathway exists that
is capable of salvaging folate released from senescent
erythrocytes.

Uptake of 5-methyl-THF by sinusoidal membrane
vesicles isolated from human liver is an electroneutral
active transport process, which is pH-dependent, sodium-
independent and appears to involve co-transport
with hydrogen ions mediated by the reduced
folate carrier (Horne et al., 1993). This would require
a mechanism for maintaining a gradient of H+ across
the basolateral membrane, but how this is accomplished
is not known for certain. Sinusoidal membrane
vesicles isolated from rat hepatocytes contain a
Na+–H+ exchange system (Arias & Forgac, 1984) and
it can be speculated that the H+ could be conducted
along the membrane and interact with the carrier,
thereby generating a ‘localized’ proton gradient that
could energize active transport of 5-methyl-THF.

Absorption of dietary folate

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In the human, the entire small intestine is capable of
absorbing monoglutamyl folate. Absorption is somewhat
greater in the proximal than in the distal jejunum
which, in turn, is much greater than in the ileum.
Folate transport across the brush-border membrane
of the enterocyte proceeds by two parallel processes
(Selhub & Rosenberg, 1981). At physiological concentrations
(<5 μM) of luminal folate, transport occurs
primarily by a saturable process, whereas at higher
concentrations, transport occurs by a non-saturable
process with characteristics of simple diffusion. Zimmerman
et al. (1986) produced data which suggest
that the latter process occurs in part through a conductance
pathway that involves anionic folate and a
cation (perhaps Na+) whose membrane permeation
properties affect the rate of folate transport. The saturable
component is discussed in the following with no
further mention of unsaturable transport.
Transport of folate is mediated by the reduced folate
carrier and is markedly infl uenced by changes in pH
(Schron, 1991). Folate exists primarily as an anion at
the pH of the lumenal contents. In vitro studies using
everted rat jejunal rings showed that absorption was
maximal at pH 6.3 and fell off sharply between pH
6.3 and 7.6 (Russell et al., 1979). In studies using
brush-border membrane vesicles (Said et al., 1987),
folate uptake increased as the pH of the incubation
buffer was decreased from 7.4 to 5.5. This increase in
folate uptake appeared to be partly mediated through
folate–/OH– exchange and/or folate–/H+ co-transport
mechanisms driven by the proton gradient across the
membrane and partly through a direct effect of acidic
pH on the carrier. Inhibition of folate transport by
the anion transport inhibitor DIDS suggested the
involvement of the folate–/OH– exchange mechanism.
Data reported by Mason et al. (1990) suggest that
the effect of pH on the carrier is attributable to an
increased affi nity of the carrier for its folate substrate.
The physiological relevance of the pH dependency
may be related to the existence of the acidic microclimate
at the luminal surface of the jejunum. This socalled
‘unstirred layer’ has a pH that is approximately
2 units lower than the bulk luminal pH and therefore
provides the necessary extracellular acidic conditions
for folate uptake.
Said et al. (1987) also found that folate transport
across the brush-border membrane was saturable,
competitively inhibited by structural analogues of
folic acid, unaffected by transmembrane electrical potential,
and Na+-independent. The human intestinal
reduced folate carrier has been cloned and characterized
at the molecular level (Nguyen et al., 1997).
Said et al. (1997) studied the intracellular regulation
of intestinal folate uptake using monolayers of
cultured mature IEC-6 epithelial cells. These cells are
derived from the proximal small intestine of a normal
rat and possess all of the cellular structures of native
enterocytes. Uptake of folic acid by IEC-6 cells was
similar to that of the native small intestine. Intracellular
cyclic AMP was found to affect the uptake of
folic acid independently of protein kinase A. Protein
tyrosine kinase also affected uptake, but protein kinase
C and Ca2+/calmodulin mediated pathways had
no signifi cant effect.
During intestinal transport some of the folate is
converted within the enterocyte to 5-methyl-THF in
a pH-dependent manner (Strum, 1979). This conversion
is extensive at pH 6.0 and negligible at pH 7.5
presumably because dihydrofolate reductase, the
rate-limiting enzyme in the reduction and methylation
process, has an acidic pH optimum. The percent
conversion is reduced by increasing the concentration
of folate in the mucosal medium, thus indicating saturation
of the process. Since at higher concentrations
most transported folate remains unmodifi ed, intestinal
conversion of absorbed folic acid is not obligatory
for transport into the circulation.
The mechanism of folate exit from the enterocyte
into the lamina propria of the villus is also carriermediated
and sensitive to the effect of anion exchange inhibition. In addition, the exit mechanism is electroneutral
and Na+-independent and has a higher
affi nity for the substrate than has the system at the
brush-border membrane (Said & Redha, 1987).
Absorption of milk folate by the suckling infant
Milk from humans and several species of other mammals
contains a folate-binding protein (FBP) which
may be important for folate absorption by the suckling
infant. In neonates, the uptake of folate bound
to milk FBP occurs preferentially in the ileum as opposed
to the jejunum. The incomplete development
of pancreatic and intestinal absorptive functions
could allow the FBP to reach the ileum without being
digested. This situation was demonstrated by Salter
& Mowlem (1983) who showed that a proportion
of goat’s milk FBP administered orally to neonatal
goats survived along the length of the small intestine.
Protease inhibitors inherent to colostrum may assist
the passage of bound folate along the small intestine
(Laskowski & Laskowski, 1951). In vitro, the addition
of goat’s milk FBP to the medium enhanced the transport
of 5-methyl-THF in brush-border membrane
vesicles isolated from the small intestine of neonatal
goats (Salter & Blakeborough, 1988). Mason & Selhub
(1988) observed that the characteristics of FBP-bound
folate absorption in the suckling rat resemble in some
respects the characteristics of endocytotic absorption
of macromolecules – a well-documented feature of
the suckling mammal’s intestinal physiology.
Adaptive regulation of folate absorption
Said et al. (2000) induced folate defi ciency in rats
by feeding a folate-defi cient diet that contained an
antibiotic to decrease the bacterial synthesis of folate
in the intestine. Using everted intestinal sacs and
brush-border membrane vesicles, they showed that
folate deprivation causes a specifi c up-regulation in
the transport of physiological concentrations of folic
acid across the brush-border membrane of both the
small and large intestines. The effect in the small intestine
took place not only in the jejunum, but also in
the ileum, a region that does not usually absorb folate.
The up-regulation was mediated through an increase
in the number and/or activity of functional reduced
folate carriers (increased Vmax) with no signifi cant effect
on the affi nity of the transport system (unchanged
Km). The up-regulation was associated with a marked
increase in the levels of carrier mRNA and protein,
suggesting a possible involvement of transcriptional
regulatory mechanisms. In addition to the up-regulation
of transepithelial folate transport, folate defi -
ciency was associated with a 10-fold increase in the
activity of brush-border membrane conjugase. The
intestine is therefore able to maximize its ability to
extract the limited amount of folate ingested during
periods of deprivation.

Deconjugation of polyglutamyl folate

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The folates naturally present in foods exist largely in
protein-bound form, the predominant vitamers being
polyglutamyl forms of THF, 5-methyl-THF and 10-
formyl-THF (Gregory, 1984). Folylpolyglutamates,
being large and strongly electronegative molecules,
are not transportable into cells and, before they can be
absorbed, they must be hydrolysed to monoglutamate
forms. None of the known proteases in saliva, gastric
juice or pancreatic secretions are capable of splitting
the γ-peptide bonds in the polyglutamyl side chain.
Polyglutamyl folate can, however, be hydrolysed by
folate conjugase, which is a trivial name for pteroylpolyglutamate
hydrolase, EC 3.4.12.10 (also known
as folylpoly-γ-glutamyl carboxypeptidase). As much
as 50–75% of dietary polyglutamyl folate can be absorbed
after deconjugation to monoglutamyl folate
(Butterworth et al., 1969). The presence of conjugase
activity in many raw foods of both plant and animal
origin results in a high proportion of the dietary folate
being already monoglutamyl when presented to the
intestinal mucosa (Gregory, 1989).
Two folate conjugases have been found in human
jejunal tissue fractions. One, a brush-border exopeptidase,
has a pH optimum of 6.7–7.0 and is activated by
Zn2+. The other, an intracellular endopeptidase of
mainly lysosomal origin, has a pH optimum of 4.5
and no defi ned metal requirement. The brush-border
conjugase splits off terminal glutamate residues one
at a time and is thought to be the principal enzyme in
the hydrolysis of polyglutamyl folate. Brush-border
conjugases from the jejunum of the human and pig
possess similar enzymatic properties (Gregory et al.,
1987) and thus the porcine enzyme can be used to
study folate bioavailability in humans. Interestingly,
the human and the pig are the only species in which
intestinal brush-border conjugase activity has been
demonstrated. Bhandari & Gregory (1990) showed
that extracts from certain foods (e.g. legumes, tomatoes
and orange juice) can inhibit brush-border conjugase
activity from human and porcine intestine in vitro. Organic acids may be responsible for this inhibition
(Wei & Gregory, 1998). Such inhibition may be
a factor affecting the bioavailability of polyglutamyl
folates in diets containing these foods. The intracellular
conjugase may play no role in the digestion of
dietary folate, being, instead, concerned with folate
metabolism within the enterocyte (Halsted, 1990).
Signifi cant conjugase activity has also been reported
in the pancreatic juice of pigs and humans (Gregory,
1995). Bhandari et al. (1990) found the porcine pancreatic
enzyme to be Zn2+-dependent with maximum
activity at pH 4.0–4.5. Feeding stimulated secretion of
pancreatic juice, including conjugase activity. Chandler
et al. (1991) calculated that conjugase activity
in porcine pancreatic juice was minor relative to the
activity of the jejunal brush-border conjugase.

Folate

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In 1931, a research group led by Lucy Wills showed
that an autolysed yeast preparation (Marmite),
which was therapeutically ineffective against the
pernicious anaemia caused by vitamin B12 defi ciency,
was effective against nutritional megaloblastic anaemia
in pregnant women. These researchers induced
a similar anaemia in monkeys which then responded
to crude liver extracts. Other substances that cured
specifi c defi ciency anaemias in monkeys and chicks
were isolated from yeast by different research groups
and assigned the names ‘vitamin M’ and ‘vitamin Bc’.
Another substance isolated from liver was shown to
be essential to the growth of Lactobacillus casei and
therefore called the ‘L. casei factor’. In 1941, Mitchell
and co-workers processed four tons of spinach leaves
to obtain a purifi ed substance with acidic properties
which was an active growth factor for rats and L. casei.
They named the factor ‘folic acid’ (from folium, the
Latin word for leaf). Eventually, all of the above substances
proved to be the same when Angier’s group in
1946 accomplished the synthesis and chemical structure
of folic acid.

The term ‘folate’ is used as the generic descriptor
for all derivatives of pteroic acid that demonstrate
vitamin activity in humans. The structure of the
parent folate compound, folic acid, comprises a bicyclic
pterin moiety joined by a methylene bridge to
p-aminobenzoic acid, which in turn is coupled via an
α-peptide bond to a single molecule of L-glutamic
acid (Fig. 17.1, top).
(Note: In the present context, the term ‘folic acid’
refers specifi cally to pteroylmonoglutamic acid
which, with reference to the pteroic acid and glutamate
moieties, can be abbreviated to PteGlu. ‘Folate’ is
a non-specifi c term referring to any folate compound
with vitamin activity. ‘Folacin’ is a non-approved term
synonymous with ‘folate’.)
Folic acid is not a common natural physiological
form of the vitamin. In most natural foods, the pteridine
ring is reduced to give either the 7,8-dihydrofolate
(DHF) or 5,6,7,8-tetrahydrofolate (THF) (see Fig. 17.1).
These reduced forms can be substituted with a covalently
bonded one-carbon adduct attached to nitrogen positions 5 or 10 or bridged across both positions. The
following substituted forms of THF are important
intermediates in folate metabolism: 10-formyl-THF, 5-
methyl-THF, 5-formimino-THF, 5,10-methylene-THF
and 5,10-methenyl-THF (see Fig. 17.1).
An important structural aspect of the 5,6,7,8-tetrahydrofolates
is the stereochemical orientation at
the C-6 asymmetric carbon of the pteridine ring. Of
the two stereoisomers, 6S and 6R (formerly called 6l
and 6d), only the 6S is biologically active and occurs in
nature. Methods of chemical synthesis of tetrahydrofolates,
whether by catalytic hydrogenation or chemical
reduction, yield a racemic product (i.e. a mixture
of both stereoisomers).
All folate compounds exist predominantly as polyglutamates,
containing typically from fi ve to seven
glutamate residues in γ-peptide linkage. The γ-peptide
bond is unique in mammalian biochemistry.
Folate conjugates are abbreviated to PteGlun derivatives,
where n is the number of glutamate residues; for
example, 5-CH3-H4PteGlu3 refers to triglutamyl-5-
methyltetrahydrofolic acid.
Methotrexate (4-amino-10-methylfolic acid; Fig.
17.2) is a folate antagonist which is used as an anticancer
drug.

Dietary sources
Polyglutamyl folate is an essential biochemical constituent
of living cells, and most foods contribute some
folate. The folates generally exist in nature bound to
proteins (Baugh & Krumdieck, 1971) and they are
also bound to storage polysaccharides (various types
of starch and glycogen) in foods (Cerná & Káš, 1983).
In the United States, dried beans, eggs, greens, orange
juice, sweet corn, peas and peanut products are good
sources of folate that are inexpensive and available all
the year round.

Humans and other mammals cannot synthesize
folate in their tissues and thus they must obtain the
vitamin from exogenous sources via intestinal absorption.
The intestine is exposed to two sources of
folate: (1) dietary folate and (2) folate synthesized
by bacteria in the large intestine. The latter source is
available to the host tissues through direct absorption
in the colon.
It is fundamental in folate metabolism that folate
monoglutamates are the circulatory and membrane-
transportable forms of the vitamin, whereas
polyglutamates are the intracellular biochemical and
storage forms.
17.4.1 Folate transport proteins
Cellular uptake of folate involves two functionally different
membrane transport proteins: (1) the reduced
folate carrier, which is an organic anion exchange
protein present in the plasma membrane of a wide
variety of cells, and (2) the less ubiquitous folate receptor,
which internalizes folate by a receptor-mediated
process. The affi nities of these proteins for folates
and antifolates differ signifi cantly (Table 17.2). After
internalization, folates are retained in the cytoplasm
by polyglutamylation.
In certain specifi c cell types, such as human placental
trophoblast cells, a functional coordination between
the two transport proteins has been proposed.
Enterocytes and hepatocytes lack the folate receptor
and so folate transport in the human intestine and
liver is mediated solely by the reduced folate carrier.

Dietary deficiency of Biotin

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Biotin is so widely distributed in foods that it is doubtful
whether a true dietary defi ciency of the vitamin
has ever occurred in human adults capable of utilizing
it. Artificial biotin defi ciency states have been induced
in healthy volunteers by feeding low-biotin diets containing
a high proportion of raw egg white. An initial
dry scaly dermatitis was followed by non-specifi c
symptoms that included anorexia and extreme lassitude.
All of the symptoms responded to injections
of 150–300 μg of biotin per day.

A unique opportunity to study dietary biotin
defi ciency was presented to Baugh et al. (1968) by a
62-year-old female patient who had consumed six raw
eggs and 4 pints of skimmed milk daily for 18 months.
This diet had been recommended by a physician (illadvisedly,
as we now know) as a dietary supplement
to provide a high intake of essential amino acids to
aid liver regeneration following a diagnosis of liver
cirrhosis. During this dietary period, the patient took
vitamin supplements, which included biotin, and
she also received 100 μg of vitamin B12 by injection
monthly. Thus the stage was set, unintentionally, for
the development of biotin defi ciency due to the avidin
content of raw egg whites, uncomplicated by defi ciencies
of other vitamins or common nutrients. Clinical
manifestations included anorexia, nausea, vomiting,
glossitis, pallor, depression, lassitude, substernal pain,
scaly dermatitis and desquamation of the lips. All
symptoms cleared or improved markedly after 2 to
5 days of parenteral (by injection) vitamin therapy
providing 200 μg of biotin daily, while the patient
continued her pre-treatment diet. In contrast to other
case reports, the patient did not exhibit anaemia,
muscle pains, hypercholesterolaemia or electrocardiographic
abnormalities.
Seborrhoeic dermatitis of the scalp and a more
generalized dermatitis known as Leiner’s disease have
been reported in breast-fed infants when the mother
is malnourished. These symptoms are relieved when
biotin is administered to the mother.

Inherited defects of biotin metabolism

There are two known congenital disorders of biotin
metabolism: (1) holocarboxylase synthetase (HCS)
defi ciency and (2) biotinidase defi ciency. Both disorders
are inherited as an autosomal recessive trait
and both lead to defi ciency of the four biotin-dependent
carboxylases, a condition known as multiple
carboxylase defi ciency (MCD). Because of the vital
role of these enzymes in protein, carbohydrate and
lipid metabolism, their defi ciency leads to severe lifethreatening
disease.
The two forms of MCD usually become symptomatic
in early infancy or childhood. The incidence of
biotinidase defi ciency is about one in 60 000 and that
of HCS defi ciency seems to be even lower. The underlying
cause of HCS defi ciency is decreased affi nity
of HCS for biotin resulting in reduced formation of
holocarboxylases with physiological concentrations
of biotin. In biotinidase defi ciency, MCD results from
progressive development of biotin defi ciency due to
inability to liberate biotin from the biocytin or short
biotinyl peptides that remain after metabolic degradation
of biotin-containing carboxylases. The recycling
of biotin salvaged from degraded enzymes is essential
to maintain an adequate supply of the vitamin. A lack
of biotinidase results in excessive urinary excretion of
biocytin and this raises the requirement for biotin to
above normal intakes.
The two forms of MCD differ biochemically in that
the HCS-defi cient patients have normal plasma biotin
concentrations but decreased carboxylase activities,
whereas patients with biotinidase defi ciency have
subnormal plasma biotin concentrations and normal
carboxylase activities. MCD arising from either inherited
defect causes a block in the biotin-dependent
metabolic pathways with characteristic accumulation
and urinary excretion of organic acids such as lactate,
3-hydroxyisovalerate, 3-methylcrotonylglycine and
methylcitrate.

The clinical presentation and age of onset of MCD
are extremely variable. HCS defi ciency may present
in the fi rst days of life, while biotinidase defi ciency
usually becomes manifest between the second and
fi fth months of age, depending on the amount of free
biotin in the diet. However, onset of HCS defi ciency
is delayed (2–21 months) in some cases and therefore
classifying the two disorders as neonatal- or earlyonset
and infantile- or late-onset MCD should be discouraged.
Clinical symptoms common to both disorders
include neurological abnormalities (hypotonia,
seizures, ataxia) and cutaneous changes (skin rash,
alopecia). In healthy persons receiving an adequate
diet, biotinidase activity in the brain is relatively very
low (Suchy et al., 1985) and so the brain relies largely
on biotin that is transferred across the blood–brain
barrier. This feature of the brain could explain the
rapid onset of neurological symptoms observed in
biotinidase deficiency.

Biotin

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The discovery and recognition of biotin as a member
of the water-soluble vitamin B complex resulted
from several independent lines of investigation. In
1933, Franklin E. Allison and his colleagues at the US
Department of Agriculture reported that the growth
and respiration of Rhizobium trifolii, a nitrogen-fi xing
bacterium found in the root nodules of legumes,
were stimulated by a factor, ‘coenzyme R’, extractable
from various organic sources. By the early 1920s, several investigators had isolated from various organic
sources crude fractions that contained a novel
growth factor for yeast. Eventually, in 1936, Fritz
Kögl and B. Tönnis, organic chemists at the University
of Utrecht in Germany, isolated from dried egg
yolk a crystalline substance that strongly stimulated
the growth of yeast. This growth factor, which Kögl
and Tönnis named ‘biotin’, was later shown to have
exactly the same stimulatory effect on Rhizobium as
coenzyme R. In this respect, at least, the two factors
were identical.

Further progress came from the fi eld of animal nutrition.
In 1927, Margaret A. Boas at the Lister Institute
of Preventive Medicine in London observed toxicity
in rats when raw egg white was used as a source of
protein in the animals’ diet. After a few weeks the rats
developed dermatitis and haemorrhages of the skin,
their hair fell out, their limbs became paralysed, they
lost considerable weight, and eventually they died.
Only raw or cold-dried egg white produced the toxicity;
cooking made the egg white harmless. This toxicity,
which Boas called egg white injury, was prevented
by a ‘protective factor X’ present in liver and other
sources. Paul György showed that biotin had the same
protective action against egg white injury as did protective
factor X, which he renamed ‘vitamin H’ (German
Haut, skin). György also showed that vitamin H
concentrates supported the growth of biotin-requiring
bacteria. In 1940 György and Vincent du Vigneaud
independently isolated crystalline vitamin H from
liver concentrates. It was soon proven that biotin and
vitamin H were one and the same compound.
The chemical structure of biotin was established by
du Vigneaud’s group in 1942 and in the following year
the vitamin was synthesized at the Merck Company,
USA.

Biotin is present in all natural foodstuffs, but the
content of even the richest sources is very low when
compared with the content of most other water-soluble
vitamins. Biotin is not commonly used in fortifi ed
foods, apart from infant formulas. Typical values of
some rich natural sources of biotin include ox liver
(33 μg per 100 g), whole eggs (20 μg per 100 g), dried
soya beans (65 μg per 100 g) and peanuts (72 μg per
100 g) (Holland et al., 1991). Other good sources include
yeast, wheat bran, oatmeal and some vegetables.
Muscle meats, fi sh, dairy products and cereals contain
smaller amounts, but are important contributors to
the dietary intake. Most of the biotin content of animal
products, nuts, cereals and yeast is in a protein-bound
form. A higher percentage of free, water-extractable
biotin occurs in vegetables, green plants, fruit, milk
and rice bran (Lampen et al., 1942).

