Ascorbyl palmitate as a carrier of ascorbate into neural tissues

We have investigated the hypothesis that a lipid-soluble derivative of ascorbic acid, ascorbyl-6-palmitate (AP), could serve as a carrier of ascorbate into neural tissues. Ascorbate could then exert its physiological effects in the biomembranes that are the target sites of the cellular signaling pathways which are normally hardly accessible to this water-soluble compound. The potential role of AP would require that it penetrates into tissues. The major objective of the study was to determine whether ascorbate could be recovered from cerebral cortex and carotid body tissues, both sensitive to the hypoxic stimulus, after AP given by gavage. Biological samples were analyzed by HPLC for the determination of ascorbate. We found that ascorbate was recovered from the tissues studied. Its content was higher in both tissues, by nearly an order of magnitude, after ingestion of AP than after ingestion of ascorbic acid, and the ascorbate level was higher in the carotid body than in the cortex. Hypoxia decreased the ascorbate content which implies physiological activity of ascorbate carried alongside the AP molecule. The lipophilic AP was able to cross biological barriers and satisfied the tissue demand for ascorbate better than the hydrophilic form. AP should be considered as the preferred form of transport of ascorbate into neural tissues. The results of this study suggest wider pharmacological applications of ascorbyl palmitate.     

Pulmonary bioavailability of ascorbic acid in an ascorbate-synthesising species,the horse

Vitamin C (ascorbic acid) is a non-enzymatic antioxidant important in protecting the lung against oxidative damage and is decreased in lung lining fluid of horses with airway inflammation. To examine possible therapeutic regimens in a species with ascorbate-synthesising capacity, we studied the effects of oral supplementation of two forms of ascorbic acid, (each equivalent to 20 mg ascorbic acid per kg body weight) on the pulmonary and systemic antioxidant status of six healthy ponies in a 3 x 3 Latin square design. Two weeks supplementation with ascorbyl palmitate significantly increased mean plasma ascorbic acid concentrations compared to control (29 +/- 5 and 18 +/- 7 micromol/l, respectively; p < 0.05). Calcium ascorbyl-2-monophosphate, a more stable form of ascorbic acid, also increased mean plasma ascorbic acid concentrations, but not significantly (23 +/- 1 micromol/l; p = 0.07). The concentration of ascorbic acid in bronchoalveolar lavage fluid increased in five out of six ponies following supplementation with either ascorbyl palmitate or calcium ascorbyl-2-monophosphate compared with control (30 +/- 10, 25 +/- 4 and 18 +/- 8 micromol/l, respectively; p < 0.01). Neither supplement altered the concentration of glutathione, uric acid or alpha-tocopherol in plasma or bronchoalveolar lavage fluid. In conclusion, the concentration of lung lining fluid ascorbic acid is increased following ascorbic acid supplementation (20 mg/kg body weight) in an ascorbate-synthesising species.     

An isocratic HPLC method for the simultaneous determination of novel stable lipophilic ascorbic acid derivatives and their metabolites

2-O-alpha-D-glucopyranosyl-6-O-hexadecanoyl-L-ascorbic acid (6-sPalm-AA-2G), a novel stable lipophilic ascorbic acid derivative, was hydrolyzed to 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G), ascorbyl 6-palmitate (6-sPalm-AA) and ascorbic acid (AA) with alpha-glucosidase and lipase. An HPLC method for the simultaneous determination of AA, AA-2G, 6-sPalm-AA and 6-sPalm-AA-2G was developed using a cyanopropyl column with an isocratic solution of methanol-phosphate buffer (pH 2.1) (65:35, v/v) containing 20mg/l of dithiothreitol at a detection wavelength of 240 nm. The calibration curves were found to be linear in the range of 10-200 microM. Linear regression analysis of the data demonstrated the efficacy of the method in terms of precision and accuracy. This method was satisfactorily applied to the determination of 6-sPalm-AA-2G and its three metabolites in a 6-sPalm-AA-2G solution treated with purified enzymes or a small intestine post-mitochondrial supernatant and to the separation of novel stable lipophilic AA derivatives other than 6-sPalm-AA-2G and their metabolites. AA, AA-2G and other well-known stable AA derivatives, ascorbic acid 2-phosphate and ascorbic acid 2-sulfate, were also separated under the same conditions. The results show that the procedure is rapid and simple and that it can be employed for in vitro metabolic analysis of various AA derivatives.     

