High Levels Of Ascorbic Acid Research Articles

Gastroduodenal Mucosal Vitamin-C Levels in Helicobacter pylori Infection

Background: Vitamin C is an important endogenous antioxidant, and epidemiologic evidence suggests that it may protect against the development of gastric cancer. We therefore determined mucosal vitamin-C levels in the stomach and duodenum of subjects with and without Helicobacter pylori infection. Methods: The patients were 30 subjects undergoing routine gastroscopy for investigation of dyspepsia. High-performance liquid chromatography with electrochemical detection was used to determine mucosal ascorbic acid and total vitamin-C levels. Results: In H. pylori-negative subjects with normal gastroduodenal histology the antrum contained significantly higher levels of ascorbic acid and total vitamin C than the corpus or duodenum (P < 0.05). No significant changes were seen in gastric mucosal ascorbic acid or total vitamin-C levels in the presence of H. pylori infection and related inflammation. The presence of gastric atrophy did not affect mucosal ascorbic acid or total vitamin C levels. Duodenal ascorbic acid and total vitamin-C levels did not change significantly in the presence of gastric H. pylori or duodenal inflammation. Conclusions: Although high levels of vitamin C are present in the gastroduodenal mucosa, these are not altered in the presence of H. pylori infection and inflammation. These observations suggest that the mucosal antioxidant potential of vitamin C is not impaired by H. pylori infection.

Measurement of Hydrogen Peroxide in Biological Samples Containing High Levels of Ascorbic Acid

The physiological concentration of hydrogen peroxide in the aqueous humor was reported to range between 25 and 60 μM, and conditions leading to elevated levels could have important damaging effects such as cataract formation. However, the high concentration of ascorbic acid in aqueous humor, which is 20 times that of plasma, was recently shown to interfere in the dichlorophenol–indophenol assay for hydrogen peroxide. The actual concentration of hydrogen peroxide in this fluid has become a controversial issue. In the present study, we used the method of ferrous oxidation of xylenol orange (FOX1 assay) performed in a nitrogen atmosphere to accurately measure low levels of hydrogen peroxide, even in the presence of ascorbic acid at concentrations normally found in aqueous humor. Contrary to values reported in the literature, we observed that the concentration of hydrogen peroxide in the rabbit aqueous humor is less than 5 μM, which is the detection limit of the method.

Ascorbic Acid-Dependent Cytoprotection of Ovarian Cells by Leukocyte and Nonleukocyte Peroxidases

Luteal cells contain high levels of ascorbic acid that is secreted by stimulation with agents like luteinizing hormone (LH) and prostaglandin F 2α (PGF 2α). One role for interstitial ascorbic acid, we propose, may be the detoxification of H 2O 2 by regeneration of catalytically active peroxidase. By serving as a preferred secondary substrate, ascorbic acid regenerates the catalytically active peroxidase that is inhibited irreversibly by H 2O 2 alone. To test this hypothesis, luteal cells were incubated in the absence and presence of peroxidases and H 2O 2, and the maximal cyclic AMP and steroidogenic response to LH was examined. In luteal cells, H 2O 2 is known to severely inhibit LH-sensitive cyclic AMP accumulation and steroidogenesis, and the addition of lactoperoxidase, myeloperoxidase, eosinophil peroxidase, or ascorbic acid (1 mM) alone had no effect on these responses to H 2O 2. However, co-incubation of ascorbic acid and the peroxidases completely reversed the inhibition of cyclic AMP accumulation and steroidogenesis produced by H 2O 2. These findings and the results that show direct oxidation of ascorbic acid in the presence of peroxidase and H 2O 2, but not with H 2O 2 alone, support the conclusion that ascorbic acid released from cells may detoxify H 2O 2 by regenerating the catalytically active state of peroxidases.

Accumulation of ascorbate by endocrine-regulated and glucose-sensitive transport of dehydroascorbic acid in luteinized rat ovarian cells.