Vitamin B6 deficiency

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Vitamin B6 is widely distributed in foods, and any diet
so poor as to be insuffi cient in this vitamin would
most likely lack adequate amounts of other B-group
vitamins. For this reason, a primary clinical defi ciency
of B6 in the adult human is rarely encountered under
normal circumstances.
In a well-controlled study conducted by Hodges et
al. (1962), six healthy male volunteers were divided
into pairs and given a basic formulated diet administered
by nasogastric tube twice daily. The fi rst pair of
men received a complete formula including pyridoxine;
the second pair received the same diet without
pyridoxine; and the third pair were given the anti-vitamin
deoxypyridoxine in addition to the pyridoxinefree
diet. The men receiving pyridoxine-free diets, but
not those receiving complete diets, developed adverse
symptoms and signs of illness, which were more
severe in the men given the anti-vitamin. The most
obvious symptoms were gastrointestinal disturbances
and epithelial changes. Both men in the anti-vitamin
group had scaling of the skin, foul breath, severe gingivitis,
soreness and discoloration of the tongue and
dry cracked lips. No objective neurological changes
could be demonstrated. After vitamin B6 was restored
to their diet and the anti-vitamin discontinued, one
man recovered promptly and the other recovered
gradually.
Vitamin B6 deprivation imposed at certain stages of
brain development interferes with the orderly process
of neuronal development (Kirksey et al., 1990). In the
1950s, an occurrence of convulsions in infants was
traced to an unfortifi ed liquid milk-based canned formula
that had undergone autoclaving in manufacture
(Coursin, 1954). There is some circumstantial evidence
that convulsions resulting from vitamin B6 defi
ciency may be caused by an insuffi cient production
of γ-aminobutyric acid, the major neurotransmitter
in the brain (Ebadi, 1978). However, a meaningful
correlation among vitamin B6 defi ciency, concentration
of γ-aminobutyric acid and convulsion has not
been established.

Animal studies for Vitamin B6

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Kumar & Axelrod (1968) reported a lowered level of
circulating antibodies and a dramatic reduction in
the number of antibody-forming cells in the spleens
of vitamin B6-defi cient rats immunized with sheep
erythrocytes. This decreased cellular immune response
was independent of the inanition associated
with the defi ciency and was restored to normal by the
administration of PN shortly before immunization.
Robson & Schwarz (1975) reported a dramatic
85–95% reduction in the number of thoracic duct
lymphocytes and a signifi cant reduction in cellular
immunocompetence in vitamin B6-defi cient rats.
These conclusions were based on the results of two
tests: (1) the in vitro mixed lymphocyte reaction
(MLR) and (2) the in vivo normal lymphocyte transfer
reaction (NLT). In the MLR, lymphocytes from test
Lewis strain rats (in this case, vitamin B6-defi cient and
control rats) are cultured with genetically dissimilar
lymphocytes taken from normally nourished F1 hybrid
rats. If the lymphocytes are immunocompetent,
they will become activated and then they will proliferate
and transform into the larger lymphoblasts. The
extent of blastogenesis is quantitated by exposing
the cultures to [3H]thymidine and then measuring
the incorporation of the radioactivity into DNA. In
the NLT, lymphocytes from donor Lewis rats (the test
rats) are injected into the ventral abdominal wall of F1
hybrid rats. Immunologically competent donor cells
produce a graft-versus-host reaction in the skin of the
F1 rat. The impaired proliferation of lymphocytes and
loss of cellular immunocompetence may perhaps be
attributed to a cessation of T-lymphocyte development
within the thymus of the vitamin B6-defi cient
animal.
The development of functional T lymphocytes
depends on humoral factors secreted by thymic epithelial
(TE) cells. To investigate the effects of dietary
vitamin B6 defi ciency on TE cell function, Willis-Carr
& St. Pierre (1978) used three groups of Lewis strain
rats as cell donors: (1) normal (control) rats, (2) rats
maintained for 2 weeks on a vitamin B6-defi cient diet
and (3) rats whose thymus glands had been surgically
removed 24 hours after birth (neonatally thymectomized
rats). Spleen, bone marrow and mesenteric
lymph nodes were removed from each donor and
washed cells from these lymphoid tissues were exposed
to monolayers of TE cells. The TE monolayers
were made from (1) normal, (2) vitamin B6-defi cient
and (3) ‘post B6’ rats, i.e. rats placed back on a regular
diet for 3 weeks after the original 2-week B6-defi cient
diet. Exposure of T-lymphocyte precursors from
B6-defi cient or neonatally thymectomized donors to
normal TE monolayers resulted in their conversion to
functional T lymphocytes, as measured by their response
in MLR and to mitogens. However, TE monolayers
from B6-defi cient rats were unable to effect such
a maturation of T lymphocytes. When the defi cient
rats were returned to a normal diet, TE cell function
was restored. The authors suggested that the cause
of defective cellular immunocompetence following
vitamin B6 deprivation is the inability of TE cells to
effect the differentiation of T-lymphocyte precursors
to functional T lymphocytes. Vitamin B6 defi ciency
did not impair T-lymphocyte precursors, which could
be stimulated to differentiate by exposure to normal
TE cell monolayers. Presumably, the observed effect
of vitamin B6 defi ciency is due to a blocking of the
biosynthesis and/or release of a humoral factor that is
produced by TE cells. Chandra & Puri (1985) found
signifi cantly reduced serum thymic factor activity in
rats fed diets restricted in vitamin B6 and given 4-deoxypyridoxine
hydrochloride in their drinking water.
Vitamin B6-defi cient mice exhibited impaired
production and reduced activity of cytotoxic T lymphocytes
(Sergeev et al., 1978; Ha et al., 1984). Antibody-
mediated cytotoxicity, macrophage phagocytosis
and natural killer cell activity were not affected by
the level of vitamin B6 intake (Ha et al., 1984).

Regulation of steroid hormone action

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Steroid hormones (androgens, oestrogens, progestins
and the corticosteroids) are able to enter cells, bind
to receptors and directly regulate gene transcription.
The synthesized proteins carry out the ultimate effect
of the hormone. Evidence accrued in the following
studies suggest a physiological role for vitamin B6 in
modulating steroid hormone action.
The infl uence of B6 vitamers and analogues on the
physical properties of the glucocorticoid receptor
has been studied using in vitro receptor preparations
(Allgood et al., 1990a). The combined data showed
that, among these compounds, only PLP can directly
associate with the glucocorticoid receptor and alter
several of its properties, including molecular conformation,
surface charge, susceptibility to exogenous
proteolysis, DNA binding capacity and subcellular
localization. The last two properties are requisites for
regulation of target gene expression.
In a number of rat studies, Bender’s group produced
in vivo evidence that vitamin B6 may be involved in the
normal physiological action of steroid hormones. In
male rats, Symes et al. (1984) showed that the uptake
and accumulation of tracer [1,2,6,7–3H]testosterone
in the nucleus of the prostate gland were signifi cantly
increased in vitamin B6-defi cient animals compared
with vitamin B6-adequate controls. In a corresponding
study of female rats (Bowden et al., 1986), the
animals were segregated according to the phase of
the oestrous cycle to avoid the inherent variations of
both plasma concentration of oestrogen and the concentration
of oestrogen receptor in the uterus during
the course of the oestrous cycle. As for testosterone in
the male, uptake and accumulation of tracer [2,4,6,7–
3H]17β-oestradiol in uterine nuclei were signifi cantly
increased in vitamin B6-defi cient animals throughout
the oestrous cycle; there were no signifi cant differences
at anoestrus.
Bender’s group also found evidence of enhanced
sensitivity to steroid hormone action in vitamin B6-
defi cient rats of both sexes. In the male, testosterone is
secreted by the interstitial cells of Leydig in the testes,
but only when these cells are stimulated by luteinizing
hormone (LH) released by the anterior pituitary
gland in response to hypothalamic gonadotropin-releasing
hormone (Gn-RH). Circulating testosterone
exerts negative feedback control at the level of the
hypothalamus, switching off the supply of pituitary
LH and thereby stopping testicular secretion of testosterone
(Fig. 14.9). Symes et al. (1984) found that
the plasma concentration of testosterone in vitamin
B6-defi cient male rats was only 25% of that in vitamin B6-adequate controls. This unexplainable reduction
in plasma testosterone was not accompanied by a
reduction in the relative weight of the prostate gland
as might have been expected; neither was it accompanied
by a rise in plasma LH. These two observations
suggest that there may be enhanced sensitivity of the
hypothalamus to negative feedback by testosterone in
vitamin B6 defi ciency, leading to normal (or reduced)
plasma concentrations of LH and normal growth of
the prostate despite considerably reduced circulating
concentrations of testosterone.
In the female rat, ovarian secretion of oestrogen is
stimulated by LH and follicle-stimulating hormone
(FSH) released from the anterior pituitary gland in
response to hypothalamic Gn-RH. During most of
the oestrous cycle, circulating oestrogen exerts negative
feedback control at the level of the hypothalamus,
suppressing the release of LH and FSH. The major
event of ovulation is preceded by a massive outfl ow
of LH from the pituitary (the pre-ovulatory surge)
caused by positive feedback of oestrogen upon the
hypothalamus. Bowden et al. (1986) reported that in
ovariectomized rats, doses of ethynyl-oestradiol that
had no effect on circulating LH in control animals
(i.e. submaximal doses) lowered plasma LH levels in
vitamin B6-defi cient animals. As in the male rat, this
suggests that vitamin B6 defi ciency leads to enhanced
sensitivity of the hypothalamus to negative feedback
by steroid hormone.
Allgood et al. (1990b) investigated the infl uence of
PLP on glucocorticoid receptor-dependent gene expression
by introducing a reporter gene with a defi ned promoter into a cell culture line. The results showed
that, under conditions of moderate vitamin B6 defi -
ciency, the glucocorticoid receptor becomes a more
effi cient activator of gene transcription. Conversely,
high concentrations of vitamin B6 suppress activation
of transcription. The modulatory effects of PLP
concentration occurred through a novel mechanism
that did not involve changes in glucocorticoid receptor
mRNA or protein levels, or the receptor’s ligand
binding capacity. Analogous effects of PLP were
found with the oestrogen, androgen and progesterone
receptors (Allgood & Cidlowski, 1992). Vitamin
B6 appears to modulate steroid hormone-mediated
gene expression through its infl uence on a functional
or co-operative interaction between steroid hormone
receptors and the transcription factor NF1 (Allgood
et al., 1993).

Brain homeostasis for Vitamin B6

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Vitamin B6 levels in brain are homeostatically regulated.
Although it is relatively easy to produce symptomatic
vitamin B6 deficiency in animals, levels of the
vitamin in brain (and heart) are somewhat better
maintained in defi ciency states than they are in liver
and kidney. Conversely, massive parenteral doses
(daily intravenous injections of 200 mg kg–1 of PN for
3 days to rabbits) elevated the brain levels of PLP by an
average of only 39% (Spector & Shikuma, 1978).
In brain, vitamin B6 exists predominantly in the enzymatically
active forms PLP and PMP at concentrations
much higher than in plasma and cerebrospinal
fl uid (CSF). In rabbits, the concentrations of vitamin
B6 in plasma, CSF, brain and choroid plexus were, respectively,
0.30, 0.39, 8.90 and 15.10 μmol L–1 or kg–1
(Spector, 1978a).
Spector studied the in vitro uptake and release of
tritium-labelled vitamin B6 in rabbit brain slices and
isolated choroid plexuses (Spector, 1978b; Spector
& Greenwald, 1978). Uptake of [3H]PN by both tissues
was inhibited by (1) low temperature (2°C) and
dinitrophenol, demonstrating energy dependence;
(2) pyridoxal azine, demonstrating dependence on
the activity of intracellular pyridoxal kinase; and (3)
unlabelled non-phosphorylated B6 vitamers and, to
lesser extent, phosphorylated B6 vitamers, demonstrating
saturability of the uptake system. There was
no detectable metabolism of [3H]PN to [3H]pyridoxic
acid in brain slices or choroid plexus. From 70 to 80%
of the labelled vitamin B6 in both tissues was phosphorylated
after a 30-minute incubation in [3H]PN.
Phosphorylated B6 vitamers were taken up much less
readily than non-phosphorylated vitamers. These
studies are not conclusive in separating active transport
from metabolic trapping because both pyridoxal
kinase and active transport require ATP, and therefore
depletion of ATP could affect either process. Furthermore,
dinitrophenol is known to inhibit mammalian
pyridoxal kinase as well as preventing ATP synthesis
by uncoupling oxidative phosphorylation from electron
fl ow through the electron-transport chain.
The activity of pyridoxal kinase in brain is unimpaired
by moderate and severe vitamin B6 defi ciency
(McCormick et al., 1961). Spector & Shikuma (1978)
showed that pyridoxal kinase activity and vitamin B6
accumulation by brain slices and choroid plexus are
not affected by various drugs that alter the concentrations
of PLP or biogenic amines in brain.
Spector (1978a) showed that, during one pass
through the cerebral circulation, [3H]PN was cleared
from the circulation no more rapidly than mannitol.
Mannitol, a molecule of similar size and shape to
PN, is known to be transported by diffusion. Spector
(1978a) also confi rmed in vivo, by injection directly
into the ventricular CSF of rabbits, that non-phosphorylated
B6 vitamers enter brain cells by a saturable
accumulation process. Kinetic studies conducted by
Spector & Greenwald (1978) revealed marked differences
in the uptake of vitamin B6 by choroid plexus
and brain. The half-saturation concentrations and
rate maxima for accumulation were ~0.2 μM and
1.0–2.0 μmol kg–1 per 30 min for brain slices and
7.0 μM and 40 μmol kg–1 per 30 min for isolated
choroid plexus. Assuming the absence of a membrane
carrier for vitamin B6 uptake, the kinetic constants
refer to the binding of substrate to pyridoxal kinase
and are therefore values of Km and Vmax. The differences
in the constants for vitamin B6 uptake by brain
cells and choroid plexus are presumably due to factors
(e.g. intracellular pH) that cause variations in enzyme
activity.
This makes the choroid plexus, rather than brain cells,
the likely source of the phosphorylated B6 vitamers
in CSF.
Vitamin B6 is transported in the reverse direction
(i.e. from brain and/or CSF into blood) more rapidly
than mannitol (Spector, 1978a). This suggests that the
transport mechanism for vitamin B6 in this direction
involves a mechanism other than simple diffusion.
In conclusion, Spector’s data show that circulating
vitamin B6 can enter the brain via the blood–CSF
barrier (choroid plexus). The fi nding that PN was
extracted no more rapidly than mannitol during one
pass through the cerebral circulation argues against
signifi cant entry of PN via the blood–brain barrier.
There is a saturable transport system (Km = 0.7 μM)
within the choroid plexus that regulates the entry
of free (unbound) non-phosphorylated B6 vitamers
from plasma into the CSF. The vitamers fi nds their
way into the extracellular space of brain and enter
brain cells by a high-affi nity (Km = ~0.2 μM) saturable
accumulation system. The transport system in both
choroid plexus and brain cells appears to be simple
(or possibly facilitated) diffusion accelerated by the
concentration gradient created by phosphorylation of
the transported B6 vitamers (metabolic trapping). PN,
PL and PM have comparable affi nity for the vitamin
B6 transport systems, as they also do for pyridoxal
kinase. Excessive concentrations of phosphorylated
B6 vitamers within brain cells are dephosphorylated
intracellularly and transported out of the cells.

Digestion and absorption of dietary vitamin B6

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Vitamin B6 is present in foods mainly as the PN, PLP
and PMP vitamers. In many fruits and vegetables,
30% or more of the total vitamin B6 is present as PNglucoside.
The binding of PLP to protein through aldimine
(Schiff base) and substituted aldamine linkages
is reversibly dependent on pH, the vitamin–protein
complexes being readily dissociated under normal
gastric acid (low pH) conditions. The release of PLP
from its association with protein is an important step
in the subsequent absorption of vitamin B6, as binding
to protein inhibits the next step, hydrolysis of PLP by
alkaline phosphatase (Middleton, 1986). It would appear,
therefore, that the widespread practice of raising
the post-prandial gastric and upper small intestinal
pH by the use of pharmaceutical antacids may impair
vitamin B6 absorption.
Physiological amounts of PLP and PMP are largely
hydrolysed by alkaline phosphatase in the intestinal
lumen before absorption of free PL and PM (Hamm
et al., 1979; Mehanso et al., 1979). When present in
the lumen at non-physiological levels which saturate
the hydrolytic enzymes, substantial amounts of PLP
and PMP are absorbed intact, but at a slower rate than
their non-phosphorylated forms.
The absorption of PN, PL and PM takes place
mainly in the jejunum and is a dynamic process involving
several interrelated events. The vitamers cross
the brush-border membrane by simple diffusion as
shown, for example, in everted intestinal sacs (Tsuji et al., 1973), brush-border membrane vesicles (Yoshida
et al., 1981) and isolated intestinal loops (Middleton,
1979). In humans, PM is absorbed more slowly or
metabolized differently, or both, than either PL or
PN (Wozenski et al., 1980). Middleton (1983) noted a
signifi cant positive correlation between PLP luminal
disappearance and both alkaline phosphatase activity
and net water absorption in perfused segments of rat
jejunum. It is conjectural that increased water absorption
results in a greater concentration of PL within the
lumen, allowing absorption to proceed more rapidly.
Within the enterocyte PN, PL and PM are converted
to their corresponding phosphates by the catalytic action
of cytoplasmic pyridoxal kinase, and transaminases
interconvert PLP and PMP. The conversion of
a particular vitamer to other forms by intracellular
metabolism creates a concentration gradient across
the brush border for that vitamer, thus enhancing
its uptake by diffusion (Middleton, 1985). The phosphorylated
vitamers formed in the cell are largely
dephosphorylated by non-specific phosphatases, thus
permitting easy diffusion of vitamin B6 compounds
across the basolateral membrane. The major form
of vitamin B6 released to the portal circulation is the
non-phosphorylated form of the vitamer predominant
in the intestinal lumen.
Absorption capacity in rats was not affected directly
by dietary vitamin B6 supply (Roth-Maier et al., 1982)
and so it is supposed that homeostatic regulation of
vitamin B6 is not due to a variation of absorption.

Bioavailability of Vitamin B6

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Losses of vitamin B6 content caused by thermal instability
occur during food processing, but the remaining
vitamin B6 does not necessarily exhibit incomplete
bioavailability.

The bioavailability of vitamin B6 in foods is highly
variable, owing largely to the presence of poorly utilized
PN-glucoside in plant tissues. As expected, vitamin
B6 generally has a lower availability from plantderived
foods than from animal tissues (Nguyen &
Gregory, 1983). Based on plasma PLP levels in male
human subjects, the bioavailability of the vitamin in
an average American diet ranged from 61% to 81%,
with a mean of 71% (Tarr et al., 1981).
Gregory et al. (1991) determined the bioavailability
of PN-glucoside in humans through the use
of a stable-isotope method. The utilization of orally
administered deuterated PN-glucoside was 58 ± 13%
(mean ± SEM) relative to that of deuterated PN.
Intravenously administered PN-glucoside underwent
approximately half the metabolic utilization
of oral PN-glucoside, which suggested a role of β-
glucosidase(s) of the intestinal mucosa, microfl ora, or
both, in the release of free PN from dietary PN-glucoside.
Stable isotope methodology provided evidence
that PN-glucoside weakly retards the metabolic utilization
of non-glycosylated forms of vitamin B6 in humans
(Gilbert et al., 1991). Despite the relatively high
consumption of glycosylated vitamin B6, vegetarian
women did not demonstrate any signifi cant difference
in vitamin B6 status compared with non-vegetarian
women (Shultz & Leklem, 1987; Löwik et al.,
1990). In addition, the intake of glycosylated vitamin
B6 had little, if any, effect upon maternal plasma PLP
concentration and maternal urinary excretion of total
vitamin B6 and 4-pyridoxic acid in lactating women
(Andon et al., 1989). These observations suggest that
there may be little practical signifi cance to the human
consumption of glycosylated vitamin B6.

14.4 Absorption, transport and metabolism
Humans cannot synthesize vitamin B6 and thus must
obtain the vitamin from exogenous sources via intestinal
absorption. The intestine is exposed to vitamin
B6 from two sources: (1) the diet and (2) the bacterially
synthesized vitamin B6 in the large intestine. Whether
the latter source of vitamin B6 is available to the host
tissues (apart from the colonic epithelial cells) in nutritionally
signifi cant amounts is unknown.

Dietary sources of Vitamin B6

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Vitamin B6 is present in all natural unprocessed foods,
with yeast extract, wheat bran and liver containing
particularly high concentrations. Other important
sources include whole-grain cereals, nuts, pulses, lean
meat, fi sh, kidney, potatoes and other vegetables. In
cereal grains over 90% of the vitamin B6 is found in
the bran and germ (Polansky & Toepfer, 1969), and
75–90% of the B6 content of the whole grain is lost in
the milling of wheat to low-extraction fl our (Sauberlich,
1985). Thus, white bread is considerably lower in
vitamin B6 content than is whole wheat bread. Milk,
eggs and fruits contain relatively low concentrations
of the vitamin.
In raw animal and fi sh tissue the major form of
vitamin B6 is PLP. Apart from very low concentrations
in liver, PN and PNP are virtually absent in animal
tissues.
Plant tissue contains mostly PN, a proportion of
which may be present as PN-glucoside and/or other
conjugates. PN-glucoside has not been found in
animal products. No generalizations can be made
as to one group of foods consistently having a high PN-glucoside content. Typical sources of PN-glucoside
(expressed as a percentage of the total vitamin B6
present) are bananas (5%), raw broccoli (35%), raw
green beans (58%), raw carrots (70%) and orange juice
(69%) (Gregory & Ink, 1987). PN-glucoside accounted
for 10–15% of the total vitamin B6 in the typical mixed
diets used in an American human study (Gregory et al.,
1991), but would be proportionally higher in vegetarian
diets.

Vitamin B6

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In 1934 Paul György observed the appearance of a
scaly dermatitis (acrodynia) in rats fed on diets free
from the whole vitamin B complex and supplemented
with thiamin and ribofl avin. This observation led to
the establishment of a ‘rat acrodynia-preventative factor’
and its designation as vitamin B6. The isolation of the pure crystalline vitamin was first reported by Lepkovsky in 1938, and the synthesis of pyridoxine
was accomplished by Harris and Folkers in the following
year. Discovery of the existence of pyridoxal
and pyridoxamine and the recognition of their phosphorylated
forms as coenzymes is largely credited to
Esmond E. Snell during 1944–1948.

Vitamin B6 is the generic descriptor for all 3-hydroxy-
2-methylpyridine derivatives which exhibit qualitatively
in rats the biological activity of pyridoxine.
Six B6 vitamers are known, namely pyridoxine or
pyridoxol (PN), pyridoxal (PL) and pyridoxamine
(PM), which possess, respectively, alcohol, aldehyde
and amine group in the 4-position; their respective
5´-phosphate esters are designated as PNP, PLP and
PMP (Fig. 14.1).
In its role as a coenzyme, PLP is attached to the
apoenzyme by a Schiff base (aldimine) linkage
(–N=CH–) formed through condensation of the 4-
carbonyl group with the ε-amino group of specifi c
lysine residues A ubiquitous bound form of PN that occurs in
plant tissues is a glucoside conjugate, 5´-O-(β-Dglucopyranosyl)
pyridoxine (Fig. 14.3), designated
in this text as PN-glucoside. A more complex derivative
of PN-glucoside containing cellobiose and
5-hydroxydioxindole-3-acetic acid moieties has been
identifi ed as a major form of vitamin B6 in rice bran
and a minor form in wheat bran and legumes (Tadera
& Orite, 1991).
All of the six B6 vitamers are considered to have approximately
equivalent biological activity in humans
as a result of their ultimate conversion to coenzymes.

