Characterisation of the non-oxidative degradation pathway of dehydroascorbic acid in slightly acidic aqueous solution.

Although l-ascorbate (vitamin C) is an important biological antioxidant, its degradation pathways in vivo remain incompletely characterised. Ascorbate is oxidised to dehydroascorbic acid, which can be either hydrolysed to diketogulonate (DKG) or further oxidised. DKG can be further degraded, oxidatively or non-oxidatively. Here we characterise DKG products formed non-enzymically and non-oxidatively at 20 °C and at a slightly acidic pH typical of the plant apoplast. High-voltage electrophoresis revealed at least five products, including two novel CPLs (epimers of 2-carboxy-l-threo-pentonolactone), which slowly interconverted with CPA (2-carboxy-l-threo-pentonate). One of the two CPLs has an exceptionally low pKa. The CPL structures were supported by MS [(C6H7O7)-] and by 1H and 13C NMR spectroscopy. Xylonate and its lactone also appeared. Experiments with [1-14C]DKG showed that all five products (including the 5-carbon xylonate and its lactone) retained DKG's carbon-1; therefore, most xylonate arose by decarboxylation of CPLs or CPA, one of whose -COOH groups originates from C-2 or C-3 of DKG after a 'benzilic acid rearrangement'. Since CPLs appeared before CPA, a DKG lactone is probably the main species undergoing this rearrangement. CPA and CPL also form non-enzymically in vivo, where they may be useful to researchers as 'fingerprints', or to organisms as 'signals', indicating a non-oxidative, slightly acidic biological compartment.

The oxidation of dehydroascorbic acid and 2,3-diketogulonate by distinct reactive oxygen species.

l-Ascorbate, dehydro-l-ascorbic acid (DHA), and 2,3-diketo-l-gulonate (DKG) can all quench reactive oxygen species (ROS) in plants and animals. The vitamin C oxidation products thereby formed are investigated here. DHA and DKG were incubated aerobically at pH 4.7 with peroxide (H2O2), 'superoxide' (a ∼50 : 50 mixture of [Formula: see text] and [Formula: see text]), hydroxyl radicals (•OH, formed in Fenton mixtures), and illuminated riboflavin (generating singlet oxygen, 1O2). Products were monitored electrophoretically. DHA quenched H2O2 far more effectively than superoxide, but the main products in both cases were 4-O-oxalyl-l-threonate (4-OxT) and smaller amounts of 3-OxT and OxA + threonate. H2O2, but not superoxide, also yielded cyclic-OxT. Dilute Fenton mixture almost completely oxidised a 50-fold excess of DHA, indicating that it generated oxidant(s) greatly exceeding the theoretical •OH yield; it yielded oxalate, threonate, and OxT. 1O2 had no effect on DHA. DKG was oxidatively decarboxylated by H2O2, Fenton mixture, and 1O2, forming a newly characterised product, 2-oxo-l-threo-pentonate (OTP; '2-keto-l-xylonate'). Superoxide yielded negligible OTP. Prolonged H2O2 treatment oxidatively decarboxylated OTP to threonate. Oxidation of DKG by H2O2, Fenton mixture, or 1O2 also gave traces of 4-OxT but no detectable 3-OxT or cyclic-OxT. In conclusion, DHA and DKG yield different oxidation products when attacked by different ROS. DHA is more readily oxidised by H2O2 and superoxide; DKG more readily by 1O2 The diverse products are potential signals, enabling organisms to respond appropriately to diverse stresses. Also, the reaction-product 'fingerprints' are analytically useful, indicating which ROS are acting in vivo.

Metabolites of 2,3-diketogulonate delay peroxidase action and induce non-enzymic H2O2 generation: Potential roles in the plant cell wall.