Generation and recycling of radicals from phenolic antioxidants

Hindered phenols are widely used food preservatives. Their pharmacological properties are usually attributed to high antioxidant activity due to efficient scavenging of free radicals. Butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) also cause tissue damage. Their toxic effects could be due to the production of phenoxyl radicals. If phenoxyl radicals can be recycled by reductants or electron transport, their potentially harmful side reactions would be minimized. A simple and convenient method to follow phenoxyl radical reactions in liposomes and rat liver microsomes based on an enzymatic (lipoxygenase + linolenic acid) oxidation system was used to generate phenoxyl radicals from BHT and its homologues with substitutents in m- and p-positions. Different BHT-homologues display characteristic ESR signals of their radical species. In a few instances the absence of phenoxyl radical ESR signals was found to be due to inhibition of lipoxygenase by BHT-homologues. In liposome or microsome suspensions addition of ascorbyl palmitate resulted in disappearance of the ESR signal of phenoxyl radicals with concomittant appearance of the ascorbyl radical signal. After exhaustion of ascorbate, the phenoxyl radical signal reappears. Comparison of the rates of ascorbyl radical decay in the presence or absence of BHT-homologues showed that temporary elimination of the phenoxyl radical ESR signal was due to their reduction by ascorbate. Similarly, NADPH or NADH caused temporary elimination of ESR signals as a result of reduction of phenoxyl radicals in microsomes. Since ascorbate and NADPH might generate superoxide in the incubation system used, SOD was tested. SOD shortened the period, during which the phenoxyl radicals ESR signal could not be observed. Both ascorbyl palmitate and NADPH exerted sparing effects on the loss of BHT-homologues during oxidation. These effects were partly diminished by SOD. These data indicate that reduction of phenoxyl radicals was partly superoxide-dependent. It is concluded that redox recycling of phenoxyl radicals can occur by intracellular reductants like ascorbate and microsomal electron transport.     

Ascorbate 6-palmitate protects human erythrocytes from oxidative damage

Lipid-soluble antioxidants, such as α-tocopherol, protect cell membranes from oxidant damage. In this work we sought to determine whether the amphipathic derivative of ascorbate, ascorbate 6-palmitate, is retained in the cell membrane of intact erythrocytes, and whether it helps to protect the cells against peroxidative damage. We found that ascorbate 6-palmitate binding to erythrocytes was dose-dependent, and that the derivative was retained during the multiple wash steps required for preparation of ghost membranes. Ascorbate 6-palmitate remained on the extracellular surface of the cells, because it was susceptible to oxidation or removal by several cell-impermeant agents. When bound to the surface of erythrocytes, ascorbate 6-palmitate reduced ferricyanide, an effect that was associated with generation of an ascorbyl free radical signal on EPR spectroscopy. Erythrocyte-bound ascorbate 6-palmitate protected membrane α-tocopherol from oxidation by both ferricyanide and a water-soluble free radical initiator, suggesting that the derivative either reacted directly with the exogenously added oxidant, or that it was able to recycle the α-tocopheroxyl radical to α-tocopherol in the cell membrane. Ascorbate 6-palmitate also partially protected cis-parinaric acid from oxidation when this fluorescent fatty acid was intercalated into the membrane of intact cells. These results show that an amphipathic ascorbate derivative is retained on the exterior cell surface of human erythrocytes, where it helps to protect the membrane from oxidant damage originating outside the cells.     

Enzymatic synthesis of palm-based ascorbyl esters

The synthesis of palm-based ascorbyl esters through transesterification of ascorbic acid and palm oil in tert-amyl alcohol catalyzed by immobilized lipase is described. Highest conversion (70–75%) was determined after 16 h reaction at 40 °C using lipase (Novozyme 435 from Candida antartica) with an ascorbic acid to palm oil mole ratio of 1:8. The purified product was further characterized by ¹³C NMR and GC–MS and the mixture of ascorbyl monoesters obtained were identified as ascorbyl monooleate (61%), ascorbyl monopalmitate (30%) and ascorbyl monostearate (9%). The antioxidant activity of palm-based ascorbyl esters was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) test. The results showed that pure palm-based ascorbyl esters have an antioxidant activity with an IC₅₀ value of 0.1 mg/mL.     

The hydroxyl free radical reactions of ascorbyl palmitate as measured in various in vitro models.

