Báo cáo khoa học: Vitamin C Biosynthesis, recycling and degradation in mammals doc

REVIEW ARTICLE
Vitamin C
Biosynthesis, recycling and degradation in mammals
Carole L. Linster and Emile Van Schaftingen
Universite
´
Catholique de Louvain, Christian de Duve Institute of Cellular Pathology, Brussels, Belgium
Keywords
ascorbate; dehydroascorbate;
2,3-diketogulonate; glucuronate;
gulonolactonase;
L-gulonolactone oxidase;
semidehydroascorbate; UDP-
glucuronosyltransferases; vitamin C;
xenobiotics
Correspondence
E. Van Schaftingen, Laboratory of
Physiological Chemistry, UCL-ICP, Avenue
Hippocrate 75, B-1200 Brussels, Belgium
Fax: +32 27647598
Tel: +32 27647564
E-mail:
C. L. Linster, The Department of Chemistry
and Biochemistry and the Molecular Biology
Institute, University of California, Los
Angeles, CA 90095-1569, USA
Fax: +1 310 825 1968
Tel: +1 310 825 3137
E-mail:
(Received 12 September 2006, revised
1 November 2006, accepted 21 November

2006)
doi:10.1111/j.1742-4658.2006.05607.x
Vitamin C, a reducing agent and antioxidant, is a cofactor in reactions cata-
lyzed by Cu
+
-dependent monooxygenases and Fe
2+
-dependent dioxygenas-
es. It is synthesized, in vertebrates having this capacity, from d-glucuronate.
The latter is formed through direct hydrolysis of uridine diphosphate
(UDP)-glucuronate by enzyme(s) bound to the endoplasmic reticulum mem-
brane, sharing many properties with, and most likely identical to, UDP-
glucuronosyltransferases. Non-glucuronidable xenobiotics (aminopyrine,
metyrapone, chloretone and others) stimulate the enzymatic hydrolysis of
UDP-glucuronate, accounting for their effect to increase vitamin C forma-
tion in vivo. Glucuronate is converted to l-gulonate by aldehyde reductase,
an enzyme of the aldo-keto reductase superfamily. l-Gulonate is converted
to l-gulonolactone by a lactonase identified as SMP30 or regucalcin, whose
absence in mice leads to vitamin C deficiency. The last step in the pathway
of vitamin C synthesis is the oxidation of l-gulonolactone to l-ascorbic acid
by l-gulonolactone oxidase, an enzyme associated with the endoplasmic ret-
iculum membrane and deficient in man, guinea pig and other species due to
mutations in its gene. Another fate of glucuronate is its conversion to
d-xylulose in a five-step pathway, the pentose pathway, involving identified
oxidoreductases and an unknown decarboxylase. Semidehydroascorbate, a
major oxidation product of vitamin C, is reconverted to ascorbate in the
cytosol by cytochrome b
5
reductase and thioredoxin reductase in reactions
involving NADH and NADPH, respectively. Transmembrane electron

transfer systems using ascorbate or NADH as electron donors serve to
reduce semidehydroascorbate present in neuroendocrine secretory vesicles
and in the extracellular medium. Dehydroascorbate, the fully oxidized form
of vitamin C, is reduced spontaneously by glutathione, as well as enzymati-
cally in reactions using glutathione or NADPH. The degradation of vita-
min C in mammals is initiated by the hydrolysis of dehydroascorbate to
2,3-diketo-l-gulonate, which is spontaneously degraded to oxalate, CO
2
and
l-erythrulose. This is at variance with bacteria such as Escherichia coli,
which have enzymatic degradation pathways for ascorbate and probably
also dehydroascorbate.
Abbreviations
BSO,
L-buthionine-(S,R)-sulfoximine; DHA, dehydroascorbate; 2,3-DKG, 2,3-diketo-L-gulonate; FAD, flavin adenine dinucleotide; GLO,
L-gulonolactone oxidase; GSH, glutathione (reduced form); GST, glutathione S-transferase; GSTO, Omega class glutathione S-transferase;
PDI, protein disulfide isomerase; SDA, semidehydroascorbate; UDP, uridine diphosphate; UGT, UDP-glucuronosyltransferase.
FEBS Journal 274 (2007) 1–22 ª 2006 The Authors Journal compilation ª 2006 FEBS 1
Vitamin C (or l-ascorbic acid; hereafter, ‘ascorbic
acid’ and ‘ascorbate’ will always refer to ‘l-ascorbic
acid’ and ‘l-ascorbate’) is unique among vitamins for
several reasons. It is present in various foods, partic-
ularly of plant origin, in quantities (typically 10–
100 mg ⁄ 100 g [1]) that are several orders of magnitude
higher than those of other vitamins. This is certainly
related to the facts that it is formed from sugars,
which are abundant compounds, and that its biochemi-
cal synthesis is rather simple. Another unique aspect of
ascorbic acid is that it is a vitamin for only a few ver-
tebrate species, those which have lost the capacity to

synthesize it. From a structural point of view, it is also
one of the rare compounds containing a hydroxyl
group that is so acidic as to be completely dissociated
at neutral pH (carbon-3 hydroxyl pK
a
¼ 4.2). This is
related to the fact that ascorbic acid comprises two
conjugated double bonds and that a resonance form
can be written for the deprotonated monoanionic form
(Fig. 1). Resonance forms can also be written for the
form of vitamin C that has lost one electron (Fig. 1),
making the radical semidehydroascorbate (SDA) much
more stable, and thus much less reactive, than most
other free radicals [2]. Vitamin C is therefore able to
play the role of a free-radical scavenger [3], reacting
with highly ‘aggressive’ (oxidizing) species to replace
them by a much less reactive and, moreover, enzymati-
cally recyclable one, SDA. Ascorbate is certainly the
most abundant water-soluble compound acting in
one-electron reactions, and this is most probably
why it plays the role of a cofactor in reactions cata-
lyzed by a number of metal-dependent oxygenases.
The Cu
+
-dependent monooxygenases peptidylglycine
a-amidating monooxygenase and dopamine b-hydroxy-
lase convert two ascorbate molecules to two SDAs per
catalytic cycle [4]. In the case of Fe
2+
⁄ a-ketoglutarate-

dependent dioxygenases (e.g. collagen prolyl and lysyl
hydroxylases, the two hydroxylases involved in carni-
tine biosynthesis [5], the asparaginyl hydroxylase
that modifies hypoxia-inducible factor 1 (HIF-1) [6]),
ascorbate most probably serves to reconvert inactive,
Fe
3+
-containing enzyme (which results from abortive
catalytic cycles) to the active, Fe
2+
-containing form
[5]. Because of these important roles, it is not surpri-
sing that vitamin C deficiency leads to a debilitating
disorder, scurvy, in man and in animals unable to syn-
thesize the vitamin.
Important progress has been made recently in our
understanding of the synthesis and the recycling of
vitamin C, and a novel pathway has been described
for the degradation of vitamin C in bacteria. This
forms the subject of this review. Vitamin C transport,
which has also witnessed important developments
lately, is only briefly alluded to in the following para-
graph, as other recent reviews are available [7–9].
Ascorbate entry into mammalian cells is energy-
dependent, being effected by two distinct Na
+
-depend-
ent cotransporters, SVCT1 and SVCT2, which show
distinct tissue distributions. Interestingly, targeted dele-
tion of the widely distributed SVCT2 is lethal in mice

Fig. 1. The three redox states of vitamin C
(ascorbate, fully reduced form; SDA, mono-
oxidized form; DHA, fully oxidized form),
and stabilization of the ascorbate monoanion
and SDA by electron delocalization. SDA,
semidehydroascorbate; DHA, dehydroascor-
bate.
Vitamin C metabolism and recycling in mammals C. L. Linster and E. Van Schaftingen
2 FEBS Journal 274 (2007) 1–22 ª 2006 The Authors Journal compilation ª 2006 FEBS
[10], further underlining the importance of vitamin C.
Dehydroascorbate (DHA; see Fig. 1) is transported by
glucose transporters, particularly GLUT1, GLUT3
and GLUT4 [9], and is therefore not energetically dri-
ven. However, intracellular DHA is readily converted
to ascorbate (see ‘Recycling of vitamin C’) and this
highly favourable reductive step drives DHA uptake
by the cell. There are also mechanisms allowing the
efflux of ascorbate from cells [7], e.g. from enterocytes
during intestinal absorption and from liver cells, which
in many mammals produce ascorbate, but the molecu-
lar identity of the proteins involved in this process is
not yet firmly established.
Formation of vitamin C in mammals
and other vertebrates
Outline of the pathway
Ascorbate is synthesized by many vertebrates. The
occurrence of ascorbate biosynthesis in sea lamprey
[11] suggests that this trait appeared early in the evolu-
tionary history of fishes (590–500 million years ago),
i.e. prior to terrestrial vertebrate emergence [12]. The