A proportion of the plant's l-ascorbate (vitamin C) occurs in the apoplast, where it and its metabolites may act as pro-oxidants and anti-oxidants. One ascorbate metabolite is 2,3-diketogulonate (DKG), preparations of which can non-enzymically generate H2O2 and delay peroxidase action on aromatic substrates. As DKG itself generates several by-products, we characterised these and their ability to generate H2O2 and delay peroxidase action. DKG preparations rapidly produced a by-product, compound (1), with λmax 271 and 251 nm at neutral and acidic pH respectively. On HPLC, (1) co-eluted with the major H2O2-generating and peroxidase-delaying principle. Compound (1) was slowly destroyed by ascorbate oxidase, and was less stable at pH 6 than at pH 1. Electrophoresis of an HPLC-enriched preparation of (1) suggested a strongly acidic (pKa ≈ 2.3) compound. Mass spectrometry suggested that un-ionised (1) has the formula C6H6O5, i.e. it is a reduction product of DKG (C6H8O7). In conclusion, compound (1) is the major H2O2-generating, peroxidase-delaying principle formed non-enzymically from DKG in the pathway ascorbate → dehydroascorbic acid → DKG → (1). We hypothesise that (1) generates apoplastic H2O2 (and consequently hydroxyl radicals) and delays cell-wall crosslinking - both these effects favouring wall loosening, and possibly playing a role in pathogen defence.

Oxidation of dehydroascorbic acid and 2,3-diketogulonate under plant apoplastic conditions.

The rate of L-ascorbate catabolism in plants often correlates positively with the rate of cell expansion. The reason for this correlation is difficult to explore because of our incomplete knowledge of ascorbate catabolism pathways. These involve enzymic and/or non-enzymic oxidation to dehydroascorbic acid (DHA), which may then be hydrolysed to 2,3-diketogulonate (DKG). Both DHA and DKG were susceptible to further oxidation under conditions of pH and H2O2 concentration comparable with the plant apoplast. The kinetics of their oxidation and the identity of some of the products have been investigated here. DHA, whether added in pure form or generated in situ by ascorbate oxidation, was oxidised non-enzymically to yield, almost simultaneously, a monoanion (cyclic-oxalyl-threonate; cOxT) and a dianion (oxalyl-threonate; OxT). The monoanion was resistant to periodate oxidation, showing that it was not oxalic threonic anhydride. The OxT population was shown to be an interconverting mixture of 3-OxT and 4-OxT, differing in pK(a). The 3-OxT appeared to be formed earlier than 4-OxT, but the latter predominated at equilibrium. DKG was oxidised by H2O2 to two partially characterised products, one of which was itself further oxidised by H2O2 to yield threonate. The possible occurrence of these reactions in the apoplast in vivo and the biological roles of vitamin C catabolites are discussed.

Alternative pathways of dehydroascorbic acid degradation in vitro and in plant cell cultures: novel insights into vitamin C catabolism.

L-Ascorbate catabolism involves reversible oxidation to DHA (dehydroascorbic acid), then irreversible oxidation or hydrolysis. The precursor-product relationships and the identity of several major DHA breakdown products remained unclear. In the presence of added H2O2, DHA underwent little hydrolysis to DKG (2,3-dioxo-L-gulonate). Instead, it yielded OxT (oxalyl L-threonate), cOxT (cyclic oxalyl L-threonate) and free oxalate (~6:1:1), essentially simultaneously, suggesting that all three product classes independently arose from one reactive intermediate, proposed to be cyclic-2,3-O-oxalyl-L-threonolactone. Only with plant apoplastic esterases present were the esters significant precursors of free oxalate. Without added H2O2, DHA was slowly hydrolysed to DKG. Downstream of DKG was a singly ionized dicarboxy compound (suggested to be 2-carboxy-L-xylonolactone plus 2-carboxy-L-lyxonolactone), which reversibly de-lactonized to a dianionic carboxypentonate. Formation of these lactones and acid was minimized by the presence of residual unreacted ascorbate. In vivo, the putative 2-carboxy-L-pentonolactones were relatively stable. We propose that DHA is a branch-point in ascorbate catabolism, being either oxidized to oxalate and its esters or hydrolysed to DKG and downstream carboxypentonates. The oxidation/hydrolysis ratio is governed by reactive oxygen species status. In vivo, oxalyl esters are enzymatically hydrolysed, but the carboxypentonates are stable. The biological roles of these ascorbate metabolites invite future exploration.

Photostability and interaction of ascorbic acid in cream formulations.