The OH(*) free radical scavenging properties of ascorbyl palmitate (AP), water-solubilized in the presence of a surfactant (Brij 35), were tested in various systems: (1) The inhibition of polymerization of bovine serum albumin by OH(*) free radicals generated by the Fenton reaction indicated AP exerts a considerable protective effect against polymerization by scavenging the OH(*) free radicals. (2) ESR spin trapping comparisons of DMPO with AP were conducted. Using the Fenton reaction as a source of OH(*) free radicals, AP was 1 order of magnitude faster in scavenging these radicals than DMPO. (3) Oxidative modification of BSA by (60)Co-gamma irradiation of 80 krad, results in a strong increase in protein carbonyl content. AP inhibits carbonyl formation very efficiently, indicating that AP may be utilized as a biological OH(*) free radical scavenger in human therapy.     

Ascorbyl free radical as a reliable indicator of free-radical-mediated myocardial ischemic and post-ischemic injury. A real-time continuous-flow ESR study

The real-time kinetics of the release of ascorbyl free radicals in the coronary perfusate from isolated rat hearts submitted to an ischemia/reperfusion sequence has been achieved by continuous-flow ESR using high-speed acquisition techniques. Enhanced ESR detection of ascorbyl free radicals was obtained by addition of dimethyl sulfoxide (Me2SO), a strong cation chelator and oxidizing agent. A continuous-flow device allowed a direct monitoring of the ascorbyl free radical and/or ascorbate leakage in coronary perfusate by observation of the ascorbyl radical doublet (aH = 0.188 mT and g = 2.0054). 1. The results showed that ascorbyl free radical release occurred mainly during sequences of low-flow ischemia (90 min) coupled or not with 30 min of zero-flow ischemia followed by reperfusion (60 min). The kinetic profiles of ascorbyl-free-radical detection confirm in quantitative terms the expected correlation between the duration of the ischemic insult and the magnitude of ascorbate extracellular release upon reperfusion. There is indication that ascorbyl free radical depletion could be secondary to oxygen-derived-free-radical-induced cellular damage. 2. The amount of residual ascorbic acid was quantitated on myocardial tissue at the end of reperfusion using Me2SO as extracting solvent. Intense oxidation of ascorbate and chemical stabilization of the resulting free radical species provided by Me2SO allowed ESR measurement of a marked tissue ascorbate depletion related to the duration of ischemia. 3. Perfusion of superoxide dismutase during low-flow ischemia and the first 10 min of reperfusion greatly inhibited both extracellular release and endogenous ascorbate depletion. These results suggest that the ascorbate redox system constitutes a major protective mechanism against free-radical-induced myocardial injury. 4. The proposed direct ESR detection of ascorbyl free radicals in the coronary perfusates or in tissue extracts does not require extensive chemical preparation and conditioning of effluent or tissue samples. It provides an interesting straightforward alternative to the evaluation of detrimental free radical processes affecting the myocardium during ischemia and reperfusion.     

Enzymatic synthesis and anti-oxidative activities of plant oil-based ascorbyl esters in 2-methyltetrahydrofuran-containing mixtures

Ascorbyl fatty acid esters are commercially interesting fat-soluble antioxidants. In this work, enzymatic synthesis of ascorbyl esters from less expensive and readily available plant oils, and their anti-oxidative activities are described. Among the immobilized lipases tested, Candida antarctica lipase B was the best for the synthesis of plant oil-based ascorbyl esters. The enzyme showed much better catalytic performances in the binary mixtures of biomass-based 2-methyltetrahydrofuran (MeTHF) and t-butanol than the previously preferred t-butanol. The conversions of 70–73% were obtained under the optimal reaction conditions after 24?h, with the unsaturated fatty acid esters (oleate and linoleate, 80–90%) as the major products. The immobilized lipase kept the relative activity of 80% after reuse for 6 batches in MeTHF-containing system. Besides, anti-oxidative activities of plant oil-based ascorbyl esters and ascorbic acid were comparable, which could remove α,α-diphenyl-β-picrylhydrazyl (DPPH) free radical of >87%.     