biosynthetic capacity has, however, subsequently been
lost in a number of species, such as teleost fishes, pas-
seriform birds, bats (intriguingly, not only the fruit-
eating ones, but also others, feeding on blood or
insects [13]), guinea pigs, and primates including
humans, for whom ascorbate has thus become a vita-
min. Fish, amphibians and reptiles synthesize ascor-
bate in the kidney, whereas mammals produce it in the
liver [11,14].
Vitamin C is also formed by all plant species studied
so far [15] and yeasts produce d-erythroascorbate, a C
5
analogue of ascorbate [16]. Interestingly, very different
pathways have evolved for vitamin C biosynthesis in
animals, plants and fungi. In animals, d-glucuronate,
derived from UDP-glucuronate, is reduced to l-gulo-
nate, which leads to inversion of the numbering of the
carbon chain (‘inversion of configuration’) since the
aldehyde function of d-glucuronate (C1) becomes a
hydroxymethyl group in the resulting l-gulonate
(Fig. 2; see [16] for a review of the early literature). The
latter is converted to its lactone, which is oxidized to
l-ascorbate by l-gulonolactone oxidase (GLO). In
plants, the pathway starts with GDP-d-mannose, which
is converted (without change in carbon numbering) to
l-galactonolactone, the substrate for the plant homo-
logue of GLO, l-galactonolactone dehydrogenase [15].
The synthesis of d-erythroascorbate in yeasts proceeds
from d-arabinose [16], but the mechanisms of forma-
tion of the latter have not been elucidated.

Effect of xenobiotics on vitamin C formation
The regulation of vitamin C formation by xenobiotics
is described here, because it helps to understand the
mechanism of d-glucuronate formation. Other aspects
of this regulation are described in a separate section.
It was already observed in the 1940s that administra-
tion of a series of xenobiotics to animals was followed
by enhanced urinary excretion of ascorbate. The stimu-
latory effect is shared by a wide variety of structurally
unrelated substances such as barbiturates, paraldehyde,
chloretone, aminopyrine, antipyrine, 3-methylcholanth-
rene, polychlorinated biphenyls (PCB) and 1,1,1-tri-
chloro-2,2-bis(p-chlorophenyl)ethane (DDT) [17–19].
Turnover rate studies using radiolabelled ascorbate
indicated that the amount of ascorbate synthesized per
day was four- to eight-fold higher in chloretone- or
pentobarbital-treated rats than in untreated animals
[20]. Furthermore, chloretone and barbital were shown
to greatly stimulate the incorporation of radiolabelled
glucose into urinary glucuronate and ascorbate [21]. As
barbital was found to be neither metabolized nor
conjugated, but excreted unchanged in urine, its stimu-
latory effect on urinary glucuronate and ascorbate
excretion was proposed to be unrelated to any detoxifi-
cation mechanism. This view was further supported
by the observation that compounds such as borneol,
a-naphthol and phenolphthalein, known to be primar-
ily excreted as glucuronides, had essentially no effect
on ascorbate excretion [21]. Furthermore, the findings
that the in vivo conversion of both d-glucose and

d-galactose to glucuronate and ascorbate was increased
by xenobiotics [22], but that this was not the case for
the conversion of radiolabelled d-glucuronolactone
or l-gulonolactone to ascorbate [23], suggested that
stimulation occurs at a step between UDP-glucose
and d-glucuronolactone in the ascorbate biosynthesis
pathway.
Many of the following investigations on this subject
studied the effect of agents stimulating vitamin C for-
mation on the activity levels of several enzymes
potentially implicated in ascorbate synthesis. UDP-glu-
cose dehydrogenase and UDP-glucuronosyltransferases
(UGTs) were found to be induced by some agents,
although not by all of them [18,24]. A study using
Gunn rats [25] provided highly suggestive evidence for
the involvement of UGTs in the formation of vita-
min C. Gunn rats are deficient in UGT isoforms of the
UGT1A family, but not of the UGT2 family [26,27].
3-Methylcholanthrene, an inducer of UGTs of the
UGT1A family, increased urinary excretion of ascor-
bate in normal rats (five-fold) and heterozygous Gunn
rats (two-fold), but not in homozygous Gunn rats.
C. L. Linster and E. Van Schaftingen Vitamin C metabolism and recycling in mammals
FEBS Journal 274 (2007) 1–22 ª 2006 The Authors Journal compilation ª 2006 FEBS 3
However, treatment with phenobarbital (an inducer of
isoforms of the UGT2 family) increased the urinary
excretion of ascorbate in normal and homozygous
Gunn rats. Taken together, these results indicate
that UGT isoforms of the UGT1A, but probably also
of the UGT2 family are involved in ascorbate bio-

synthesis, possibly by forming a glucuronidated inter-
mediate that would be hydrolyzed by microsomal
b-glucuronidase or, as suggested below, by catalyzing
the hydrolysis of UDP-glucuronate to UDP and
Fig. 2. Vitamin C synthesis pathway and pentose pathway in animals. The reactions are catalyzed by the following enzymes: 1, UDP-glucose
pyrophosphorylase; 2, UDP-glucose dehydrogenase; 3, nucleotide pyrophosphatase; 4, UDP-glucuronosyltransferase; 5, UDP-glucuronidase;
6, phosphatase; 7, b-glucuronidase; 8, glucuronate reductase; 9, gulonolactonase; 10,
L-gulonolactone oxidase; 11, L-gulonate 3-dehydroge-
nase; 12, decarboxylase; 13,
L-xylulose reductase; 14, xylitol dehydrogenase; 15, D-xylulokinase. Three possible mechanisms for glucuronate
formation (a, b and c) are shown (see text). For the sake of clarity, the linear form of glucuronate is represented. SMP30 KO mice, senes-
cence marker protein 30 knockout mice; ODS rats, osteogenic disorder Shionogi rats; od ⁄ od pigs, mutant pigs deficient in
L-gulonolactone
oxidase; GLO KO mice,
L-gulonolactone oxidase knockout mice.
Vitamin C metabolism and recycling in mammals C. L. Linster and E. Van Schaftingen
4 FEBS Journal 274 (2007) 1–22 ª 2006 The Authors Journal compilation ª 2006 FEBS
glucuronate (mechanisms b and c described in the next
subsection).
These effects on enzyme levels require increased gene
transcription and new protein synthesis, which implies
that the effect of xenobiotics on vitamin C formation
is a long-term effect. However, recent work on isolated
hepatocytes demonstrated that the effect of xenobiotics
(e.g. aminopyrine, metyrapone, chloretone) on the for-
mation of vitamin C and of its precursor, free glucuro-
nate, occurs in a matter of minutes [28]. The increase
in free glucuronate formation, which was best observed
in the presence of an inhibitor of the downstream
enzyme, glucuronate reductase, was already apparent

after 5 min and reached up to 15-fold. It was accom-
panied by a decrease in the UDP-glucuronate level,
but little if any change in the concentration of UDP-
glucose, indicating that the effect of xenobiotics on
vitamin C formation consists in a short-term effect
involving an increase of the conversion of UDP-glu-
curonate to glucuronate (Fig. 2) and not an increase in
the concentration of upstream precursors of glucuro-
nate (‘push-effect’). Most of the stimulating agents did
not give rise to detectable amounts of b-glucuronides,
arguing against the involvement of a glucuronidation-
deglucuronidation cycle in the stimulation of ascorbate
formation (see below). It may be interesting to notice
that up to 100 nmol hexose unitsÆmin
)1
Æg
)1
liver are
channelled towards glucuronate formation in the pres-
ence of saturating concentrations of stimulating xeno-
biotics [28].
Formation of glucuronate from UDP-glucuronate
The formation of glucuronate from UDP-glucuronate
could hypothetically involve (a) the cleavage of UDP-
glucuronate to glucuronate 1-phosphate, followed by
dephosphorylation of the latter by a glucuronate-1-
phosphatase [29,30]; (b) the formation of a glucuroni-
dated intermediate, on an exogenous or endogenous
acceptor, followed by its hydrolysis by b-glucuronidase
[31] or esterases (which could hydrolyze acyl-glucuro-