Nutritional aspects for Niacin

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Human requirement

Requirements for niacin are related to energy intake
because of the involvement of NAD and NADP as
coenzymes in the oxidative release of energy from
food. Estimation of niacin requirement is complicated
by the conversion of tryptophan to the vitamin. The
effi ciency of the conversion is affected by a variety of
infl uences, including the amounts of tryptophan and
niacin ingested, protein and energy intake, hormonal
status, and vitamin B6 and ribofl avin nutriture. A
normal intake of protein will probably provide more
than enough tryptophan to meet the body’s requirement
for niacin without the need for any preformed
niacin in the diet.

A notable exception to the 60:1 conversion ratio of
L-tryptophan to niacin is the state of pregnancy, in
which the conversion is about twice as efficient. This
increased conversion is presumably due to the stimulation
by oestrogen of tryptophan oxygenase, which
is a rate-limiting enzyme in the biosynthetic pathway.
Conversion is also increased when contraceptive pills
are used.

Effects of high intake
Nicotinic acid administered orally at doses as low
as 100 mg per day causes peripheral vasodilatation,
with the appearance of skin fl ushing. In high doses,
nicotinic acid competes with uric acid for excretion,
leading to an increase in the incidence of gouty arthritis.
Of greatest concern is possible liver damage,
and in one report severe jaundice occurred at doses
of 750 mg per day for only 3 months. Nicotinamide
does not cause vasodilatation, but is otherwise two to
three times as toxic as the acid (Miller & Hayes, 1982;
Alhadeff et al., 1984).

Niacin deficiency

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A deficiency in niacin results in pellagra, which is a
nutritional disease endemic among poor communities
who subsist chiefl y on maize. The classical features
of endemic pellagra are dermatitis, infl ammation of
the mucous membranes, diarrhoea and psychiatric
disturbances. The dermatitis often appears after exposure
to sunlight and resembles sunburn. The skin
becomes red and blistered and frequently peels off in
large areas. In chronic cases the skin becomes rough
and thickened with a brown pigmentation. In acute
pellagra, the mucous membranes of the gastrointestinal
and genitourinary tracts are severely infl amed.
The mouth becomes extremely sore and the tongue
is swollen and scarlet in colour. Chewing and swallowing
are painful and even liquids may be refused.
Infl ammation of the small and large intestine is manifested
by diarrhoea, abdominal pain and soreness of
the rectum. Hypermotility of the gastrointestinal
tract and the loss of appetite lead to profound loss of
weight. Infl ammation of the lower urinary tract causes
urethritis with increased micturition accompanied
by a burning sensation. In the female, severe vaginitis
is observed and amenorrhoea is common. Bender
(1984) vividly described neurological and neuropsychiatric
signs. Early signs include tremor, irritability,
anxiety and depression, with delirium and dementia
sometimes occurring in severe and chronic cases.
Unless the disease is treated, the inevitable outcome
is death. Fortunately, the response to nicotinamide
therapy is rapid and dramatic.
The prognosis is complicated by signs of proteinenergy
malnutrition and by an imbalance of amino
acid intake, particularly low levels of tryptophan and
high levels of leucine. Because most proteins contain
at least 1.0% tryptophan, it is theoretically possible
to maintain adequate niacin status on a diet devoid
of niacin but containing >100 g of protein. Primary
defi ciencies are rare (at least in industrialized countries),
but secondary defi ciencies may arise from
gastro intestinal disorders or alcoholism.

Role of NAD in ADP-ribosylation

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NAD functions in ADP-ribosylation, a reversible
post-translational modifi cation of proteins in which
the ADP-ribose moiety of NAD is transferred to
acceptor proteins, thereby altering their function.
ADP-ribosylation reactions are classifi ed into two
major groups: mono-ADP-ribosylation and poly-
ADP-ribosylation.
Mono-ADP-ribosylation by bacterial toxins
The transfer of ADP-ribose to the acceptor protein
(Fig. 13.5) is catalysed by ADP-ribosyltransferases,
which are found in the cytosol, plasma membrane and
nuclear envelope of eukaryotic cells. The ADP-ribose
reacts with specifi c amino acid residues on the acceptor
protein to form N-glycosides. Certain bacterial
toxins also possess ADP-ribosyltransferase activity
(Ueda & Hayaishi, 1985) and, since more is known
about them than eukaryotic ADP-ribosyltransferases,
they will be selected as examples.
Two bacterial exotoxins, diphtheria toxin and
Pseudomonas aeruginosa exotoxin A, prevent protein
synthesis in bacterially infected eukaryotic cells by
inactivating elongation factor 2, a protein required
for polypeptide chain elongation. The uncontrolled
action of these exotoxins results in death of the host
cells. A mammalian cellular ADP-ribosyltransferase
also inactivates elongation factor 2 (Iglewski, 1994),
but this is a controlled action required for normal
protein synthesis.
Cholera toxin and Escherichia coli heat-labile enterotoxin
ADP-ribosylate the α subunit of the stimulatory
G protein, Gs, which relays the signal from a
hormone-activated cell surface receptor to an intracellular
effector, in this case adenylyl cyclase (see Section
3.7.5). Cholera toxin-catalysed ADP-ribosylation
inhibits the intrinsic GTPase activity of Gsα, resulting
in stabilization of an active GTP-bound subunit and
persistent activation of adenylyl cyclase. ADP-ribosyltransferase
activity of cholera toxin is enhanced
by ADP-ribosylation factor (ARF), a GTP-dependent
eukaryotic protein that functions in intracellular vesicular
transport (Moss & Vaughn, 1995).
Pertussis toxin ADP-ribosylates the α subunit of
the inhibitory G protein, Gi. The modifi ed G protein
uncouples from the receptor, thereby maintaining
the protein as its inactive heterotrimer. Because this
inhibitory G protein is inactivated, inhibition of
adenylyl cyclase is removed and the result is increased
cyclase activity.

Brain homeostasis for Niacin

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Niacin and NAD levels in brain are homeostatically
regulated. In niacin-defi cient animals, levels of niacin
and NAD in the brain are much better maintained
than they are in the liver. Conversely, even massive
doses (500 mg kg–1) of nicotinamide injected intravenously
result in only a 50% increase in brain NAD
levels (Spector, 1981).
In plasma and cerebrospinal fl uid (CSF), nicotinamide
is normally the predominant if not the only
form of niacin present. In rabbits, the concentration
of nicotinamide in plasma and CSF is 0.5 μM and
0.7 μM, respectively. Phosphorylated forms of nicotinamide
or nicotinic acid cannot penetrate brain cells
without fi rst being dephosphorylated.
The choroid plexus, the anatomical locus of the
blood–CSF barrier, contains separate saturable uptake
systems for nicotinic acid and nicotinamide
(Spector & Kelley, 1979). The half-saturation concentrations
for 14C accumulation by the isolated choroid
plexus with [14C]nicotinic acid and [14C]nicotinamide
in the medium were 18.1 μM and 0.23 μM, respectively;
the respective rate maxima were 439 μmol kg–1
per 30 min and 18.6 μmol kg–1 per 30 min. Nicotinic
acid uptake appeared to depend completely on its
immediate intracellular conversion to NAD, whereas
nicotinamide uptake was thought to depend partly on
its incorporation into intracellular NAD in exchange
for the nicotinamide released from NAD by the action
of NAD glycohydrolase (EC 3.2.2.5). The intracellular
concentration of [14C]nicotinamide was fi ve times the
medium concentration, so it seems that nicotinamide
is actively transported into choroid plexus before
being incorporated into NAD. The isolated choroid
plexus released predominantly [14C]nicotinamide
whether pre-incubated in [14C]nicotinic acid or
[14C]nicotinamide.
Transport studies using rabbit brain slices
(Spector & Kelley, 1979) showed that uptake of
[14C]nicotinamide by brain cells in vitro was saturable
and dependent on the production of intracellular energy;
the half-saturation concentration of the uptake
system was 0.80 μM. Spector (1979) reported that
when [14C]nicotinamide was injected into the ventricle
of the brain of conscious rabbits, some of the radioactivity
was incorporated into intracellular NAD
and some left the brain and CSF extremely rapidly
by a nonsaturable system. This rapid equilibration
of nicotinamide between CSF and plasma suggests
that there is no control of the concentration of nicotinamide
in the CSF and extracellular space of brain:
nicotinamide concentrations in these compartments
refl ect concentrations in plasma. Once within the
extracellular space, nicotinamide enters brain cells by
a concentration-dependent, saturable accumulation
system. On entry into the brain cells, much of the
nicotinamide is incorporated into NAD.
Spector (1987) measured the unidirectional infl ux
of [14C]nicotinamide across cerebral capillaries (the
anatomical locus of the blood–brain barrier) using
an in situ rat brain perfusion technique. Transport
of nicotinamide was much faster than could be
explained by simple diffusion alone and was not
saturable with 10 mM nicotinamide in the perfusate.
However, with periods of infusion longer than 30 s,
there was substantial backfl ow of [14C]nicotinamide
into the perfusate. At a concentration of 1.7 μM,
nicotinamide transport was not inhibited by 3-
acetylpyridine. The non-saturability and lack of
inhibition in the presence of a structural analogue
indicate that nicotinamide transport is not carriermediated.
The data suggested that most, if not all, of
the nicotinamide that enters brain from blood gains
access to the extracellular space of brain directly via
the blood–brain barrier.
From the above fi ndings the following inferences
can be made. The saturability seen in the brain slices
and in vivo studies is due not to saturation of carrier
(probably no carrier exists) but to saturation of the
enzymes involved in the intracellular conversion of
nicotinamide to NAD. This metabolic conversion
requires ATP and so accounts for the observed energy
dependency in these experiments. Niacin levels
in brain are controlled by the saturable system of
nicotinamide uptake by brain cells. Since CSF has a
nicotinamide concentration of ~0.7 μM, the uptake
system (with half-saturation concentration 0.8 μM)
is normally approximately half-saturated. This means
that the entry of excessive amounts of nicotinamide
into the brain is prohibited. As to the initial uptake
mechanism, it can be speculated that nicotinamide
that has entered the extracellular space of brain
mainly via the blood–brain barrier enters brain cells
by diffusion, accelerated by the favourable concentration
gradient created by metabolic trapping.

Dietary sources and bioavailability of Niacin

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Niacin can be synthesized in the human body from
the essential amino acid L-tryptophan. Approximately
60 mg of L-tryptophan yield 1 mg of niacin;
therefore, to calculate the niacin equivalent of a diet,
one adds one-sixtieth of the weight of tryptophan
present to the weight of preformed niacin.
Because of the contribution of tryptophan, foods
containing balanced protein are important contributors
to total niacin equivalent intake. Lean red meat,
poultry and liver contain high levels of both niacin
and tryptophan and, together with legumes, are important
sources of the vitamin. Peanut butter is an excellent
source of niacin. Cheese and eggs are relatively
poor sources of preformed niacin, but these highprotein
foods contain ample amounts of tryptophan
and therefore have a high niacin equivalent. Fruits and
vegetables provide useful amounts, depending upon
the dietary intake. Other useful sources are whole
grain cereals, bread, tea and coffee.

In mature cereal grains most of the niacin is present
as bound nicotinic acid and is concentrated in the
aleurone and germ layers. Milling to produce white
fl our removes most of the vitamin with the bran. In
the UK it is compulsory by law to add niacin to white
fl our (mostly 70% extraction rate) at 16 mg kg–1. All
fl our other than wholemeal (100% extraction) must
be enriched (Bender, 1978).
As discussed later, some plant-derived foods contain
niacin in chemically bound forms that result in
their bioavailabilities being low. Most food composition
tables give total niacin, and are compiled from the
results of analyses in which nicotinic acid is liberated
from unavailable bound forms by hydrolysis with
acid or alkali. Therefore, tabulated niacin contents
for many plant foods, particularly mature cereals,
over-estimate their value in providing biologically
available niacin.

Bioavailability
The majority of the bound nicotinic acid in mature
cereal grains is biologically unavailable after conventional
cooking (Wall & Carpenter, 1988). Nicotinoyl
glucose itself is readily utilized, so why should this
compound be unavailable when present in plant
tissues? Mason & Kodicek (1973) suggested that its
incorporation within indigestible celluloses and
hemicelluloses prevents access of the gastrointestinal
esterases to the nicotinoyl ester bonds. Alternatively,
esterase activity may be poor: the methyl ester of nicotinic
acid was only 15% as effective as the free acid in
supporting the growth of rats (Wall & Carpenter,
1988). About 10% of the total niacin was released as
free nicotinic acid after extraction of sorghum meal
with 0.1 N HCl (Magboul & Bender, 1982). This suggests
that a small proportion of bound nicotinic acid
can be hydrolysed by gastric juice and made available.
Pellagra, the disease caused by a defi ciency of both
niacin and tryptophan, has commonly been found in
population groups having maize as their staple food.
The generally accepted explanation for this association
is the unavailability of niacin in maize, coupled
with a very low proportion of tryptophan in zein
(the major protein in maize). Mexican and Central
American peasants, and also Hopi Indians in Arizona,
rely upon maize as a staple food and yet do not experience
pellagra. The explanation for this paradox lies
in the way in which these people prepare the maize
for bread-making. In the traditional preparation of
Mexican tortillas (Cravioto et al., 1945), the maize is
soaked at alkaline pH in lime-water before baking and
this process releases the nicotinic acid from its bound
forms. In the making of piki bread the Hopi Indians
use wood ash, which is alkaline and also results in the
liberation of nicotinic acid. The availability of nicotinic
acid in tortillas baked from maize treated with
lime-water has been demonstrated in pigs by Kodicek
et al. (1959).

Niacin: Nicotinic Acid and Nicotinamide

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The human disease of pellagra was first described in
Spain by Casal in 1735 after the introduction of maize
into Europe from the Americas. In the 1920s, Goldberger
in the USA reported that pellagra and black
tongue in dogs responded to treatment with animal
protein and also to boiled protein-free extracts of
yeast. In 1937, Elvehjem found that the active component
in liver extracts used to successfully treat canine
black tongue was nicotinamide, and reports that nicotinic
acid cured pellagra soon followed.
Nicotinic acid and nicotinamide had been isolated
from the coenzymes now known as NAD and NADP
by 1934–1935; hence knowledge of their biochemical
roles in electron-transfer reactions preceded the discovery
of their nutritional signifi cance. By 1946, the
metabolism of dietary tryptophan to an active form
of the vitamin had been demonstrated.

In living tissues nicotinamide is the reactive moiety
of the coenzymes NAD and NADP. The structure of
NAD can be envisaged as the adenosine diphosphateribosyl
moiety, hereafter abbreviated as ADP-ribose,
attached covalently to nicotinamide through a β-Nglycosidic
linkage (Fig. 13.1). This linkage constitutes
a high-energy bond, the energy of which supplies the
driving force for various ADP-ribosylation reactions
(see Section 13.5.2). NAD glycohydrolases hydrolyse
the N-glycosidic linkage of NAD, yielding free ADPribose,
nicotinamide and a proton. Most cellular NAD
and NADP is stored in the cytoplasm, bound to protein
(Weiner & van Eys, 1983).

Renal reabsorption and excretion for Riboflavin

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In the kidney, riboflavin that has been fi ltered through
the glomerulus can undergo either reabsorption or
secretion in the proximal tubule before it is fi nally
excreted in the urine. Reabsorption is the process
by which ribofl avin in the tubular lumen enters the
epithelial cell via the brush-border membrane, exits
the cell at the basolateral membrane and diffuses into
the peritubular capillary. Secretion is the process by
which ribofl avin in the peritubular capillary is transported
across the epithelial cell and into the tubular lumen in the opposite direction to reabsorption. As
at least 40% of plasma ribofl avin is thought to be unbound
in humans (Rose, 1988), renal reabsorption is
an important conservation mechanism. Renal secretion
removes excess ribofl avin from the body at a rate
several times higher than the glomerular fi ltration
rate. The kidney, together with the intestine, therefore
plays an important role in maintaining ribofl avin
homeostasis in the body.

Kumar et al. (1998) studied riboflavin uptake by
cultures of human-derived renal proximal tubule
epithelial cells. They demonstrated uptake via an energy-
dependent, Na+-independent, carrier-mediated
system that adapted according to the concentration of
ribofl avin in the growth medium. The adaptive regulatory
effect of ribofl avin was mediated via changes
in the number and/or activity as well as affi nity of the
ribofl avin uptake carriers. Using specifi c modulators
of intracellular signal transduction pathways, it was
shown that protein kinase A, protein kinase C and
protein tyrosine kinase were not involved in regulating
ribofl avin uptake. In contrast, inhibition of the
Ca2+/calmodulin signal transduction pathway resulted
in a signifi cant inhibition of ribofl avin uptake,
implicating this system in the regulation of ribofl avin
transport. The effect of one inhibitor, calmidazolium,
appeared to be mediated through decreases in both
the number/activity and affi nity of the ribofl avin
uptake carriers.

Yanagawa et al. (2000) studied riboflavin transport
in rabbit renal proximal tubules by using the in vitro
isolated perfused tubule. This technique is ideal for
studying bi-directional tubular transport processes
because it allows unidirectional fl uxes to be measured
separately in a defi ned tubular segment. Both reabsorption
and secretion were found to be infl uenced by
ribofl avin concentration. At 0.1 μM ribofl avin concentration,
secretion was higher than reabsorption so
that net ribofl avin transport occurred in the direction
of secretion. Lowering the ribofl avin concentration
to 0.01 μM reduced both reabsorption and secretion,
but the two fl uxes were not signifi cantly different and
so no net ribofl avin transport occurred. In contrast,
both reabsorption and secretion were increased when
the ribofl avin concentration was raised to 1 μM, leading
to a signifi cantly greater net ribofl avin secretion.
Both fl uxes were abolished by the metabolic inhibitor
iodoacetate and signifi cantly lowered by lumichrome,
indicating dependence on energy and a carrier, respectively.
Secretion, but not reabsorption, was inhibited
by the anion inhibitor probenecid and paraaminohippuric
acid (an organic anion), indicating
that the organic anion transport system is involved in
tubular ribofl avin secretion. Changes in luminal pH
over the physiological range (7.0–8.0) did not affect
reabsorption, but secretion was inhibited when the
bath pH was increased to 8.0. Secretion was inhibited
by trifl uoperazine, indicating that the intracellular
Ca2+/calmodulin-dependent pathway may play an
important role in mediating the regulation of tubular
transport through its effect on ribofl avin secretion.
For normal adults eating varied diets, ribofl avin
accounts for 60–70% of fl avin compounds in the
urine; the remainder are riboflavin metabolites (Mc-
Cormick, 1994). Urinary excretion studies carried
out in humans have suggested that any ribofl avin
secreted into the bile is almost fully reabsorbed, i.e.
the vitamin is subject to enterohepatic cycling (Jusko
& Levy, 1967).

Brain homeostasis for riboflavin

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The concentration of total riboflavin in brain, unlike
in liver and kidney, is maintained relatively constant
even in the face of severe vitamin B2 defi ciency or
after massive doses of intravenous ribofl avin. As in
other tissues, ribofl avin that enters the brain is enzymatically
phosphorylated to FMN, which can then
be converted to FAD. In rat brain, more than 90% of
the total ribofl avin is present as FAD and FMN. About
10% of the total ribofl avin in the brain of normal rats
turns over per hour.

The concentration of total riboflavin in cerebrospinal
fl uid (CSF) is about 50% of that in plasma.
However, because roughly 50% of the total ribofl avin
in plasma is bound to serum proteins, the concentrations
of unbound vitamin in plasma and CSF are approximately equal. Because CSF is constantly leaving
the central nervous system, ribofl avin must be
continually supplied to the newly formed fl uid.
Ribofl avin, but not FMN or FAD, enters the central
nervous system principally through the blood–brain
barrier (Spector, 1980a). Uptake of [14C]ribofl avin by
rabbit brain slices in vitro took place by a saturable
system that depended on the conversion of accumulated
ribofl avin to FMN and FAD, i.e. intracellular
trapping of ribofl avin (Spector, 1980b). These enzymatic
conversions require ATP. Energy dependency
was shown by the inhibition of uptake by dinitrophenol
and low-temperature (1°C) incubation. The
system was one-half saturated at the normal plasma
concentration of riboflavin (~0.03 μM). This means
that the entry of excessive amounts of ribofl avin from
blood to brain is prohibited.

Spector & Boose (1979) studied the capacity of
the isolated choroid plexus, the anatomical locus of
the blood–CSF barrier, to transport [14C]ribofl avin.
With concentrations of [14C]ribofl avin of 0.7 μM or
greater in the incubation medium, the choroid plexus
accumulated [14C]ribofl avin against a large concentration
gradient, thus demonstrating active transport.
Uptake did not depend on intracellular binding or
phosphorylation of the vitamin. The half-saturation
concentration (Km) was 78 μM (cf. normal plasma
concentration of ribofl avin of ~0.03 μM), which
means that the system has the potential to transport
high concentrations of ribofl avin before it becomes
saturated. Studies using the isolated choroid plexus do
not show direction of transport.
Spector (1980a) injected [14C]ribofl avin directly
into the ventricular CSF of anaesthetized rabbits.
Some of the ribofl avin entered the brain by a saturable
accumulation system that depended in part on
phosphorylation of the ribofl avin, but the majority
of the labelled vitamin left the CSF extremely rapidly
and was not found in the brain. The disappearance of
the injected ribofl avin means that the choroid plexus
must have actively transported the vitamin from the
CSF into the bloodstream. Direct pictorial evidence of
this property of the choroid plexus was provided by
fl uorescence microscopy (Spector, 1980c).