The kinetics of photolysis of ascorbic acid in cream formulations on UV irradiation has been studied using a specific spectrophotometric method with a reproducibility of ± 5%. The apparent first-order rate constants (k(obs)) for the photolysis of ascorbic acid in creams have been determined. The photoproducts formed in the cream formulations include dehydroascorbic acid and 2,3-diketogulonic acid. The photolysis of ascorbic acid appears to be affected by the concentration of active ingredient, pH, and viscosity of the medium and formulation characteristics. The study indicates that the ionized state and redox potentials of ascorbic acid are important factors in the photostability of the vitamin in cream formulations. The viscosity of the humectant present in the creams appears to influence the photostability of ascorbic acid. The results show that the physical stability of the creams is an important factor in the stabilization of the vitamin. In the cream formulations stored in the dark, ascorbic acid undergoes aerobic oxidation and the degradation is affected by similar factors as indicated in the photolysis reactions. The rate of oxidative degradation in the dark is about seventy times slower than that observed in the presence of light.

Effect of ascorbate and its oxidation products on H2O2 production in cell-suspension cultures of Picea abies and in the absence of cells.

Dehydroascorbate and traces of ascorbate were present apoplastically in living spruce (Picea abies) twigs. Since the proposed apoplastic ascorbate degradation pathway contains several steps that possibly generate H(2)O(2), the effects of ascorbate and some of its degradation products were tested on apoplastic H(2)O(2) concentrations in a cell culture of P. abies as a model and on non-enzymic H(2)O(2) production in vitro. Ascorbate scavenged H(2)O(2) in the culture medium of lignin-producing Picea cells and in spent and boiled spent medium; in the presence of Cu(2+) or fresh medium, ascorbate led to the non-enzymic generation of H(2)O(2). Preparations of dehydroascorbate (the initial oxidation-product of ascorbate), and diketogulonate (the hydrolysis-product of dehydroascorbate) induced H(2)O(2) accumulation both non-enzymically and enzymically in Picea cell-suspensions. Paper electrophoresis showed that the dehydroascorbate and diketogulonate preparations contained several degradation products; some of these probably contributed to H(2)O(2) production and/or scavenging in these experiments, and would also do so in vivo. These results indicate a complex ability of apoplastic ascorbate, dehydroascorbate, diketogulonate, and further products to modulate H(2)O(2) concentrations, with potential consequences for the control of growth, development and lignification.

[The contents of ascorbic acid and its oxidized forms at the aging of red blood cells produced in conditions of the normal and intense of erythropoesis].

Levels of ascoroic acid, its oxidized forms, dehydroascorbic acid (DHA) and diketogulonic aci (DKGA), and the sum of all forms of ascorbic acid (SAA) were investigated in aging red blood cells produced under physiological and stress conditions (massive hemorrhage) Aging of red blood cells o intact animals is accompanied by accumulation of DKGA and decrease of AA, DHA and SAA concentrations. Comparison of these parameters between young and old red blood cells produced befor and after hemorrhage revealed decrease of AA concentration (52%) and accumulation of DKGA (27%) on the 7th day. On the 7th, 20th, 30th days after hemorrhage red blood cells are characterize by increased concentrations of DHA, DKGA, SAA with simultaneously decreased contents of the AA as compared with red blood cells of intact animals. Similar changes were the most pronounced o the 7th day; on the 20th day they decreased and on the 30th day they returned to control levels.

[Levels of serum ascorbate and its metabolites in hemodialysis patients].