The corpus luteum is notable for very high levels of ascorbic acid. In luteal cells, ascorbic acid depletion occurs as a result of consumption during radical scavenging, inhibition of ascorbic acid uptake, and stimulation of its secretion. Oxidation of ascorbic acid generates dehydroascorbic acid (DHAA). Although levels of DHAA in blood are much lower than those of ascorbic acid, DHAA serves as the major transportable form of ascorbate for certain cell types. The aim of the present studies was to investigate whether DHAA transport is a potential mechanism for conserving ascorbic acid in the corpus luteum. DHAA uptake by rat luteal cells precultured for 24 h was linear for up to 30 min. Kinetics studies showed that uptake of DHAA was a concentration-dependent and saturable process with an estimated Michaelis constant (Km) of 830 microM and a maximum velocity (Vmax) of 700 pmol/min per 10(6) cells, a rate 50 times that of ascorbate transport. More than 90% of DHAA was reduced to ascorbic acid within 2 h of cellular uptake. DHAA uptake was energy- and microfilament-dependent, as transport was inhibited by 2,4-dinitrophenol (1 mM) and cytochalasin B (10 microM). Menadione (50 microM), an intracellular generator of reactive oxygen species, also markedly reduced DHAA uptake. In contrast to ascorbic acid transport, DHAA uptake was potently inhibited by glucose and phloretin, an inhibitor of glucose transporters, with IC50s of approximately 5 mM and 10 microM, respectively. DHAA uptake appears to occur via an insulin-insensitive transporter, as insulin (10 nM) had no effect on uptake. However, 24-h preincubation with insulin-like growth factor (IGF)-I dose-dependently (10-100 ng/ml) stimulated DHAA uptake; similar concentrations of IGF-II had no effect. The secretion of radioactivity by cells preloaded with radiolabeled DHAA was significantly increased by prostaglandin F2alpha (1 microM). The ability of luteal cells to transport DHAA in a regulated manner may serve to maintain vital levels of ascorbic acid within the corpus luteum.

Element contents and stress-physiological characterization of Pinus canariensis needles in Mediterranean type field stands in Tenerife

Contents of Ca, Mg, K, Na, S, Cl, P, N, pigments, ascorbic acid, glutathione, and tocopherol together with chlorophyll fluorescence were measured in needles of Pinus canariensis trees growing at four field stands at 4 different altitudes at the south-eastern slope of the Teide in Tenerife. S contents reflected impacts of SO 2 at lower altitudes. Via Na and Cl contents the influence from the sea was assessed up to 1000 m. Above 1000 m we found higher levels of ascorbic acid, less chlorophyll, higher carotenoid/chlorophyll-ratios, and lower α/β-carotene ratios. This pattern suits in a picture of elevated oxidative stress these trees are responding to. Decreases in Fv/Fm-ratios of chlorophyll fluorescence below 0.80 reflecting damages in the photosynthetic apparatus were only found at one site at 1500 m, where altitude stress was probably amplified by drought stress.

Effectiveness of ascorbic acid as an oxygen scavenger in improving viability of probiotic bacteria in yoghurts made with commercial starter cultures

Abstract The viability of yoghurt and probiotic bacteria was assessed during manufacture and 35 days storage of yoghurt supplemented with four levels of ascorbic acid using four commercial starter cultures. There were no marked differences in titratable acidity and pH of the yoghurt, whereas oxygen content and redox potential varied in yoghurts with various levels of ascorbic acid. Hydrogen peroxide content was initially higher in the products prepared with starter cultures that contained Lactobacillus delbrueckii ssp. bulgaricus. During storage of yoghurt, increase in oxygen content and redox potential and percentage retention of ascorbic acid were identical for all four starter cultures, but different at various levels of ascorbic acid. The oxygen content and redox potential gradually increased during storage in plastic cups and remained lower with higher levels of ascorbic acid. Loss of ascorbic acid occurred during manufacture and storage, with an average retention of only 15–20% after about 35 days storage of yoghurt at 4 °C. The viable counts of Streptococcus thermophilus were lower, whereas those of L. delbrueckii ssp. bulgaricus were higher, with increasing concentration of ascorbic acid. The counts of Lactobacillus acidophilus during storage decreased less rapidly with increasing concentration of acsorbic acid for all four starter cultures, whereas the counts of bifidobacteria remained unchanged.