nides); or (c) direct hydrolysis of UDP-glucuronate to
UDP and glucuronate (Fig. 2). As explained below,
recent work performed on liver microsomes supports
the third mechanism.
As a follow-up of the work showing that a series of
nonglucuronidable xenobiotics rapidly stimulate the
formation of glucuronate in isolated hepatocytes [28],
it was found that the same xenobiotics also stimulated
the formation of glucuronate from UDP-glucuronate
in liver cell-free extracts enriched with ATP or in liver
microsomes supplemented with ATP and a heat-stable
cofactor identified as coenzyme A [32]. Quantitatively,
the formation of glucuronate observed under these
conditions accounted for the formation of glucuronate
observed in intact cells, indicating that glucuronate is
formed from UDP-glucuronate by a microsomal
enzyme. This enzyme is most probably present in the
endoplasmic reticulum as, similarly to UGTs, it is sti-
mulated by UDP-N-acetylglucosamine, which enhances
the transport of UDP-glucuronate into vesicles derived
from the endoplasmic reticulum [33].
Although rat liver microsomal preparations hydro-
lyze UDP-glucuronate to glucuronate 1-phosphate
(presumably because they are contaminated with
plasma membrane fragments, which contain a highly
active nucleotide pyrophosphatase [34,35]), their glu-
curonate 1-phosphate phosphatase activity is insuffi-
cient to account for the formation of free glucuronate
by this preparation [32]. Furthermore, the formation
and hydrolysis of glucuronate 1-phosphate are unaffec-

ted by the nonglucuronidable xenobiotics under condi-
tions under which glucuronate formation is stimulated
approximately three-fold. These and other arguments
[32] exclude mechanism (a).
Mechanism (b), which is supported by the observa-
tions made on Gunn rats [25], is ruled out by the fact
that glucuronate formation from UDP-glucuronate
occurs in rat liver microsomes in the absence of UGT
substrates and is actually inhibited by such substrates
(see below). Furthermore, inhibitors of b-glucuronidase
and esterases do not affect the formation of glucuro-
nate from UDP-glucuronate by microsomal prepara-
tions [32], ruling out also the involvement of an
endogenous glucuronide acceptor that would still be
present in washed liver microsomes.
Taken together, these observations lead to the con-
clusion that glucuronate is formed by direct hydro-
lysis of UDP-glucuronate by a UDP-glucuronidase
(mechanism c). The findings that UDP-glucuronidase
is similarly sensitive to various detergents as UGTs
and that it is inhibited by UGT substrates suggest
that it is a side-activity of these transferases (repre-
senting 5% of the transferase activity) [32]. The
identification of UGTs as the UDP-glucuronidase also
allows one to reconcile mechanism (c) with the obser-
vations made on Gunn rats [25]. In the only study
that focuses on the UDP-glucuronate hydrolase activ-
ity of a purified UGT, Hochman and Zakim [36]
found that GT
2P

had a minor UDP-glucuronidase
activity, which could be stimulated by phenylethers
and lysophosphatidylcholines up to 0.03% of the
transferase activity. When transfected into human
embryonic kidney cells, human UGT1A6 displayed a
hydrolase to transferase activity ratio of 0.4% under
certain conditions [32], which is still about one order
C. L. Linster and E. Van Schaftingen Vitamin C metabolism and recycling in mammals
FEBS Journal 274 (2007) 1–22 ª 2006 The Authors Journal compilation ª 2006 FEBS 5
of magnitude lower than the ratio observed in liver
microsomes. It is likely that the ability of UGTs to
hydrolyze UDP-glucuronate varies among isoforms
and depends on the phospholipidic environment. This
last point may explain the observation that UDP-
glucuronidase is inhibited in rat liver microsomes by
the addition of ATP and coenzyme A [32], as the lat-
ter combination of cofactors could allow the reesteri-
fication of lipids in a subcellular fraction known to
contain free fatty acids, acyl-CoA synthetase and
acyltransferases. Further work is obviously needed to
establish which UGT isoforms are involved in the
formation of free glucuronate and the conditions
under which they are able to do so.
Glucuronate reductase (aldehyde reductase)
The reduction of d-glucuronate to l-gulonate is cata-
lyzed by an NADPH-dependent reductase, with broad
specificity, known as aldehyde reductase or TPN
l-hexonate dehydrogenase in the older literature [37]
and now referred to as aldo-keto reductase 1A1
(AKR1A1) for the human enzyme [38]. K

m
values of
0.33 and 0.69 mm were obtained for d-glucuronate
and d-glucuronolactone, respectively, and these com-
pounds are converted by aldehyde reductase to l-gulo-
nate and l-gulonolactone, respectively [37].
Aldehyde reductase belongs to the large group
of monomeric NADPH-dependent oxidoreductases,
known as aldo-keto reductases, which comprise many
members in the human genome, including aldose
reductases (the closest homologues of human aldehyde
reductase, sharing 65% sequence identity [39]) and
hydroxysteroid dehydrogenases [38]. These enzymes
display broad substrate specificities and it would there-
fore not be surprising that, besides aldehyde reductase,
other members of the aldo-keto reductase superfamily
participate in the reduction of d-glucuronate. Aldose
reductase appears to be much less efficient than alde-
hyde reductase in this respect [40]. Furthermore, as it
is barely expressed in liver [41], it is unlikely that it con-
tributes significantly to vitamin C formation. Aldose
reductase inhibitors, which have been developed in
the hope of preventing diabetic complications by
blocking the enhanced formation of sorbitol from glu-
cose in hyperglycaemic states, usually cross-react with
aldehyde reductase [42,43]. Thus, sorbinil, an inhibitor
of both aldehyde reductase and aldose reductase
[42,43], was shown to block the conversion of glucuro-
nate to downstream metabolites (Fig. 2) and inhibit
the formation of vitamin C in isolated rat hepatocytes

[28], supporting the involvement of aldehyde reductase
in ascorbate synthesis.
Urono- and gulonolactonase
As discussed in the next paragraph, the enzyme that
forms vitamin C acts on the lactone form of l-gulonate.
The conversion of d-glucuronate to l-gulonolactone
requires the action of two enzymes, a reductase and a
lactonase, proceeding either via d-glucuronolactone if
the lactonization is the first step, or via l-gulonate if the
first reaction is the reduction. Three different types of
lactonases acting on sugar derivatives have been charac-
terized in mammalian tissues: 6-phosphogluconolacto-
nase, uronolactonase and aldonolactonase. The first one
is an enzyme of the pentose phosphate pathway, which
belongs, in mammals, to the same family of proteins as
glucosamine 6-phosphate isomerase [44] and has no
(direct) role to play in the formation of vitamin C.
Uronolactonase, a microsomal enzyme, hydrolyzes
d-glucurono-3,6-lactone (K
m
 8mm), but is inactive
against aldonolactones [45]. It is a metal-dependent
enzyme, but its sequence is presently unknown.
Aldonolactonase (gulonolactonase) is also a metal-
dependent enzyme, acting best with Mn
2+
, which is pre-
sent in the cytosol and hydrolyzes a number of c- and
d-lactones of a variety of 5-, 6-, and 7-carbon aldonates,
including l-gulono-1,4-lactone and d-glucono-1,5-

lactone [45]. It also catalyzes the lactonization of aldo-
nates, e.g. of l-gulonate, and can therefore participate in
the formation of vitamin C [46]. It has recently been
identified as SMP30 (senescence marker protein 30) [47],
also known as regucalcin, a protein homologous to bac-
terial gluconolactonases [48], and which was initially
thought to regulate liver cell functions related to Ca
2+
[49] and to be possibly involved in senescence because of
its decreased expression with age in liver, kidney and
lung [50,51]. The finding that targeted inactivation of
the SMP30 gene leads to vitamin C deficiency [47]
strongly argues in favour of the involvement of gulono-
lactonase in the ascorbate synthesis pathway in mam-
mals. This conclusion is in line with earlier findings
indicating that l-gulonate rather than d-glucuronolac-
tone is an intermediate in this pathway, such as the fact
that ascorbate is more readily formed from d-glucuro-
nate than from d-glucuronolactone in liver extracts [52],
and with the lower K
m
of glucuronate reductase for
d-glucuronate than for d-glucuronolactone (see above).
L-Gulonolactone oxidase
Characterization of the enzyme and of the catalyzed
reaction
GLO, a microsomal enzyme, catalyzes aerobically the
conversion of l-gulonolactone to l-ascorbate with pro-
duction of H
2