The status of ascorbic acid (AA) in dialysis patients is the subject of debate. Some reports have found AA to be deficient in dialysis patients, while others have found that AA is not deficient. In an attempt to confirm AA serum concentrations in dialysis patients, we analyzed the concentrations of AA as well as its metabolites using the specific determination of AA with chemical derivatization and the HPLC method. We studied 131 patients under maintenance hemodialysis therapy (HD), 23 patients with chronic renal failure (CRF) and 48 healthy controls (C). Serum concentrations of AA and the AA metabolites dehydroascorbic acid (DHA) and 2, 3-diketogulonate (DKG) were measured by HPLC. Nine HD patients were taking AA supplements. Seventy-six (62.3%) of the 122 HD patients not taking AA supplements exhibited deficient levels of AA (< 20 microM), while 13 (56.5%) of the 23 CRF patients and 9 (18.8%) of the 48 C showed deficient levels of AA. Analysis of AA metabolites in the normal-range AA (20-80 microM) group revealed that the DHA/AA ratio in HD patients was significantly higher than in C (3.3 +/- 2.6% and 1.2 +/- 2.2%, respectively). The DKG/AA ratio in HD patients was higher than in CRF patients (3.6 +/- 5.2% vs. 0.9 +/- 1.9%), whereas DKG was not detected in C. When compared to serum levels before the start of dialysis, serum AA, DHA and DKG concentrations at the end of the dialysis session decreased by an average of 74.2, 84.0 and 78.8% respectively. In HD patients, serum levels of thiobarbituric reactive substances (TBARS) were significantly lower in the higher AA (> 80 microM) group than in the deficient and normal-range AA groups. In 12 AA-deficient patients, after 1 month of taking AA supplements (200 mg/day), serum AA levels rose to 79.9 microM, while serum TBARS level declined when compared with levels before supplementation. In conclusion, the frequency of AA deficiency in dialysis patients is extremely high. AA deficiency in HD patients may result in high TBARS levels, which reflect increased oxidative stress. Adequate AA supplementation should therefore be considered in such patients.

Ascorbic acid in blood serum of patients with pulmonary tuberculosis and pneumonia.

Ascorbic acid plays a major role in pulmonary antioxidant defense. Sufficient amounts of ascorbic acid are necessary to maintain normal metabolic processes in the lung. We measured the levels of ascorbic, dehydroascorbic and diketogulonic acids in blood serum of patients with pulmonary tuberculosis (PTB) and pneumonia. The serum levels of ascorbic acid were decreased in PTB and pneumonia, and those of dehydroascorbic acid were decreased in PTB, but not in pneumonia. The serum diketogulonic acid levels were not significantly changed in either PTB or pneumonia. The ratio of ascorbic to dehydroascorbic acid levels in serum were increased in PTB, but in pneumonia we observed a significant decrease in this index. The ratio of dehydroascorbic to diketogulonic acid in PTB was decreased, but in pneumonia this index did not significantly differ from the control value. Thus, in PTB the rate of ascorbic acid oxidation is decreased and the rate of dehydroascorbic acid oxidation is increased. By contrast, in pneumonia the rate of ascorbic acid oxidation is increased, but the rate of dehydroascorbic acid oxidation did not differ from control values.

Light-induced byproducts of vitamin C in multivitamin solutions.

BACKGROUND: When solutions of multivitamin preparations (MVPs) are exposed to light, H(2)O(2) as well as organic peroxides are generated and the concentration of vitamin C decreases. The aim of this study was to determine, using mass spectrometry, whether the generation of oxidative byproducts of vitamin C, such as dehydroascorbate (DHA) and 2,3-diketogulonic acid (DKG), accounted for the reported decrease in ascorbic acid in MVPs exposed to light. METHODS: Mass spectrometry was used to document the formation of byproducts of ascorbic acid in solutions containing a MVP, vitamin C + riboflavin, and vitamin C + H(2)O(2) + Fe(2+). The involvement of ascorbic acid and H(2)O(2) in the formation of organic peroxides was tested by measuring peroxide concentrations in solutions containing H(2)O(2) with or without ascorbic acid and with or without Fe(2+) before and after addition of catalase. RESULTS: The loss of ascorbic acid in photo-exposed MVPs was associated with the concomitant generation of byproducts different from DHA and DKG. Among them, one mass fingerprint was particularly observed with solutions of vitamin C + riboflavin exposed to ambient light as well as with the solution of vitamin C + H(2)O(2) + Fe(2+), suggesting a Fenton-like reaction. This fingerprint was associated with the formation of catalase-resistant peroxides. CONCLUSION: Exposure of MVPs to light leads to the rapid loss of ascorbic acid and generation of specific byproducts that differ from DHA and DKG. The conversion of vitamin C into byproducts could be of biological importance in accounting for the decrease in ascorbic acid concentrations and the generation of organic peroxides in light-exposed MVPs.

Prenatal infection obliterates glutamate-related protection against free hydroxyl radicals in neonatal rat brain.