Pyridoxal isonicotinoyl hydrazone inhibits iron-induced ascorbate oxidation and ascorbyl radical formation

Previous work from our laboratory demonstrated that pyridoxal isonicotinoyl hydrazone (PIH) has in vitro antioxidant activity against iron plus ascorbate-induced 2-deoxyribose degradation due to its ability to chelate iron; the resulting Fe(III)-PIH(2) complex is supposedly unable to catalyze oxyradical formation. A putative step in the antioxidant action of PIH is the inhibition of Fe(III)-mediated ascorbate oxidation, which yields the Fenton reagent Fe(II) [Biochim. Biophys. Acta 1523 (2000) 154]. In this work, we demonstrate that PIH inhibits Fe(III)-EDTA-mediated ascorbate oxidation (measured at 265 nm) and the formation of ascorbyl radical (in electron paramagnetic resonance (EPR) studies). The efficiency of PIH against ascorbate oxidation, ascorbyl radical formation and 2-deoxyribose degradation was dose dependent and directly proportional to the period of preincubation of PIH with Fe(III)-EDTA. The efficiency of PIH in inhibiting ascorbate oxidation and ascorbyl radical formation was also inversely proportional to the Fe(III)-EDTA concentration in the media. When EDTA was replaced by the weaker iron ligand nitrilotriacetic acid (NTA), PIH was much more effective in preventing ascorbate oxidation, ascorbyl radical formation and 2-deoxyribose degradation. Moreover, the replacement of EDTA with citrate, a physiological chelator with a low affinity for iron, also resulted in PIH having a higher efficiency in inhibiting iron-mediated ascorbate oxidation and 2-deoxyribose degradation. These results demonstrate that PIH removes iron from EDTA (or from either NTA or citrate), forming an iron-PIH complex that cannot induce ascorbate oxidation effectively, thus inhibiting iron-mediated oxyradical formation. These results are of pharmacological relevance because PIH has been considered for experimental chelating therapy in iron-overload diseases.     

Synthesis and antioxidant properties of a new lipophilic ascorbic acid analogue

4-(4-Hydroxyphenyl)-5-(4-hydroxyphenylmethyl)-2-hydroxyfurane-2-one 1 was prepared by an acidic dimerisation of 4-hydroxyphenylpyruvic acid and some of its antioxidant and spectroscopic properties have been measured and compared to that of ascorbic acid. 1 is as good an antioxidant as ascorbic acid in the DPPH (2,2-diphenyl-1-picryl hydrazyl radical) test and the inhibition of hydroxyl radical and a powerful inhibitor of the Cu(2+) or AAPH (2,2'-azobis-(2-amidinopropane) dihydrochloride) induced oxidation of human LDL. 1 gives a stable radical characterised by its ESR spectrum similarly to ascorbic acid but in lower concentration and with a different reactivity towards nitroxides. Theoretical calculations allow us to propose the structure for the radical formed from 1, to explain its lower stability than ascorbyl radical and to evaluate the lipophilicity of 1.     

Optimal EPR detection of weak nitroxide spin adduct and ascorbyl free radical signals.

We have investigated the optimal nominal power settings for the electron paramagnetic resonance detection of typical nitroxides, nitroxide spin adducts, and the ascorbyl free radical. In room temperature aqueous solution, we find that, for all the nitroxides examined, saturation effects begin at approx. 25 mW nominal power with maximum signal intensity achieved at approx. 100 mW power when using a TM110 cavity. For the ascorbyl free radical, we find that saturation effects begin at approx. 16 mW nominal power and that maximum peak-to-peak signal amplitude is obtained at approx. 40 mW microwave power. For the ascorbyl free radical, we find that a modulation amplitude of approx. 0.65 G yields the maximum signal height for the doublet signal. This information will help researchers maximize the EPR signal height of minimally detectable free radicals such as encountered in biological systems.     

Optimized enzymatic synthesis of ascorbyl esters from lard using Novozym 435 in co-solvent mixtures

A mild and efficient method for the conversion of fatty acid methyl esters from lard into ascorbyl esters via lipase-catalyzed transesterification in co-solvent mixture is described. A solvent engineering strategy was firstly applied to improve fatty acid ascorbyl esters production. The co-solvent mixture of 30% t-pentanol:70% isooctane (v/v) was optimal. Response surface methodology (RSM) and central composite design (CCD) were employed to estimate the effects of reaction parameters, such as reaction time (12–36 h), temperature (45–65 °C), enzyme amount (10–20%, w/w, of fat acid methyl esters), and substrate molar ratio of fatty acid methyl esters to ascorbic acid (8:1–12:1) for the synthesis of fatty acid ascorbyl esters in co-solvent mixture. Based on the RSM analysis, the optimal reaction conditions were determined as follows: reaction time 34.32 h, temperature 54.6 °C, enzyme amount 12.5%, substrate molar ratio 10.22:1 and the maximum conversion of fatty acid ascorbyl esters was 69.18%. The method proved to be applicable for the synthesis of ascorbyl esters using Novozym 435 in solvent.     