Seasonal Variation in the Antioxidant Defense System of the Brain of the Ground Squirrel ( Citellus citellus) and Response to Low Temperature Compared with Rat

Seasonal variation in the activity of antioxidant enzymes (superoxide dismutase (EC 1.15.1.1.; SOD), catalase (EC 1.11.1.6; CAT), glutathione peroxidase (EC 1.11.1.9; GSH-Px), glutathione reductase (EC 1.6.4.2; GR), glutathione-S-transferase (EC 2.5.1.18; GST) and low-molecular-weight antioxidants: ascorbic acid (AsA), vitamin E (VIT E) and glutathione (GSH+GSSG) were examined in the brain of the ground squirrels ( Citellus citellus) maintained at 30°C during the whole year. The highest activity (per mg protein) of antioxidant defense (AD) enzymes was found in the spring and was much lower in the summer. A further decrease in activity of CAT, GSH-Px and GST was observed in the winter. The highest levels of AsA and glutathione were recorded in winter in comparison with spring and summer. AD system in the brain of the ground squirrel and rats (maintained at thermoneutrality) exposed to low temperature (4°C) for 3, 6 or 24 hr during the summer was studied as well. Summer was chosen as a period of stable euthermia for ground squirrels and in thermoregulation similar to rats. Consumption of free fatty acid and glucose during the acute exposure to low temperature was found to be species specific. In the ground squirrel, an increase in the specific activities of SOD, after 3, 6 and 24 hr, CAT after 3 and 6 hr and GR after 6 hr of exposure to low temperature was detected. When activities were expressed in U/g wet mass, an increase of SOD after 3, 6 and 24 hr ( P < 0.02, P < 0.02, P< 0.005) and CAT and GSH-Px 3 hr ( P < 0.01) upon exposure to low temperature was observed. In the rats, no changes in the specific activities of these enzymes after exposure to low temperature were recorded and only an increase in GST activity (U/g wet mass) after 6 hr exposure was registered. Low-molecular-weight AD components in both animal species were unchanged upon short-term exposure to low temperature. The species-specific differences in brain AD between the rats and the ground squirrels after short exposure to low temperature may be ascribed to seasonal changes of the brain activity in the latter.

Evidence for the involvement of a muscarinic receptor in ascorbic acid secretion in the rat stomach

It has been demonstrated that ascorbic acid is present in human gastric juice at a very high level even in the fasting state. In this study, we confirmed such a physiological event in pylorus-ligated rats and investigated the mechanism for gastric ascorbic acid secretion using the rat in vivo perfusion method. Gastric juice from fasting rats after a 4-hr ligation contained a 6-fold higher level of ascorbic acid than that in sera, indicating the presence of its active transport system across the gastric wall. To identify an endogenous mediator, four kinds of gastric stimulants were compared for their secretory abilities. Although all four secretagogues used stimulated acid secretion to comparable levels, ascorbic acid secretion was stimulated only by carbamylcholine chloride (carbachol). Carbachol-stimulated ascorbic acid secretion was abolished by atropine pretreatment, showing that it is mediated via a muscarinic cholinergic receptor. This cholinergic stimulation was also demonstrated in a rat mutant that genetically lacks the ability to synthesize ascorbic acid in the liver, similar to humans. In addition, a potent inhibitor of the gastric proton pump, which is a final regulatory component in the mechanism of acid secretion, caused no inhibition of ascorbic acid secretion in Osteogenic Disorder Shionogi (ODS) rats. These results are the first evidence indicating that ascorbic acid, the reduced form of vitamin C, is actively and independently secreted into the gastric juice under regulation of a cholinergic receptor system.

Redox changes in perfusates following intracerebral penetration of microdialysis probes.

Microdialysis probe insertion into rat cerebral cortex significantly affects the levels of redox-active substances in brain extracellular fluid. Ascorbic acid levels are high immediately after probe insertion, decline rapidly, and then rise as the rat recovers from anesthesia 5-8 hours after surgery. Uric acid is at a low level for 5 hours and then rapidly increases in parallel with ascorbic acid. High ascorbic acid levels immediately after probe insertion are likely due to a shift from intracellular to extracellular fluids, whereas the delayed increase in uric acid may be due to increased enzymatic formation. After removal from the brain, hydrogen peroxide (H2O2) in microdialysis samples produces catalase-sensitive oxidative chemiluminescence. Microdialysis samples also produce high level catalase-resistant chemiluminescence associated with ascorbic acid levels after penetration injury. Although ascorbic acid is likely an antioxidant at concentrations estimated to be in brain extracellular fluid, it may have prooxidant effects when complexed with transition metals released into the neuronal microenvironment during traumatic brain injury.

Dietary ascorbic acid requirements during the hatchery production of turbot larvae

The effect of high ascorbic acid (AA) levels transferred through enriched live food was evaluated for turbot Scophthalmus maximus larvae in two consecutive feeding experiments. The same feeding strategy was applied to all treatments, except for the AA content of the live food which was manipulated through bioencapsulation with ascorbyl palmitate. This resulted finally in a low, medium and high‐AA treatment. The AA incorporation levels in the turbot larvae (up to 1400 μg AA g DW−1) were correlated with the AA content of the live food administered. However, feeding the high AA concentration resulted in the same values as for the medium treatment, indicating a saturation of the body AA reserves. Under standard culture conditions, no differences in growth nor overall survival could be detected among the different groups, illustrating that the dietary AA requirements of larval turbot are met by non‐enriched live food containing already 500 μg AA g DW−1. The larvae of the high‐AA treatment, however, showed a better pigmentation rate (47 and 32% for experiments 1 and 2, respectively) compared to the other groups (35 and 25%, respectively). Evaluation of the physiological condition applying a salinity stress test revealed an improvement by feeding extra AA, significantly in the medium‐AA treatment. Though not significantly different, cumulative mortalities after challenge with Vibrio anguillarum amounted to 50% for the control v. 40% for the fish fed medium and high‐AA diets, respectively. Moreover, the onset of mortalities in this study was slower (not significantly) for the fish fed the extra AA.

Dietary ascorbic acid requirements during the hatchery production of turbot larvae

The effect of high ascorbic acid (AA) levels transferred through enriched live food was evaluated for turbot Scophthalmus maximus larvae in two consecutive feeding experiments. The same feeding strategy was applied to all treatments, except for the AA content of the live food which was manipulated through bioencapsulation with ascorbyl palmitate. This resulted finally in a low, medium and high-AA treatment. The AA incorporation levels in the turbot larvae (up to 1400 μg AA g DW−1) were correlated with the AA content of the live food administered. However, feeding the high AA concentration resulted in the same values as for the medium treatment, indicating a saturation of the body AA reserves. Under standard culture conditions, no differences in growth nor overall survival could be detected among the different groups, illustrating that the dietary AA requirements of larval turbot are met by non-enriched live food containing already 500 μg AA g DW−1. The larvae of the high-AA treatment, however, showed a better pigmentation rate (47 and 32% for experiments 1 and 2, respectively) compared to the other groups (35 and 25%, respectively). Evaluation of the physiological condition applying a salinity stress test revealed an improvement by feeding extra AA, significantly in the medium-AA treatment. Though not significantly different, cumulative mortalities after challenge with Vibrio anguillarum amounted to 50% for the control v. 40% for the fish fed medium and high-AA diets, respectively. Moreover, the onset of mortalities in this study was slower (not significantly) for the fish fed the extra AA.

Influence of dietary vitamin C dosage on turbot ( Scophthalmus maximus) and European sea bass ( Dicentrarchus labrax) nursery stages

Stable forms of vitamin C were verified as dietary sources of ascorbic acid (AA) for the nursery stages of European sea bass and turbot. In a first experiment, various concentrations of ascorbyl palmitate (AP) and one level of AA 2-polyphosphate (ApP) were evaluated in a semi-purified diet for European sea bass, Dicentrarchus labrax. A significantly lower AA concentration was detected in the fry fed the AP-supplemented diets compared to the ApP-fed group, providing evidence that AP is an inferior source of vitamin C for young stages of sea bass. ApP was subsequently selected for the determination of the AA requirements of both sea bass and turbot ( Scophthalmus maximus) fry. The AA levels incorporated in the body tissues reflected the levels of ApP in the diet, providing evidence of the bioavailability of this stable phosphate ester. Reduced production results were only obtained in the AA-free treatment, indicating that a level of 20 mg AA/kg is sufficient for sea bass and turbot during nursery rearing. This was supported with data from the ultrastructural evaluation of the hepatocytes. Results of challenge tests with pathogenic Vibrio strains remained inconclusive and did not provide evidence for an immunostimulative effect of high AA levels during the early post-weaning phase of these species.

Evaluation of vitamin C-enriched Artemia nauplii for larvae of the giant freshwater prawn

The effect of high levels of ascorbic acid (AA) delivered through enriched live food has been verified through the successful culture of larval giant freshwater prawn, Macrobrachium rosenbergii. Two successive feeding trials were set up using a control (550 Μg AA g−1 DW) and two different AA-enrichment levels in Artemia (1300 and 2750 Μg AA g−1 DW). Under standard culture conditions, no differences in growth nor survival could be observed demonstrating that the nutritional requirements are below 550 Μg AA g−1 DW, which is the normal level occurring in freshly-hatched Artemia. However, a significantly positive effect could be demonstrated on the physiological condition of the postlarvae, measured by means of a salinity stress test, when vitamin C-boosted live food was administered. Since the AA levels in the predator larvae are linked with the enrichment levels in the live prey, it may be assumed that a positive influence on stress resistance was caused by feeding vitamin C-enriched Artemia. It is expected that under suboptimal conditions, supplementation of high vitamin C levels might also enhance production characteristics.

The Effect of Four Levels of Ascorbic Acid and Two Levels of Calcium on Eggshell Quality of Forced-Molted White Leghorn Hens

The present experiment evaluated the effect of four levels of ascorbic acid (0, 100, 250, and 500 ppm) and two levels of calcium (3.0 and 3.5%) on the productivity and eggshell quality of molted Single Comb White Leghorn hens, housed four per 30.5 cm wide × 45.7 cm deep cage. The 4 × 2 factorial treatment combinations were randomly assigned to eight blocks of cages.Egg production increased (P < .06) an average of 5% for the levels of 250 and 500 ppm ascorbic acid. No differences were observed for feed consumption, egg weight, and shell thickness for the different levels of ascorbic acid or calcium in the diet. An increase (P < .01) was observed for specific gravity, as the levels of ascorbic acid increased. Increasing the level of calcium from 3.0 to 3.5% increased (P < .01) specific gravity from 1.073 to 1.075. Shell weight increased (P < .01) with the higher levels of ascorbic acid (250 and 500 ppm). No interactions were observed between the levels of ascorbic acid and calcium. Results suggest that supplementing ascorbic acid to molted laying hens can be beneficial to egg production and eggshell quality.

High Levels of Ascorbic Acid, Not Glutathione, in the CNS of Anoxia‐Tolerant Reptiles Contrasted with Levels in Anoxia‐Intolerant Species

Ascorbic acid and glutathione (GSH) are antioxidants and free radical scavengers that provide the first line of defense against oxidative damage in the CNS. Using HPLC with electrochemical detection, we determined tissue contents of these antioxidants in brain and spinal cord in species with varying abilities to tolerate anoxia, including anoxia-tolerant pond and box turtles, moderately tolerant garter snakes, anoxia-intolerant clawed frogs (Xenopus laevis), and intolerant Long-Evans hooded rats. These data were compared with ascorbate and GSH levels in selected regions of guinea pig CNS, human cortex, and values from the literature. Ascorbate levels in turtles were typically 100% higher than those in rat. Cortex, olfactory bulb, and dorsal ventricular ridge had the highest content in turtle, 5-6 mumol g-1 of tissue wet weight, which was twice that in rat cortex (2.82 +/- 0.05 mumol g-1) and threefold greater than in guinea pig cortex (1.71 +/- 0.03 mumol g-1). Regionally distinct levels (2-4 mumol g-1) were found in turtle cerebellum, optic lobe, brainstem, and spinal cord, with a decreasing anterior-to-posterior gradient. Ascorbate was lowest in white matter (optic nerve) in each species. Snake cortex and brainstem had significantly higher ascorbate levels than in rat or guinea pig, although other regions had comparable or lower levels. Frog ascorbate was generally in an intermediate range between that in rat and guinea pig. In contrast to ascorbate, GSH levels in anoxia-tolerant turtles, 2-3 mumol g-1 of tissue wet weight, were similar to those in mammalian or amphibian brain, with no consistent pattern associated with anoxia tolerance. GSH levels in pond turtle CNS were significantly higher (by 10-20%) than in rat for several regions but were generally lower than in guinea pig or frog. GSH in box turtle and snake CNS were the same or lower than in rat or guinea pig. The distribution GSH in the CNS also had a decreasing anterior-to-posterior gradient but with less variability than ascorbate: levels were similar in optic nerve, brainstem, and spinal cord. The paradoxically high levels of ascorbate in turtle brain, which has a lower rate of oxidative metabolism than mammalian, suggest that ascorbate is an essential cerebral antioxidant. High levels may have evolved to protect cells from oxidative damage when aerobic metabolism resumes after a hypoxic dive.

Influence of low dose allopurinol on ischaemia — Reperfusion injury during abdominal aortic surgery

To ascertain whether surgery causes ischaemia-reperfusion (I-R) related injury, if this injury is augmented by preoperative shock, and reduced with low dose allopurinol. Randomised blind placebo controlled trial. Surgical laboratory. 22 pigs were randomly allocated to four groups; OP = operation/placebo, OA = operation/ allopurinol, SOP = shock + operation/placebo, SOA = shock + operation/allopurinol. An aortic tube prosthesis was inserted in all. In groups SOP and SOA preoperative shock was induced by exsanguination. Allopurinol was administered in group OA on the preoperative day and peroperatively, in group SOA during shock and peroperatively. Perioperative blood concentrations of thiobarbituric acid reactive species (TBARS), ascorbic acid (AA), albumin, 99mTc-albumin and creatine phosphokinase (CPK) as indicators of oxidative membrane damage, antioxidant activity, microvascular permeability changes and muscular cell damage respectively. In the OP and OA groups TBARS gradually increased, while AA, 99mTc-albumin and CPK remained unchanged and albumin decreased. No effect of allopurinol was observed in these groups. In the SOP group TBARS and AA were not significantly different from groups OP and OA. Yet, albumin, 99mTc-albumin and CPK decreased significantly more in the SOP group. Compared with the SOP group, allopurinol treatment (SOA) produced lower TBARS and higher AA levels, and reduced the effect of shock on albumin, 99mTc-albumin and CPK concentrations. Aortic surgery causes no I-R related damage. Pre-operative shock produces I-R related damage, which is reduced by allopurinol.

Ascorbic acid in exotic fruits: a liquid chromatographic investigation

The levels of ascorbic acid (AA) have been measured by means of an HPLC method in 11 different exotic fruits (avocado pear, babaco, feijoa, grapefruit, kiwi, kumquat, litchi, mango, papaya, passion fruit, pineapple) and, for comparative purposes, in two citrus fruits (lemon and orange). They were measured in the exotic fruits at two different stages of ripening: (i) immediately after purchasing from a local fresh fruit market, and (ii) after a one-week period of artificial ripening. The results show that all tropical fruits contain relatively high levels of AA (varying between 20 and 90 mg/100g), with the exceptions of avocado pear and feijoa (whose AA levels are markedly affected by oxidation processes). Moreover, the results show that there is a remarkable loss of AA content (usually 30–40%) after the one-week period of artificial ripening, in all the different tropical fruits considered. They seem to indicate that the process of artificial ripening, which is usually carried out during the short-term storage of exotic fruits, can affect the nutritional value of this kind of food as far as the concentration of the reduced form of vitamin C is concerned.

Elevated concentrations of ascorbate and normoxia suppress testosterone production in cultured guinea pig leydig cells

In recent years, several metabolic roles have been proposed for vitamin C. Recent information suggests a strong causal relationship between high endogenous levels of ascorbic acid and changes in normal reproductive biology. Using highly enriched populations of guinea pig Leydig cells, we have found that elevated levels (50 to 500 μM) of ascorbate significantly ( P < 0.01) depressed testosterone production in a dose-dependent manner while low levels (0 to 10 μM) were without effect. Leydig cells incubated under hypoxic (3% oxygen) culture conditions produced significantly ( P < 0.01) more testosterone than similar cells cultured under normoxic (19% oxygen) conditions. The results of this study suggest that high concentrations of ascorbate and normoxic culture conditions suppress testosterone production in isolated Leydig cells. Thus, it would seem that there exists a delicate balance between normal metabolic requirements for vitamin C and excessive ascorbate levels that might alter normal gonadal reproductive events.

Amperometric determination of urea using an NADH-dependent coupled enzyme

Enzyme electrodes for the amperometric measurement of urea were prepared by co-immobilizing l-glutamate dehydrogenase and urease onto an Immobilon-AV affinity membrane with attachment to a glassy carbon electrode. Reduced nicotinamide adenine dinucleotide (NADH) was used as the electroactive species. The electrochemical oxidation of NADH was monitored at +1.0 V vs. Ag/AgCl. The enzyme immobilized electrode was linear over the range of 2.0 × 10 −5 to 2 × 10 −4 M. The response time of the electrode was 3 min and the optimum pH of enzyme immobilized membrane was pH 7.4–7.6 (Dulbecco's buffer solution). It was stable for at least two weeks and 50 assays. There were no interferences from other physiological material, except for high levels of ascorbic acid.

Regulation of saturation and depletion of ascorbic acid in rainbow trout

Rainbow trout (Oncorhynchus mykiss) juveniles were fed fish meal-based diets supplemented with 0, 30, 60, 120, 240, and 480 mg ascorbic acid kg −1 dry diet or 15, 60, and 240 mg ascorbate monophosphate (expressed as ascorbate equivalents) kg −1. Significant differences in both growth and instantaneous mortality rate between two compared strains (London, OH USA and Mount Shasta, CA USA) were found in fish fed the vitamin C-free diet. Deficiency symptoms included anorexia and lethargy, but no acute signs of lordosis or scoliosis were observed before heavy mortality after 9 weeks. Diets with supplemental ascorbate monophosphate supported significantly better growth and resulted in lower mortality than diets supplemented with equivalent amounts of ascorbic acid. Ascorbate concentrations in tissues (liver, kidney, and intestine) during the saturation period were higher in the groups fed 60 and 240 mg ascorbate monophosphate/kg than in groups supplemented with corresponding or higher levels of ascorbic acid. Significant differences in mortalities were experienced in fish with a different ascorbate status during the depletion phase. Kaplan-Meier analysis indicates that initial ascorbate level during the depletion phase can influence survival of fish. Analyses of ascorbate concentration during depletion revealed that the half-life ( t 1 2 amounted to 42.8 to 50.2 days for the liver and 47.8 to 79.6 days for the kidney. These data imply that a conservation of tissue ascorbate is not operative in juvenile rainbow trout. We propose a two-compartmental model as describing ascorbate depletion pattern in juvenile rainbow trout.