In conclusion, the controlled entry and exit of ribofl
avin from the central nervous system provide the
means for maintaining total riboflavin homeostasis
in brain cells. The main entry is via the blood–brain
barrier followed by high-affinity saturable uptake by
brain cells and metabolic trapping. The choroid plexus
actively transports ribofl avin from blood into the
CSF, but perhaps more importantly it has the capacity
to transport excess ribofl avin in the reverse direction

Thyroid hormone regulation of flavocoenzyme biosynthesis

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The tight binding of FMN or FAD to their respective
apoenzymes confers stability to the holoenzyme and
an insuffi ciency of coenzyme leads to loss of enzyme
activity through proteolysis. There is evidence (Rivlin
& Langdon, 1966; Rivlin, 1970; Lee & McCormick,
1985) that thyroid hormone regulates the conversion
of ribofl avin to its functional coenzyme forms.
The mechanism of regulation is on biosynthetic
rather than degradative steps. Hepatic fl avokinase
activity was diminished in hypothyroid rats, causing
decreased FMN and FAD synthesis and consequent
decreases in the activities of a number of FMN- and
FAD-dependent enzymes. FAD synthetase activity
was also diminished, but to a lesser degree than fl avokinase.
Furthermore, ribofl avin levels were reduced
in the livers of hypothyroid rats. Treatment of the
hypothyroid rats with thyroid hormone restored
coenzyme levels and enzyme activities to normal. In
the case of hyperthyroid rats, hepatic levels of FMN
and FAD were not increased above normal despite a
two-fold increase in the activity of fl avokinase. Even
when supplemental ribofl avin was administered,
coenzymes levels were not increased above normal.
Cimino et al. (1987) reported that in hypothyroid
adult humans, the activity of the FAD-containing enzyme
erythrocyte glutathione reductase was reduced
to levels observed during ribofl avin defi ciency. After
2 weeks of therapy with thyroxine and without supplementation
with ribofl avin, the enzyme activity
reverted to normal.
It is well established that thyroid hormone increases
the synthesis of protein at the transcriptional and
translational levels. Yet the restoration of fl avoprotein
enzyme activity produced by thyroid hormone
treatment in hypothyroidism was not prevented by
inhibiting protein synthesis with actinomycin-D
(Rivlin & Langdon, 1966). Apparently, therefore,
thyroid hormone does not induce the synthesis of
fl avokinase apoenzyme; rather it appears to stimulate
the conversion of an inactive precursor form of fl avokinase
to the active form or, alternatively, decrease
the proteolytic conversion of active to inactive form
(Lee & McCormick (1985). The reduced levels of substrate
(ribofl avin) could explain the reduced activity
of fl avokinase in hypothyroidism.
Rivlin & Langdon (1966) offered a plausible explanation
for the apparent upper limit in the hepatic
concentration of FMN and FAD in hyperthyroidism.
The concentration of FMN/FAD remaining in the
liver cells is restricted by the quantity of apoenzyme
to which it can be stably bound. Excess free coenzyme
would not be stored but would be destroyed enzymatically.
Thus the elevated fl avokinase activity seen in
hyperthyroidism is not accompanied by abnormally
high levels of coenzyme.
From these data it appears that thyroid hormone
regulates FMN and FAD synthesis by altering the
activity of fl avokinase. Excessive concentrations of
FMN/FAD are prevented by the enzymatic destruction
of coenzyme that is not stably bound to the limited
amounts of apoenzyme.

Adaptive regulation of ribofl avin absorption

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Feeding rats with a vitamin B2-defi cient diet caused
a signifi cant and specifi c up-regulation in ribofl avin
uptake by rat intestinal brush-border membrane vesicles
compared with controls. Conversely, over-supplementing
rats with ribofl avin caused a down-regulation
in the vitamin’s uptake. Both up- and down-regulation
were mediated through changes in the number of the
functional ribofl avin uptake carriers and/or their activity
(decreased or increased Vmax) with no effect on
the affi nity of the transport system (unchanged Km)
(Said & Mohammadkhani, 1993). Similarly, growing
Caco-2 monolayers in a ribofl avin-defi cient medium
caused signifi cant enhancement of ribofl avin uptake,
whereas an over-supplemented medium suppressed
ribofl avin uptake (Said & Ma, 1994). This adaptive upor
down-regulation of ribofl avin uptake was further
studied by Said et al. (1994) who established a role for
the ‘second messenger’ cyclic AMP (cAMP) in the regulation
mechanism. They found that compounds that
increased intracellular cAMP concentration through
different mechanisms caused a signifi cant and concentration-
dependent inhibition (down-regulation)
in ribofl avin uptake. This inhibition of ribofl avin
uptake was in contrast to other fi ndings of stimulation
of intestinal uptake of D-glucose by these compounds
(Sharp & Debnam, 1994), indicating that the effect of
cAMP-stimulating compounds on ribofl avin intestinal
uptake is not generalized in nature.
12.4.2 Absorption of bacterially
synthesized ribofl avin in the large intestine
The normal microfl ora of the large intestine synthesize
considerable amounts of vitamin B2, a signifi cant
portion of which exists as free ribofl avin. The amount
of vitamin B2 synthesized depends on the diet, being
signifi cantly higher following consumption of a vegetable-
based diet compared with a meat-based diet
(Iiuma, 1955). In a study with human subjects, Sorrell
et al. (1971) showed that ribofl avin instilled directly
into the lumen of the mid-transverse colon was absorbed,
as judged by an increase in plasma ribofl avin
concentrations. Colonic absorption of FMN sodium
has also been demonstrated in the rat (Kasper, 1970).
Said et al. (2000) demonstrated the existence of a
high-affi nity, carrier-mediated transport system in the
large intestine using cultured human colonic epithelial
cells (NCM460 cells). Saturable uptake of ribofl avin by
these cells was energy-dependent and Na+-independent.
Transport was regulated by the Ca2+/calmodulin
cell signalling pathway but not by the protein kinase
C pathway. An adaptive up- and down-regulation of
ribofl avin uptake took place when NCM460 cells were
grown in a ribofl avin-defi cient or over-supplemented
medium, respectively. These fi ndings are similar to
those in the small intestine and suggest that the same
mechanism may be operating in the large intestine to
absorb the bacterially synthesized ribofl avin.
12.4.3 Post-absorptive metabolism
Following absorption, B2 vitamers are carried by the
portal blood to the liver. About 50% of circulating
fl avins is ribofl avin, with somewhat less FAD and less
than 10% FMN. The concentration of ribofl avin in
human plasma is about 0.03 μM on average (McCormick,
1989). A proportion of the circulating fl avins is
bound loosely to albumin and tightly to some immunoglobulins.
The extent to which fl avins are bound
to plasma proteins is not believed to be crucial in
regulating tissue availability of the vitamin (White &
Merrill, 1986). Erythrocytes contain four to fi ve times
more fl avin than plasma. There is a relatively slow
equilibration of free riboflavin between the plasma
and erythrocytes, hence flavin levels in erythrocytes
are less subject to recent dietary intake. For this
reason, the activity of an erythrocyte enzyme, glutathione
reductase, is used as an indicator of vitamin
B2 status.

Uptake of riboflavin by human-derived cultured
liver cells is by means of a carrier-mediated, energydependent,
Na+-independent system which appears
to be regulated by an intracellular Ca2+/calmodulinmediated
transduction pathway and by substrate level
in the growth medium (Said et al., 1998). The liver
is the major storage site of the vitamin and contains
about one-third of the total body fl avins, 70–90% of
which is in the form of FAD. Free ribofl avin constitutes
less than 5% of the stored fl avins. Other storage
sites are the spleen, kidney and cardiac muscle. These
depots maintain signifi cant amounts of the vitamin
even in severe defi ciency states.

Absorption, transport and metabolism of Vitamin B2

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Humans cannot synthesize vitamin B2 and thus must
obtain the vitamin from exogenous sources via intestinal
absorption. The intestine is exposed to fl avins
from two sources: (1) the diet and (2) the bacterially
synthesized fl avins in the large intestine. Whether the
latter source of vitamin B2 is available to the host tissues
(apart from the colonic epithelial cells) in nutritionally
signifi cant amounts is unknown.
12.4.1 Digestion and absorption of dietary
vitamin B2
The FMN and FAD present in the ingested food are
released from noncovalent binding to fl avoproteins as
a consequence of acidifi cation in the stomach and gastric
and intestinal proteolysis. Ribofl avin is similarly
released from its association with binding proteins
(Merrill et al., 1981).
The fl avin coenzymes are hydrolysed in the upper
small intestine to free ribofl avin, which is then absorbed.
Hydrolysis of both FMN and FAD is effected
by alkaline phosphatase (EC 3.1.3.1), which has a
broad specifi city and is located on the brush-border
membrane of the enterocyte (Daniel et al., 1983a).
Two additional brush-border enzymes, FMN phosphatase
and FAD pyrophosphatase, participate in
the degradation of the fl avin coenzymes (Akiyama
et al., 1982). The considerably smaller amounts of
covalently bound fl avins are released as 8α-(peptidyl)
ribofl avins, which are absorbed along with the free
ribofl avin (Chia et al., 1978).
In vitro studies using rat everted jejunal sacs have
shown that absorption of ribofl avin takes place by a
saturable, energy-dependent process at physiologically
relevant concentrations and by simple diffusion
at higher concentrations (Daniel et al., 1983b; Said et
al., 1985; Middleton, 1990). This dual process of absorption
has been confi rmed under in vivo conditions
(Feder et al., 1991). Casirola et al. (1994) showed that
the ribitol side chain and the NH group at position 3
of the isoalloxazine moiety are essential for ribofl avin
binding to specifi c sites on the brush-border membrane
of rat small intestine.
Transport of ribofl avin across brush-border and
basolateral membrane vesicles prepared from rabbit
small intestine was found to be independent of sodium
and electroneutral in nature (Said et al., 1993a,b).
Using human-derived Caco-2 intestinal epithelial
cells, Said & Ma (1994) confi rmed the involvement of
a carrier-mediated process in the initial phase of ribofl
avin uptake (a 3-min incubation time). Ribofl avin
uptake was Na+- and pH-independent and the initial
phase occurred without metabolic alteration of the
transported ribofl avin. Inhibitors of anion transport
did not produce inhibition of ribofl avin uptake by
Caco-2 cells, thus ribofl avin does not appear to act as
an anion with regard to its intestinal transport.
Some of the absorbed ribofl avin is phosphorylated
to FMN within the cytosol of the enterocyte by fl avokinase
(ATP:ribofl avin 5´-phosphotransferase, EC
2.7.1.26) and most of the FMN is further converted
to FAD by FAD synthetase (ATP:FMN adenylyltransferase,
EC 2.7.7.2). Both of these metabolic steps require
ATP, i.e. they are energy-dependent. Gastaldi et
al. (1999) investigated the energy dependency of the
ribofl avin uptake process in isolated rat enterocytes
by comparing de-energized cells (cells treated with
rotenone) with normal cells. Short (3 min) and long
(20 min) incubation times were selected as these
times represent membrane events and intracellular
metabolic events, respectively. The results showed
that in the initial (3 min) phase, the saturable uptake
of [3H]ribofl avin is mainly an energy-independent
process with high affi nity and low capacity, whereas in
the later (20 min) phase the saturable uptake is strictly
energy-dependent and has an increased capacity. The
presence of a saturable mechanism even when intracellular
metabolism is blocked, as in de-energized
cells, suggests that the transport across the membrane
is due solely to ribofl avin binding to carrier proteins
on the brush-border membrane. Saturable uptake in
the later phase is due to high-affi nity binding of ribofl
avin to the cytosolic enzymes fl avokinase and FADsynthetase.
The conversion of ribofl avin to FMN
and FAD by these enzymes accounts for the energy
dependency of the transport process.
Additional evidence that intracellular phosphorylation
is important for the absorption of physiologically
relevant concentrations of ribofl avin is the observation
that ribofl avin analogues that are absorbed at low
concentrations by the saturable transport process are
good substrates for fl avokinase, whereas analogues
that are absorbed solely through simple diffusion at
all concentrations are poor substrates for this enzyme
(Kasai et al., 1990). Moreover, both membrane and
intracellular events in ribofl avin absorption are inhibited
by ribofl avin analogues that are readily phosphorylated
(Gastaldi et al., 1999).
To summarize, the small intestine is well adapted to
completely extracting the small amounts of ribofl avin
that are largely bound within the ingested fl avin coenzymes.
The coenzymes are dephosphorylated in the
lumen and the liberated ribofl avin is extracted very effi
ciently by a high-affi nity, carrier-mediated transport
system, which is distributed along the entire length of
the small intestine. The uptake mechanism is Na+-independent
and electroneutral in nature. After uptake,
some of the ribofl avin is metabolically trapped within
the enterocyte as FMN. The energy used in ribofl avin
absorption is not required for membrane uptake, but
rather for ribofl avin metabolism within the enterocyte.
Thus, intracellular metabolism is probably the
driving force behind the internalization of ribofl avin.
The vitamin is dephosphorylated to permit exit of
ribofl avin across the basolateral membrane; this also
takes place by a carrier-mediated, Na+-independent
and electroneutral mechanism.

Dietary sources of Vitamin B2

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Living cells require FMN and FAD as the prosthetic
groups of a variety of enzymes, and hence the fl avins
are found, at least in small amounts, in all natural unprocessed
foods. In most foods the predominant form
of vitamin B2 is protein-bound FAD. Yeast extract is
exceptionally rich in vitamin B2, and liver and kidney
are also rich sources. Wheat bran, eggs, meat, milk and
cheese are important sources in diets containing these
foods. Cereal grains contain relatively low concentrations
of fl avins, but are important sources in those
parts of the world where cereals constitute the staple
diet. The milling of cereals results in considerable loss
(up to 60%) of vitamin B2, so white fl our is enriched
by addition of the vitamin. The enrichment of bread
and breakfast cereals contributes signifi cantly to the
dietary supply of vitamin B2. Polished rice is not usually
enriched, since the yellow colour of the vitamin
would make the rice visually unacceptable. However,
most of the fl avin content of the whole brown rice is
retained if the rice is steamed prior to milling. This
process drives the water-soluble vitamins in the germ
and aleurone layers into the endosperm (Cooperman
and Lopez, 1991).

Flavins: Riboflavin, FMN and FAD (Vitamin B2)

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It became apparent during the 1920s that the
polyneuritis preventive factor, which was later designated
‘water-soluble B’ and subsequently ‘vitamin B’,
could prevent pellagra in humans as well as beriberi.
This discovery led to the replacement of the term
‘vitamin B’ by the terms ‘vitamin B1’ (the heat-labile,
anti-neuritic factor) and ‘vitamin B2’ (the heat-stable,
pellagra-preventive factor). Vitamin B2, present in
yeast extracts, was needed to prevent human pellagra
and an apparently similar canine disease called ‘black
tongue’. It was also required by rats to prevent a pellagra-
like dermatitis and to promote growth. At that
time vitamin B2 was assumed to be a single substance,
but it was later found that there are several vitamins
present in this heat-stable fraction of yeast.

The presence of water-soluble, fl uorescent, yellow
pigments in natural materials had been known for
some time. These pigments, known generically as
fl avins, were found in milk, liver, kidney, muscle, yeast
and plant materials. They were given specifi c names
according to their sources, e.g. lactofl avin (milk)
and hepatofl avin (liver). In 1933, Kuhn, György and
Wagner-Jauregg isolated from egg white a fl uorescent,
yellow, crystalline compound (‘ovofl avin’) which was
a growth-promoting factor for rats. The isolation of
other growth-promoting fl avins followed. By 1934,
Kuhn’s group had determined the structures of these
various fl avins and found them to be chemically identical.
Because each molecule contained a ribose-like
(ribitol) side chain, the term ‘ribofl avin’ was adopted.
Thus ribofl avin was the component responsible for
the rat growth-promoting activity in the aforementioned
vitamin B2 complex. The pellagra-preventive
factor and the rat anti-dermatitis factor subsequently
became known as niacin and vitamin B6, respectively.
Meanwhile, by 1932, Warburg and Christian had
isolated from yeast an enzyme, which dissociated into
a protein apoenzyme and a yellow prosthetic group
that was chemically similar to a fl avin. The yellow
pigment isolated from this ‘old yellow enzyme’ was a
vitamin-inactive, photo-derivative of a fl avin (lumifl
avin). Determination of this compound’s structure
proved useful to Kuhn for elucidating the structure
of ribofl avin. The synthesis of ribofl avin was accomplished
independently by Kuhn’s group and Karrer’s
group in 1935. By 1938, Warburg and Christian had
isolated and characterized fl avin adenine dinucleotide
(FAD) and shown it to be a coenzyme of D-amino
acid oxidase. The structure of the simpler coenzyme,
ribofl avin 5´-phosphate (FMN), was secured in the
previous year by Theorell.

The principal vitamin B2-active fl avins found in nature
are ribofl avin, ribofl avin-5´-phosphate (fl avin
mononucleotide, FMN) and ribofl avin-5´-adenosyldiphosphate
(fl avin adenine dinucleotide, FAD).
The structures of these compounds are depicted in
Fig. 12.1. The parent ribofl avin molecule comprises
a substituted isoalloxazine moiety with a ribitol side
chain.
The ‘mononucleotide’ and ‘dinucleotide’ designations
for FMN and FAD, respectively, are actually
incorrect but are nevertheless still accepted. FMN is
not a nucleotide, as the sugar group is not ribose, and
the isoalloxazine ring is neither a purine nor a pyrimidine.
FAD is composed of a nucleotide (adenosine
monophosphate, AMP) and the so-called fl avin pseudonucleotide.
In biological tissues, FAD and, to a lesser extent,
FMN occur almost entirely as prosthetic groups for
a large variety of fl avin enzymes (fl avoproteins). In
most fl avoproteins the fl avins are bound tightly but
noncovalently to the apoenzyme. In mammalian tissues
less than 10% of the FAD is covalently attached
to specifi c amino acid residues of four important
apoenzymes. These are found within succinate and
sarcosine dehydrogenases, monoamine oxidase and
gulonolactone oxidase in which FAD is peptidelinked
to an N-histidyl or S-cysteinyl residue via the
8-methyl group.

Wernicke–Korsakoff syndrome

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The Wernicke–Korsakoff syndrome is a vitamin B1 defi
ciency disease particularly associated with chronic
alcoholics who derive more than half their daily calories
from ethanol every day. Binge drinkers who eat
normally between bouts and who are in a reasonable
state of health are not prone to the disease. Alcoholic
beverages contain water, ethanol, variable amounts of
carbohydrate, and little else of nutritive value. Apart
from beers, protein and vitamin content of these
beverages is extremely low. Vitamin B1 defi ciency in
the alcoholic is a combination of several factors. The
main factor is reduced food intake due to depressed
consciousness during inebriation and hangover. Suppression
of appetite by alcohol has been postulated,
but not studied much. Gastritis and diarrhoea caused
by alcohol will impair digestion and absorption, and
alcoholic pancreatitis results in decreased secretion of
digestive enzymes. Impairment of thiamin absorption
has been reported in severely alcoholic patients
(Tomasulo et al., 1968). In cases of alcoholic cirrhosis
of the liver, there may be a decreased conversion of
thiamin to TPP and a decreased capacity of the liver to
store vitamin B1 (Leevy & Baker, 1968). The mortality
of the Wernicke–Korsakoff syndrome is 90% without
therapy, heart failure being the usual cause of death.
The Wernicke–Korsakoff syndrome has two components,
Wernicke’s encephalopathy (or Wernicke’s
disease) and Korsakoff ’s psychosis. The former is
specifi c to vitamin B1 defi ciency, whereas the latter
may be seen in association with other disorders of the
nervous system.

Wernicke’s disease is characterized by a triad of
clinical signs: eye abnormalities, incoordination and
altered state of consciousness. They occur simultaneously,
or one may precede the other by days or weeks.
The eye signs are caused by paralysis of one or more
of the eye muscles (ophthalmoplegia). Typical signs
are photophobia, nystagmus (oscillation of the eyeballs),
strabismus (crossed eyes) and diplopia (double
vision). In advanced cases, there may be complete loss
of eye movement and the pupils may become non-reactive.
The disorder of coordination is seen as a broadbased
stance and ataxia (staggering gait). Common
mental signs are listlessness, inattentiveness, apathy
and confusion. There is sometimes delirium and, in
extreme cases, stupor and coma.

Following the administration of thiamin, the
symptoms of Wernicke’s disease are alleviated and the
features of Korsakoff ’s psychosis become evident. The
most notable features are a form of amnesia in which
events of ordinary daily life are forgotten as quickly
as they occur but events in the distant past are well
remembered. The patient is alert and can converse,
think and solve problems, but is unable to memorize
new information. The symptom of confabulation
(story-telling) is an attempt by the patient to hide the
amnesia.

Neuropathological changes are seen as brain lesions
distributed in a bilaterally symmetrical fashion
in the mammillary bodies, superior vermis of the
cerebellum, hypothalamic nuclei and other diencephalic
structures (Reuler et al., 1985). Histology
shows necrosis of neurons and glial cells. There is also
capillary damage with endothelial proliferation and
pin-point haemorrhages. Damage to specifi c regions
of the brain can account for the clinical features of
286 Vitamins: their role in the human body
Wernicke–Korsakoff syndrome. Thus, for example,
the nystagmus is due to damage of the sixth cranial
nerve; the ataxia is related to loss of neurons in the
superior vermis of the cerebellum; and the amnesia is
associated with atrophy of the mammillary bodies.
Although the Wernicke–Korsakoff syndrome results
from a lack of dietary vitamin B1, two clinical observations
suggest that genetic factors are important
in its pathogenesis: it develops in only a small majority
of alcoholics and other chronically malnourished
persons, and it occurs much more frequently among
Europeans than among Asians. The possibility of
a genetic effect was investigated by Blass & Gibson
(1977) who found that transketolase in tissue-cultured
cells from patients with Wernicke–Korsakoff
syndrome was abnormal in that the binding of TPP
to the apoenzyme was diminished. The abnormality
persisted through more than 20 generations of
culture in medium containing excess thiamin and no
ethanol, and therefore appeared to be genetic rather
than dietary. Thus the abnormal enzyme appears to
be a structural mutant. Persistent aberrations had
previously been found in erythrocyte transketolase
from these patients, even after they had been treated
with thiamin for months while in hospital. The abnormality
appeared to be specifi c for transketolase as
pyruvate dehydrogenase and α-ketoglutarate dehydrogenase
were unaffected.

Because the symptoms of Wernicke’s disease can be
alleviated following the administration of thiamin,
the abnormal transketolase is presumably clinically
unimportant if the diet is adequate. This type of genetic
abnormality is an example of an inborn predisposition
to metabolic disorders. Unlike inborn errors
of metabolism, inborn predispositions are likely to
be clinically silent unless the person with the predisposition
faces an appropriate stress. In cases of Wernicke–
Korsakoff syndrome, the stress is a defi ciency of
vitamin B1. Nixon et al. (1984) demonstrated a highly
signifi cant association between a particular variant
of erythrocyte transketolase and the Wernicke–Korsakoff
syndrome, supporting the concept that the
syndrome has a genetic as well as a dietary origin.

Beriberi is treated with a proprietary thiamin
preparation, the dosage and route depending on the
patient’s condition. To prevent further recurrences,
a good diet containing all of the B-group vitamins
should be instituted. Severe cardiac (shoshin) beriberi
and Wernicke–Korsakoff syndrome constitute
medical emergencies requiring immediate treatment
with thiamin, given intravenously. Treatment of Wernicke–
Korsakoff syndrome will eradicate the symptoms
of encephalopathy (with abstinence of alcohol),
but the psychosis is irreversible.