O
2
[53,54]. The immediate oxidation
Vitamin C metabolism and recycling in mammals C. L. Linster and E. Van Schaftingen
6 FEBS Journal 274 (2007) 1–22 ª 2006 The Authors Journal compilation ª 2006 FEBS
product of GLO is 2-keto-l-gulonolactone, an interme-
diate that spontaneously isomerizes to l-ascorbate [54].
The preferred substrate of the enzyme is l-gulono-
1,4-lactone, but it also acts on l-galactono-, d-man-
nono- and d-altrono-1,4-lactone [55]. Other c-lactones,
including l-idono- and d-gluconolactone, were not
oxidized by the enzyme, indicating its configurational
specificity for the hydroxyl group at C2. K
m
values for
l-gulonolactone ranging from 0.007 to 0.15 mm have
been reported [55,56]. The enzyme transfers electrons
not only to O
2
, but also to artificial electron acceptors
such as phenazine methosulfate and ferricyanide,
although not to cytochrome b
5
and cytochrome c [57].
The production of H
2
O
2
is unusual for an enzyme of
the endoplasmic reticulum, and one may wonder if this

membrane-bound oxidoreductase does not transfer its
electrons to another acceptor in intact cells, most par-
ticularly because its plant homologues do so. The
latter share 30% sequence identity with mammalian
GLO and differ from this enzyme in three main
aspects: (1) they act specifically on l-galactono-1,4-lac-
tone [58,59], which is their physiological substrate; (2)
they are bound to the inner mitochondrial membrane
[60]; and (3) they do not transfer electrons directly to
O
2
, but to cytochrome c [59].
GLO is a 50.6 kDa protein [61], which, as indicated
by sequence comparisons, is related to plant l-galacton-
olactone dehydrogenase and fungal d-arabinonolactone
oxidase, and more distantly to 6-hydroxynicotine oxid-
ase, d-2-hydroxyglutarate dehydrogenase and 24-dehy-
drocholesterol reductase, all flavin adenine dinucleotide
(FAD)-linked enzymes. Each monomer of GLO binds
one molecule of FAD, which is covalently linked to a
histidyl residue [55,62]. This residue is presumably
His54, which aligns with the histidine (His72) that cov-
alently links FAD in Arthrobacter nicotinovorans 6-hyd-
roxy-d-nicotine oxidase, as indicated by inspection of
the structure (pdb file 2BVH) of this bacterial enzyme.
The requirement of detergents for the solubilization
of GLO from the microsomal fraction [55–57] strongly
suggests its membrane localization. Accordingly, the
amino acid sequence of the protein contains several
strongly hydrophobic regions, which are thus possibly

associated with the endoplasmic reticulum membrane
[61]. These regions are predicted to form b-sheets rather
than the typical transmembrane helical structure. The
orientation of the catalytic site towards the lumen of
the endoplasmic reticulum is indicated by the intralumi-
nal accumulation of ascorbate and the preferential
intraluminal glutathione oxidation (presumably by
hydrogen peroxide) in rat liver microsomes incubated
with gulonolactone [63]. It should be noted that the
GLO sequence is apparently devoid of targeting motifs
for the endoplasmic reticulum, as indicated by analysis
of the sequence with the TargetP program [64].
Molecular defects in man and other species
Early enzymological studies identified GLO deficiency
as the reason for the inability of some species such as
man and guinea pig to synthesize their own vitamin C
[65]. Man [66] and guinea pig [67] both have a gene
homologous to the rat GLO gene, but they are highly
mutated. Compared with the rat gene, which comprises
12 exons, two coding exons (I and V) are missing in its
guinea pig homologue [67].
Nucleotide sequence alignment of one exon of the
GLO gene from rat with the corresponding exon in the
highly mutated, nonfunctional GLO gene of several
primates revealed that nucleotide substitutions have
occurred at random throughout the primate sequence,
as expected for the exon of a gene that ceased to be
active during evolution and subsequently evolved with-
out functional constraint [68]. From these two exam-
ples, and the finding that ascorbate-deficient species

are also observed in several other lineages, it appears
that inactivation of the GLO gene occurred several
times during evolution, suggesting that the loss of this
gene may be advantageous to some species. It has been
proposed that the formation of hydrogen peroxide by
GLO and the glutathione depletion that ensues are
detrimental [69] and that the selective pressure to keep
the ability of forming vitamin C is lost in species with
ample dietary supply of ascorbic acid.
Rat, pig and mouse models of vitamin C deficiency
are known. The osteogenic disorder Shionogi rat is a
mutant rat of the Wistar strain deficient in GLO activ-
ity and thus unable to synthesize ascorbate. The GLO
cDNA of this mutant was found to contain a single
base mutation leading to a Cys fi Tyr substitution at
position 61 in the amino acid sequence [70]. Overex-
pression experiments in COS-1 cells suggested that this
mutation leads to instability of the mutant GLO pro-
tein and is responsible for the enzymatic defect in the
osteogenic disorder Shionogi rat. The latter manifests
deformity, shortening of the legs, multiple fractures,
osteoporosis, growth retardation and haemorrhagic
tendency when it is fed an ascorbate-deficient diet;
these symptoms are largely prevented by providing
vitamin C in the food [71]. A similar symptomatology
is found in the od⁄ od pig, in which the GLO gene is
inactivated due to an intragenic deletion removing
exon 8 [72].
GLO knockout mice have also been generated
through homologous recombination and the effects of

vitamin C deficiency in these mice have been studied
C. L. Linster and E. Van Schaftingen Vitamin C metabolism and recycling in mammals
FEBS Journal 274 (2007) 1–22 ª 2006 The Authors Journal compilation ª 2006 FEBS 7
[73]. The mutant mice depend on dietary vitamin C
supplementation for survival. The most striking effects
of a low vitamin C diet on the knockout mice were
alterations in their aortic walls (for instance fragmenta-
tion of elastic lamina), which were proposed to be
caused by defects in the crosslinking of collagen and
elastin.
An alternative fate for glucuronate: the
pentose pathway
As described above, d-glucuronate can be metabolized
to ascorbate in most vertebrates, well known excep-
tions being primates and the guinea pig. By contrast,
in all animal species examined, glucuronate can be
converted to the pentose l-xylulose in a pathway
known as the pentose pathway or the glucuronic acid
oxidation pathway [74]. The reactions involved in this
pathway are represented in Fig. 2. The reduction step
catalyzed by glucuronate reductase (see previous sec-
tion) is shared by the ascorbate synthesis pathway and
the pentose pathway.
In the latter, l-gulonate is then oxidized to 3-keto-
l-gulonate by an NAD-dependent dehydrogenase [75].
The cDNA encoding rabbit liver l-gulonate 3-dehy-
drogenase has recently been cloned [76]. The enzyme,
shown to be identical to lens k-crystallin, displays a
K
m

of 0.2 mm for l-gulonate. 3-Keto-l-gulonate is
decarboxylated to l-xylulose by a poorly characterized
decarboxylase [77] whose molecular identity is
unknown. l-Xylulose is then converted to xylitol by an
NADPH-dependent l-xylulose reductase. A cDNA
encoding a dicarbonyl ⁄ l-xylulose reductase has been
cloned from a mouse kidney cDNA library [78]. This
reductase displays a marked preference for NADPH
over NADH and is ubiquitously expressed in several
mammalian species. A K
m
value of 0.21 mm for l-xy-
lulose has been reported for the human recombinant
enzyme.
Xylitol is oxidized to d-xylulose by an NAD-
dependent enzyme identical to sorbitol dehydrogenase
[79]. Finally, d-xylulose can enter the pentose phos-
phate pathway after its phosphorylation by d-xylulo-
kinase. The latter has been purified to homogeneity
from bovine liver and was shown to be a monomeric
enzyme of 51 kDa [80]. The pure enzyme acted on
d-xylulose and d-ribulose with respective K
m
values
of 0.14 and 0.27 mm. A human cDNA encoding a
‘xylulokinase-like’ protein of 528 amino acids has been
isolated [81]. The predicted gene product bears 22%
identity to the xylulokinase of Haemophilus influenzae.
The occurrence of the pentose pathway in humans is
indicated by the fact that rare individuals excrete

abnormal quantities of l-xylulose (1–4 gÆday
)1
) in the
urine. This benign condition, known as essential pen-
tosuria [74], was recognized by Garrod, almost a cen-
tury ago, as an inborn error of metabolism. In 1929,
Margolis [82] noted that ingestion of aminopyrine
leads to a marked increase in pentose excretion in
pentosuric subjects and some years later it was shown
that this effect could be mimicked by the administra-
tion, not only of a series of other drugs, but also of
glucuronic acid [74]. This effect, which is very reminis-
cent of the stimulation exerted by nonglucuronidable
hydrophobic drugs on vitamin C formation in animals
[28], is most probably also due to a stimulation of the
UDP-glucuronidase activity of UGTs.
Pentosuria, an autosomal recessive trait, is due to
l-xylulose reductase deficiency [83]. Lane [84] reported
the separation of a major and a minor isozyme for
l-xylulose reductase in human erythrocytes. In pentos-
uric subjects, only the minor isozyme, which displayed
a K
m
for l-xylulose of 100 mm, was detected upon
electrophoresis and ion-exchange chromatography.
This suggests that homozygosity for the pentosuria
allele results in deficiency of the major isozyme, which
most probably corresponds to the recently cloned
dicarbonyl ⁄ l-xylulose reductase (see above). The gen-
etic defect underlying pentosuria has not yet been