Prenatal infection constitutes an important risk factor for brain injury, in both premature and full-term infants. Unfortunately, as the mechanisms involved are far from understood, no therapeutic strategy emerges to prevent the damage. We tested the hypothesis that administration of lipopolysaccharide (LPS) to gravid female rats enhanced glutamate-induced oxidative stress in brain of pups. A microdialysis probe was implanted into the striatum of 14-day-old animals and the release of hydroxyl radicals (.OH) in the perfusion medium was evaluated. Glutamate promoted a delayed.OH release in the offspring of dams given LPS, contrasting with the.OH decreases observed in control animals. A similar response occurred after infusion of (R,S)-3,5-dihydroxyphenylglycine (DHPG), a Group I metabotropic glutamate receptor (mGluR) agonist. This response was not consecutive to a remote activation of N-methyl-D-aspartate (NMDA) receptors, as it was unaffected by an NMDA receptor antagonist. Furthermore, the response to NMDA itself decreased in the offspring of dams given LPS. Massive amounts of DHPG, however, likely internalizing the mGlu receptor, still blunted the response to NMDA, as in controls. No quantitative variation occurred in mGluR1, mGluR5, or the NR1 subunit of the NMDA receptor between controls and neonates born from LPS-treated dams. Direct LPS injection into age-matched pups, by contrast, affected the response to neither glutamate nor DHPG. These results confirm that normally during perinatal development, the brain is protected from any oxidative stress resulting from excess glutamate, and the results support the hypothesis that maternal infection before delivery may lead to critical brain damage via the release of toxic free radicals.

Vitamin C metabolomic mapping in experimental diabetes with 6-deoxy-6-fluoro-ascorbic acid and high resolution 19F-nuclear magnetic resonance spectroscopy.

Metabolomic mapping is an emerging discipline geared at providing information on a large number of metabolites as a complement to genomics and proteomics. Here we have probed ascorbic acid homeostasis and degradation in diabetes using 6-deoxy-6-fluoro ascorbic acid (F-ASA) and 750 MHz (19)F-nuclear magnetic resonance (NMR) spectroscopy with proton decoupling In vitro, Cu(2+)-mediated degradation of F-ASA revealed the formation of 4 major stable degradation products at 24 hours. However, when normal or diabetics rats were injected with F-ASA intraperitoneally (IP) for 4 days, up to 20 fluorine-labeled compounds were observed in the urine. Their composition resembled, in part, metal catalyzed degradation of F-ASA and was not explained by spontaneous degradation in the urine. Diabetes led to a dramatic increase in urinary F-ASA loss and a relative decrease in most other urinary F-compounds. Diabetes tilted F-ASA homeostasis toward oxidation in liver (P <.01), kidney (P <.01), spleen (P <.01), and plasma (P <.01), but tended to decrease oxidation in brain, adrenal glands, and heart. Surprisingly, however, besides the major oxidation product fluoro-dehydroascorbic acid (F-DHA), no F-ASA advanced catabolites were detected in tissues at 5 micromol/L sensitivity. These findings not only confirm the key role of the kidney in diabetes-mediated loss of ascorbic acid, but demonstrate that only selected tissues are prone to increased oxidation in diabetes. While the structure of most degradation products needs to be established, the method illustrates the power of high resolution (19)F-NMR spectroscopy for the mapping of complex metabolomic pathways in disease states.

Vitamin C metabolomic mapping in the lens with 6-deoxy-6-fluoro-ascorbic acid and high-resolution 19F-NMR spectroscopy.

