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

R E V I E W A R T I C L E

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: vanschaftingen@bchm.ucl.ac.be 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: linster@chem.ucla.edu

(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 Fe2+-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 b5 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, CO2 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.

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[5],

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 Fe2+ ⁄ a-ketoglutarate- dependent dioxygenases (e.g. collagen prolyl and lysyl hydroxylases, the two hydroxylases involved in carni- the asparaginyl hydroxylase tine biosynthesis that modiﬁes hypoxia-inducible factor 1 (HIF-1) [6]), ascorbate most probably serves to reconvert inactive, Fe3+-containing enzyme (which results from abortive catalytic cycles) to the active, Fe2+-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

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- in quantities (typically 10– ularly of plant origin, 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 pKa 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 is most probably one-electron reactions, and this

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.

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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.

[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.

(DDT)

Formation of vitamin C in mammals and other vertebrates

Outline of the pathway

[13]), guinea pigs, and primates

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- feeding on blood or eating ones, but also others, insects 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].

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 [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 the in vivo conversion of both d-glucose and that d-galactose to glucuronate and ascorbate was increased by xenobiotics [22], but that this was not the case for radiolabelled d-glucuronolactone the conversion of or l-gulonolactone to ascorbate [23], suggested that stimulation occurs at a step between UDP-glucose and d-glucuronolactone in the ascorbate biosynthesis pathway.

Vitamin C is also formed by all plant species studied so far [15] and yeasts produce d-erythroascorbate, a C5 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 resulting l-gulonate hydroxymethyl group in the (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.

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.

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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.

these results

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, 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

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glucuronate (mechanisms b and c described in the next subsection).

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- to glucuronate 1-phosphate lyze UDP-glucuronate (presumably because contaminated with they are 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).

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- indicating that the effect of xenobiotics on glucose, 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 (cid:2)100 nmol hexose unitsÆmin)1Æg)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

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.

the transferase activity)

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.

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 (cid:2)5% of [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 GT2P had a minor UDP-glucuronidase activity, which could be stimulated by phenylethers and lysophosphatidylcholines up to (cid:2)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

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,

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Urono- and gulonolactonase

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 free glucuronate and the conditions formation of under which they are able to do so.

Glucuronate reductase (aldehyde reductase)

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 (Km (cid:3) 8 mm), but is inactive against aldonolactones [45]. It is a metal-dependent enzyme, but its sequence is presently unknown.

l-gulono-1,4-lactone

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]. Km 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

in the human genome,

Aldonolactonase (gulonolactonase) is also a metal- dependent enzyme, acting best with Mn2+, 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, and d-glucono-1,5- including 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 Ca2+ [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 Km of glucuronate reductase for d-glucuronate than for d-glucuronolactone (see above).

L-Gulonolactone oxidase

Characterization of the enzyme and of the catalyzed reaction

to the large group of monomeric NADPH-dependent oxidoreductases, known as aldo-keto reductases, which comprise many members including aldose reductases (the closest homologues of human aldehyde reductase, sharing (cid:2)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.

GLO, a microsomal enzyme, catalyzes aerobically the conversion of l-gulonolactone to l-ascorbate with pro- duction of H2O2 [53,54]. The immediate oxidation

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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].

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. Km values for l-gulonolactone ranging from 0.007 to 0.15 mm have been reported [55,56]. The enzyme transfers electrons not only to O2, but also to artiﬁcial electron acceptors such as phenazine methosulfate and ferricyanide, although not to cytochrome b5 and cytochrome c [57]. The production of H2O2 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 (cid:2)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 O2, but to cytochrome c [59].

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.

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.

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].

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

GLO knockout mice have also been generated through homologous recombination and the effects of vitamin C deﬁciency in these mice have been studied

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[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

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, ingestion of aminopyrine Margolis [82] noted that 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,

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.

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 Km for l-xylulose of (cid:2)100 mm, was detected upon electrophoresis chromatography. and ion-exchange 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.

identity

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

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 Km of (cid:2)0.2 mm for l-gulonate. 3-Keto-l-gulonate is decarboxylated to l-xylulose by a poorly characterized is [77] whose molecular decarboxylase 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 Km value of 0.21 mm for l-xy- lulose has been reported for the human recombinant enzyme.

Xylitol

Outline on the regulation of vitamin C synthesis

that

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 Km 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 that rare individuals excrete

indicated by the fact

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 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

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including,

deﬁciency have (when fed an ascorbate-free diet) a plasma ascorbic acid level amounting to (cid:2)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 (cid:2)30% of l-gulonate is directed towards ascorbate formation [28].

Effect of xenobiotics

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 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].

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.

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

the generation of

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 reactive oxygen species when inserted in membranes with active electron transport, the increased vitamin C availability playing then a protective role.

Effect of glutathione

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 b5 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.

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 led to a two-fold inhibitor of glutathione synthesis,

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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.

NADH-cytochrome b5 reductase

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 b5 reductase gene were shown to be deﬁcient in NADH-dependent SDA reductase activity [96], which conﬁrms the involvement of this enzyme in SDA reduction.

reductase

Early studies showed that oxidation of NADH by liver microsomes was stimulated in the presence of SDA, and microsomal NADH-cytochrome b5 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 suggested participation of with speciﬁc antibodies NADH-cytochrome b5 cyto- and of chrome b5-like protein of the outer mitochondrial membrane [90].

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 b5 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 Km value (in the presence of thioredoxin, which acts as an activator) of (cid:2)3 lm for this radical [98]. NADPH-dependent reduction of SDA was also demonstrated in dialyzed cytosolic

NADH-cytochrome b5 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 the enzyme is apparently also associated with of the plasma membrane [94]. Its C-terminal catalytic is oriented domain ((cid:2)275 amino acid residues)

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contributions of

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 (cid:2)75%) in liver cytosolic fractions derived from selenium-deﬁcient rats, which have low (<10% of control rats) thioredoxin reduc- tase activity.