Beriberi

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The development of beriberi, its symptoms and its
pathology are extremely variable, making it diffi cult
to describe a clinical picture or sequence of development.
Many of the early writers described three forms
of beriberi in adult humans: dry (wasting) and wet
(oedematous) beriberi, which are chronic forms, and
acute, fulminating (cardiac) beriberi. Which of these
forms predominates depends on the circumstances.
Vitamin B1 deprivation accompanied by malnutrition
and low physical activity tends to favour beriberi presenting
in the dry form, whereas high carbohydrate
intake and high physical activity during vitamin B1
deprivation predispose to wet beriberi. It should be
emphasized that any one of these forms may merge
into another.
Dry beriberi is a disease of the peripheral nervous
system involving bilateral impairment of sensory,
motor and refl ex functions. The pathological fi ndings
are segmental thinning of myelin in peripheral
nerves, progressing to degeneration of fi bre tracts.

The neuropathy begins in the feet and legs and then
extends up the body. Early signs of dry beriberi often
include sensations of pins and needles and numbness
in the feet. The legs, especially the calves, feel heavy
and weak so that walking becomes uncomfortable.
As the disease progresses, there is a marked wasting
of the leg muscles and even slight pressure applied to
the calves elicits severe pain. The characteristic foot
and wrist drop develop and there may be complete
fl accid paralysis of the lower, and occasionally upper,
extremities.
In wet beriberi, vitamin B1 defi ciency affects the
cardiovascular system by causing arteriolar dilation
throughout the circulatory system and by weakening
the heart muscle. The vasodilation causes a two-fold
increase in the venous return of blood to the heart.
Physical signs of wet beriberi are indicative of highoutput
cardiac failure; they include tachycardia, rapid
circulation time, elevated peripheral venous pressure
and widespread oedema. Pathological changes are
an enlarged heart, swollen liver, and the presence of
fl uid in the pericardial, pleural and abdominal cavities.
Microscopically, the cardiac muscle fi bres show
fragmentation and hydropic degeneration with interstitial
oedema.

The acute form of beriberi, known in Japan as
‘shoshin’, usually results in sudden death from heart
failure. The sufferer experiences severe dyspnoea,
violent palpitation of the heart and intense precordial
pain. The heart and liver become enlarged and there is
neck vein distension, cyanosis and extreme tachycardia.
Oedema is variable in this form of beriberi.
Infantile beriberi occurs in breast-fed babies between
the second and fi fth month of life. The mother
may display no signs of beriberi although obviously
her milk must be of low vitamin B1 content through
poor diet. As in adults, several clinical syndromes may
occur and the condition may be chronic or acute.
Vomiting is one of the most important early signs of
infantile beriberi. In severe cases the child appears to
be crying but no sound is heard or only a thin whine.
This characteristic feature, aphonia, is due either to
paralysis of the laryngeal nerve or to oedema of the
vocal cords. A chronic, pseudomeningeal form of
beriberi which affects the central nervous system and
may cause convulsions occurs in older infants aged 7
to 9 months.

Vitamin B1 deficiency

2007-07-03T19:45:00.001-07:00

11.7.1 Causes and effects
A defi ciency of vitamin B1 may occur in situations of
poor diet, chronic alcoholism, excessive diarrhoea or
vomiting, malabsorption and genetic metabolic defects.
Overloading the tissues with glucose without
adequate thiamin coverage can precipitate defi ciency,
as can the use of diuretics. Diseases in which the metabolic
rate is elevated (e.g. hyperthyroidism) can also
lead to defi ciency. Some researchers have demonstrated
that secondary defi ciency of a particular B-group
vitamin can be induced by excessive dosing with
another vitamin of this group. On the other hand,
defi ciencies in vitamins B6 and B12 induced vitamin
B1 defi ciency in rats, even when dietary thiamin levels
were normal (Nishino & Itokawa, 1977). Howard et al.
(1974) confi rmed that folate defi ciency in rats impairs
thiamin absorption.
In humans, a lack of vitamin B1 has widespread
effects, causing anorexia and associated weight loss,
gastrointestinal disturbances, peripheral and central
neuropathy, muscle weakness, and cardiovascular
irregularities. With severe vitamin B1 deprivation,
mental changes develop such as loss of emotional
control, paranoid trends, manic or depressive episodes
and confusion. The classic disease resulting
from a gross defi ciency of vitamin B1 in humans is
Thiamin (vitamin B1) 283
beriberi, which is prevalent in Far Eastern populations
where polished rice is the staple diet. Not only is this
diet defi cient in vitamin B1, but the high carbohydrate
intake increases the requirement for the vitamin, and
the consumption of antithiamin factors will exacerbate
the defi ciency. In other parts of the world where
such a diet is not consumed, chronic alcoholism gives
rise to the Wernicke–Korsakoff syndrome, a form of
beriberi that affects the brain. Subclinical vitamin B1
defi ciencies are characterized by mental disturbances,
fatigue, and loss of weight resulting from anorexia and
digestive problems.
Note: The term ‘polyneuritis’ applies to the involvement
of the peripheral nervous system only, while the
symptoms of central nervous system dysfunction are
more appropriately called ‘encephalopathy’ (Dreyfus,
1976).
11.7.2 Metabolic consequences of vitamin
B1 defi ciency
Proper functioning of the nervous and cardiovascular
systems relies upon normal carbohydrate metabolism,
and this in turn depends upon a dietary supply
of vitamin B1 for conversion to TPP. The pyruvate
dehydrogenase complex and α-ketoglutarate dehydrogenase,
which require TPP as a coenzyme, are
important in the main energy-yielding pathway of
carbohydrate metabolism, the tricarboxylic acid cycle.
The activities of these enzymes are decreased in selective
regions of the brain that are reversibly damaged
as a result of vitamin B1 deprivation (Butterworth et
al., 1985, 1986). The decreased enzyme activities are
due not only to a decrease in the level of coenzyme,
but also of apoenzyme. It is logical to suppose that
impairment of these dehydrogenases due to vitamin
B1 defi ciency will lead to loss of energy production
in the form of ATP. Actually, this does not happen
– at least not in the brain. The brain fi nds alternative
metabolic pathways to bypass the TPP-dependent
steps, thereby maintaining and even increasing energy
production. One possible pathway is the GABA shunt,
which bypasses α-ketoglutarate dehydrogenase (Page
et al., 1989) (Fig. 11.7). Thus energy deprivation is not
believed to be the direct cause of brain lesions resulting
from vitamin B1 defi ciency, although the compensatory
metabolic changes necessary for maintaining
energy may affect biosynthetic processes.
Preventing the synthesis of acetylcholine through
lack of conversion of pyruvate to acetyl-CoA will
impair nerve impulse transmission at synapses, and
blocking the pentose phosphate pathway through
impaired transketolase activity will lead to a reduced
synthesis of DNA through lack of ribose. Dreyfus &
Hauser (1965) showed that transketolase is more
severely affected by vitamin B1 deficiency than is
pyruvate dehydrogenase, and the reduction of transketolase
activity is anatomically correlated to brain
lesions.

Neurophysiological functions for Vitamin B1

2007-07-03T19:44:00.000-07:00

11.6.1 Introduction
There is increasing evidence that vitamin B1, specifi -
cally thiamin triphosphate, is somehow involved in
nerve membrane function. This property appears
to be independent of the known coenzyme role of
TPP. The evidence is substantiated by the fi nding that
thiamin triphosphate, which accounts for 1% of total
thiamin in rat brain, makes up 90% of total thiamin in
the electric organ of the electric eel (Bettendorff et al.,
1987). In the lamb, vitamin B1 deprivation for 4 weeks
led to a 20% depletion of total thiamin in the brain,
with a similar percentage loss of free thiamin, thiamin
monophosphate and TPP. There was, however, no
appreciable fall in thiamin triphosphate (Thornber
et al., 1980).
Most of the vitamin B1 present in the brain and
peripheral nerves is in the coenzyme form, TPP. The
1% or so of thiamin triphosphate present in whole
brain is largely concentrated in the membrane fraction
(Matsuda & Cooper, 1981). Fluorescence microscopy
shows that the vitamin is localized in the
membranes of peripheral nerves rather than in the
axoplasm (Tanaka & Cooper, 1968). A complete set
of enzymes catalysing the interconversion of thiamin
and its phosphate esters has been isolated and purifi ed
from nervous tissue (Fox & Duppel, 1975).
As discussed in the following, vitamin B1 may play
a direct role in nerve conduction or it may be implicated
in nerve transmission.

11.6.2 Nerve conduction
When Eichenbaum & Cooper (1971) UV-irradiated
electrically stimulated vagus nerves dissected from a
rabbit, action potentials were completely abolished
after 2 hours and never spontaneously reappeared
when the irradiated nerve was immersed in physiological
solution. The irradiation almost completely
destroyed endogenous thiamin in the nerve, as expected.
Immersion of the irradiated nerve in physiological
solution containing 1 mM thiamin restored
the action potentials after about 1 hour. Thiamin is
known to be rapidly taken up by the vagus nerve,
so the delay may be explained by the two enzymatic
steps required to convert thiamin to its triphosphate
ester.
CH2OH
C
HOCH
HCOH
O
CH2OPO3H2
C
HOCH
H O
HOCH
HOCH
CH2OPO3H2
CH2OH
C
HOCH
O
HCOH
HCOH
HCOH
CH2OPO3H2
C
HOCH
H O
CH2OPO3H2
CH2OH
C
HOCH
HCOH
O
CH2OPO3H2
C
HCOH
H O
HCOH
CH2OPO3H2
CH2OH
C
HOCH
O
HCOH
HCOH
CH2OPO3H2
C
HCOH
H O
CH2OPO3H2
+
TPP. Mg2+
+
+
TPP. Mg2+
+
D-Xylulose-5-phosphate D-Ribose-5-phosphate D-Sedoheptulose-7-phosphate D-Glyceraldehyde-3-phosphate
D-Xylulose-5-phosphate D-Erythrose-4-phosphate D-Fructose-6-phosphate D-Glyceraldehyde-3-phosphate
Fig. 11.6 Two transketolation reactions in the pentose phosphate pathway.
Thiamin (vitamin B1) 281
Sasa et al. (1976) studied the effects of thiamin, thiamin
triphosphate and pyrithiamin on the excitability
of the perfused giant axon of the crayfi sh. A stimulating
current was delivered to the axon every 2 s. The
following treatments were performed sequentially.
Addition of thiamin to the perfusate produced an
increase in the rising rate (dV/dt) of the action potential
as early as 5 min; there was no accompanying
effect on either the resting membrane potential or the
threshold potential. Perfusing the axon with physiological
solution restored dV/dt to control rates in
40 min. Addition of pyrithiamin elicited a decrease of
dV/dt and prolongation of the duration of the action
potential in 60 to 90 min; again the resting membrane
potential and threshold potential were unaffected.
Perfusing with physiological solution did not restore
the action potential within 30 min, but addition of
thiamin did so in 30 min. Addition of thiamin triphosphate
produced a similar effect to thiamin. The
additional fi nding that protein-binding thiamin was
reduced in axons treated with pyrithiamin indicates
a replacement of thiamin located in the membrane.
Since dV/dt of the action potential refl ects the sodium
conductance, the fi nding that thiamin and thiamin
triphosphate increased dV/dt could be construed
as an increase in sodium conductance. Conversely,
the fi nding that pyrithiamin decreased dV/dt could
mean an impairment of sodium conductance. It was
inferred from these experiments that thiamin plays
an essential role in the membrane excitability of the
crayfi sh giant axon.
In a series of experiments summarized by Itokawa
(1977), treatment of perfused spinal cords or sciatic
nerves with a variety of neuroactive agents released
free thiamin and thiamin monophosphate. The nerve
preparations were obtained from either bullfrogs or
rats made defi cient in thiamin by dietary restriction
and injected with radioactive thiamin. The neuroactive
agents also released free thiamin and thiamin
monophosphate from the membrane-myelin fraction
obtained from homogenized rat brain, spinal
cord and sciatic nerves. As the bulk of thiamin in the
membrane fraction consisted of TPP and thiamin
triphosphate, the release phenomenon presumably
involved dephosphorylation. One of the neuroactive
agents, tetrodotoxin, acts as a nerve poison by inhibiting
the early inward current of sodium. Itokawa
(1976) postulated that the propagation of an action
potential involves the shift of TPP or thiamin triphosphate
through dephosphorylation from a specifi c
site in the sodium channel to allow the early inward
current of sodium. Tetrodotoxin, by displacing these
phosphate esters, would occupy the site and prevent
the fl ow of current.
Goldberg et al. (1975), using voltage-clamped squid
giant axons, showed that the magnitudes of both sodium
and potassium conductance were decreased by
treatment with thiamin antimetabolites. However,
there was no appreciable change in the kinetics of the
conductance changes, hinting that the mechanism of
ion channel operation was unaffected. Most probably,
the antimetabolites prevented the operation of
a certain percentage of channels: those channels that
remained open during perfusion with the antimetabolites
functioned normally.
Fox & Duppel (1975) showed that B1 vitamers (thiamin
triphosphate > TPP thiamin) applied internally
to the cut internodes of frog sciatic nerve preparations
prevented the exponential decline of sodium and potassium
currents in the node of Ranvier. Neither thiamin
triphosphate nor TPP was active when applied
to the node externally. Tetrodotoxin did not alter this
property of the thiamin compounds, implying that
the tetrodotoxin-induced release of thiamin from
nerve membranes (shown by other investigators)
is not related to the mechanism by which tetrodotoxin
blocks the sodium channels. Fox and Duppel
reasoned that the thiamin dephosphorylation and
rephosphorylation process is probably not directly
coupled to the excitation process. They suggested that
the thiamin phosphates control the number of functioning
voltage-gated ion channels by stabilizing the
density of negative surface charges at the inner side of
the nerve membrane.
Further evidence for a role of thiamin triphosphate
as a membrane electrical fi eld stabilizer was provided
by Bettendorff et al. (1990). These authors reported
that thiamin triphosphate (1 μM) increased the uptake
of radioactive chloride (36Cl–) by rat brain membrane
vesicles, while TPP, thiamin monophosphate
and thiamin had no signifi cant effect. The opening
of chloride channels to allow an infl ux of negatively
charged chloride ions into the postsynaptic neuron
contributes to neuronal inhibition. Thus, if thiamin
triphosphate controls the opening of chloride channels,
it could act as a membrane stabilizer.
282 Vitamins: their role in the human body
11.6.3 Nerve transmission
One of the characteristics of thiamin-defi ciency
encephalopathy is its predilection for specifi c brain
structures with sparing of neighbouring ones. This
selective vulnerability of certain brain regions to thiamin
defi ciency has been suggested to have a metabolic
basis.
Butterworth (1982) reviewed evidence that the
neurological signs of thiamin defi ciency may involve
a defect in synaptic transmission. Changes in
the concentration or metabolism of catecholamines
(noradrenaline and adrenaline), serotonin (5-hydroxytryptamine),
γ-aminobutyric acid (GABA)
and acetylcholine were reported in certain regions
of the brain of pyrithiamin-treated animals. In addition
to these recognized neurotransmitters, changes
were also observed in the dicarboxylic amino acids
glutamic acid and aspartic acid. These amino acids act
like neurotransmitters in that they open sodium and
potassium ionic channels and cause a rapid, powerful,
excitatory response.
The synthesis of GABA, acetylcholine, glutamic
acid and aspartic acid is directly associated with the
metabolism of glucose in the brain. There is evidence
to suggest that a decreased activity of TPP-dependent
pyruvate and α-ketoglutarate dehydrogenases and
transketolase due to vitamin B1 defi ciency may be
ultimately responsible for a block in the synthesis of
one or more of these neurotransmitters.
11.6.4 Subacute necrotizing
encephalomyelopathy
Subacute necrotizing encephalomyelopathy (Leigh’s
disease) is a rare recessively inherited degenerative
disease of the central nervous system which generally
becomes symptomatic in the fi rst year of life and
is fatal. Diagnosis is complicated by the wide variety
of symptoms, which include swallowing diffi culties,
abnormal respiration, ataxia, ophthalmoplegia,
hypotonia, convulsions and progressive mental deterioration.
The neuropathology at autopsy shows
characteristic lesions of the brain stem and spinal
cord, notable features being capillary infi ltration and
demyelination of axons. Dietary vitamin B1 defi ciency
is not a factor in the aetiology of Leigh’s disease. Early
reports describing encouraging responses to large
doses of thiamin or thiamin derivatives have not been
borne out by further experience (Blass, 1981).
Infants with Leigh’s disease exhibit a defi ciency
of thiamin triphosphate in the brain. A substance,
possibly a lipoprotein, that inhibits the synthesis
of thiamin triphosphate from TPP via the thiamin
pyrophosphate-ATP phosphotransferase has been
detected in the blood, urine and cerebrospinal fl uid
of these patients (Cooper & Pincus, 1979). This substance
only inhibits the brain enzyme; the phosphotransferase
that catalyses the formation of thiamin
triphosphate in the liver is unaffected. Leigh’s disease
appears therefore to be the result of disordered brain
metabolism, possibly caused by the genetic lack of
some enzyme. The resultant defi ciency of thiamin
triphosphate explains the neurological symptoms and
neuropathological changes.

Historical overview of Vitamin B1

2007-06-29T06:35:00.001-07:00

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
ester.
(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

2007-06-29T06:33:00.000-07:00

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
status.
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

2007-06-29T06:32:00.002-07:00

Background information can be found in Section
4.5.8.
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.,
2000).
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

2007-06-29T06:32:00.001-07:00

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

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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
coagulation
Background information can be found in Section
4.4.3.
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
metabolism
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
reported.
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

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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
Storage
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

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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
aureus.
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
concentration.
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

2007-06-29T06:27:00.000-07:00

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.,
1996).

Vitamin E deficiency

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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
haemolysis.
Encephalomalacia
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
encephalomalacia.
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
anaemia.
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
injection.
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.
Abetalipoproteinaemia
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

2007-06-29T06:24:00.002-07:00

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.,
1993).
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
cytotoxicity
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
lesions.
9.7.11 Epidemiological studies and clinical
trials
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

2007-06-29T06:24:00.001-07:00

Background information can be found in Section
4.5.
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
transmigration
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
macrophages.
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

2007-06-29T06:22:00.002-07:00

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
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.
Osteomalacia
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
1α,25(OH)2D3.
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
hypercalcaemia.
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.

Immunoregulatory properties

2007-06-29T06:22:00.001-07:00

Background information can be found in Chapter 5.
8.7.1 Presence of the vitamin D receptor in
cells of the immune system
The VDR has been identifi ed in almost all nucleated
cell types in the body, including malignant and
non-malignant cells of haemopoietic origin. Peripheral
blood monocytes express the VDR constitutively.
Resting T and B lymphocytes do not express the VDR:
only when activated by mitogens and antigens do
these cells express the receptor. Interaction of T-cell
receptors with the peptide–MHC complex of antigenpresenting
tissue cells and cells of the immune system
leads to T-cell activation and subsequent gene expression
events (Manolagas et al., 1990).
Vitamin D 219
8.7.2 In vitro effects of 1α,25-dihydroxyvitamin
D3 on cells of the immune system
Monocytes/macrophages
1α,25-Dihydroxyvitamin D3 inhibits proliferation
and promotes differentiation of bone marrow-derived
macrophage precursors and specifi cally promotes expression
of the differentiation-associated cell membrane
receptor (mannose receptor) (Clohisy et al.,
1987). The relatively quiescent resident macrophages
are activated by immunogenic stimuli to become
activated macrophages, now with a greatly enhanced
ability to phagocytose and destroy pathogens.
1α,25-Dihydroxyvitamin D3 appears to be essential
for macrophage activation. Gavison & Bar-Shavit
(1989) reported that macrophages from vitamin
D-defi cient mice injected with activating or eliciting
agents had defective anti-tumour activity and
an impaired respiratory burst (low production of
hydrogen peroxide and superoxide). Activity of the
lysosomal enzyme acid phosphatase was unaffected
by vitamin D defi ciency. Incubation of the vitamin
D-defi cient macrophages with 1α,25(OH)2D3 markedly
enhanced their anti-tumour activity, but did not
affect the cells’ capacity to produce hydrogen peroxide
and superoxide, or acid phosphatase. Abe et al. (1984)
reported that 1α,25(OH)2D3 increases the number
of Fc receptors on the surface of macrophages and
induces cytotoxicity.
In cultured monocytes, 1α,25(OH)2D3 enhances
by two- to three-fold the IFN-γ-induced expression
of major histocompatibility complex (MHC) class
II molecules that mediate antigen presentation to T
lymphocytes (Morel et al., 1986). Pre-treatment of
normal human monocytes with 1,25(OH)2D3 enhances
their responsiveness to various chemoattractants
(Girasole et al., 1990). Augmented monocyte
chemotaxis to FMLP is associated with an increased
number of high-affi nity binding sites for this chemoattractant.
The hormone is also able to stimulate
the migratory capacity of monocytes obtained from
patients with acquired immune defi ciency syndrome
(AIDS), a condition associated with impaired monocyte
chemotaxis. 1α,25(OH)2D3 increased production
of IL-1 by human peripheral blood monocytes
(Bhalla et al., 1986) and also enhanced these cells’
capacity for lipopolysaccharide-triggered release of
tumour necrosis factor (Rook et al., 1987). Incubation
of monocytes with 1α,25(OH)2D3 at 45°C led
to an increased synthesis of heat shock proteins accompanied
by a relative preservation of total protein
synthesis (Polla et al., 1986).
It has been postulated that vitamin D may have
a protective role in tuberculosis infection (Davies,
1985). The bacterium responsible for tuberculosis is
a bacillus, Mycobacterium tuberculosis. Monocytes,
although phagocytic, have only a limited capacity
to kill M. tuberculosis and can become infected with
the bacillus in vitro. However, any bacilli released by
dying monocytes are rapidly phagocytosed by other
cells, especially macrophages, and destroyed. Rook et
al. (1986) reported that 1α,25(OH)2D3 inhibited the
growth of M. tuberculosis in cultured human monocytes
and IFN-γ enhanced this inhibition. They also
showed that incubation of monocytes with IFN-γ
led to increased ability to metabolize 25(OH)D3 to
1α,25(OH)2D3. This ability has also been demonstrated
in normal human macrophages (Koeffl er et
al., 1985). Based on these observations, a possible
scenario is that infected monocytes produce IFN-γ
which induces these cells to synthesize 1α,25(OH)2D3
from circulating 25(OH)D3. The locally produced
hormone stimulates monocytes to differentiate into
macrophages, which are better equipped to deal with
the infection.
Natural killer cells
Merino et al. (1989) showed that 1,25(OH)2D3 inhibits
the generation of cytotoxic activity from cultured
natural killer cells. The hormone was, however, unable
to interfere with the cytotoxic function of cells already
established, placing the inhibition at the level of natural
killer cell activation.
Lymphocytes
In contrast to the stimulatory effects of 1α,25(OH)2D3
on the innate arm of the immune response, the principal
action of the hormone on the acquired immune
response, mediated by lymphocytes, is immunosuppression.
1α,25(OH)2D3 inhibits T-lymphocyte
proliferation after these cells have been activated and
the VDR is expressed. The hormone prevents entry
of the cells into the S phase of the cell cycle by blocking
the RNA synthesis required for the transition of
cells from early G1 to late G1 (Rigby et al., 1985).
1α,25-Dihydroxyvitamin D3 also inhibits the growthpromoting
interleukin-2 (IL-2) (Tsoukas et al., 1984).
Inhibitors of IL-2 synthesis block the cell cycle at the
220 Vitamins: their role in the human body
same point as 1α,25(OH)2D3 blocks the cycle, suggesting
that the inhibitory effect of 1α,25(OH)2D3
on T-cell proliferation is mediated by IL-2. Alroy et
al. (1995) demonstrated that 1α,25(OH)2D3 represses
IL-2 gene transcription by a direct, VDR-dependent
effect. In the absence of intracellular 1α,25(OH)2D3,
the IL-2 gene in T lymphocytes is activated by the
binding of a T-cell-specifi c transcription factor,
NFATp, to an NF-AT-1 element and subsequent
recruitment of the ubiquitous transcription factors
Jun and Fos (AP-1). VDR–RXR heterodimers, which
would form in response to the intracellular presence
of 1α,25(OH)2D3, directly inhibit the interaction
between AP-1 and NFATp. Moreover, the stable binding
of VDR–RXR to the NF-AT-1 element blocks the
binding of any NFATp–AP-1 that may subsequently
be formed; however, prebound NFATp–AP-1 cannot
be destabilized by VDR–RXR. The suppressive effect
of 1α,25(OH)2D3 on lymphocyte proliferation
is countered indirectly by IL-1 produced by monocytes
in response to stimulation by 1α,25(OH)2D3
(Bhalla et al., 1986). IL-1 potentiates the release of
IL-2 from activated T cells and the IL-2 stimulates
lymphocyte proliferation. These differential effects of
1α,25(OH)2D3 provide a fi nely tuned mechanism for
regulating T-cell proliferation.
Lemire et al. (1984) demonstrated an inhibitory
role of 1α,25(OH)2D3 on proliferation and immunoglobulin
production by normal activated human
peripheral blood mononuclear cells in vitro. Further
studies revealed the T helper cell to be particularly
suppressed by 1α,25(OH)2D3 (Lemire et al., 1985). T
helper cells are divided into Th1 and Th2 subsets on
the basis of their pattern of cytokine secretion. Th1
cells secrete interleukin (IL-2) and interferon (IFN-γ)
and induce B cells to produce immunoglobulin IgG2a,
while Th2 cells secrete IL-4 and IL-10 and induce the
production of IgG1 and IgE by B cells. IL-12 that is
produced by macrophages and B cells induces IFN-
γ secretion by natural killer cells and Th1 cells and
promotes the differentiation of Th1 cells from their
uncommitted precursors.
Pre-incubation of T helper cells with 1α,25(OH)2D3
prevents these cells from inducing B cells to synthesize
immunoglobulin. The hormone also reduces mRNA
levels for IL-2 and IFN-γ in T helper cells. These observations
are consistent with a selective suppressive
effect of 1α,25(OH)2D3 on Th1 cells. 1α,25(OH)2D3
also inhibits the secretion of IL-12 by macrophages
and B cells, thereby preventing the differentiation of
precursor cells to Th1 cells (Lemire et al., 1995). Thus,
the immunosuppressive activity of 1α,25(OH)2D3
that takes place in vitro is aimed specifi cally at Th1
cells, preventing their expression both directly or
indirectly through inhibition of macrophage-derived
IL-12.
Meehan et al. (1992) studied the effects of
1α,25(OH)2D3 on the human mixed lymphocyte
reaction (MLR), the in vitro model of transplant
compatibility. The hormone stimulated suppressor
T-cell activity and prevented the generation of
cytotoxic T-cell activity. A signifi cant reduction in
expression of MHC class II molecules (but not class
I molecules) was also observed in the presence of the
hormone. The suppression of IFN-γ production by
1α,25(OH)2D3 could explain the latter effect. The effects
of 1α,25(OH)2D3 on the MLR are similar to those
of the potent immunosuppressive drug, cyclosporin A
(Hess & Tutschka, 1980).