PURPOSE: Metabolomics, or metabolic profiling, is an emerging discipline geared to providing information on a large number of metabolites, as a complement to genomics and proteomics. In the current study, a fluorine-labeled derivative of ascorbic acid (F-ASA), a major antioxidant- and UV-trapping molecule in the aqueous humor and the lens, was used to investigate the extent to which the lens accumulates potentially toxic degradation products of vitamin C. METHODS: Human lens epithelial cells (HLE-B3) and rat lenses were exposed to hyperglycemic or oxidative stress in vitro or in vivo and probed for accumulation of F-ASA, fluoro-dehydroascorbate (F-DHA), fluoro-2,3-diketogulonate (F-DKG), and their degradation products in protein-free extracts, by proton-decoupled 750-MHz (19)F-nuclear magnetic resonance (NMR) spectroscopy. RESULTS: F-ASA and F-DHA were taken up into HLE B-3 cells by an Na(+)-dependent transporter. Their uptake was unexpectedly only slightly affected by hyperglycemia in vitro, unless glutathione was severely depleted. Glycemic stress catalyzed oxidation of F-ASA into a single novel F-compound at -212.4 ppm, whereas F-DHA and F-DKG were the major degradation products observed after GSH depletion. In contrast, F-ASA uptake was markedly suppressed in diabetic cataractous rat lenses, which accumulated both the F-DHA and the -212.4-ppm compound. In an unexpected finding, the latter formed only from F-ASA and not F-DHA or F-DKG, suggesting a novel pathway of in vivo F-ASA degradation. Both the cells and the intact rat and human lenses were permeable to several advanced F-ASA and F-DHA degradation products, except F-DKG. The unknown compound at -212.4 ppm was the only F-ASA degradation product that spontaneously formed in rabbit aqueous humor upon incubation with F-ASA. CONCLUSIONS: These studies suggest the existence of a novel ascorbic-acid-degradation pathway in the lens and aqueous humor that is influenced by the nature of the oxidant stress. Under similar culture conditions, intact lenses are more prone to hyperglycemia-mediated oxidant stress than are lens epithelial cells, but both are permeable to various F-ASA degradation products, the structure and biological roles of which remain to be established.

Identification of 3,4-dihydroxy-2-oxo-butanal (L-threosone) as an intermediate compound in oxidative degradation of dehydro-L-ascorbic acid and 2,3-diketo-L-gulonic acid in a deuterium oxide phosphate buffer.

Dehydro-L-ascorbic acid (DAA), an oxidation product of L-ascorbic acid (vitamin C), is unstable in the neutral and basic pH regions. When DAA was incubated in a phosphate buffer with deuterium oxide (pH 7.4), it was degraded to form the main degradation compound, which was identified as 3,4-dihydroxy-2-oxobutanal (L-threosone). This compound was also formed from diketo-L-gulonic acid (DKG) in a phosphate buffer with deuterium oxide. L-threosone had reducing activity, probably due to its enolization, and is likely to have been involved in the formation of the reducing activity that was observed in aqueous DAA and DKG solutions. As a reactive dicarbonyl compound, L-threosone might also take some role in the cross-linking of tissue proteins that are formed in vivo in the Maillard reaction.

Role of biological antioxidants in benzanthrone toxicity.

Previous studies indicate that benzanthrone, an anthraquinone dye intermediate, caused significant depletion of ascorbic acid (AsA). In this investigation the effect of benzanthrone on the status of different forms of AsA and other bio-antioxidants such as glutathione (GSH) was studied. Oral administration of benzanthrone (50, 125 or 250 mg/kg body weight) resulted in a significant increase of urinary AsA levels with a concomitant decrease in the urinary dehydroascorbic acid (DHA) content in both rats and guinea-pigs. Benzanthrone caused a dose-dependent decrease in hepatic, adrenal and serum AsA levels with a subsequent increase in DHA and diketogulonic acid (DKA) levels in both rats and guinea-pigs. Following benzanthrone treatment, rats showed an increase in the scorbutic index (to 1.01-1.21) of the liver, adrenal glands and serum compared to controls (0.12-0.24). The scorbutic indices of liver, adrenal glands and serum were also substantially increased (to 3.61-11.20) in benzanthrone-treated guinea-pigs compared to controls (0.16-0.38). Single oral administration of benzanthrone to guinea-pigs caused a dose-dependent depletion of GSH in liver (15-51%), adrenal glands (27-64%) and serum (32-86%). Furthermore, the depletion of GSH by benzanthrone in rats was of a lesser degree. This suggests that continued exposure of guinea-pigs to benzanthrone may lead to scurvy-type changes in this animal species but not to the same extent in rats, since the latter has the enzymatic capacity to synthesise AsA. Therefore, it can be hypothesised that benzanthrone per se, or its metabolites, interact with reduced GSH thereby causing its depletion. Furthermore, in order to replenish the depleted GSH levels, AsA might be oxidized to DHA and hence the decrease in AsA with the simultaneous increase in DHA was observed.

