Dehydroascorbic Acid Reduction in Several Tissues and Cultured Hepatocytes of the Chicken

Dehydroascorbic acid, the oxidized form of ascorbic acid, is rapidly reduced to ascorbate in living organs (ascorbate recycling). We examined the GSH-dependent dehydroascorbate reductase activity in several tissues of the chicken. The activity was highest in the liver, and second highest in the brain. The activity was localized in the cytosol fraction of the liver. We subsequently examined the dehydroascorbate reduction in separated chiken hepatocytes. The cellular ascorbate concentration was elevated in dehydroascorbate-treated cells. It is thought that hepatocytes incorporated external dehydroascorbate and converted it into ascorbate. These findings suggest that the liver plays an important role in ascorbate recycling by the chicken.     

Dehydroascorbic acid reduction in several tissues and cultured hepatocytes of the chicken.

Dehydroascorbic acid, the oxidized form of ascorbic acid, is rapidly reduced to ascorbate in living organs (ascorbate recycling). We examined the GSH-dependent dehydroascorbate reductase activity in several tissues of the chicken. The activity was highest in the liver, and second highest in the brain. The activity was localized in the cytosol fraction of the liver. We subsequently examined the dehydroascorbate reduction in separated chicken hepatocytes. The cellular ascorbate concentration was elevated in dehydroascorbate-treated cells. It is thought that hepatocytes incorporated external dehydroascorbate and converted it into ascorbate. These findings suggest that the liver plays an important role in ascorbate recycling by the chicken.     

Ascorbate uptake is decreased in the hippocampus of ageing rats

Ascorbate, an intracellular antioxidant, has been considered critical for neuronal protection against oxidant stress, which is supported especially by in vitro studies. Besides, it has been demonstrated an age-related decrease in brain ascorbate levels. The aims of the present study were to investigate ascorbate uptake in hippocampal slices from old Wistar rats, as well as its neuroprotective effects in in vitro and in vivo assays. Hippocampal slices from male Wistar rats aged 4, 11 and 24 months were incubated with radiolabeled ascorbate and incorporated radioactivity was measured. Hippocampal slices from rats were incubated with different concentrations of ascorbate and submitted to H(2)O(2)-induced injury, cellular damage and S100B protein levels were evaluated. The effect of chronic administration of ascorbate on cellular oxidative state and astrocyte biochemical parameters in the hippocampus from 18-months-old Wistar rats was also studied. The ascorbate uptake was decreased in hippocampal slices from old-aged rats, while supplementation with ascorbate (2 weeks) did not modify any tested oxidative status in the hippocampus and the incubation was unable to protect hippocampal slices submitted to oxidative damage (H(2)O(2)) from old rats. Our data suggest that the decline of ascorbate uptake might be involved in the brain greater susceptibility to oxidative damage with advancing age and both in vitro and vivo assays suggest that ascorbate supplementation did not protect hippocampal cells.     

The Glutamate Aspartate Transporter (GLAST) Mediates l-Glutamate-Stimulated Ascorbate-Release Via Swelling-Activated Anion Channels in Cultured Neonatal Rodent Astrocytes

Vitamin C (ascorbate) plays important neuroprotective and neuromodulatory roles in the mammalian brain. Astrocytes are crucially involved in brain ascorbate homeostasis and may assist in regenerating extracellular ascorbate from its oxidised forms. Ascorbate accumulated by astrocytes can be released rapidly by a process that is stimulated by the excitatory amino acid, l-glutamate. This process is thought to be neuroprotective against excitotoxicity. Although of potential clinical interest, the mechanism of this stimulated ascorbate-release remains unknown. Here, we report that primary cultures of mouse and rat astrocytes release ascorbate following initial uptake of dehydroascorbate and accumulation of intracellular ascorbate. Ascorbate-release was not due to cellular lysis, as assessed by cellular release of the cytosolic enzyme lactate dehydrogenase, and was stimulated by l-glutamate and l-aspartate, but not the non-excitatory amino acid l-glutamine. This stimulation was due to glutamate-induced cellular swelling, as it was both attenuated by hypertonic and emulated by hypotonic media. Glutamate-stimulated ascorbate-release was also sensitive to inhibitors of volume-sensitive anion channels, suggesting that the latter may provide the conduit for ascorbate efflux. Glutamate-stimulated ascorbate-release was not recapitulated by selective agonists of either ionotropic or group I metabotropic glutamate receptors, but was completely blocked by either of two compounds, TFB-TBOA and UCPH-101, which non-selectively and selectively inhibit the glial Na⁺-dependent excitatory amino acid transporter, GLAST, respectively. These results suggest that an impairment of astrocytic ascorbate-release may exacerbate neuronal dysfunction in neurodegenerative disorders and acute brain injury in which excitotoxicity and/or GLAST deregulation have been implicated.     