Phosphate homeostasis

2007-06-29T06:21:00.000-07:00

Plasma phosphate levels are maintained within a
concentration range of 1.12–1.45 mM. The restoration
of the normal plasma calcium level in response to
hypocalcaemia is not accompanied by a rise in plasma
phosphate because PTH independently causes a
phosphate diuresis. Unlike calcium, dietary phosphate
usually exceeds the body’s nutritional requirement,
therefore a major component of phosphate
homeostasis is renal excretion. A diet that is low in
phosphorus is likely to be low also in calcium, which
complicates the picture of phosphate homeostasis.
Let us consider a hypothetical situation of a normal
plasma calcium level during hypophosphataemia.
A lowering of plasma phosphate will stimulate the
kidney to release 1α,25(OH)2D3, which elicits both
the previously mentioned rapid (nongenomic) and
long-term (genomic) responses in the kidney, leading
to increased renal reabsorption of phosphate. The
1α,25(OH)2D3 will also increase the intestinal absorption
of phosphate and calcium. The parathyroids will
not be stimulated to produce PTH. In the absence of
PTH, mobilization of phosphate from the bone will be
retarded and there will be no phosphate diuresis. The
net effect will be an elevation of plasma phosphate.
Hyperphosphataemia is countered through phosphate
excretion, governed by the fact that the blood
phosphate concentration is maintained at or near the
renal transport maximum for the ion.
8.6.11 Effects of vitamin D defi ciency
Vitamin D defi ciency can arise from lack of sunlight
exposure, lack of dietary vitamin D intake, or impaired
intestinal absorption of the vitamin. At the
onset of defi ciency, there is a decreased effi ciency of
intestinal calcium absorption and a consequent fall in
the plasma calcium level. In response to the hypocalcaemia,
the plasma Ca2+ concentration is restored to
normal, but the Pi concentration falls. The rise in Ca2+
concentration is caused principally by two effects.
Firstly, PTH, acting with whatever 1α,25(OH)2D is
still present at the onset of defi ciency, elicits the mobilization
of Ca2+ and Pi from bone; secondly, PTH, acting
alone, causes an increase in the renal reabsorption
of calcium. The decline in plasma Pi concentration is
caused by a very strong effect of PTH on the kidney in
causing excessive phosphate excretion, an effect that is
usually great enough to override increased phosphate
mobilization from the bone.
During prolonged vitamin D defi ciency, the
increase in PTH secretion necessary to maintain
calcium homeostasis causes extreme osteoclastic
resorption of bone. This in turn causes the bone to
become progressively weaker and imposes marked
physical stress on the bone, resulting in rapid osteoblastic
activity. The osteoblasts lay down large quantities
of osteoid but, because of insuffi cient Ca2+ and
Pi, calcifi cation does not occur. Thus failure to calcify
newly formed bone matrix leads ultimately to rickets
or osteomalacia.

Differentiation of osteoclast progenitors into mature osteoclasts

2007-06-29T06:20:00.000-07:00

1α,25-Dihydroxyvitamin D3 enhances bone resorption
by stimulating differentiation and fusion of
mononuclear osteoclast progenitors into mature
multinucleated osteoclasts (Clohisy et al., 1987; Takahashi
et al., 1988). This process, osteoclastogenesis,
involves a complex interaction of osteoclast progenitors,
osteoblasts and bone marrow-derived stromal
cells (Suda et al., 1992a). Studies using VDR-ablated
mice showed that stimulation of osteoclast formation
by 1α,25(OH)2D3 requires the presence of VDR in
osteoblast-like cells but not in osteoclast precursor
cells; however, if VDR is absent in the osteoblastic
cells, PTH or interleukin-1α can stimulate osteoclast
formation (Takeda et al., 1999).
Stromal cells of the bone marrow control osteoclastogenesis
through the production of cytokines capable
of promoting the proliferation and differentiation
of osteoclast progenitors. One particular cytokine, interleukin-
11 (IL-11), is a rather specifi c product of the
mesenchymal cell lineage, which includes bone marrow
stromal cells and osteoblasts (Paul et al., 1990).
IL-11 is a potent inducer of osteoclast development
(Girasole et al., 1994) and its production by primary
osteoblasts can be stimulated by 1α,25(OH)2D3 among
other osteoclastogenic factors (Romas et al., 1996).
The addition of anti-IL-11 antibody to bone marrow
cell cultures suppresses the ability of 1α,25(OH)2D3 to
induce osteoclast development (Girasole et al., 1994),
suggesting that IL-11 is an essential factor for the osteoclastogenic
effect of 1α,25(OH)2D3. Another soluble
factor, macrophage-colony stimulating factor (MCSF),
is essential for osteoclast differentiation from
progenitors, but its production by osteoblasts/stromal
cells does not appear to be regulated by 1α,25(OH)2D3
(Suda et al., 1992a).
In 1997, two independent research groups reported
the discovery of novel cytokines which inhibited the
differentiation of osteoclast progenitors into mature
osteoclasts. These secreted proteins were named
osteoclastogenesis inhibitory factor (OCIF) and
osteoprotegerin (OPG) by the respective groups
(Tsuda et al., 1997; Simonet et al., 1997). Yasuda et
al. (1998a) found these two proteins to be identical,
hence the present term, OPG/OCIF. Transgenic mice
with over-expressed OPG/OCIF and mice injected
with OPG/OCIF exhibited profound yet non-lethal
osteopetrosis, coincident with arrested osteoclast
development in the later stages (Simonet et al., 1997).
Yasuda et al. (1998a) reported that the expression of
the OPG/OCIF gene in stromal cells is down-regulated
by 1α,25(OH)2D3 and up-regulated by calcium
ions. These results imply that OPG/OCIF regulates
osteoclastogenesis in response to stimulators of bone
resorption and calcium ions released at bone-resorbing
sites.
1α,25-Dihydroxyvitamin D3, along with PTH and a
number of other factors, stimulate osteoclastogenesis
through signal transduction pathways mediated by an
osteoclast differentiation factor (ODF) on the membrane
of osteoblasts/stromal cells (Suda et al., 1992b).
OPG/OCIF inhibits in vitro osteoclastogenesis by
directly binding to the ODF, thereby interrupting
ODF-mediated signalling from osteoblast/stromal
216 Vitamins: their role in the human body
cells to osteoclast progenitors for their differentiation
into mature osteoclasts (Tsuda et al., 1997). 1α,25-Dihydroxyvitamin
D3, by down-regulating OPG/OCIF,
permits osteoclastic bone resorption.
Yasuda et al. (1998b) showed that ODF is identical
to a regulator of T lymphocyte cells and dendritic
cells designated TRANCE or RANKL. Expression of
the TRANCE/RANKL/ODF gene was up-regulated by
osteotropic factors, including 1α,25(OH)2D3.
Sato et al. (1991) reported that mouse bone marrow-
derived stromal cells and primary osteoblastic
cells in vitro produce the third component of complement
(C3) in response to 1α,25(OH)2D3. This appears
to be a bone-specifi c effect as C3 production by hepatocytes
is not dependent on 1α,25(OH)2D3. Bone C3
is also induced tissue-specifi cally by 1α,25(OH)2D3
in vivo (Jin et al., 1992). Actinomycin D completely
inhibits the effect of 1α,25(OH)2D3 on both mRNA
expression and protein production of C3, indicating
that the hormone acts at the transcriptional level
(Hong et al., 1991). The addition of anti-C3 antibody
to mouse bone marrow cultures completely inhibits
the 1α,25(OH)2D3-induced formation of osteoclastlike
cells, suggesting that the C3 produced by stromal
cells in response to 1α,25(OH)2D3 is somehow involved
in osteoclast formation (Sato et al., 1991). The
production of C3 in stromal cells and osteoblastic
cells is also stimulated by local bone-resorbing agents,
such as interleukin-1, tumour necrosis factor-α and
lipopolysaccharides (Hong et al., 1991). Sato et al.
(1993) concluded that the bone C3, acting in concert
with other factors induced by 1α,25(OH)2D3, potentiates
proliferation of bone marrow cells and induces
differentiation of these cells into osteoclasts.
Attachment of osteoclasts to the bone matrix
1,25-Dihydroxyvitamin D3 activates the transcription
of integrin αv and β3 subunit genes, resulting in an
increased number of integrin αvβ3 receptors on the
surface of osteoclast progenitors (Medhora et al.,
1993; Mimura et al., 1994). These receptors recognize
and bind to the bone matrix proteins osteopontin
and bone sialoprotein. Expression of integrin αvβ3
coincides with the differentiation of progenitors into
osteoclasts and is essential to the resorptive process.
Activation of quiescent osteoclasts
1α,25-Dihydroxyvitamin D3 stimulates osteoclastic
bone resorption in tissue culture (Raisz et al., 1972)
and in rat bones in vivo (Holtrop et al., 1981). Mc-
Sheehy & Chambers (1987) reported that isolated osteoclasts
do not respond to 1α,25(OH)2D3 if incubated
alone, but they do so if incubated in the presence of
osteoblastic-like cells. Incubation of osteoblastic cells
in the presence of 1α,25(OH)2D3 produced a soluble
factor that stimulated osteoclastic bone resorption.
Jimi et al. (1996) demonstrated that cell-to-cell contact
between osteoblastic cells and osteoclast-like cells
was required to promote pit-forming activity. Mee et
al. (1996) were the fi rst to show that active human
osteoclasts in vivo possess mRNA for the VDR. It
seems, therefore, 1α,25(OH)2D3 can act directly on
osteoclasts to stimulate bone resorption as well as
indirectly through its effects on osteoblasts.
8.6.9 Calcium homeostasis
The control of calcium homeostasis involves three
major sites: bone, the kidneys and the intestine.
Bone is the major reservoir of calcium in the body,
storing around 99% of the total. The role of bone
in calcium homeostasis is to ‘buffer’ blood calcium
level, releasing Ca2+ to the blood when the blood
level decreases and taking Ca2+ back when the level
rises. Calcium homeo stasis is coordinately regulated
by 1α,25(OH)2D3 and parathyroid hormone (PTH),
with calcitonin playing an important supporting role.
PTH is a peptide hormone produced by the parathyroid
glands in response to low plasma calcium levels.
Its action is mediated by PKA via the second messenger
cyclic AMP (cAMP) (Horiuchi et al., 1977). In the
kidney, PTH activates the 25(OH)D3-1α-hydroxylase
by increasing the enzyme’s mRNA through effects on
gene transcription (Brenza et al., 1998; Murayama et
al., 1998). Simultaneously, PTH suppresses the renal
24R-hydroxylase (Shinki et al., 1992), the enzyme responsible
for catalysing the C-24 oxidation pathway.
This reciprocal regulation by PTH in the kidney allows
for 1α,25(OH)2D3 production with minimal
concomitant catabolism. The effect of 1α,25(OH)2D3
is to restore plasma calcium levels to normal. PTH
has no effect on intestinal 24R-hydroxylase activity
(Shinki et al., 1992), which is not surprising as the intestine
lacks PTH receptors. In osteoblasts, PTH acts
synergistically with 1α,25(OH)2D3 to increase transcription
of the 24R-hydroxylase gene (Armbrecht et
al., 1998). Thus PTH exerts opposite effects in kidney
and bone with respect to 24R-hydroxylase regulation.
Vitamin D 217
Activation of the 24R-hydroxylase in bone is a means
of regulating the resorptive action of 1α,25(OH)2D3.
Cycloheximide has no effect on the capacity of PTH to
increase 24R-hydroxylase mRNA levels in osteoblasts,
showing that the action of PTH does not require new
protein synthesis. One possible mechanism by which
PTH could increase 24R-hydroxylase promoter
activity in conjunction with 1α,25(OH)2D3 is phosphorylation
of the cAMP-response element binding
protein (CREB) by PKA and consequent binding of
the CREB to a cAMP-response element (CRE). Interaction
between CREB/CRE and VDR/VDRE complexes
would synergistically increase 24R-hydroxylase
promoter activity.
PTH and 1α,25(OH)2D3 mutually regulate each
other’s synthesis and/or secretion. PTH downregulates
VDR abundance in the kidney (Reinhardt
& Horst, 1990), while 1α,25(OH)2D3 lowers preproPTH
mRNA levels in the parathyroids (Silver et
al., 1985, 1986) with a subsequent reduction in PTH
secretion (Cantley et al., 1985). In osteoblastic cells,
1α,25(OH)2D3 reduces PTH-stimulated cAMP production
(Pols et al., 1986), while PTH up-regulates
VDR abundance (Krishnan et al., 1995).
The homeostatic control of calcium is represented
schematically in Fig. 8.10. In response to a lowered
concentration of serum Ca2+, the parathyroid glands
are stimulated, within minutes, to secrete PTH. This
hormone stimulates, within hours, the activity of
renal 25(OH)D-1α-hydroxylase. The 1α,25(OH)2D3
thus produced acts by itself to initiate active calcium
transport in the intestine. In bone, 1α,25(OH)2D3
acts synergistically with PTH to mobilize Ca2+ (accompanied
by Pi) from the bone fl uid compartment
into the bloodstream. The presence of both PTH and
the vitamin D hormone are required for this system to
operate in vivo (Jones et al., 1998). In the kidney, PTH
and 1α,25(OH)2D3 act in concert to cause the reabsorption
of the last 1% of the fi ltered load of calcium
into the plasma compartment. The resultant increase
in the concentration of circulating Ca2+ provides a
powerful negative feedback signal to the parathyroids,
suppressing the secretion of PTH and ultimately stopping
the renal production of 1α,25(OH)2D3.

Renal calcium reabsorption

2007-06-29T06:19:00.000-07:00

The kidney plays a crucial role in calcium homeostasis.
To maintain a net calcium balance, more than 98%
of the fi ltered load of calcium must be reabsorbed
along the nephron. Tubular reabsorption of calcium
can take place via the transcellular and paracellular
routes by mechanisms similar to those described for
calcium transport in the intestine.
Paracellular, diffusional movement predominates
in the proximal convoluted tubule and thick ascending
limb of Henle’s loop, in which the epithelium has a
low electrical resistance and hence high permeability.
Transcellular, active transport takes place in the distal
nephron where the epithelia are less permeable. The
rate of active calcium reabsorption is controlled by
PTH and 1α,25(OH)2D3. The involvement of calbindin-
D28k in active calcium transport is suggested
by its exclusive presence in the distal nephron and its
increased production in response to 1α,25(OH)2D3
treatment of collecting duct cells (Bindels, 1993).
8.6.4 Na+/Pi co-transporters
Three families of vertebrate Na+/Pi co-transporter
have been identifi ed: type I, type II (IIa and IIb isoforms)
and type III. Type I proteins have been found
in the apical membrane of renal proximal tubules,
but their function is not yet clearly established. Type
II proteins are also located in apical membranes – type
IIa in renal proximal tubules and type IIb in small
intestinal enterocytes. Type III co-transporters are
found in many tissues and appear to be located at the
basolateral membrane. Type II (IIa and IIb) Na+/Pi
co-transporters mediate secondary active phosphate
transport in which the immediate energy source is the
downhill concentration gradient for Na+ maintained
by the action of the sodium pump at the basolateral
membrane. The parallel operation of other sodiumcoupled
transport systems will indirectly affect the
Na+/Pi co-transport rate due to competition for
driving forces (Danisi & Murer, 1991). Type II cotransporters
operate with a 3Na+ to 1Pi stoichiometry
(Murer et al., 2001). In the presence of divalent phosphate,
these transporters interact with the substrate
(Pi) followed by the loading of two Na+ ions. Thus
the translocation of the fully loaded transporter is an
electroneutral process. The observed negative charge
Vitamin D 213
transfer within the transport cycle is the result of the
reorientation of the unloaded transporter (Murer et
al., 2002).
8.6.5 Intestinal phosphate absorption
Dietary phosphorus is a mixture of inorganic phosphate
and organic phosphorus. The phosphorus in
meat and fi sh exists largely in the form of phosphoproteins
and phospholipids; over 80% of the phosphorus
in grains such as wheat, rice and maize is found as
phytic acid (hexaphosphoinositol); about 33% of the
phosphorus in milk exists as inorganic phosphate; milk
protein (casein) is particularly highly phosphorylated.
Regardless of its dietary form, most phosphorus is
absorbed in inorganic form. Organically bound phosphorus
is hydrolysed enzymatically in the lumen of the
small intestine. The phosphorus within phytic acid has
poor bioavailability owing to incomplete hydrolysis.
Phosphate absorption takes place mainly in the
jejunum by an energy-dependent transcellular route
that is regulatable and a passive paracellular route that
is not regulatable. The transcellular pathway involves
secondary active transport at the brush-border membrane
mediated by the type IIb Na+/Pi co-transporter.
Both the monovalent and divalent forms of phosphate
are transported (Quamme, 1985). The capacity (Vmax)
of the transport system is signifi cantly greater at pH
6.1 than at 7.4, thus the acidic environment of the
unstirred layer favours Pi uptake (Borowitz & Ghishan,
1989). The basolateral membrane also contains
a Na+/Pi co-transporter, which has a lower capacity
and a higher affi nity compared to the brush-border
co-transporter (Kikuchi & Ghishan, 1987).
A low-phosphorus diet leads to a rapid decrease of
plasma Pi concentration and activation of the renal
25(OH)D-1α-hydroxylase. The resultant increase
in circulating 1α,25(OH)2D3 induces an increased
capacity of the type IIb Na+/Pi co-transporter in the
brush border of the intestinal epithelium. The hormonal
response requires several hours and involves
protein synthesis. The rapid adaptive response observed
in the kidney (see below) does not take place in
the intestine (Hattenhauer et al., 1999).
8.6.6 Renal phosphate reabsorption
Renal reabsorption of phosphate takes place in the
proximal convoluted tubule and, under normal physiological
conditions, ~80% of phosphate contained in
the glomerular fi ltrate is reabsorbed. The brush-border
uptake, which is mediated by the type IIa Na+/Pi
co-transporter, is rate-limiting in most situations
and the target of physiological control mechanisms.
Basolateral exit is ill-defi ned, but may involve another
Na+/Pi co-transporter (Schwab et al., 1984). Transport
of phosphate in the opposite direction (from the peritubular
interstitium into the tubular cell) can take
place across the basolateral membrane if apical entry
is insuffi cient to satisfy the cell’s metabolic requirements.
The existence of multiple systems for transporting
phosphate across the brush-border membrane
of the tubular epithelium has been recognized since
1977. Walker et al. (1987) demonstrated two sodium-
dependent systems in the early segments of
the proximal tubule: a high-capacity, low-affi nity
system and a low-capacity, high-affi nity system. A
third transport system in the late proximal segments
was found by the same group to be independent of sodium
and mediated by an hydroxyl ion/Pi exchanger
or a proton/Pi co-transport system (Yan et al., 1988).
The renal control of acid–base balance requires secretion
of hydrogen ions into the tubular lumen by the
tubular epithelial cells, causing a progressive decrease
in luminal pH from 7.4 to 6.8 along the length of the
proximal tubule. Such a decline in pH results in a fall
of the divalent to monovalent phosphate ratio from
4:1 to 1:1 at any given Pi concentration (Quamme &
Wong, 1984). It has been demonstrated that the two
sodium-dependent systems mentioned above transport
the divalent form of phosphate, whereas the
sodium-independent system located in the late proximal
tubule has a preference for the monovalent form
(Yan et al., 1988). Accordingly, the multiple transport
systems act in concert to reclaim fi ltered phosphate
along the proximal tubule.
The rate of renal phosphate reabsorption is adjusted
in response to deviations in plasma Pi concentration to
achieve a correct phosphate homeostasis. A low-phosphorus
diet induces a rapid (2–4 hour) non genomic
response followed by a long-term genomic response
if phosphorus deprivation persists. Both types of
response involve stimulation of sodium-dependent
phosphate transport by 1α,25(OH)2D3. The rapid
response may be mediated by a microtubule-dependent
translocation of the Na+/Pi co-transporter protein
from intracellular compartments to the brush-border
214 Vitamins: their role in the human body
membrane (Lötscher et al., 1996). Rapid down-regulation
of the co-transporter in response to acute administration
of a large dose of phosphate is probably
mediated by endocytosis of the protein (Lötscher et
al., 1996). The long-term response involves transactivation
of the Na+/Pi co-transporter gene (Taketani
et al., 1997).
8.6.7 Calcium movement in bone
Bone contains a fl uid compartment that is separated
from the extracellular fl uid by the lining cells on the
surface of bone. As shown in Fig. 8.9, the bone fl uid
compartment comprises the fl uid-fi lled space between
the lining cells and bone matrix, around the
protoplasmic extensions in the canaliculi, and around
osteocytes in their lacunae. The bone fl uid is therefore
in direct contact with bone crystals or amorphous
calcium phosphate deposits (Talmage, 1970).
The Ca2+ concentration in the bone fl uid compartment
is normally about one-third that in the extracellular
fl uid. The lining cells exert the primary control
on extracellular calcium homeostasis. These cells have
open channels between them, permitting paracellular
entry of Ca2+ into the bone fl uid compartment down
the concentration gradient. The uphill movement of
Ca2+ from the bone fl uid compartment to the extracellular
fl uid involves active pump-driven transport
through the lining cells.
In the presence of vitamin D, parathyroid hormone
(PTH) increases the calcium permeability of the
lining cell plasma membrane facing the bone fl uid,
allowing Ca2+ to diffuse into the cells from the bone
fl uid. The increase in intracellular Ca2+ then activates
the calcium pump on the opposite membrane. This
action of PTH results in the rapid removal of Ca2+,
accompanied by Pi, from amorphous calcium phosphate
deposits in the vicinity of the lining cells and
transference of these ions to the extracellular fluid.