Stopped-flow ESR study on the reactivity of vitamin E, vitamin C and its lipophilic derivatives towards Fremy's salt in micellar systems

The reaction between Fremy's salt and alpha-tocopherol (VE), ascorbic acid (VC) and its lipophilic derivatives ascorbyl-6-caprylate (VC-8), 6-laurate (VC-12) and 6-palmitate (VC-16) were studied by stopped-flow ESR spectroscopy in cetyl trimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS) micelles, as a model reaction of these antioxidants with alkyl peroxy radicals in biological systems. The second order rate constants for the reaction of Fremy's salt with VE in CTAB and SDS micelles were found to be 7.9 x 10(3) and 2.2 M-1 s-1, respectively, with as high as a 3600-fold variation. Rate constants for VC, VC-8, VC-12 and VC-16 are 4.3, 35, 53 and 56 x 10(3) M-1 s-1 and 3.3, 2.7, 1.2 and 0.86 x 10(3) M-1 s-1 in CTAB and SDS micelles, respectively. The results demonstrate remarkable effects of the charge type of the micelles and the side-chain of the antioxidants on the antioxidation reactivity in the micelles. It reveals that the inter-micellar diffusion may be the rate-limiting step for antioxidation carried out in micelles.     

Ascorbic acid maintenance in HaCaT cells prevents radical formation and apoptosis by UV-B.

We have investigated the enzymatic reduction and accumulation of vitamin C in HaCaT epithelial cells. The subcellular localization and the activities of ascorbyl free radical reductase and dehydroascorbate reductase showed that mitochondrial, microsomal and plasma membranes fractions express high levels of ascorbyl free radical reductase activity, whereas dehydroascorbate reductase activity was found at low levels only in the post microsomal supernatant. We have also investigated cell proliferation and vitamin C accumulation induced by ascorbic acid 2-phosphate. This derivative caused no inhibition of cell growth, was uptaken from the extracellular medium and accumulated as ascorbic acid in mM concentrations. These results show that HaCaT cells possess very efficient systems to maintain high levels of both intracellular and extracellular ascorbic acid. The regeneration and uptake of ascorbic acid from extracellular medium contributes to the intracellular antioxidant capacity, as evaluated by 2',7'-dihydrodichlorofluorescein staining. Consequently, cells became more resistant to free radical generation and cell death induced by UV-B irradiation.     

Lucigenin chemiluminescence in human seminal plasma

Seminal plasma protects spermatozoa from the detrimental effects of reactive oxygen species such as hydrogen peroxide. We investigated the lucigenin-dependent chemiluminescence in cell-free seminal plasma from andrological patients. The seminal plasma was separated from cells by centrifugation. In all seminal plasmas studied lucigenin-dependent chemiluminescence (LCL) was detected. The LCL showed a strong pH-dependence. The signal was stable if samples were stored at +4°C for up to 4 days or up to 8 days at -80°C. Filtration of the samples (0.45 and 0.22 μm pore size) did not lower their luminescence. The addition of superoxide dismutase (SOD) and ascorbic acid oxidase (AAO) lowered LCL nearly to baseline values while trolox and desferal showed moderate effect, whereas allopurinol had no effect. Electron paramagnetic resonance spectroscopy demonstrated ascorbyl radicals in seminal plasma. Physiological concentrations of ascorbic acid yielded SOD-inhibitable lucigenin-chemiluminescence. The nitroblue-tetrazolium assay showed that ascorbic acid in buffer solution produced formazan. Superoxide-anion radicals were not detected in seminal plasma by the spin-trap DEPMPO due to their low steady state concentration. It is concluded that in seminal plasma ascorbate reacts with molecular oxygen yielding ascorbyl radicals and superoxide anion. If lucigenin is added to seminal plasma, reducing substances present, such as ascorbate, reduce lucigenin to the corresponding radical; this radical reacts with molecular oxygen and also forms O₂^-2. So LCL in human seminal plasma results from the autoxidation of ascorbate and the oxidation of the reduced lucigenin. While the physiological relevance of the former mechanism is unknown, the latter is an artifact.     

