Ascorbic Acid Depletion Research Articles

Developmental response of the beneficial predator Podisus maculiventris to change in dietary ascorbic acid concentration

We report here the effects of a range of ascorbic acid concentrations (0.07, 0.3, 3.0, and 30.0 g l−1) in artificial diets on growth rates, adult weights, fecundity, and survival over two generations of the predatory stink bug, Podisus maculiventris (Say) (Heteroptera: Pentatomidae). Overall, a dietary level of 3.0 g l−1 gave the shortest developmental times over two generations. The likelihood of egg hatch at one ascorbic acid concentration compared to another concentration suggested that egg hatch increased as the concentration of ascorbic acid increased from 0.07 to 3.0 g l−1 and then declined from 3.0 to 30.0 g l−1. The combination of the maximum egg oviposition at 0.3 and 3.0 g l−1, egg hatch at 3.0 g l−1, and survival at 0.07 and 0.3 g l−1 suggests an overall superior performance at a concentration between 0.3 and 3.0 g l−1. Depletion of ascorbic acid below 3.0 g l−1 or addition of ascorbic acid above 3.0 g l−1 lowered the likelihood of egg hatch, which became more pronounced in the second generation. This is consistent with previously published information for phytophagous insects.

The corticotrophic activity of tetracosactide in the adrenal ascorbic acid depletion test.

Abstract Using the subcutaneous adrenal ascorbic acid depletion test in hypophysectomized rats it was found that tetracosactide (synthetic 1–24 corticotrophin) could not be assayed against the International Working Standard (IWS) for natural corticotrophin because of a difference in the slopes of the dose-response curves of the preparations. Valid potency estimates were obtained when tetracosactide was assayed against a standard preparation closely similar to the 2nd International Standard for natural corticotrophin. By comparing this standard with the IWS, the best estimate of the potency of tetracosactide in terms of the IWS was 99 (92–106) units/mg. Five different batches of tetracosactide have yielded identical biological potencies. Since tetracosactide can be adequately standardized by physical and chemical-analytical criteria, labelling and dosage on a mg base instead of in units is recommended.

Biochemical mechanism of Caffeic Acid Phenylethyl Ester (CAPE) selective toxicity towards melanoma cell lines

In the current work, we investigated the in vitro biochemical mechanism of Caffeic Acid Phenylethyl Ester (CAPE) toxicity and eight hydroxycinnamic/caffeic acid derivatives in vitro, using tyrosinase enzyme as a molecular target in human SK-MEL-28 melanoma cells. Enzymatic reaction models using tyrosinase/O 2 and HRP/H 2O 2 were used to delineate the role of one- and two-electron oxidation. Ascorbic acid (AA), NADH and GSH depletion were used as markers of quinone formation and oxidative stress in CAPE induced toxicity in melanoma cells. Ethylenediamine, an o-quinone trap, prevented the formation of o-quinone and oxidations of AA and NADH mediated by tyrosinase bioactivation of CAPE. The IC 50 of CAPE towards SK-MEL-28 melanoma cells was 15 μM. Dicoumarol, a diaphorase inhibitor, and 1-bromoheptane, a GSH depleting agent, increased CAPE's toxicity towards SK-MEL-28 cells indicating quinone formation played an important role in CAPE induced cell toxicity. Cyclosporin-A and trifluoperazine, inhibitors of the mitochondrial membrane permeability transition pore (PTP), prevented CAPE toxicity towards melanoma cells. We further investigated the role of tyrosinase in CAPE toxicity in the presence of a shRNA plasmid, targeting tyrosinase mRNA. Results from tyrosinase shRNA experiments showed that CAPE led to negligible anti-proliferative effect, apoptotic cell death and ROS formation in shRNA plasmid treated cells. Furthermore, it was also found that CAPE selectively caused escalation in the ROS formation and intracellular GSH (ICG) depletion in melanocytic human SK-MEL-28 cells which express functional tyrosinase. In contrast, CAPE did not lead to ROS formation and ICG depletion in amelanotic C32 melanoma cells, which do not express functional tyrosinase. These findings suggest that tyrosinase plays a major role in CAPE's selective toxicity towards melanocytic melanoma cell lines. Our findings suggest that the mechanisms of CAPE toxicity in SK-MEL-28 melanoma cells mediated by tyrosinase bioactivation of CAPE included quinone formation, ROS formation, intracellular GSH depletion and induced mitochondrial toxicity.