Calcium and phosphate homeostasis

2007-06-29T06:18:00.000-07:00

Ionic calcium (Ca2+) is crucial for many cellular
functions including neurotransmitter release and
nerve impulse propagation; contraction of skeletal,
cardiac and smooth muscle; blood clotting; exocrine
and endocrine secretory processes; and cell proliferation.
Ca2+ also acts as a ‘second messenger’, connecting
some stimuli (certain hormones, growth factors
and neurotransmitters) with physiological responses.
Bones and teeth contain about 99% of the body’s calcium;
the other 1% is distributed in both intra- and
extracellular fl uids.
The blood level of Ca2+ is very closely regulated:
even small changes in Ca2+ concentration can be
fatal. Acute hypocalcaemia in the human causes essentially
no other signifi cant effects besides tetany,
because tetany kills the patient before other effects
can develop. In tetany, the low concentration of Ca2+
in the extracellular fl uid causes the nervous system
to become progressively more excitable because of
increased neuronal membrane permeability. Eventually,
peripheral nerve fi bres become so excitable
that they begin to discharge spontaneously, initiating
nerve impulses that pass to the skeletal muscles, where
they elicit the muscular spasms of tetany. In hypercalcaemia,
the nervous system is depressed, and refl ex
actions of the central nervous system become sluggish.
There is also constipation and lack of appetite,
probably because of depressed contractability of the
muscular walls of the gastrointestinal tract. Above a
certain high level of blood Ca2+, calcium phosphate is
likely to precipitate throughout the blood and the soft
tissues. Deposition of calcium in the kidney or heart
causes death due to renal failure or cardiac arrest.
Phosphorus is a component of hydroxyapatite in
the skeleton, of phospholipids in cell membranes,
and of the nucleic acids, DNA and RNA. In cells,
phosphorus participates in energy metabolism and
acid–base regulation. Many signalling molecules depend
on the phosphorylation of enzymes to elicit an
hormonal response. Approximately 85% of the body’s
phosphorus is in the skeleton, 14% is associated with
soft tissue such as muscle, and 1% is found in the
blood and body fl uids. Within the physiological range
of pH values, inorganic phosphate (Pi) is present in
two different ionic species, H2PO4
– (monovalent) and
HPO4
2– (divalent). The relative concentration of each
is dependent on the ambient pH. Thus variation in the
pH value may have marked effects on the transport of
phosphate by altering the concentration ratio of these
phosphate species.
1α,25-Dihydroxyvitamin D restores low plasma
concentrations of Ca2+ and Pi to normal by action at
the three major targets, namely intestine, bone and
kidney. The hormone (1) stimulates the intestinal absorption
of Ca2+ and Pi by independent mechanisms,
(2) stimulates the transport of Ca2+ (accompanied
by Pi) from the bone fl uid compartment to the extracellular
fl uid compartment, and (3) facilitates the
renal reabsorption of Ca2+. These three mechanisms
provide calcium for bone mineralization and prevent
hypocalcaemic tetany.
1α,25-Dihydroxyvitamin D3 regulates the synthesis
of two classes of calcium-binding proteins (calbindins)
found in mammalian intestine and kidney. An
intestinal 9-kDa protein (calbindin-D9k) binds two
calcium ions per molecule, and a renal 28-kDa protein
(calbindin-D28k) binds fi ve to six calcium ions per
molecule (Lowe et al., 1992). Gross & Kumar (1990)
reviewed extensive evidence that the calbindins are
involved in transcellular calcium transport.
8.6.2 Intestinal calcium absorption
Calcium is present in foods and dietary supplements
as relatively insoluble salts. Because calcium
is absorbed only in its ionized form, it must fi rst be
released from the salts. Solubilization of most calcium
salts takes place in the acidic medium of the stomach
but, on reaching the alkaline environment of the small
intestine, some of the Ca2+ may complex with minerals
or other specifi c dietary constituents, thereby limiting
calcium bioavailability.
The mammalian intestine has developed special
vitamin D-dependent mechanisms to ensure the
absorption of appropriate amounts of calcium in the
face of changing needs and varying dietary calcium
intakes. Present knowledge of these mechanisms
is rather limited, although it appears that multiple
mechanisms are involved. In view of the amount of
controversy and continued research into this subject,
any model of vitamin D action must be tentative.
Calcium absorption takes place by the translocation
of luminal Ca2+ through the enterocytes (transcellular
route) and between adjacent enterocytes via the tight
Vitamin D 209
junctions (paracellular route). Transcellular movement
is a saturable, energy-dependent process that
is subject to regulation by vitamin D and is confi ned
almost entirely to the duodenum and upper jejunum.
In the perfused chick intestine, the stimulation of
calcium transport by 1α,25(OH)2D3 is suppressed by
24R,25(OH)2D3 (Nemere, 1999). Paracellular movement
is passive, independent of vitamin D status, and
exists all along the small intestine (Pansu et al., 1983).
Two models which describe the mechanism of
transcellular absorption are the calbindin-based diffusional-
active transport model and the vesicular
transport model (Wasserman & Fullmer, 1995).
The calbindin-based diffusional-active transport
model
This transcellular pathway is a complex process involving
three steps: (1) entry by movement of Ca2+
from lumen through the brush-border membrane
of the enterocyte, (2) intracellular diffusion, and (3)
extrusion from the cell across the basolateral membrane.
The major action of vitamin D in regulating
this process is on the steps involved in Ca2+ movement
beyond brush-border entry (Roche et al., 1986; Schedl
et al., 1994). The concentration of cytosolic Ca2+ within
the enterocyte is closely controlled at about 10–7 M,
and this is ultimately a function of the rate of entry
and the rate of exit of Ca2+ across the cell boundaries.
Intracellular organelles, including mitochondria, microsomes
and lysosomes, play a major role in controlling
cytosolic Ca2+ by storing and releasing the cation
as appropriate. The overall rate of transcellular movement
is determined by the intracellular diffusion,
which is the rate-limiting step (Bronner, 1990).
One of the most striking effects of vitamin D is
the induction of calbindin-D9k, which is distributed
throughout the cytoplasm of the enterocyte. Absence
of intestinal calbindin-D9k may be considered a
molecular index of vitamin D defi ciency (Bronner,
1991).
Entry
This step involves the movement of luminal Ca2+
across the brush-border membrane of the enterocyte
into the cytosol. The downhill electrochemical gradient
permits the entry of luminal Ca2+ without the
input of metabolic energy.
Fullmer (1992) reviewed data from several laboratories
that suggested a ‘liponomic regulation’ of intestinal
calcium transport by 1α,25(OH)2D3. In this model,
1α,25(OH)2D3 alters the phospholipid structure of the
brush-border membrane, causing an increase in membrane
fl uidity, which, in turn, leads to a specifi c increase
in the permeability of the membrane to Ca2+. Among
these reports, 1α,25(OH)2D3 enhanced the synthesis of
phosphatidylcholine and also increased the incorporation
of unsaturated fatty acids into phosphatidylcholine
in chick duodenal enterocytes (Matsumoto et al.,
1981). Incorporation of methyl cis-vaccinic acid (a
fatty acid known to increase membrane fl uidity) into
brush-border membrane vesicles from vitamin D-defi
cient chickens caused an increase in rate of vesicular
calcium uptake, but there was no such effect in vesicles
from 1α,25(OH)2D3-treated chickens. Conversely,
methyl trans-vaccinic acid (a fatty acid known to decrease
membrane fl uidity) caused a decrease in rate of
calcium uptake in vesicles from 1α,25(OH)2D3-treated
chickens, but no change in vesicles from vitamin Ddefi
cient chickens (Fontaine et al., 1981). Brasitus et
al. (1986) demonstrated that the fl uidity of the brushborder
membrane from vitamin D-deprived rats was
lower than that of vitamin D-replete control animals.
Treatment with 1α,25(OH)2D3 restored fl uidity to control
levels within 1–2 hours. The changes in membrane
fl uidity were associated with appropriate changes in
lipid composition, and preceded detectable increases
in calcium absorption (demonstrable only during the
5th hour). The lack of temporal correspondence indicates
that the early changes in membrane composition
attributable to 1α,25(OH)2D3 are probably not a major
factor in calcium absorption. The same conclusion was
reached by Schedl et al. (1994), who found no signifi -
cant effect of vitamin D on saturable or nonsaturable
uptake of calcium.
The observation that Ca2+ uptake at the brush border
has a saturable component suggests an interaction
with a low-affi nity binding site, which might be associated
with a calcium channel or a carrier of some sort.
The results of in vitro and in vivo studies using calcium
channel blocking drugs suggest that voltage-activated
calcium channels, such as those found in excitable
tissues (nerve, muscle) are unlikely to be present in
intestinal brush borders (Favus & Tembe, 1992). This
does not exclude the possibility that the brush-border
membrane may contain calcium channels with properties
distinct from those found in nerve and muscle,
although no such channels have as yet been identifi ed
in this membrane.

Gene regulation

2007-06-29T06:16:00.000-07:00

The ligand-activated VDR may function to recruit
coactivators that remodel chromatin structure and
permit greater accessibility of the transcriptional machinery
to DNA. Coactivators shown to interact with
liganded VDR include mouse SUG1 (vom Baur et al.,
1996), RAC3 (Li et al., 1997), NCoA-62 (Baudino et
al., 1998; MacDonald et al., 2001) and a multiprotein
complex known as DRIP (Rachez et al., 1998, 1999).
There have been no reports as yet of co-repressor proteins
which bind to the VDR, so it is presently unclear
whether the VDR interacts with co-repressors to bring
about down-regulation of target genes. At least one
co-repressor, N-CoR, fails to interact with the VDR
(Hörlein et al., 1995). Determination of whether vitamin
D hormone action is stimulatory or inhibitory
may be tissue-specifi c or dependent on the state of
cellular differentiation. Most vitamin D-responsive
genes are up-regulated by 1α,25(OH)2D3, the human
PTH gene being one of the few that is down-regulated
(see Table 8.2).
Repression of vitamin D-induced transactivation
of the osteocalcin gene by YY1
Overlapping the recognition sequences for VDR–RXR
within the VDRE of the osteocalcin gene are two binding
motifs for the multifunctional transcription factor
YY1. This protein can either activate, repress or initiate
gene transcription (Shrivastava & Calame, 1994). In
the context of the osteocalcin promoter, YY1 represses
1α,25(OH)2D3-induced transcription by competing
with VDR–RXR for binding at the VDRE. YYI also interacts
directly with TFIIB thereby interfering with the
transactivation function of the VDR (Guo et al., 1997).
This repressive function of YY1 would prevent the precocious
induction of osteocalcin gene transcription by
VDR-mediated mechanisms under physiological conditions
in which the gene should not be expressed.
Suppression of osteocalcin gene expression
during osteoblast proliferation
The target cell for the hormonal action of vitamin
D on bone is the osteoblast. Within this cell,
1α,25(OH)2D3 enhances osteocalcin gene expression
at the three principal levels – transcription, mRNA
accumulation and protein synthesis. The enhanced
transcription is dependent upon basal levels of gene
expression (Owen et al., 1991), suggesting that there
is a coordinate transactivation involving the contribu-
Vitamin D 203
tion of activities at the VDRE and basal elements. Two
essential basal elements in the proximal promoter are
the TATA box and the osteocalcin box (OC box), a 24-
nucleotide sequence that contains a central CCAAT
motif (Lian & Stein, 1992). The VDRE is located further
upstream between nucleotides –512 and –485 in
the human osteocalcin gene promoter (Ozono et al.,
1990).
The sequential expression of vitamin D-dependent
genes associated with bone tissue development
has been studied in cultures of normal diploid rat
osteoblasts. The fi rst 10 to 12 days constitute the cell
proliferation period, characterized by the expression
of genes encoding AP-1 proteins (Jun and Fos) and
extracellular matrix proteins. The jun and fos genes
are transcribed in response to growth factors and
other cell-surface stimuli, and the protein products
are responsible for converting the transient stimuli
into a long-term transcriptional response. During the
following stages, the proliferative activity declines and
the genes encoding the AP-1 proteins and extracellular
matrix proteins are gradually down-regulated.
During the next stage of matrix maturation (days
12 through 18), alkaline phosphatase mRNA and
enzyme activity increases more than ten-fold to peak
levels, and matrix Gla protein is expressed maximally.
Days 16 through 20 are characterized by the progressive
mineralization of the extracellular matrix. As the
cellular levels of alkaline phosphatase mRNA decline,
the accumulation of calcium in the matrix increases
coordinately with the up-regulated expression of
genes encoding the calcium-binding proteins, osteocalcin
and osteopontin (Stein & Lian, 1993).
The modular organization of the human osteocalcin
gene promoter explains how gene expression
might be suppressed during cell proliferation. Overlapping
the VDRE and also within the OC box are
AP-1 binding sites which bind Jun–Fos heterodimers
and Jun homodimers. During the proliferation
of osteoblasts, the Jun and Fos proteins are present
at high levels. Occupancy of the AP-1 binding site
overlapping the VDRE by Jun and Fos dimers blocks
the binding of liganded VDR to its specifi c site and
prevents transcription of the osteocalcin gene. At the
end of the proliferation period, the levels of Fos and
Jun decline, allowing the VDR to bind to the VDRE
and the osteocalcin gene to be expressed. Thus the
competition of the VDR and the AP-1 proteins for
binding to the composite DNA element determines
the activation or suppression of the osteocalcin gene
(Lian & Stein, 1992). High levels of Jun and Fos also
suppress basal transcription of the osteocalcin gene
(Schüle et al., 1990).
Inhibitory effect of the VDR on transactivation of
the growth hormone gene by thyroid hormone
and retinoic acid
The rat growth hormone gene, located exclusively in
the somatotropic pituitary cells, contains a hormone
response element that functions both as a retinoic
acid response element (RARE) and a thyroid hormone
response element (TRE) (García-Villalba et al.,
1993). This allows thyroid hormone and retinoic acid
to interact co-operatively to stimulate transcription
of the growth hormone gene in pituitary cells (Bedo
et al., 1989). García-Villalba et al. (1996) reported that
incubation of rat pituitary cells with nanomolar concentrations
of vitamin D3 inhibits thyroid hormone
and retinoic acid transactivation of the growth hormone
gene by interference on the common response
element. The results suggested that the liganded VDR
can directly affect the pituitary response to other nuclear
receptors, thereby contributing to the growth
arrest that occurs in hypervitaminosis D. This negative
effect on the growth hormone gene is apparently
paradoxical, since a defi ciency of vitamin D produces
a rachitic state associated with a defect in growth.
Repression of VDR-mediated transcription of the
osteocalcin and osteopontin genes by the thyroid
hormone receptor
Thompson et al. (1999) demonstrated two distinct
repressive actions of the TR on VDR-mediated
transcription of the rat osteocalcin and mouse osteopontin
genes: (1) a thyroid hormone-independent
action, perhaps due to TR–RXR out-competing
VDR–RXR for binding to the VDREs and (2) a
thyroid hormone-dependent repression, likely by diversion
of limiting RXR from VDR–RXR toward the
formation of TR–RXR heterodimers. The reverse of
this phenomenon was indicated by a relatively weak
binding of VDR–RXR to the TRE of the myosin heavy
chain gene and modest repression by liganded VDR
of thyroid hormone-mediated transactivation. This
reciprocal inhibition of transactivation by VDR and
TR is permitted by the half-site homology between
the rat osteocalcin VDRE and the rat myosin heavy
chain TRE. The formation of a TR–VDR heterodimer
204 Vitamins: their role in the human body
reported by Schräder et al. (1994) was not observed
by Thompson et al. (1999) who concluded that this
heterodimer is not biochemically or physiologically
relevant. As far as is known, the only relevant heterodimerization
among nuclear receptors (excluding
RXR) is between the type I mineralocorticoid and
glucocorticoid receptors.
8.5.3 Nongenomic actions of 1α,25-
dihydroxyvitamin D3 and 24R ,25-
dihydroxyvitamin D3
Background information can be found in Sections
3.7.6 and 3.7.9.
A large number of responses occur within seconds
to minutes following addition of vitamin D metabolites
to the in vitro system – too rapid to involve
changes in gene expression controlled by the VDR.
Moreover, such responses are not blocked by inhibitors
of transcription (actinomycin D) or protein synthesis
(cycloheximide). For these reasons, the rapid
responses are described as nongenomic.
1α,25-Dihydroxyvitamin D3
Rapid nongenomic responses to 1α,25(OH)2D3 have
been observed in many cell types, including enterocytes,
colonocytes, chondrocytes, osteoblasts, hepatocytes,
skeletal and cardiac muscle cells, keratinocytes,
mammary gland epithelial cells, parathyroid cells
and pituitary cells (Norman, 1998). The responses
have been most clearly delineated in osteoblast-like
osteosarcoma cells. At a molecular level, these include
effects on membrane phospholipid metabolism
(Grosse et al., 1993), activation of voltage-sensitive
calcium channels (Caffrey & Farach-Carson, 1989)
and elevation of cytosolic (Baran et al., 1991) and nuclear
(Sorensen et al., 1993) Ca2+ concentrations.
A number of nongenomic effects of 1α,25(OH)2D3
are concerned with the regulation of intracellular
calcium, which is an important second messenger
involved in the activation of many target enzymes.
Additional effects include stimulation of prostaglandin
production (Boyan et al., 1994), increased
intra cellular pH (Jenis et al., 1993) and increased
membrane fl uidity (Brasitus et al., 1986).
Regulation of intracellular calcium
Transient increases in intracellular calcium can be
initiated in two major ways. (1) Extracellular calcium
can enter the cell down its electrochemical gradient
by the opening of voltage-gated calcium channels. (2)
Calcium can be released from intracellular storage
sites associated with mitochondria and the endoplasmic
reticulum.
The presence of L-type (dihydropyridine-sensitive)
voltage-activated calcium channels has been
demonstrated in basolateral membranes of rabbit
ileal enterocytes (Homaidan et al., 1989) and in osteosarcoma
cells (Guggino et al., 1989). The opening
of such channels is the fastest known response of
osteoblasts to treatment with nanomolar concentrations
of 1α,25(OH)2D3. The hormone enhances the
activity of voltage-gated channels in rat pituitary cells
(Tornquist & Tashjian, 1989) and rat osteosarcoma
cells (Caffrey & Farach-Carson, 1989).
The binding of 1α,25(OH)2D3 to a membrane
receptor in a wide range of cell types stimulates the
activity of phospholipase C, whose hydrolytic action
on membrane phosphoinositides results in the generation
of the two second messengers, diacylglycerol
and inositol triphosphate (see Fig. 3.31). Cell types
examined include osteosarcoma cells (Civitelli et al.,
1990), myoblasts (Morelli et al., 1993), enterocytes
(Lieberherr et al., 1989), colonocytes (Wali et al.,
1990), hepatocytes (Baran et al., 1988), parathyroid
cells (Bourdeau et al., 1990) and keratinocytes (Mac-
Laughlin et al., 1990). Diacylglycerol is an activator
of PKC, which is involved in a myriad of cellular
processes. Inositol triphosphate releases Ca2+ from
the intracellular storage sites, thereby increasing the
concentration of Ca2+ in the cytosol.
A rapid increase in calcium translocation, termed
transcaltachia, has been described using the perfused
chick duodenal loop (Nemere et al., 1984). The
involvement of 1α,25(OH)2D3 in the activation of
voltage-gated calcium channels appears to be an early
effect in transcaltachia. Introduction of a calcium
channel antagonist completely abolished the movement
of calcium, while a calcium channel agonist
mimicked the stimulatory response to 1α,25(OH)2D3
(de Boland et al., 1990). de Boland & Norman (1990)
provided evidence for the involvement of PKA and
PKC in transcaltachia. Forskolin and phorbol ester,
activators of PKA and PKC, respectively, stimulated
transcaltachia analogously to 1α,25(OH)2D3. In addition,
the transcaltachial response to 1α,25(OH)2D3
was respectively suppressed or abolished by inhibitors
of PKA and PKC. Collectively, these observations sug-
Vitamin D 205
gest that 1α,25(OH)2D3 binds to a membrane receptor
located in the basolateral membrane of the enterocyte
and signal transduction via G proteins, effector
proteins and second messengers leads to activation
of PKA and PKC. These kinases might stimulate calcium
infl ux via phosphylation-dependent activation
of voltage-gated calcium channels at the basolateral
membrane.
Evidence for a distinct membrane receptor for
1α,25(OH)2D3
There is strong evidence that the nongenomic responses
to 1α,25(OH)2D3 are mediated by a membrane
receptor that is biochemically different from the
nuclear VDR. Ligand specifi city for the rapid actions
is different from that for genomic responses (Farach-
Carson et al., 1991). Using osteoblasts from mice in
which the VDR gene had been genetically ablated,
Wali et al. (2003) showed that the 1α,25(OH)2D3-
induced rapid increases in intracellular calcium and
PKC activity are neither mediated, nor dependent
upon, a functional VDR.
A 66-kDa protein that binds vitamin D analogues
has been isolated from basolateral membranes of
chick intestinal epithelium (Nemere et al., 1994) and
from both plasma membranes and matrix vesicles
of rat chondrocytes (Nemere et al., 1998). Antibody
(Ab99) generated to a [3H]1α,25(OH)2D3 binding
protein isolated from the basolateral membrane of
chick intestinal epithelium blocked the rapid activation
of PKC by 1α,25(OH)2D3 in chondrocytes
(Nemere et al., 1998) and enterocytes (Nemere et al.,
2000). Slater et al. (1995) discovered that physiological
concentrations of 1α,25(OH)2D3 can directly activate
PKC in artifi cial membranes. This suggests that the
PKC protein itself can act as a membrane-associated
receptor for 1α,25(OH)2D3, providing an additional
signal transduction pathway to the well- established
route in which PKC is activated by diacylglycerol.
Baran et al. (2000) reported that annexin II, a 36-kDa
protein, can serve as a cell-surface receptor mediating
1α,25(OH)2D3-induced increase in intracellular
calcium in osteoblast-like ROS 24/1 cells that lack
the functional nuclear VDR. The rapid effects of
1α,25(OH)2D3 to increase intracellular calcium were
not observed in cells pre-treated with anti-annexin
II antibodies. It is likely that several membrane receptors
mediate the rapid actions of 1α,25(OH)2D3
(Brown et al., 1999).
Modulation of the genomic actions of
1α,25(OH)2D3 by nongenomic mechanisms
1β,25-Dihydroxyvitamin D3 does not interact with
the nuclear VDR and thus has no effect on basal gene
transcription. It does, however, inhibit nongenomic
effects by competing with 1α,25(OH)2D3 for the
membrane receptor. Baran et al. (1992) demonstrated
that, in osteosarcoma cells, the 1β epimer inhibits the
1α,25(OH)2D3-induced rapid rise in intracellular
calcium and accompanying increase in osteocalcin
gene transcription. The inhibition of transcription
occurred without interfering with the binding of the
1α,25(OH)2D3–VDR complex to the VDRE. Since
1β,25(OH)2D3 has no genomic effects on osteocalcin,
yet can block the transcription that accompanies the
nongenomic effect of 1α,25(OH)2D3, this study suggests
that the nongenomic effects of 1α,25(OH)2D3
can modulate the genomic actions of this hormone
in some way.
Khoury et al. (1994) showed that the 1α,25(OH)2D3-
induced transcription of osteocalcin and osteopontin
genes in osteosarcoma cells is independent of Ca2+
infl ux, suggesting that the nongenomic stimulation
of calcium channels by 1α,25(OH)2D3 is not required
for target gene activation. Jenis et al. (1993) discovered
that the nongenomic 1α,25(OH)2D3-induced
increase in intracellular pH is necessary for osteocalcin
and osteopontin expression in the osteoblast. The
effect upon pH appears to be regulated by the Na+/H+
antiporter, since the incubation of cells in a Na+-free
medium eliminated the pH effect and blocked the
hormone-induced increase in osteocalcin and osteopontin
mRNA steady-state levels.
The genomic response may be modulated by second
messengers generated at both the plasma and
nuclear membranes in response to the binding of
1α,25(OH)2D3 to membrane receptors (Baran &
Sorensen, 1994). 1α,25-Dihydroxyvitamin D3 increases
PKC activity through stimulation of plasma
membrane phosphoinositide and formation of diacylglycerol
(Wali et al., 1990). In renal epithelial cells,
1α,25(OH)2D3 specifi cally induces translocation of
PKCβ (but not PKCα) from the plasma membrane
to the nuclear membrane, an event which enhances
phosphorylation of nuclear proteins (Simboli-
Campbell et al., 1994). Phosphorylation of the nuclear
VDR by PKC has been shown to down-regulate
transcription (Hsieh et al., 1993). Sorensen & Baran
(1993) reported that 1α,25(OH)2D3 rapidly enhances
206 Vitamins: their role in the human body
phospholipase C activity in the nuclear membrane of
osteosarcoma cells, resulting in an increased level of
inositol triphosphate, which in turn releases calcium
from intranuclear storage sites.
24R,25-Dihydroxyvitamin D3
The importance of 24R,25(OH)2D3 in endochondral
bone formation is indicated by its accumulation in
growth plate cartilage when normal rats are injected
with [3H]25(OH)D3 (Seo et al., 1996).
Effect on calcium current in osteosarcoma cells
Li et al. (1996) demonstrated a dual nongenomic effect
of 24R,25(OH)2D3 on the L-type calcium channel
current in osteosarcoma cells. At a low physiological
concentration (1 × 10–8 M), 24R,25(OH)2D3 activated
the PKA signal pathway leading to an increase
in current amplitude, whereas a higher concentration
(1 × 10–5 M) reduced the current amplitude via the
PKC signal pathway. In comparison, a high concentration
of 1α,25(OH)2D3 (1 × 10–6 M) increased the
current amplitude.

Molecular action of the vitamin D hormones

2007-06-29T06:15:00.000-07:00

Introduction
1α,25-Dihydroxyvitamin D3, also known as calcitriol,
exerts its effects in cells by both genomic and
non genomic mechanisms. These involve long-term
modulation of gene expression and short-term activation
of intracellular signalling pathways, respectively.
The genomic actions are mediated by the vitamin
D receptor (VDR) which, on binding the hormone,
interacts with the DNA to induce or inhibit new protein
synthesis. Nongenomic actions are mediated by a
membrane receptor that is distinct from the VDR. The
binding of 1α,25(OH)2D3 to the membrane receptor triggers signal transduction pathways which involve
second messengers and which modulate the genomic
actions of the hormone.
Historically, 24R,25(OH)2D3 has been considered
by many to possess little or no intrinsic activity, its
formation serving only to divert the metabolism of
25(OH)D3 away from 1α,25(OH)2D3. However, several
reports indicate that 24R,25(OH)2D3 is a functionally
independent hormone and plays a crucial
role in intramembranous and endochondral bone
formation and in the repair of bone fractures. The genomic action of 1α,25(OH)2D3 is mediated
by the VDR, which functions as a ligand-activated
transcription factor in the cells of target tissues. The
sequence of events involved in the control of gene transcription
by the VDR is (1) binding of 1α,25(OH)2D3
to the VDR in the cytosol, (2) translocation of the
hormone–receptor complex to the nucleus, (3)
binding of VDR–RXR heterodimers (RXR, retinoid
X receptor) or, less commonly, VDR homodimers
to the vitamin D response element (VDRE) in the
promoter of primary vitamin D-responding genes
and (4) recruitment of other nuclear proteins into the
transcriptional pre-initiation complex. The VDRE
functions as a transcriptional enhancer.
One must always bear in mind that an increase in
the level of mRNA is not necessarily due to increased
transcription: an increased mRNA stability will also
lead to its accumulation.
Expression and regulation of the VDR
The VDR is a protein of 53 kDa which selectively
binds 1α,25(OH)2D3 with high affi nity. The protein
is a type II member of the nuclear hormone receptor
superfamily and possesses the characteristic two zinc
fi nger motifs in the DNA binding site (see Section
6.8.5). The VDR is present in most tissues that have
been examined, including activated immune cells
such as T lymphocytes, where it plays a role in modulating
the levels of cytokines such as interleukin-2.
Bone, kidney and especially small intestine have high
levels of receptor compared to other tissues. There are
no isoforms of the VDR. The level of VDR expression
in a cell determines the magnitude of the response
evoked by 1α,25(OH)2D3. Within each target tissue,
the level of VDR is not fi xed but rather it is dynamically
regulated by multiple factors. These include
1α,25(OH)2D3, which up-regulates the amount of
receptor (homologous regulation), and other hormones
and growth factors which may cause up- or
down-regulation of receptor abundance (heterologous
regulation). The mechanisms underlying the
regulation of VDR abundance include alterations in
the rate of transcription of the VDR gene, the stability
of the VDR mRNA and post-translational events. The
protein kinase A (PKA) and protein kinase C (PKC)
signal transduction pathways interact at the level of
VDR regulation. Activation of the PKA pathway by
forskolin up-regulates VDR gene expression whereas
activation of the PKC pathway by phorbol ester downregulates
VDR gene expression (Krishnan & Feldman,
1991, 1992). There are profound tissue- and cell-specifi
c variations in VDR regulation.
Post-translational modifi cation of the VDR by
phosphorylation
The binding of 1α,25(OH)2D3 to the VDR results in
a substantial increase in phosphorylation of the receptor
(Brown & DeLuca, 1990). Two sites of serine
(Ser) phosphorylation on the human VDR have been
identifi ed, each with a different function. Ser-51 in the
zinc fi nger region is phosphorylated by PKC (Hsieh et
al., 1991), a post-translational modifi cation that inhibits
the receptor’s ability to interact with the VDRE
(Hsieh et al., 1993). Phosphorylation of Ser-208 in the
ligand-binding domain by casein kinase II enhances
the transcriptional activating capacity of the receptor,
with no effect on receptor–ligand binding, receptor
partitioning into the nucleus or association of receptor
with a VDRE (Jurutka et al., 1996). Replacement of
Ser-51 or Ser-208 with amino acids that are incapable
of being phosphorylated does not affect DNA binding
or attenuate 1α,25(OH)2D3-mediated transcriptional
activation. This demonstrates that phosphorylation of
the two serine residues is not an obligatory switch for
VDR function; rather, these modifi cations represent
both positive (casein kinase II) and negative (PKC)
modulatory mechanisms that apparently govern
receptor activity under appropriate cellular conditions.
Jurutka et al. (1996) envisaged two populations
of liganded VDR: one that is hypophosphorylated at
Ser-208, yet still active in transcriptional enhancement,
and a superactive, hyperphosphorylated form
that is even more effective at co-operatively recruiting
coactivators and/or basal transcription factors.
Desai et al. (1995) used staurosporine, an inhibitor
of PKC and related protein kinases, and okadaic
acid, an inhibitor of protein phosphatases, to investigate
the contribution of VDR phosphorylation/
dephosphorylation to vitamin D-stimulated transcription
of the rat osteocalcin gene. The results
suggested the presence of at least two functionally
distinct phosphorylation sites on the VDR. At one site,
staurosporine inhibits a phosphorylation event that is
specifi cally required for the intrinsic transactivation
function of VDR–RXR heterodimers. At the other
site, okadaic acid inhibits a post-translational dephosphorylation
event that is required for VDRE binding
of VDR-containing transcription factor complexes.
200 Vitamins: their role in the human body
Nuclear localization of the liganded VDR
The unoccupied VDR exists in equilibrium between
the cytosolic and nuclear compartments of the target
cell. Receptor–hormone binding shifts this equilibrium
to favour nuclear localization (Walters et
al., 1986). The hormone–receptor complex rapidly
translocates to the nucleus along microtubules facilitated
by specialized motor proteins. Disruption of
microtubular integrity impairs the genomic response
to 1α,25-dihydroxyvitamin D3 in human monocytes,
which clearly underlines the physiological importance
of the intracellular tubulin transport system
(Kamimura et al., 1995).
Role of ligands in VDR–RXR heterodimer binding
to DNA
Thompson et al. (1998) investigated the molecular
function of 1α,25(OH)2D3 and 9-cis retinoic acid ligands
in the binding of the VDR and RXR to mouse osteopontin
and rat osteocalcin VDREs. Effi cient binding
to either VDRE occurred as a VDR–RXR heterodimer,
not as a VDR homodimer. 1α,25-Dihydroxyvitamin
D3 dramatically enhanced heterodimer–VDRE interaction,
whereas somewhat higher concentrations of
9-cis retinoic acid inhibited this association. A possible
explanation for this inhibition is that the binding
of 9-cis retinoic acid to the RXR partner destabilizes
the heterodimer and induces RXR homodimer formation.
MacDonald et al. (1993) offered this explanation
when they demonstrated that 9-cis retinoic acid
repressed vitamin D3-induced transactivation of the
osteocalcin gene. Thus liganded RXR is diverted from
vitamin D-activated transcription toward expression
of vitamin A-dependent genes.
Thompson et al. (1998) showed that the transcriptional
response to hormone depends on the sequential
order in which the components assemble. They
proposed the existence of two alternative allosteric
pathways for VDR–RXR association and response to
ligand. (1) An unliganded VDR associates with RXR
to form an apo-heterodimer in solution. Subsequent
binding of 1α,25(OH)2D3 induces a conformational
change in the heterodimer, which results in enhanced
binding to the VDRE. The RXR partner can readily be
dissociated from the DNA-bound heterodimer by the
addition of 9-cis retinoic acid, leading to the formation
of RXR homodimers that mediate retinoid-responsive
pathways. This 9-cis retinoic acid-receptive
conformation is proposed to exist also in monomeric
RXR and in the apo-VDR–RXR. (2) When VDR
binds 1α,25(OH)2D3 before heterodimer formation,
it is postulated to acquire a conformation distinct
from that in the fi rst pathway. After heterodimerization,
the liganded VDR allosterically alters the ligandbinding
domain of its RXR partner, rendering the
RXR unable to bind its own ligand and thus making
the heterodimer resistant to 9-cis retinoic acid-elicited
dissociation.
Interaction of the VDR with basal transcription
factor TFIIB
In the formation of the pre-initiation complex on a
gene promoter, the binding of the basal transcription
factor TFIIB to the TATA-binding protein is a requisite
for the recruitment of RNA polymerase II (Section
6.5.3). VDR interacts with TFIIB through a highly
specifi c, ligand-independent, direct protein–protein
contact and enhances transcription in the manner
of an activator (Blanco et al., 1995; MacDonald et al.,
1995).

Historical overview of Vitamin D

2007-06-29T06:14:00.001-07:00

The discovery of vitamin D arose from research into
rickets – a bone disease of infancy and early childhood
that reached epidemic proportions in the industrial
cities of Europe and the United States of America during
the industrial revolution. The benefi cial effect of
sunlight in curing rickets had been recognized in
the early nineteenth century. It was the work of Sir
Edward Mellanby published in 1919 that fi nally led
to the acceptance of rickets as a nutritional disease.
In the same year, Huldschinsky cured four children
with severe rickets by exposing them to the rays of a
mercury quartz lamp, thus demonstrating that UV radiation
from an artifi cial source was equally effective
as solar radiation. Huldschinsky further showed that
the effect was not localized, since exposing one of each
child’s arms to the radiation resulted in the healing of
both arms. Two years later, Powers showed that codliver
oil and UV radiation had similar curative effects
on rachitic rats, thus establishing the dual source for
antirachitic activity – diet and UV radiation.
In 1922 McCollum and associates published the results
of experiments designed to determine whether
the antirachitic factor in cod-liver oil was identical to
or distinct from the previously discovered ‘fat-soluble
vitamin A’. They found that cod-liver oil retained its
antirachitic properties after destruction of the vitamin
A by heating and aeration. Thus, in addition to
vitamin A, cod-liver oil contained a new fat-soluble
vitamin, which McCollum later (1925) called ‘fatsoluble
vitamin D’. Zucker and co-workers in 1922
found that vitamin D was present in the unsaponifi -
able fraction of cod-liver oil, and suggested that it was
closely related to cholesterol.
In 1923, Goldblatt and Soames irradiated rachitic
rats and fed their livers to other rachitic rats which
were not irradiated. The latter rats were cured of their
rickets, thereby demonstrating that exposure to UV
radiation induces the production of an antirachitic
substance in the liver. Later, in a similar experiment,
Hess and Weinstock fed small portions of irradiated
skin to rachitic rats and noted a curative effect. In
1924, Steenbock and Black discovered that rat rations
exposed to UV radiation had the same benefi cial effects
as when rachitic rats were irradiated. A year later,
Hess and Weinstock induced antirachitic activity
by irradiating such foods as milk, butter, bread and
meats. It was further demonstrated that it is the sterols
in foods that are activated and converted to vitamin D.
It was fi nally realized that skin and certain foods contain
a precursor of vitamin D that can be converted to
the active vitamin by exposure to UV irradiation.
Irradiation of ergosterol, a sterol obtained from
yeast, led to the isolation of a photo-product that was
originally designated as vitamin D1. It was later realized
that vitamin D1 was a mixture of substances, which
explains its non-existence as a D vitamer in present
nomenclature. Further purifi cation of the irradiation
mixture by Askew in 1931 yielded a single compound
which was called ergocalciferol or vitamin D2. It was
assumed at the time that the vitamin D produced in
human skin during exposure to UV radiation was vitamin
D2. However, in the following year, Steenbock
noted that rachitic chickens did not respond to irradiated
ergosterol, but did so to irradiated cholesterol
preparations and cod-liver oil. This observation led to
the discovery of 7-dehydrocholesterol as the vitamin
D precursor in the cholesterol preparations and the
isolation of cholecalciferol (vitamin D3) by Windaus
in 1936. Vitamin D3, unlike vitamin D2, had antirachitic
activity in both chicks and rats. It was concluded
that 7-dehydrocholesterol, rather than ergosterol, was
the precursor for vitamin D3 in the skin.
A major breakthrough in our understanding of vitamin
D function arose from the discovery of the biologically
active metabolite, 1α,25-dihydroxyvitamin
D3. This event was initiated by Carlsson who, in 1952,
noted a lag between the time of vitamin D administration
and the appearance of its physiological response,
namely intestinal calcium transport. The discovery of
a metabolite of vitamin D3 in intestinal mucosa chromatin
was published by Norman’s group in 1968, and
its biological signifi cance was reported in 1970 by the
same group. In 1970, Fraser and Kodicek showed that
the kidney was the source of synthesis of the newly
discovered metabolite. The chemical characterization
of 1α,25-dihydroxyvitamin D3 was reported in 1971
simultaneously by three independent laboratories,
those of Norman, Kodicek, and Deluca, all within
a six-week period in February/March of that year.
The major circulating metabolite of vitamin D3, 25-
hydroxyvitamin D3, was identifi ed by DeLuca’s group
in 1968 and subsequently was shown to be produced
primarily in the liver.
In 1969, Norman’s group reported the existence of
the vitamin D receptor in the chromatin fraction of
the intestinal mucosa. The interaction of the receptor
190 Vitamins: their role in the human body
with the transcriptional machinery inside vitamin D
target cells demonstrated that 1α,25-dihydroxyvitamin
D3 has a similar mechanism of action to that of
steroid hormones.

Dietary sources
The vitamin D activity in the human diet is contributed
mainly by vitamin D itself and its immediate
metabolite, 25(OH)D. The proportion of vitamin D
obtained from the diet is normally very small compared
with that synthesized in skin in response to
sunlight. The richest natural sources of vitamin D3
are fi sh-liver oils, especially halibut-liver oil. Fatty fi sh,
such as herring, sardines, pilchards and tuna, are rich
natural food sources; smaller amounts of the vitamin
are found in mammalian liver, eggs and dairy products.
Cereals, vegetables and fruit contain no vitamin
D, whilst meat, poultry and white fi sh contribute insignifi
cant amounts.
Foodstuffs commonly enriched with vitamin D include
margarine, skimmed milk powder, evaporated
milk, milk-based beverages, breakfast cereals, dietetic
products of all kinds, baby foods and soup powders.
8.4 Cutaneous synthesis, intestinal
absorption, transport and metabolism
8.4.1 Overview
Solar radiation converts 7-dehydrocholesterol in the
skin to previtamin D3, which in turn is converted by
CH2
H
HO
H
H3C OH
H3C
OH
CH2
H
HO
H
H3C
H3C
CH2
H
HO
H
H3C OH
H3C
CH2
H
HO
H
H3C OH
H3C
OH
Liver Kidney
Vitamin D3 25-Hydroxyvitamin D3 1α,25-Dihydroxyvitamin D3
Kidney
24R,25-Dihydroxyvitamin D3
Fig. 8.2 Conversion of vitamin D3 to hormonal metabolites.
192 Vitamins: their role in the human body
body heat to vitamin D3. Vitamins D2 and D3 can be
obtained orally from natural and fortifi ed foods, commercial
fi sh-liver oil preparations and vitamin tablets.
Unlike excessive ingestion of vitamin D supplements,
the cutaneous source of vitamin D3 does not result
in toxicity when the skin is overexposed to sunlight.
Vitamin D of both cutaneous and dietary origin is
converted in the liver to 25(OH)D, the major circulating
form of the vitamin. The 25(OH)D is converted
in the kidney to 1α,25(OH)2D, which circulates at low
concentrations and acts in the manner of a steroid
hormone.
8.4.2 The vitamin D-binding protein
In the plasma, 25(OH)D, and indeed all vitamin D
metabolites, are mainly bound to a specifi c glycoprotein,
known as the vitamin D-binding protein (DBP).
Much smaller amounts of circulating vitamin D metabolites
are bound with low affi nity to albumin. DBP
is synthesized principally in the liver and belongs to
the same gene family as albumin. It has a molecular
weight of 58 kDa and contains a single binding site for
vitamin D metabolites. At normal circulating concentrations
of vitamin D metabolites, less than 5% of the
available binding sites on DBP are occupied. The features
of the secosteroid molecule necessary for binding
activity are the three conjugated double bonds and
a hydroxyl group at C-25. The binding affi nities of the
DBP for vitamin D and its metabolites are 25(OH)D3
= 24R,25(OH)2D3 = 25,26(OH)2D3 > 1α,25(OH)2D3
> vitamin D3. The difference in the dissociation constants
for 25(OH)D3 and 1α,25(OH)2D3 is about 10-
fold. In mammals, the metabolites of vitamins D2 and
D3 exhibit the same relative affi nitites for DBP (Brown
et al. 2000).