Lucigenin chemiluminescence in human seminal plasma

Seminal plasma protects spermatozoa from the detrimental effects of reactive oxygen species such as hydrogen peroxide. We investigated the lucigenin-dependent chemiluminescence in cell-free seminal plasma from andrological patients. The seminal plasma was separated from cells by centrifugation. In all seminal plasmas studied lucigenin-dependent chemiluminescence (LCL) was detected. The LCL showed a strong pH-dependence. The signal was stable if samples were stored at +4°C for up to 4 days or up to 8 days at -80°C. Filtration of the samples (0.45 and 0.22 μm pore size) did not lower their luminescence. The addition of superoxide dismutase (SOD) and ascorbic acid oxidase (AAO) lowered LCL nearly to baseline values while trolox and desferal showed moderate effect, whereas allopurinol had no effect. Electron paramagnetic resonance spectroscopy demonstrated ascorbyl radicals in seminal plasma. Physiological concentrations of ascorbic acid yielded SOD-inhibitable lucigenin-chemiluminescence. The nitroblue-tetrazolium assay showed that ascorbic acid in buffer solution produced formazan. Superoxide-anion radicals were not detected in seminal plasma by the spin-trap DEPMPO due to their low steady state concentration. It is concluded that in seminal plasma ascorbate reacts with molecular oxygen yielding ascorbyl radicals and superoxide anion. If lucigenin is added to seminal plasma, reducing substances present, such as ascorbate, reduce lucigenin to the corresponding radical; this radical reacts with molecular oxygen and also forms O₂^-2.. So LCL in human seminal plasma results from the autoxidation of ascorbate and the oxidation of the reduced lucigenin. While the physiological relevance of the former mechanism is unknown, the latter is an artifact.     

Direct detection of circulating free radicals in the rat using electron spin resonance spectrometry.

We developed a new technique for directly observing in vivo free radical formation in the circulating blood of living rats using electron spin resonance (ESR) spectrometry without any labeling or trapping agents. It was found that a doublet peak spectrum was obtained following ferric citrate and ascorbic acid injection. The signals were confirmed in different ways to be due to ascorbic acid radicals. These results provide evidence to support the involvement of free radical intermediates in iron-ascorbic acid reactions, and further confirm the suggested mechanisms of both the adverse and protective effects of ascorbic acid in biological systems. Furthermore, this method of direct observation is a new application of ESR spectrometry to living animals.     

Tocopheroxyl radical persistence and tocopherol consumption in liposomes and in vitamin E-enriched rat liver mitochondria and microsomes

Substantial loading of rat liver mitochondrial and microsomal membranes with D-alpha-tocopherol was achieved by dietary supplementation with no adverse effects of this loading being apparent, e.g. on treadmill exercise endurance. The tocopheroxyl radical was readily detected by ESR in the enriched microsomes and mitochondria. Continuous enzymatic oxidation with horseradish peroxidase and a hydrophilic phenol, to favor selective oxidation of tocopherol without the involvement of lipid peroxidation, allowed the tocopheroxyl radical to be observed for up to 1 h in liposomes of dioleoylphosphatidylcholine and for about 15 min in the subcellular membranes. Total alpha-tocopherol decreased throughout this period, but a significant residual fraction remained after all the ESR signal of tocopheroxyl had disappeared. Decay kinetics of the tocopheroxyl radical ESR signal produced by a burst of intense UV irradiation consisted of a rapid initial phase and a slower exponential decay. A more narrow and more persistent ESR signal, not yet chemically identified, was observed after the tocopheroxyl radical had disappeared under prolonged oxidation. Ascorbic acid prevented formation of the tocopheroxyl radical until the ascorbyl radical ESR signal had decayed, whereas uric acid, up to saturating concentration in phosphate buffer, had no effect.     

Effect of dietary ascorbic acid supplementation and chronic hypoxia on sea bream growth and vitamin C status

Hypoxia (55% DO) and ascorbic acid (AA) supplementation level (0, 10, 25 and 50 mg AA equivalent kg^—1 diet, supplied as ascorbyl polyphosphate) did not significantly alter juvenile sea bream Sparus aurata growth, body composition, survival and skin hydroxyproline concentration. Liver, head kidney and spleen AA concentrations significantly decreased with decreasing dietary AA level, but were not influenced by hypoxia. This study showed that sea bream AA requirements were not influenced by chronic hypoxic stress.