Modulation of copper accumulation and copper-induced toxicity by antioxidants and copper chelators in cultured primary brain astrocytes

Copper is essential for several important cellular processes, but an excess of copper can also lead to oxidative damage. In brain, astrocytes are considered to play a pivotal role in the copper homeostasis and antioxidative defence. To investigate whether antioxidants and copper chelators can modulate the uptake and the toxicity of copper ions in brain astrocytes, we used primary astrocytes as cell culture model. These cells accumulated substantial amounts of copper during exposure to copper chloride. Copper accumulation was accompanied by a time- and concentration-dependent loss in cell viability, as demonstrated by a lowering in cellular MTT reduction capacity and by an increase in membrane permeability for propidium iodide. During incubations in the presence of the antioxidants ascorbate, trolox or ebselen, the specific cellular copper content and the toxicity in copper chloride-treated astrocyte cultures were strongly increased. In contrast, the presence of the copper chelators bathocuproine disulfonate or tetrathiomolybdate lowered the cellular copper accumulation and the copper-induced as well as the ascorbate-accelerated copper toxicity was fully prevented. These data suggest that predominantly the cellular content of copper determines copper-induced toxicity in brain astrocytes.     

Cellular assay optimization: part I: the use of large-scale transiently transfected cryobanks and introduction of a c-Myc tag to design a standardized ELISA process

This study investigated the use of large-scale transiently transfected cryopreserved cells for medium-throughput cellular screening. The data generated indicated that preprepared transiently transfected cryobanks can be used for cell-based assays and in fact can greatly enhance the consistency of data generated by cellular screens. In addition to this, a generic enzyme-linked immunosorbent assay method was designed that introduced a c-Myc tag to four different targets and allowed all four cell assays to be run using a standardized process. These process improvements yielded cost savings and greatly reduced the required resource, as well as reducing timelines for developing cellular assays.     

The active role of vitamin C in mammalian iron metabolism: Much more than just enhanced iron absorption!

Ascorbate is a cofactor in numerous metabolic reactions. Humans cannot synthesize ascorbate owing to inactivation of the gene encoding the enzyme l-gulono-γ-lactone oxidase, which is essential for ascorbate synthesis. Accumulating evidence strongly suggests that in addition to the known ability of dietary ascorbate to enhance nonheme iron absorption in the gut, ascorbate within mammalian systems can regulate cellular iron uptake and metabolism. Ascorbate modulates iron metabolism by stimulating ferritin synthesis, inhibiting lysosomal ferritin degradation, and decreasing cellular iron efflux. Furthermore, ascorbate cycling across the plasma membrane is responsible for ascorbate-stimulated iron uptake from low-molecular-weight iron–citrate complexes, which are prominent in the plasma of individuals with iron-overload disorders. Importantly, this iron-uptake pathway is of particular relevance to astrocyte brain iron metabolism and tissue iron loading in disorders such as hereditary hemochromatosis and β-thalassemia. Recent evidence also indicates that ascorbate is a novel modulator of the classical transferrin–iron uptake pathway, which provides almost all iron for cellular demands and erythropoiesis under physiological conditions. Ascorbate acts to stimulate transferrin-dependent iron uptake by an intracellular reductive mechanism, strongly suggesting that it may act to stimulate iron mobilization from the endosome. The ability of ascorbate to regulate transferrin iron uptake could help explain the metabolic defect that contributes to ascorbate-deficiency-induced anemia.     