reported.
The benign nature of this condition (the only symp-
tom is the elevated urinary pentose excretion) shows
that the pentose pathway does not play an indispens-
able role in human metabolism. While in most mam-
malian species, this pathway produces a precursor for
the formation of ascorbate (l-gulonate), in humans
and some other species it probably essentially allows
the return of a portion of glucuronate carbon to main-
stream carbohydrate metabolism.
Control of vitamin C synthesis
Outline on the regulation of vitamin C synthesis
The main control is apparently exerted at the level of
the formation of glucuronate from UDP-glucuronate,
as enhancement of this conversion is accompanied by
an increase in the formation of vitamin C [28]. How-
ever, the pathway is branched at the level of l-gulo-
nate and the proportion of l-gulonate that is
converted to vitamin C or l-xylulose must depend on
the relative activities of the rate-limiting enzymes
downstream in the pathways. In the case of vitamin C
formation, the rate-limiting step downstream is cata-
lyzed by l-gulonolactone oxidase, as indicated by the
observation that heterozygous (OD ⁄ od) pigs for GLO
Vitamin C metabolism and recycling in mammals C. L. Linster and E. Van Schaftingen
8 FEBS Journal 274 (2007) 1–22 ª 2006 The Authors Journal compilation ª 2006 FEBS
deficiency have (when fed an ascorbate-free diet) a
plasma ascorbic acid level amounting to 50% of that
found in control (OD ⁄ OD) pigs [72]. A comparison
between the amount of d-glucuronate that accumulates

in isolated rat hepatocytes incubated with various
xenobiotics in the presence of the glucuronate reduc-
tase inhibitor sorbinil (vitamin C formation is then
blocked!) and the amount of ascorbate that is formed
under similar conditions but in the absence of sorbinil
indicates that 30% of l-gulonate is directed towards
ascorbate formation [28].
Effect of xenobiotics
As mentioned above, the stimulatory effect of xenobio-
tics has been ascribed, at least partially, to a short-term
effect on UDP-glucuronidase, i.e. most likely UGTs.
How the various (always nonglucuronidable) xenobio-
tics act is still unknown. Their important structural
diversity and their hydrophobic character suggest that
they could act by perturbing locally the phospholipidic
environment of UGTs and by thus inducing a conform-
ational change favouring a hydrolase activity of these
transferases. Alternatively, these compounds, which are
hydrophobic but lack a suitable glucuronosyl acceptor
function, could stimulate the hydrolase activity through
a pseudosubstrate mechanism. Besides this short-term
action, the effect of some xenobiotics to stimulate the
expression of UGTs [18,24,25] or GLO [85] is also con-
ducive to a stimulated formation of ascorbic acid.
One may wonder what advantage organisms may
derive from the stimulation of vitamin C biosynthesis
caused by nonglucuronidable xenobiotics. There is
no answer at present to this question, but an inter-
esting possibility would be that the stimulatory xeno-
biotics are membrane-perturbing agents which could

favour the generation of reactive oxygen species
when inserted in membranes with active electron
transport, the increased vitamin C availability playing
then a protective role.
Effect of glutathione
From a quantitative point of view, glutathione and
vitamin C are the most abundant reducing agents in
cells. Furthermore, GSH is implicated in vitamin C
recycling from DHA (see next section). It would there-
fore make sense for glutathione to exert a control on
vitamin C synthesis. Several experiments performed
with glutathione-depleting agents indicate that gluta-
thione depletion favours vitamin C synthesis. Adminis-
tration to adult mice of buthionine sulfoximine, an
inhibitor of glutathione synthesis, led to a two-fold
increase in the amount of vitamin C in liver within 4 h
[86]. Similarly, incubation of rat hepatocytes with
1-bromoheptane or phorone, which are conjugated
with GSH, caused a more than two-fold increase in
vitamin C content after 2 h of incubation [87]. On the
basis of the observation that a series of glutathione-
depleting agents including, surprisingly, dibutyryl
cyclic AMP, enhanced vitamin C formation and also
glycogenolysis in murine hepatocytes, it was proposed
that increased ascorbate synthesis is the result of a
‘push effect’ involving an increase in the concentration
of UDP-glucose [88]. However, no measurements of
UDP-glucose or UDP-glucuronate were made to sub-
stantiate this hypothesis. Furthermore, the potent
glycogenolytic agent glucagon does not stimulate glu-

curonate [28] or vitamin C [87] formation in rat
hepatocytes, and the effects of some of the compounds
that were tested by Braun et al. [88] could not be
reproduced by other authors [28,87].
The mechanism of the effect of glutathione-depleting
agents is therefore presently not understood. One may
wonder to what extent some of the agents used to
deplete glutathione do not act like xenobiotics, by
stimulating the formation of glucuronate from UDP-
glucuronate through a direct action on UDP-glucu-
ronidase. A more exciting possibility would be that the
control is exerted downstream on GLO. If this were
the case, glutathione depletion should enhance the con-
version of l-gulonolactone to vitamin C. Finally, one
has also to consider the theoretical possibility that the
glutathione-depleting agents might act by slowing
down vitamin C degradation.
Recycling of vitamin C
As described in the introduction, ascorbate plays
major roles as a water-soluble antioxidant and as a
cofactor of several enzymes, which lead to its one-
electron oxidation to SDA. Disproportionation of
SDA (2 SDA fi ascorbate + DHA), in turn, results
in the formation of DHA. Both SDA and DHA are
reduced back to ascorbate by several enzymatic sys-
tems that are briefly reviewed below and schematized
in Fig. 3.
Reduction of semidehydroascorbate
The reduction of SDA in the cytosol has been assigned
to enzymes using NADH (NADH-cytochrome b

5
reductase) or NADPH (thioredoxin reductase). SDA
can also be reduced in the lumen of neuroendocrine
secretory vesicles or in the external medium by trans-
membrane electron transfer systems.
C. L. Linster and E. Van Schaftingen Vitamin C metabolism and recycling in mammals
FEBS Journal 274 (2007) 1–22 ª 2006 The Authors Journal compilation ª 2006 FEBS 9
NADH-cytochrome b
5
reductase
Early studies showed that oxidation of NADH by liver
microsomes was stimulated in the presence of SDA,
and microsomal NADH-cytochrome b
5
reductase was
proposed to participate in the electron transfer system,
since the purified enzyme itself was able to reduce
SDA in the presence of NADH [89]. Ito et al. [90]
showed that, in rat liver, most of the NADH-depend-
ent SDA reductase activity is localized in the outer
mitochondrial membrane. Some activity could be
detected in the nuclear and microsomal fractions. Inhi-
bition of the mitochondrial SDA reductase activity
with specific antibodies suggested participation of
NADH-cytochrome b
5
reductase and of a cyto-
chrome b
5
-like protein of the outer mitochondrial

membrane [90].
NADH-cytochrome b
5
reductase is an FAD-con-
taining enzyme [91,92], which exists as a 300 amino
acid membrane-bound form and a 275 amino acid
soluble form [93]. The membrane-bound protein is
located mainly in the endoplasmic reticulum and the
outer mitochondrial membranes, but a small fraction
of the enzyme is apparently also associated with
the plasma membrane [94]. Its C-terminal catalytic
domain (275 amino acid residues) is oriented
towards the cytosol [95]. The soluble form (identical
to the catalytic domain of the membrane-bound form)
of the protein is found mainly in erythrocytes, where
it is involved in the reduction of methaemoglobin
[95]. Fibroblasts of a patient with methaemoglobinae-
mia due to a mutation in the NADH-cytochrome
b
5
reductase gene were shown to be deficient in
NADH-dependent SDA reductase activity [96], which
confirms the involvement of this enzyme in SDA
reduction.
An NADH-linked soluble SDA reductase was also
purified from rabbit lens, but the N-terminal sequence
of a peptide fragment prepared from this protein
did not show significant similarity with any known pro-
tein sequence [97]. This suggests that, besides NADH-
cytochrome b