Ascorbic acid depletion enhances expression of the sodium-dependent vitamin C transporters, SVCT1 and SVCT2, and uptake of ascorbic acid in livers of SMP30/GNL knockout mice

In this study, we examined whether ascorbic acid (AA) and dehydroascorbic acid (DHA), the oxidized form of AA, levels in tissues regulate the AA transporters, sodium-dependent vitamin C transporters (SVCT) 1 and SVCT2 and DHA transporters, glucose transporter (GLUT) 1, GLUT3, GLUT4 mRNA by using senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice. These mice are incapable of synthesizing AA in vivo. AA depletion enhanced SVCT1 and SVCT2 mRNA expression in the liver and SVCT1 and GLUT4 mRNA expression in the small intestine, but not in the cerebrum or kidney. Next, we examined the actual impact of AA uptake by using primary cultured hepatocytes from SMP30/GNL KO mice. In the AA-depleted hepatocytes from SMP30/GNL KO mice, AA uptake was significantly greater than in matched cultures from wild-type mice. These results strongly affirm that intracellular AA is an important regulator of SVCT1 and SVCT2 expression in the liver.

Comparison of the antioxidant status in tracheal and bronchoalveolar epithelial lining fluids in recurrent airway obstruction

Following a period of airway inflammation the clearance of inflammatory cells along the mucociliary escalator may impose a considerable oxidant load on the trachea. To determine the degree of oxidative stress in tracheal epithelial lining fluid (ELF) in comparison to that present in peripheral airways after an acute exposure to organic dust. Tracheal wash fluid and bronchoalveolar lavage fluid (BALF) were collected for cytology and antioxidant analyses from 6 recurrent airway obstruction (RAO)-affected horses and 6 healthy control horses before and after stabling on straw bedding for 24 h. In RAO-affected horses, organic dust exposure resulted in a significant decrease in ascorbic acid concentration in tracheal ELF (P<0.0001), which was greater than the decrease in bronchoalveolar ELF (P = 0.0003). The percentage decrease in tracheal ELF ascorbic acid correlated with the percentage decrease in bronchoalveolar ELF ascorbic acid (r = 0.76; P = 0.004) following exposure. Acute organic dust exposure results in significant antioxidant depletion in the trachea, which may reflect inflammation and oxidative processes in peripheral airways. Further work is required to evaluate the role of ascorbic acid depletion in the pathogenesis of RAO.

Inhibition of spermatogenesis in house rat (Rattus rattus Rufescens) following the administration of alpha- chlorohydrin.

Chronic administration of alpha-chlorohydrin (25 mg/kg body wt.) caused lesions in the testis of house rat. The changes in the germ cells were degenerative. The seminiferous tubules and Leydig cell nuclear diameter were reduced. Epididymal cell height was greatly reduced. The lumen was devoid of spermatozoa. alpha-Chlorohydrin administration inhibited the synthesis of RNA, protein, sialic acid in the testis and accessory sex organs. Total cholesterol of the testis was increased. Adrenal ascorbic acid depletion was conspicuous. alpha-Chlorohydrin anatagonizes the stimulatory action of testosterone in castrated house rats. alpha-Chlorohydrin can be considered as a biologically effective compound for the control of house rat population in human dwellings. It's dose dependent reaction is discussed.

Assessment of the biopotency of follitropin alfa and lutropin alfa combined in one injection: a comparative trial in Sprague-Dawley rats

BackgroundThe current study was designed to determine if follitropin alfa (recombinant human follicle-stimulating hormone; r-hFSH) and lutropin alfa (recombinant human luteinizing hormone; r-hLH) biopotencies were unchanged by reconstituting in sterile water for injection and mixing prior to injection.MethodsThe biopotencies of r-hFSH and r-hLH were determined following injection of female Sprague-Dawley rats with a mixture of follitropin alfa revised formulation female (RFF) and lutropin alfa (1:1, r-hFSH:r-hLH). Biopotencies of follitropin alfa RFF and lutropin alfa were measured using ovarian weight and ascorbic acid depletion assays, respectively, and compared with a reference standard. Stock mixtures of follitropin alfa RFF and lutropin alfa (1:1) were prepared within 1 h prior to each respective assay's injection and stored at 6 +/- 2°C. Separate low dose (follitropin alfa RFF 1.5 IU/rat, lutropin alfa 2 IU/rat) and high dose (follitropin alfa RFF 3 IU/rat, lutropin alfa 8 IU/rat) treatments were prepared from stock mixtures or individual solutions by diluting with 0.22% bovine serum albumin saline solution and injected within 1 h of preparation. The main outcome measures were ovarian weight and ovarian ascorbic acid depletion.ResultsFSH bioactivities were similar (p > 0.10) between the individual follitropin alfa RFF test solution (84.2 IU) and follitropin alfa RFF/lutropin alfa (87.6 IU) mixtures prepared within 1 h of injection and stored at 6 +/- 2°C. LH bioactivities were similar (p > 0.10) between lutropin alfa (94.7 IU) test solution and lutropin alfa/follitropin alfa RFF (85.3 IU) mixtures prepared within 1 h of injection and stored at 6 +/- 2°C for not more than 1 h prior to injection.ConclusionMixing follitropin alfa RFF and lutropin alfa did not alter the bioactivity of either FSH or LH.