Ascorbate Modulates 5-[3H]Hydroxytryptamine Binding to Central 5-HT3 Sites in Bovine Frontal Cortex

Ascorbate is present in millimolar concentrations in mammalian brain and can be released from cellular stores by membrane depolarization. We report here that physiologically relevant concentrations of ascorbate modulate 5-[3H]hydroxytryptamine ([3H]5-HT) binding to bovine frontal cortex membranes. Under conditions where [3H]5-HT binding is reversible and saturable, ascorbate causes a concentration-dependent increase in the affinity of [3H]5-HT for central 5-HT3 binding sites. At pH 7.4, increasing ascorbate from 0 to 5.7 mM changes the equilibrium affinity constant (KD) of binding to 5-HT3 sites from 125 nM to 30 nM, without affecting binding site number. These ascorbate-induced effects are pH dependent. At pH 7.1 binding to central 5-HT3 sites is essentially eliminated in the presence of ascorbate. These studies suggest that ascorbate and hydrogen ion concentration interactions may modulate serotonergic function.     

The biosynthesis of ascorbate protects isolated rat hepatocytes from cumene hydroperoxide-mediated oxidative stress

Most animals synthesize ascorbate. It is an essential enzymatic cofactor for the synthesis of a variety of biological molecules and also a powerful antioxidant. There is, however, little direct evidence supporting an antioxidant role for endogenously produced ascorbate. Recently, we demonstrated that incubation of rat hepatocytes with 1-bromoheptane or phorone simultaneously depleted glutathione (GSH) and triggered rapid ascorbate synthesis. The present study investigates the hypothesis that endogenous ascorbate synthesis can confer protection against oxidative stress. Rat and guinea pig hepatocytes were depleted of GSH with 1-bromoheptane and subsequently treated with the oxidative stressor cumene hydroperoxide (CHP) in the presence or absence of the ascorbate synthesis inhibitor sorbinil. In rat hepatocytes, ascorbate content increased linearly (from 15.1 to 35.8 nmol/10(6) cells) over a 105-min incubation. Prior depletion of GSH increased CHP-induced cellular reactive oxygen species (ROS) production, lipid peroxidation, and cell death in rat and guinea pig hepatocytes. Inhibiting ascorbate synthesis, however, further elevated ROS production (2-fold), lipid peroxidation (1.5-fold), and cell death (2-fold) in rat hepatocytes only. This is the first time that endogenous ascorbate synthesis has been shown to decrease cellular susceptibility to oxidative stress. Protection by endogenously produced ascorbate may therefore need to be addressed when extrapolating data to humans from experiments using rodents capable of synthesizing ascorbate.     

Transport of vitamin C in animal and human cells

The transport systems of animal and human tissues for vitamin C are reviewed with respect to their properties. It emerges that pure diffusion plays only a very minor role, while a variety of more or less specific transporters is found on cellular membranes. Although most tissues prefer the reduced ascorbate over the oxidized dehydroascorbic acid and have high-affinity transporters for it, there are several examples for the reversed situation. Special attention is given to similarity or identity with glucose transporters, especially the GLUT-1 and the sodium-dependent intestinal and renal transporters, and to the very widespread dependence of ascorbate transport on sodium ions. The significance of ascorbate transport for vitamin C-requiring and nonrequiring species as well as alterations in states of disease can be seen from ample experimental evidence.     

Quantitative data on the contribution of GSH and Complex II dependent ascorbate recycling in plant mitochondria

The reduction of dehydroascorbate, the oxidized form of ascorbate plays important role in the maintenance of sufficient level of ascorbate. In plant mitochondria two DHA reducing mechanisms, the GSH-dependent and the mitochondrial electron transfer chain dependent ascorbate recycling have been characterized. Although both pathways have been extensively studied quantitative information about the electron fluxes from one or another direction for the reduction of DHA is not known. The cellular, mitochondrial glutathione pools and mitochondrial DHA reducing capacity was measured in BSO treated and control tobacco cells. While BSO caused dramatic decrease of cellular GSH content the difference was much smoother at mitochondrial level. The difference in DHA reduction capacity was even smoother affirming the existence of alternative, non-GSH dependent DHA reducing mechanism(s) in plant mitochondria. On the base of the parallel determination of mitochondrial GSH content and ascorbate production upon DHA addition, GSH (consumption) is responsible for the ~ 20 % of ascorbate production. Almost 90 % enhancement of ascorbate production could be provoked by the addition of Complex II substrate succinate which could be almost totally prevented by the concomitant addition of malonate or TTFA. On the base of these results, the importance of mitochondrial Complex II compared to GSH-dependent mechanisms in mitochondrial ascorbate recycling has been underestimated so far.     