5
reductase, additional NADH-dependent
enzymes may participate in SDA reduction.
Thioredoxin reductase
Purified rat liver thioredoxin reductase, a selenopro-
tein, was shown to catalyze the NADPH-dependent
reduction of SDA with a K
m
value (in the presence
of thioredoxin, which acts as an activator) of 3 lm
for this radical [98]. NADPH-dependent reduction of
SDA was also demonstrated in dialyzed cytosolic
Fig. 3. Recycling of vitamin C. Vitamin C is transported into the cell under its reduced (ascorbate) and oxidized (DHA) forms by active and
facilitative transport systems (shown in blue), respectively. The utilization of ascorbate as an antioxidant or enzyme cofactor leads to the for-
mation of SDA in the cytosol, neuroendocrine secretory vesicles and the extracellular medium. Various enzymatic systems (represented in
green) reconvert SDA to ascorbate. Intracellular DHA, arising through disproportionation of SDA or import from external sources, can also be
reduced back to ascorbate by several enzymes (shown in red) or through spontaneous reaction with GSH (not shown). DHA, dehydroascor-
bate; GSTO, Omega class glutathione S-transferase; 3a-hydroxysteroid DH, 3a-hydroxysteroid dehydrogenase; PDI, protein disulfide iso-
merase; SDA, semidehydroascorbate.
Vitamin C metabolism and recycling in mammals C. L. Linster and E. Van Schaftingen
10 FEBS Journal 274 (2007) 1–22 ª 2006 The Authors Journal compilation ª 2006 FEBS
fractions prepared from rat liver, where it was found
to be enhanced by selenocystine and inhibited by
aurothioglucose (an inhibitor of selenoenzymes), indi-
cating that it is contributed by thioredoxin reductase
[98]. This interpretation was further supported by the
finding that the NADPH-dependent SDA reduction
was markedly decreased (by 75%) in liver cytosolic
fractions derived from selenium-deficient rats, which
have low (<10% of control rats) thioredoxin reduc-

tase activity.
Cytochrome b
561
-mediated reduction of intravesicular
SDA
Ascorbate is a cofactor of dopamine b-hydroxylase
and of peptidylglycine a-amidating monooxygenase
(see Introduction). Both enzymes are localized in neu-
roendocrine secretory vesicles (catecholamine-storing
vesicles for the first enzyme and peptide-storing vesi-
cles for the second) and catalyze reactions generating
SDA [99]. As ascorbate and SDA do not cross the
membrane of the storage vesicles, the reduction of
SDA to ascorbate occurs inside the vesicles and
involves the transmembrane transfer of electrons by
cytochrome b
561
, the second most abundant protein in
the chromaffin granule membrane [100,101]. Electrons
are furnished to cytochrome b
561
by cytosolic ascor-
bate, which is maintained under its reduced form by
the mitochondrial outer membrane SDA reductase
[102]. The intravesicular reduction of SDA to ascor-
bate is thermodynamically driven by the pH gradient
(SDA reduction involves the binding of a proton and
is therefore favoured at acidic pH) and the membrane
potential (positive inside) created across the chromaffin
vesicle membrane by an inwardly directed proton-

translocating ATPase [103]. The sequence of cyto-
chrome b
561
is known [104], but this protein has not
yet been crystallized. Current structural models suggest
that cytochrome b
561
forms six transmembrane a-heli-
ces and contains two hemes with differing redox poten-
tials, each anchored by a pair of well-conserved
histidyl residues, one near the cytosolic and the other
near the intravesicular face of the membrane [105–
107].
Reduction of extracellular SDA involving transfer
of electrons across the plasma membrane
Erythrocytes are able to transfer electrons from intra-
cellular donors to extracellular SDA. In a system
where SDA was generated extracellularly from ascor-
bate by ascorbate oxidase, it was shown that erythro-
cytes decreased both the ascorbate oxidation rate and
the steady-state extracellular SDA concentration [108].
Using a similar experimental model, Van Duijn et al.
[109] showed that erythrocytes prevented the degrada-
tion of extracellular ascorbate by reactions that were
driven by intracellular ascorbate and NADH. The rel-
ative contributions of endogenous ascorbate and
NADH could not be established, but the results clearly
indicated that intracellular ascorbate donates electrons
to extracellular SDA via a plasma membrane redox
system. The electron transport system still needs to be

identified. It does not involve cytochrome b
561
, which
is absent from the erythrocyte plasma membrane [110].
Isolated rat liver plasma membranes catalyze SDA
reduction in the presence of NADH and this activity is
inhibited by lectins [111], indicating the involvement of
glycoprotein(s). Furthermore, HL-60 cells slow down
the (chemically induced) oxidation of external ascor-
bate [112]. This effect is increased when NADH forma-
tion is stimulated by the addition of lactate to the cells
and is inhibited by lectins. These and other observa-
tions suggest the existence of an NADH-dependent
trans-plasma membrane redox system reducing SDA to
ascorbate in several cell types [113], although part of
the observed stabilization of external ascorbate by cells
could be explained by transition ion chelation and
thus inhibition of ascorbate autoxidation [114,115].
The enzymatic system seems to be distinct from the
plasma membrane NADH-dependent ferricyanide
reductase [116] and to involve coenzyme Q [117], and
NADH-cytochrome b
5
reductase [118], as well as
other, as yet unidentified, factors [119]. A system
allowing the utilization of reducing equivalents in the
extracellular medium (experimentally, by ferricyanide)
derived from intracellular ascorbate appears also to be
present in HL-60 cells [120].
Reduction of dehydroascorbate

This process is probably less important quantitatively
than the reduction of SDA, which is the most
important oxidation product of ascorbate. Intracellu-
lar DHA may, however, derive from SDA through
disproportionation or through import from extracel-
lular sources.
Spontaneous reaction with GSH
DHA can be nonenzymatically reduced to ascorbate
by GSH [121]. This reaction, which presumably pro-
ceeds through a glutathione-ascorbate thiohemiketal
adduct, is thermodynamically favourable (DE°¢ ¼
0.16 V; K
eq
 2.5 · 10
5
m
)1
at pH 7), but is relatively
slow at physiological concentrations of GSH. From
C. L. Linster and E. Van Schaftingen Vitamin C metabolism and recycling in mammals
FEBS Journal 274 (2007) 1–22 ª 2006 The Authors Journal compilation ª 2006 FEBS 11
the data reported by Winkler et al. [121], it can be cal-
culated that at 5 mm GSH and 0.1 m m DHA (pH 7.0;
20–22 °C), this reaction would be half-complete after
10 min, which compares with a half-life of 100 min
for the spontaneous hydrolysis of DHA to 2,3-diketo-
gulonate at the same temperature [122]. However, as
described in the next section, the hydrolysis of DHA is
enhanced by bicarbonate and by an intracellular lacto-
nase. These considerations lead to the conclusion that

an important (irreversible) loss of vitamin C would
occur if the spontaneous reaction with GSH were
solely responsible for the reduction of DHA to ascor-
bate. However, as detailed below, several enzymes
facilitate this reaction with GSH. Furthermore,
NADPH-dependent reductases appear also to be
involved in DHA reduction.
Glutaredoxin and protein disulfide isomerase
Highly purified preparations of glutaredoxin (also
known as thioltransferase) from pig liver, beef thymus
and human placenta, as well as of bovine liver protein
disulfide isomerase (PDI) display DHA reductase activ-
ity [123]. Glutaredoxin and PDI are thiol-disulfide
oxidoreductases, which both possess active-site cysteine
residues. Glutaredoxins are small (12 kDa) cytosolic
enzymes that physiologically use GSH as a reductant,
whereas PDI is a larger (57 kDa) protein, bound to the
endoplasmic reticulum membrane, which serves to cre-
ate and rearrange disulfide bonds [123]. Kinetic analysis
of their DHA reductase activity revealed K
m
values in
the range of 0.2–2.2 mm and 1.6–8.7 mm for DHA and
GSH, respectively. Based on the subcellular localization
of these two types of enzymes, it was proposed that
glutaredoxin could contribute to ascorbate recycling
from DHA in the cytosol, whereas PDI might catalyze
this reaction in the endoplasmic reticulum. The finding
that PDI can act on DHA led to the proposal that the
latter could be implicated as an oxidant in protein

disulfide formation catalyzed by PDI [124,125].
More recently, experiments with cultured human
lens epithelial cells in which thioltransferase (glutare-
doxin) was overexpressed or knocked down, indicated
that this enzyme plays a major role in ascorbate recyc-
ling in these cells [126]. Other studies have shown that
PDI is intrinsically much poorer (250-fold lower
catalytic efficiency) as a DHA reductase than gluta-
redoxin [127].
Omega class glutathione transferase
Maellaro et al. [128] purified a cytosolic rat liver DHA
reductase, which was clearly different from glutaredox-
in and PDI. SDS ⁄ PAGE analysis gave a single band at
31 kDa and K
m
values of 0.25 and 2.8 mm were repor-
ted for DHA and GSH, respectively. The enzyme
had a specific requirement for GSH as hydrogen
donor. Immunotitration experiments with polyclonal
antibodies revealed that it accounted for 70% of the
GSH-dependent DHA reductase activity of a rat liver
cytosol [129]. Other studies with the same enzyme
purified from human erythrocytes showed it to have a
catalytic efficiency comparable to that of glutaredoxin
in its DHA reductase activity (k
cat
⁄ K
m
ratio of
 25 000 m