Ascorbic acid deficiency in bariatric surgical population

Background To determine the prevalence of ascorbic acid deficiency in the surgical population, whether the body mass index (BMI) has an effect on ascorbic acid concentrations; and whether an association exists between ascorbic acid deficiency and adverse surgical outcomes. Methods Preoperative plasma ascorbic acid concentrations were prospectively assessed in 20–60-year-old patients undergoing elective abdominal surgery. Ascorbic acid deficiency was defined as any concentration ≤0.3 mg/dL and depletion as any concentration >0.3–0.59 mg/dL. Results Of the 266 patients evaluated, 167 had a BMI ≥35 kg/m 2. A greater BMI was associated with lower mean ascorbic acid concentrations ( P = .021). Of the 266 patients, 96 (36%) had abnormally low ascorbic acid concentrations, with 57 (21%) depleted and 39 (15%) deficient. The factors associated with decreased mean ascorbic acid concentrations included younger age ( P = .004) and limited vegetable and fruit intake ( P = .026). Ascorbic acid supplementation was associated with lower depletion and deficiency rates ( P = .001). Conclusion Ascorbic acid depletion and deficiency occur within the surgical population. The contributing factors included younger age, limited intake of fruits and vegetables, lack of vitamin supplementation, and greater BMI. Low concentrations of ascorbic acid did not affect the surgical outcome.

Blood and liver concentration of ascorbic acid in Sparus aurata fed Rovimix Stay C-25

The study aimed at evaluating the ascorbic acid (AA) concentrations in the liver and blood of the Mediterranean sea bream (Sparus aurata L.) after being fed Rovimix Stay C-25 (RS C-25) at three different levels (0, 200 and 800 mg kg−1 of AA) over 45 days. RS C-25 is a mixture of equal parts of AA monophosphate, diphosphate and polyphosphate, containing a 25% AA equivalent. Increasing RS C-25 levels in the diet yielded an increasing ascorbic acid content in the plasma and liver, showing a good sea bream utilization of this source of vitamin C. Dietary vitamin C did not affect the growth rate or feed efficiency during the 45-day experiment. After 75 days, fish fed a vitamin C-free diet displayed a severe depletion of AA in the liver.

Alterations in redox potential of glutathione/glutathione disulfide and cysteine/cysteine disulfide couples in plasma of dropsy patients with argemone oil poisoning

Several incidences of adverse effects on human health have been reported in many countries, due to consumption of edible oil adulterated with argemone oil (AO). The clinical manifestation of the disease is commonly referred to as epidemic dropsy. In the present study, we determined the relationship between redox potentials ( E h) of glutathione/glutathione disulfide (GSH/GSSG), cysteine/cysteine disulfide (Cys/CySS) couples and non-enzymatic antioxidants such as α-tocopherol and ascorbic acid status in plasma of dropsy patients ( n = 14) from an outbreak of argemone oil poisoning in Lucknow (March, 2005), India. Depleted GSH (55%) and concomitant enhancement (163%) of plasma GSSG content was observed in patients ( P < 0.05). Furthermore, lower content of Cys (42%) and CySS (25%) was noticed in patients ( P < 0.05) when compared to control subjects. E h GSH and E h Cys values were shifted by +46 mV and +12 mV towards more oxidizing environment in patients ( P < 0.05). In addition, α-tocopherol and ascorbic acid contents were found to be depleted significantly ( P < 0.05) in plasma of patients (59–58%). The alterations in redox potentials and antioxidants in plasma, which are synthesized in liver, may be responsible for histopathological changes in hepatic tissue of patients showing swelling of hepatocytes, fluid accumulation in spaces of Desci along with mild kupfur cell hyperplasia. Over all the present study shows that redox state of GSH/GSSG and Cys/CySS pools become oxidized which inturn causes depletion of α-tocopherol and ascorbic acid, thus providing a strategy to distinguish pro-oxidant and antioxidant events in patients.