Use of ascorbate in the preparation and maintenance of brain slices.

Ascorbate and glutathione (GSH) are normally concentrated in brain cells at millimolar levels. However, both of these low-molecular-weight antioxidants are washed out of mammalian brain tissue during slice preparation and subsequent incubation. Ascorbate, which is not synthesized in the brain, can be added back to slices by active uptake from the incubation medium. Levels of GSH, on the other hand, are regulated by synthesis rather than uptake, and cannot be readily maintained in slices. Importantly, maintenance of brain slice ascorbate content at at least 50% of that in vivo, prevents the increase in slice water content that normally occurs during incubation. Slices with maintained ascorbate levels also have better histological characteristics than ascorbate-depleted tissue. The medium concentration of ascorbate sufficient to maintain content and inhibit edema formation is 400 microM, which is the normal concentration in brain extracellular fluid. This paper describes methods to maintain ascorbate levels in brain slices, including procedures to minimize oxidation in oxygenated incubation media. Also described is an HPLC analysis for ascorbate and GSH that is based on direct injection rather than extraction of samples.     

Vitamin C function in the brain: vital role of the ascorbate transporter SVCT2

Ascorbate (vitamin C) is a vital antioxidant molecule in the brain. However, it also has a number of other important functions, participating as a cofactor in several enzyme reactions, including catecholamine synthesis, collagen production, and regulation of HIF-1α. Ascorbate is transported into the brain and neurons via the sodium-dependent vitamin C transporter 2 (SVCT2), which causes accumulation of ascorbate within cells against a concentration gradient. Dehydroascorbic acid, the oxidized form of ascorbate, is transported via glucose transporters of the GLUT family. Once in cells, it is rapidly reduced to ascorbate. The highest concentrations of ascorbate in the body are found in the brain and in neuroendocrine tissues such as adrenal, although the brain is the most difficult organ to deplete of ascorbate. Combined with regional asymmetry in ascorbate distribution within different brain areas, these facts suggest an important role for ascorbate in the brain. Ascorbate is proposed as a neuromodulator of glutamatergic, dopaminergic, cholinergic, and GABAergic transmission and related behaviors. Neurodegenerative diseases typically involve high levels of oxidative stress and thus ascorbate has been posited to have potential therapeutic roles against ischemic stroke, Alzheimer's disease, Parkinson's disease, and Huntington's disease.     

Oxidative stress following traumatic brain injury in rats: quantitation of biomarkers and detection of free radical intermediates

Oxidative stress may contribute to many pathophysiologic changes that occur after traumatic brain injury. In the current study, contemporary methods of detecting oxidative stress were used in a rodent model of traumatic brain injury. The level of the stable product derived from peroxidation of arachidonyl residues in phospholipids, 8-epi-prostaglandin F(2alpha), was increased at 6 and 24 h after traumatic brain injury. Furthermore, relative amounts of fluorescent end products of lipid peroxidation in brain extracts were increased at 6 and 24 h after trauma compared with sham-operated controls. The total antioxidant reserves of brain homogenates and water-soluble antioxidant reserves as well as tissue concentrations of ascorbate, GSH, and protein sulfhydryls were reduced after traumatic brain injury. A selective inhibitor of cyclooxygenase-2, SC 58125, prevented depletion of ascorbate and thiols, the two major water-soluble antioxidants in traumatized brain. Electron paramagnetic resonance (EPR) spectroscopy of rat cortex homogenates failed to detect any radical adducts with a spin trap, 5,5-dimethyl-1-pyrroline N:-oxide, but did detect ascorbate radical signals. The ascorbate radical EPR signals increased in brain homogenates derived from traumatized brain samples compared with sham-operated controls. These results along with detailed model experiments in vitro indicate that ascorbate is a major antioxidant in brain and that the EPR assay of ascorbate radicals may be used to monitor production of free radicals in brain tissue after traumatic brain injury.     

Effect of direct and indirect dopamine agonists on brain extracellular ascorbate levels in the striatum and nucleus accumbens of awake rats

Systemic administration of direct and indirect dopamine agonists resulted in increased extracellular ascorbic acid levels in the striatum and, to a lesser degree, in the nucleus accumbens as measured by in vivo voltammetry. Intraperitoneal d-amphetamine sulfate (5mg/kg) increased ascorbate concentrations in striatal extracellular fluid. Amphetamine also increased extracellular ascorbate levels in the nucleus accumbens although more gradually and to a lesser extent. Intraperitoneal phenethylamine hydrochloride (20 mg/kg) following pargyline hydrochloride pretreatment (20 mg/kg) increased extracellular ascorbate levels in the striatum significantly above the small increase seen in the nucleus accumbens. The direct acting dopamine agonists Ly-141865 and Ly-163502 when given i.p. at 1 mg/kg, resulted in increased extracellular ascorbate concentrations in both brain areas, again with a significantly greater effect in the striatum. These results indicate that brain extracellular ascorbate levels can be modulated by dopaminergic neuro-transmission and that this modulation is quantitatively different in different dopamine-containing brain structures.     

Changes in the ascorbate metabolism of apoplastic and symplastic spaces are associated with cell differentiation

Ascorbate levels and redox state, as well as the activities of the ascorbate related enzymes, have been analysed both in the apoplastic and symplastic spaces of etiolated pea (Pisum sativum L.) shoots during cellular differentiation. The ascorbate pool and the ascorbate oxidizing enzymes, namely ascorbate oxidase and ascorbate peroxidase, were present in both pea apoplast and symplast, whereas ascorbate free radical reductase and dehydroascorbate reductase were only present in the symplastic fractions. During cell differentiation the ascorbate redox enzymes changed in different ways, since a decrease in ascorbate levels, ascorbate peroxidase and ascorbate free radical reductase occurred from meristematic to differentiated cells, whereas ascorbate oxidase and dehydroascorbate reductase increased. The activity of secretory peroxidases has also been followed in the apoplast of meristematic and differentiating cells. These peroxidases increased their activity during differentiation. This behaviour was accompanied by changes in their isoenzymatic profiles. The analysis of the kinetic characteristics of the different peroxidases present in the apoplast suggests that the presence of ascorbate and ascorbate peroxidase in the cell wall could play a critical role in regulating the wall stiffening process during cell differentiation by interfering with the activity of secretory peroxidases.     

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.     

The osmotic/calcium stress theory of brain damage: Are free radicals involved?

This overview presents data showing that glucose use increases and that excitatory amino acids (i.e., glutamate, aspartate), taurine and ascorbate increase in the extracellular fluid during seizures. During the cellular hyperactive state taurine appears to serve as an osmoregulator and ascorbate may serve as either an antioxidant or as a pro-oxidant. Finally, a unifying hypothesis is given for seizure-induced brain damage. This unifying hypothesis states that during seizures there is a release of excitatory amino acids which act on glutamatergic receptors, increasing neuronal activity and thereby increasing glucose use. This hyperactivity of cells causes an influx, of calcium (i.e. calcium stress) and water movements (i.e., osmotic stress) into the cells that culminate in brain damage mediated by reactive oxygen species.Special issue dedicated to Dr. Frederick E. Samson     

Some characteristics of rabbit muscle phosphofructokinase-1 inhibition by ascorbate

These studies relate to a working hypothesis that glycogen storage is facilitated in resting muscle by inhibiting glycolysis via inhibition of LDH, AK, and PFK-1 by ascorbate; when muscle is active, these isozymes combine with muscle proteins and are released and protected from inhibition by ascorbate and glycolysis proceeds. Focus in these studies is on the ability of G-actin and aldolase to prevent PFK-1 inhibition by ascorbate. We found that inhibition by ascorbate was PFK-1 concentration dependent; ascorbate does not inhibit above 200 nM PFK-1. We conclude that ascorbate inhibits PFK-1 dimers (and perhaps monomers) but not PFK-1 tetramers. Separation of PFK-1 dimers from tetramers was achieved with centrifugal filter devices and differences in their sensitivity to ascorbate inhibition were demonstrated. Some comparisons are made with attributes of AK inhibitions by ascorbate that, like PFK-1, are also enzyme concentration dependent. Discussions relate findings to cellular infrastructure and the role of ascorbate in glycogen synthesis.