Effects of 2,3-diketo-L-gulonic acid on the oxidation of yolk lipoprotein.

2,3-Diketo-L-gulonic acid (DKG) is an important intermediate product of oxidative degradation of L-ascorbic acid (AsA) in both biological and food systems, but the physiological function of DKG is still unclear. In this study, it was found that DKG had a strong antioxidative effect on copper-dependent oxidative modification of yolk lipoprotein (YLP), on the basis of both the decreased electrophoretic mobility and longer lag time of conjugated diene formation in a concentration-dependent manner. DKG is known to be very unstable and easily converts into two delta-lactones of DKG, the 3,4-enediol form of DKG delta-lactone (3,4-DKGL) and 2,3-enediol form of DKG delta-lactone (2,3-DKGL) depending on both pH and temperature. 3,4-DKGL was thought to be the first degradation product of DKG and could play an antioxidative role in the oxidation of lipoproteins induced by copper ion or peroxyl radicals in neutral aqueous solution.

[The excretion of ascorbic acid and its metabolites in uremia and hemodialysis]. / Vyvedenie askorbinovoi kisloty i ee metabolitov pri uremii i gemodialize.

Renal excretion of ascorbic, dehydroascorbic and diketogulonic acids in uremia and relevant loss in hemodialysis are measured in comparison with those in patients with uremic syndrome (prior to hemodialysis) and in healthy subjects (control). Renal elimination of ascorbic acid was higher while of dehydroascorbic acid lower vs control. Elimination of diketogulonic acid was similar to control. In a session of hemodialysis, the organism loses 132.0 +/- 13.6 mg of ascorbic, 132.0 +/- 10.0 mg of dehydroascorbic and 204.0 +/- 9.0 mg of diketogulonic acid. 48-hour urinary losses of the patients reached 8.4 +/- 1.4, 19.6 +/- 1.1, 75.6 +/- 1.5 mg, respectively. Compared to control, hemodialysis patients lose the above acids 24.3, 2.7 and 4.6 times more.

The non-oxidative degradation of ascorbic acid at physiological conditions.

The degradation of L-ascorbate (AsA) and its primary oxidation products, L-dehydroascorbate (DHA) and 2,3-L-diketogulonate (2, 3-DKG) were studied under physiological conditions. Analysis determined that L-erythrulose (ERU) and oxalate were the primary degradation products of ASA regardless of which compound was used as the starting material. The identification of ERU was determined by proton decoupled (13)C-nuclear magnetic resonance spectroscopy, and was quantified by high performance liquid chromatography, and enzymatic analysis. The molar yield of ERU from 2,3-DKG at pH 7.0 37 degrees C and limiting O(2)97%. This novel ketose product of AsA degradation, was additionally qualitatively identified by gas-liquid chromatography, and by thin layer chromatography. ERU is an extremely reactive ketose, which rapidly glycates and crosslinks proteins, and therefore may mediate the AsA-dependent modification of protein (ascorbylation) seen in vitro, and also proposed to occur in vivo in human lens during diabetic and age-onset cataract formation.

An electrospray ionization ion trap mass spectrometric (ESI-MS-MSn) study of dehydroascorbic acid hydrolysis at neutral pH.

The hydrolysis of dehydroascorbic acid (DAAH) at neutral pH and 27 degrees C was investigated by direct infusion electrospray ionisation ion trap mass spectrometry (ESI-MS). This approach permitted derivatisation and elution procedures to be avoided, reducing to the minimum extent sample manipulation and allowing a rapid and direct observation of the species involved in the reaction. Six main peaks, related to hydrated dehydroascorbate (HyDAA-) and diketogulonate (HyDKG-) anions, were observed in the mass spectra of DAAH solutions at different times of incubation and were characterised by MSn experiments. The relevant signal intensities changed with time and a model, based on the irreversible pseudo-first order HyDAA(-)-->HyDKG- conversion, fitted successfully the data obtained for dehydroascorbate. The kinetic constant of the process was (3.2 +/- 0.5) x 10(-2) min-1. The influence of metal ion traces on the hydrolysis rate was also checked, performing experiments in the presence of EDTA, and was found to be negligible.