Tissue content, fecundity and quality of eggs and phyllosoma larvae after supplementing the diet of spiny lobster Jasus edwardsii broodstock with ascorbic acid-enriched Artemia biomass

Ascorbic acid (AA)-enriched Artemia in alginate pellets and unenriched pellets were fed to Jasus edwardsii broodstock to supplement AA intake of the basal diet (mussels, squid and compound prawn pellets) during ovarian development before egg extrusion. Pellet AA content ranged from 150 μg g−1 (unenriched) to 9153 μg g−1 (enriched). The basal diet (150 μg AA g−1) was compared with low (150 μg AA g−1), medium (450 μg AA g−1) and high (1350 μg AA g−1) AA supplementation. Dietary AA content was obtained using combinations of unenriched and AA-enriched Artemia in combination with the basal diet. Supplementation resulted in ovarian AA saturation at ∼240 μg g−1, a significant increase over 152 μg g−1 at time 0. Digestive gland concentrations were 76–92 μg AA g−1 for diets containing ≤450 μg AA g−1, but reached 270 μg AA g−1 for the high supplement. The considerable AA store in tail muscle appeared to be translocated to the ovary during maturation. There was no significant AA depletion in eggs during embryogenesis suggesting minimal AA utilization during this phase. Jasus edwardsii spawns once annually, unlike other multiple spawning crustaceans. Therefore, AA supplementation did not alter fecundity or phyllosoma quality, but resulted in a dose-dependent increase (up to 33%) in AA content of eggs and phyllosoma.

Systemic administration of adenosine ameliorates pentylenetetrazol‐induced chemical kindling and secondary behavioural and biochemical changes in mice

Adenosine is one of the inhibitory neuromodulators in the brain and is considered to be responsible for seizure arrest and postictal refractoriness. Adenosine, adenosine receptor agonists, and adenosine uptake blockers are known to reduce the severity and duration of amygdala-kindled seizures. The present study was carried out to elucidate the anticonvulsant and neuromodulatory effect of systemic adenosine on the pentylenetetrazol (PTZ)-induced chemical kindling in mice. Kindling was induced by chronic administration of a subconvulsive dose of PTZ (40 mg/kg, i.p.) on every other day for a total period of 9 days. Adenosine was administered daily, 30 min before PTZ or vehicle. The kindling score was recorded immediately following PTZ administration according to a prevalidated scoring scale. Various behavioral and biochemical estimations were performed on day 10 (i.e. 24 h after the last dose of PTZ). Chronic PTZ treatment progressively increased the seizure score with the maximum score reached on day 9. Behavioral analysis found hyperlocomotor activity, anxiogenic response, hyperalgesia and amnesia in kindled mice. Biochemical analysis revealed that chronic treatment with PTZ significantly increased lipid peroxidation (malondialdehyde levels), nitrite (NO(2-) levels), adenosine deaminase (ADA) and total RNA levels and decreased catalase, reduced glutathione (GSH) levels in brain homogenates, and a depletion of adrenal ascorbic acid. Daily treatment with adenosine (25 and 50 mg/kg, i.p.) for 9 days led to a significant decrease in PTZ-induced kindling score and also reversed various behavioral and biochemical alterations produced by PTZ. The results of the present study suggested that systemic adenosine administration reversed the behavioral and biochemical alterations induced by chronic PTZ.

Intracellular ascorbic acid inhibits transport of glucose by neurons, but not by astrocytes

It has been demonstrated that glutamatergic activity induces ascorbic acid (AA) depletion in astrocytes. Additionally, different data indicate that AA may inhibit glucose accumulation in primary cultures of rat hippocampal neurons. Thus, our hypothesis postulates that AA released from the astrocytes during glutamatergic synaptic activity may inhibit glucose uptake by neurons. We observed that cultured neurons express the sodium-vitamin C cotransporter 2 and the facilitative glucose transporters (GLUT) 1 and 3, however, in hippocampal brain slices GLUT3 was the main transporter detected. Functional activity of GLUTs was confirmed by means of kinetic analysis using 2-deoxy-d-glucose. Therefore, we showed that AA, once accumulated inside the cell, inhibits glucose transport in both cortical and hippocampal neurons in culture. Additionally, we showed that astrocytes are not affected by AA. Using hippocampal slices, we observed that upon blockade of monocarboxylate utilization by alpha-cyano-4-hydroxycinnamate and after glucose deprivation, glucose could rescue neuronal response to electrical stimulation only if AA uptake is prevented. Finally, using a transwell system of separated neuronal and astrocytic cultures, we observed that glutamate can reduce glucose transport in neurons only in presence of AA-loaded astrocytes, suggesting the essential role of astrocyte-released AA in this effect.

Depletion of Ascorbic Acid Restricts Angiogenesis and Retards Tumor Growth in a Mouse Model

Angiogenesis requires the deposition of type IV collagen by endothelial cells into the basement membrane of new blood vessels. Stabilization of type IV collagen triple helix depends on the hydroxylation of proline, which is catalyzed by the iron-containing enzyme prolyl hydroxylase. This enzyme, in turn, requires ascorbic acid to maintain the enzyme-bound iron in its reduced state. We hypothesized that dietary ascorbic acid might be required for tumor angiogenesis and, therefore, tumor growth. Here, we show that, not surprisingly, ascorbic acid is necessary for the synthesis of collagen type IV by human endothelial cells and for their effective migration and tube formation on a basement membrane matrix. