Tuesday, July 3, 2007

Vitamin C deficiency

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

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
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
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

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
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

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

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

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

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.