Cytochrome b561-mediated reduction of intravesicular SDA

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 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 b561, 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 b5 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.

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 b561, the second most abundant protein in the chromafﬁn granule membrane [100,101]. Electrons are furnished to cytochrome b561 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 b561 is known [104], but this protein has not yet been crystallized. Current structural models suggest that cytochrome b561 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].

Spontaneous reaction with GSH

Reduction of extracellular SDA involving transfer of electrons across the plasma membrane

thermodynamically favourable

DHA can be nonenzymatically reduced to ascorbate by GSH [121]. This reaction, which presumably pro- ceeds through a glutathione-ascorbate thiohemiketal (DE(cid:2)¢ ¼ adduct, 0.16 V; Keq (cid:3) 2.5 · 105 m)1 at pH 7), but is relatively slow at physiological concentrations of GSH. From

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

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this

in and PDI. SDS ⁄ PAGE analysis gave a single band at 31 kDa and Km 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 (cid:2)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 (kcat ⁄ Km ratio of (cid:2) 25 000 m)1Æs)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 data reported by Winkler et al. [121], it can be cal- culated that at 5 mm GSH and 0.1 mm DHA (pH 7.0; 20–22 (cid:2)C), this reaction would be half-complete after (cid:2)10 min, which compares with a half-life of (cid:2)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 reaction with GSH. Furthermore, facilitate NADPH-dependent reductases appear also to be involved in DHA reduction.

Glutaredoxin and protein disulﬁde isomerase

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 (cid:2)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 the orthologue of rat structure of human GSTO1, 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].

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 Km 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].

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 (cid:2)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].

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 ((cid:2)250-fold lower catalytic efﬁciency) as a DHA reductase than gluta- redoxin [127].

3a-Hydroxysteroid dehydrogenase

Omega class glutathione transferase

Del Bello et al. [134] puriﬁed an NADPH-dependent DHA reductase from rat liver cytosol. This enzyme had, however, a lower afﬁnity for DHA (Km ¼ 4.6 mm) than the GSH-dependent enzymes mentioned above.

Maellaro et al. [128] puriﬁed a cytosolic rat liver DHA reductase, which was clearly different from glutaredox-

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The monomeric structure, the relatively low molecular weight ((cid:2)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

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 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 Km value for this substrate (0.7 mm), though >200-fold higher than that for SDA (3 lm), is in the same range as the Km 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 than the total to three-fold higher typically two- NADPH-dependent reductase activity and that it was not affected by selenium deﬁciency [135].

Assessment of the role of the reductive systems

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

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-

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 (cid:2)C [122]. Incubation of DHA or 2,3-DKG in the presence of phosphate buffer (pH 7) for several hours at 37 (cid:2)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 H2O2 (which may be formed

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Fig. 4. Spontaneous breakdown of dehydroascorbate (oxidation product of ascorbate) in the absence or in the presence of hydrogen peroxide.

supplementation and increased risk of kidney stone formation remains a matter of controversy [142].

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 H2O2 [140], indicating that the reaction proceeds by successive cleavage of the bonds between C1 and C2, and C2 and C3.

cataract

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 formation [139].

Degradation of vitamin C in mammals

In vivo studies

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 (cid:2)4–5 times longer in man than in guinea pigs and rats. Second, in guinea pigs and rats, radiolabelled CO2 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

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 ((cid:2)20%) and DHA (< 2%), and (cid:2)20% of the total urinary radioactivity was present as ascorbate [141]. Little if any radioactiv- ity was recovered as CO2. 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- the association between ascorbate uals. However,

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

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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 is homologue of a metal-dependent hydrolase that 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

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.

excretion

ascorbate

the divergently transcribed operons ulaABCDEF and ulaG encode proteins allowing the uptake of ascorbate and its conversion to d-xylulose 5-phosphate and CO2 (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].

hepatocytes

causes

As for the formation of oxalate, which is one of in products the major 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 H2O2 levels are kept low) is unknown. The ﬁnding that addition of ascorbate or DHA to glycogen-depleted mouse 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

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.

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

A search for similar operons in other bacterial ge- nomes using the SEED database (http://theseed.uchicago. edu) indicates that the ﬁrst (ula) occurs not only in Enterobacteriae (E. coli, Shigella dysenteriae, Shigella sonnei, Shigella ﬂexneri, Salmonella enterica, Salmonella

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C. L. Linster and E. Van Schaftingen

Vitamin C metabolism and recycling in mammals

A

B

C

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 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).

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

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Vitamin C metabolism and recycling in mammals

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

Vitamin C

Biosynthesis, recycling and degradation in mammals

Formation of vitamin C in mammals and other vertebrates

An alternative fate for glucuronate: the pentose pathway

Control of vitamin C synthesis

Recycling of vitamin C

Catabolism of vitamin C

A

B

C

Acknowledgements

References

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

Vitamin C

Biosynthesis, recycling and degradation in mammals

Formation of vitamin C in mammals and other vertebrates

An alternative fate for glucuronate: the pentose pathway

Control of vitamin C synthesis

Recycling of vitamin C

Catabolism of vitamin C

A

B

C

Acknowledgements

References

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Tài liêu mới

AI tóm tắt

Giới thiệu tài liệu

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Chính sách

Thoả thuận sử dụng

Chính sách bảo mật

Chính sách hoàn tiền

DMCA

Hỗ trợ

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093 303 0098

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