)1
Æs
)1
in both cases) [127]. The enzyme was
found to be broadly distributed among organs, as
revealed by activity measurement, immunoblot analysis
[129] and northern blot analysis [130].
The cDNA of rat liver GSH-dependent DHA reduc-
tase has been cloned and found to encode a protein
with a predicted molecular weight of 25 kDa [130].
DHA reductase is not homologous to glutaredoxin or
PDI, but belongs to the superfamily of glutathione
S-transferases (GSTs), more precisely to the Omega
class of GSTs (GSTO; this class of GSTs has recently
been reviewed in [131]). GSTs generally catalyze the
transfer of GSH to a variety of substrates including
aromatic compounds and leukotrienes. The crystal
structure of human GSTO1, the orthologue of rat
GSH-dependent DHA reductase, shows the presence
in the active site of a cysteine (Cys32), conserved in
GSTOs, at a position where GSTs from other classes
contain a tyrosine or a serine residue serving to stabil-
ize bound GSH as a thiolate via the hydroxyl group
[131,132]. In contrast, Cys32 of GSTO1 was found to
form a disulfide bond with GSH [132].
GSTOs have very low glutathione conjugating activ-
ity, but they act as thioltransferases, and as reductases
for DHA and monomethylarsonate (an intermediate in
the arsenic biotransformation pathway) [131]. Con-
cerning DHA reduction, human GSTO2 (which shares

64% identity with human GSTO1, but has a more
restricted tissue distribution) was found to be 70–100
times more active than GSTO1 [133]. The role of
GSH-dependent DHA reductase in the recycling of
vitamin C was substantiated by showing that CHO
cells expressing rat liver DHA reductase accumulated
more ascorbate than nontransfected cells when incuba-
ted in the presence of DHA [130].
3a-Hydroxysteroid dehydrogenase
Del Bello et al. [134] purified an NADPH-dependent
DHA reductase from rat liver cytosol. This enzyme
had, however, a lower affinity for DHA (K
m
¼ 4.6 mm)
than the GSH-dependent enzymes mentioned above.
Vitamin C metabolism and recycling in mammals C. L. Linster and E. Van Schaftingen
12 FEBS Journal 274 (2007) 1–22 ª 2006 The Authors Journal compilation ª 2006 FEBS
The monomeric structure, the relatively low molecular
weight (37 kDa), the NADPH-dependence, as well as
the cytosolic localization of the enzyme suggested that
it belongs to the aldo-keto reductase superfamily. The
substrate specificity as well as the inhibition pattern of
the enzyme (reduction of 5a-androstane-3,17-dione and
inhibition by steroidal and nonsteroidal anti-inflamma-
tory drugs) pointed to a possible identity with 3a-hy-
droxysteroid dehydrogenase, which was finally proven
by microsequence analysis.
Thioredoxin reductase
In addition to its activity on SDA (see previous subsec-
tion), purified rat liver thioredoxin reductase was also

shown to catalyze the NADPH-dependent reduction of
DHA [135]. The K
m
value for this substrate (0.7 mm),
though >200-fold higher than that for SDA (3 lm), is
in the same range as the K
m
values of the GSH-depend-
ent DHA reductases. Arguments mentioned above to
support the role of thioredoxin reductase in the reduc-
tion of SDA (decreased NADPH-dependent reductase
activity in liver cytosolic fractions of selenium-deficient
rats; inhibition by aurothioglucose) also apply to the
reduction of DHA and indicate that thioredoxin reduc-
tase is responsible for at least 75% of the NADPH-
dependent reduction of DHA in a rat liver cytosol. It
should be mentioned that GSH-dependent DHA reduc-
tase activity in cytosolic fractions was found to be
typically two- to three-fold higher than the total
NADPH-dependent reductase activity and that it was
not affected by selenium deficiency [135].
Assessment of the role of the reductive systems
As mentioned above, SDA can be either directly
reduced in a one-electron step to ascorbate or further
oxidized (e.g. by disproportionation) to DHA before
the latter is reduced in a two-electron step. Coassin
et al. [136] compared the NADH-dependent SDA
reductase and the GSH-dependent DHA reductase
activities in homogenates of pig tissues and concluded
that, at the low physiological concentrations (micro-

molar range) of DHA, recycling of ascorbate is effect-
ively accomplished by reduction of the ascorbyl
radical, DHA reduction occurring only at a negligible
rate under these conditions. No DHA reductase acti-
vity was detected at 0.1 mm DHA. Reduction of DHA
is, however, certainly important when this compound
is imported from the extracellular medium.
Studies in which glutathione was depleted have poin-
ted to the importance of the GSH-dependent reduction
of DHA. Administration of l-buthionine-(S,R)-sulfoxi-
mine (BSO), a transition-state inactivator of c-glut-
amylcysteine synthetase, to newborn rats decreased
tissue levels of GSH, but also of ascorbate and mark-
edly increased DHA [137]. Severe tissue damage was
observed especially at the level of mitochondria and
the animals died within a few days. Simultaneous
administration of ascorbate decreased their mortality
and increased glutathione levels. Newborn rats given
lower doses of BSO developed cataracts. This could
also be partially prevented by the administration of
ascorbate. These in vivo observations showed that one
of the roles of glutathione is to maintain ascorbate in
its reduced state and that ascorbate can partially
replace glutathione in some of its antioxidant func-
tions. Adult rats and mice are much less sensitive to
BSO treatment than newborn animals. Mortality was
not increased by BSO administration to adult mice
and much less severe tissue damage was observed [86].
The higher sensitivity of newborn animals was pro-
posed to be due to lower ascorbate synthesis in the

first days of life, but may also partly be explained by
the higher retention of BSO in tissues of newborn ani-
mals than of adult animals [137]. These and other
studies emphasizing an in vivo relationship between
glutathione and ascorbate in animals that can or
cannot synthesize ascorbate have been reviewed by
Meister [138].
Catabolism of vitamin C
In mammals, the degradation of ascorbate appears to
proceed via DHA, an unstable molecule, and to
involve spontaneous and possibly enzyme-catalyzed
reactions. A specific pathway for the degradation of
vitamin C has only been fully described in bacteria.
Spontaneous breakdown of dehydroascorbate
Ascorbate is oxidized to DHA in a variety of enzymat-
ic and nonenzymatic reactions (see Introduction).
DHA can be reduced back to ascorbate (see previous
section), but can also be hydrolyzed to 2,3-diketo-l-
gulonate (2,3-DKG). The latter reaction is irreversible
and 2,3-DKG, unlike DHA, is therefore devoid of
antiscorbutic activity. DHA is unstable and nonenzy-
matic hydrolysis occurs rather rapidly at neutral pH,
the half-time of decay being about 5–15 min at 37 °C
[122]. Incubation of DHA or 2,3-DKG in the presence
of phosphate buffer (pH 7) for several hours at 37 °C
leads to the formation of l-erythrulose and oxalate
[139], indicating cleavage between the second and the
third carbon (Fig. 4). When the incubation is per-
formed in the presence of H
2

O
2
(which may be formed
C. L. Linster and E. Van Schaftingen Vitamin C metabolism and recycling in mammals
FEBS Journal 274 (2007) 1–22 ª 2006 The Authors Journal compilation ª 2006 FEBS 13
during chemical oxidation of ascorbate), l-threonate is
the major degradation product formed (Fig. 4) and no
l-erythrulose is detected [139]. A five-carbon interme-
diate (3,4,5-trihydroxy-2-ketopentanoate) is also transi-
ently formed in the presence of H
2
O
2
[140], indicating
that the reaction proceeds by successive cleavage of the
bonds between C1 and C2, and C2 and C3.
It was speculated that the highly reactive ketose,
l-erythrulose, if formed in vivo, could play a role in
ascorbate-dependent modifications of protein observed
in vitro and proposed to occur in vivo in human lens
during diabetic and age-onset cataract formation
[139].
Degradation of vitamin C in mammals
In vivo studies
In human subjects injected with ascorbate labelled on
C1, an average of 44% of the total radioactivity
excreted in urine was recovered as oxalate; other urin-
ary metabolites were 2,3-DKG (20%) and DHA
(< 2%), and 20% of the total urinary radioactivity
was present as ascorbate [141]. Little if any radioactiv-

ity was recovered as CO
2
. These findings indicate that a
major catabolic event in man is the cleavage of the C6
molecule (presumably a spontaneous cleavage of 2,3-
DKG) between C2 and C3, with little if any decarboxy-
lation. The oxalate formed in this way may contribute
to the formation of kidney stones in susceptible individ-
uals. However, the association between ascorbate
supplementation and increased risk of kidney stone
formation remains a matter of controversy [142].
The global human metabolism of ascorbic acid dif-
fers from that of animal species in several respects
[141,143]. Firstly, the half-life of ascorbate was shown
to be 4–5 times longer in man than in guinea pigs
and rats. Second, in guinea pigs and rats, radiolabelled
CO
2
is produced from ascorbate labelled on C1. Third,
whereas in humans DHA is almost completely conver-
ted to ascorbate, this process is less efficient in other
animals where DHA is rapidly catabolized. Finally, on
vitamin C-free diet, the onset of scurvy is less rapid in
man than in guinea pigs. These findings indicate that
man is better able than rodents to save vitamin C,
probably because of both a better ability to reduce
DHA to ascorbate and a lower dehydroascorbatase
activity (see below). We speculate that this also applies
to bats, which are unable to synthesize ascorbic acid
and some of which have a low supply of this vitamin

in their food [13].
In vitro studies
As mentioned above, DHA is unstable and its non-
enzymatic hydrolysis occurs rather rapidly at pH 7.0
[122]. It is accelerated by bicarbonate, which is respon-
sible for the rapid disappearance of DHA in blood
plasma [144]. An enzyme catalyzing the delactonization
of DHA has been detected and partially purified from
beef liver [143,145]. This enzyme hydrolyzes a series of
Fig. 4. Spontaneous breakdown of dehydroascorbate (oxidation product of ascorbate) in the absence or in the presence of hydrogen peroxide.
Vitamin C metabolism and recycling in mammals C. L. Linster and E. Van Schaftingen
14 FEBS Journal 274 (2007) 1–22 ª 2006 The Authors Journal compilation ª 2006 FEBS
other lactones and shares many properties with gulo-
nolactonase, from which it could not be separated
[143]. Lower activities of both dehydroascorbatase
[145] and gulonolactonase (aldonolactonase) [45,146]
are present in tissues of primates compared to other
mammals. Assuming that both activities are contribu-
ted by the same enzyme, this lower activity may be
beneficial in primates because it would slow down the
irreversible loss of DHA while not having any detri-
mental effect on ascorbate formation (which is absent).
It would be interesting to know if the bone loss
observed in transgenic rats overexpressing regucalcin
[147,148] (which, as noted above, has recently been
identified as the aldonolactonase involved in ascorbate
synthesis [47]) is related to vitamin C deficiency.
As for the formation of oxalate, which is one of
the major excretion products of ascorbate in
humans, no enzyme has yet been described that cata-

lyzes the cleavage of 2,3-DKG between the second
and the third carbon. It is therefore likely that this
reaction takes place spontaneously. The metabolic
fate of the other product (most probably l-erythru-
lose, since H
2
O
2
levels are kept low) is unknown.
The finding that addition of ascorbate or DHA to
glycogen-depleted mouse hepatocytes causes an
increase in the formation of glucose and in the con-
centration of d-xylulose 5-phosphate [149] suggests
that they can be converted to a metabolizable sugar
or sugar derivative.
Utilization of vitamin C by Escherichia coli
E. coli can ferment ascorbate and the operon respon-
sible for ascorbate utilization has recently been iden-
tified [150]. This identification was based on the
hypothesis that the catabolic pathway of ascorbate
could lead to the formation of the pentose phos-
phate intermediate d-xylulose 5-phosphate, via 4-epi-
merization of l-ribulose 5-phosphate. The latter
reaction is also involved in the catabolic pathway of
l-arabinose, where it is catalyzed by AraD. Two
multigene operons of unknown function were found
to comprise an AraD homologue in the genome of
E. coli K-12.
The deletion of one of these operons generated
mutants that failed to ferment ascorbate [150]. The

‘utilization of
L-ascorbate’ operon (ula operon) thus
identified contains six genes (designated ulaA, B, C, D,
E and F) (Fig. 5A). UlaA, UlaB and UlaC are compo-
nents of a PTS system involved in the uptake of
ascorbate and its phosphorylation to l-ascorbate
6-phosphate [151]. The proteins encoded by ulaD,
ulaE and ulaF were functionally characterized as
3-keto-l-gulonate 6-phosphate decarboxylase, l-xylu-
lose 5-phosphate 3-epimerase and l-ribulose 5-phos-
phate 4-epimerase, respectively [150]. A gene located
upstream of the ula operon, ulaG, encodes a distant
homologue of a metal-dependent hydrolase that is
essential for ascorbate utilization [151]. It was proposed
to encode a cytoplasmic ascorbate 6-phosphate lacto-
nase catalyzing the conversion of ascorbate 6-phos-
phate to 3-keto-l-gulonate 6-phosphate.
All together the divergently transcribed operons
ulaABCDEF and ulaG encode proteins allowing the
uptake of ascorbate and its conversion to d-xylulose
5-phosphate and CO
2
(Fig. 5C). They are both regula-
ted by the nearby ulaR gene and together constitute a
regulon [152]. ulaR encodes a repressor belonging to
the DeoR family of bacterial regulatory proteins and
inactivation of this gene leads to constitutive expres-
sion of the ula operons. A detailed study of the regula-
tion of expression of the ula operons by UlaR and
other factors has been reported [153].

Disruption of the other operon (yia) containing an
AraD homologue (Fig. 5B) did not affect the fermen-
tation of ascorbate [150]. This operon encodes nine
different proteins, five of which have been over-
expressed and characterized [150]. On this basis, it
has been proposed that the yia operon allows the
metabolism of 2,3-diketo-l-gulonate through a path-
way in which it is successively reduced to 3-keto-
l-gulonate by YiaK (a dehydrogenase belonging to a
novel family of pyridine nucleotide-linked oxidoreduc-
tases [154]) and phosphorylated to 3-keto-l-gulonate
6-phosphate by an ATP-dependent kinase (YiaP)
(Fig. 5C). 3-Keto-l-gulonate 6-phosphate would then
be converted to d-xylulose 5-phosphate in a sequence
of reactions similar to that described above for the
ula operon. The gene products involved are YiaQ
(3-keto-l-gulonate 6-phosphate decarboxylase), YiaR
(l-xylulose 5-phosphate 3-epimerase) and YiaS (l-rib-
ulose 5-phosphate 4-epimerase). It should be noted
that no enzymatic activity could be detected in the
case of YiaR despite its high degree of identity with
UlaE (l-xylulose 5-phosphate 3-epimerase) [150]. The
operon also encodes three proteins of a putative ABC
transport system (YiaM, YiaN and YiaO), possibly
involved in the transport of 2,3-DKG [150]. However,
the definitive proof that this operon is involved in the
utilization of the hydrolyzed form of DHA is still
lacking.
A search for similar operons in other bacterial ge-
nomes using the SEED database (icago.

edu) indicates that the first (ula) occurs not only in
Enterobacteriae (E. coli, Shigella dysenteriae, Shigella
sonnei, Shigella flexneri, Salmonella enterica, Salmonella
C. L. Linster and E. Van Schaftingen Vitamin C metabolism and recycling in mammals
FEBS Journal 274 (2007) 1–22 ª 2006 The Authors Journal compilation ª 2006 FEBS 15
paratyphi, Salmonella typhimurium, and Klebsiella
pneumoniae), but also in Lactobacillales (Enterococcus
faecium, Streptococcus agalactiae, Streptococcus pneu-
moniae, Streptococcus pyogenes, and Streptococcus
uberis) and in Mycoplasma penetrans, Mycoplasma pneu-
moniae, and Mycoplasma synoviae. The second type of
operon (yia) appears to be much less frequent. We noted
its presence only in E. coli, S. enterica, S. typhimurium,
and Serratia marcescens. Mammalian genomes appar-
ently do not encode an orthologue of 3-keto-l-gulonate
6-phosphate decarboxylase, the critical enzyme in these
vitamin C degradation pathways, as indicated by blast
searches.
Acknowledgements
This work was supported by the Concerted Research
Action Program of the Communaute
´
Franc¸ aise de Bel-
gique; the Interuniversity Attraction Poles Program,
Belgian Science Policy; and the Fonds de la Recherche
Scientifique Me
´
dicale. CLL was a fellow of the Fonds
National de la Recherche Scientifique (FNRS).
A

B
C
yyyyy yyyy
Fig. 5. Degradation of vitamin C in bacteria. Operons in the E. coli K-12 genome encoding proteins involved in the utilization of ascorbate
(ula) (A) and, hypothetically, 2,3-diketo-
L-gulonate (yia) (B). The reactions catalyzed by the gene products of these operons are shown in (C).
Vitamin C metabolism and recycling in mammals C. L. Linster and E. Van Schaftingen
16 FEBS Journal 274 (2007) 1–22 ª 2006 The Authors Journal compilation ª 2006 FEBS
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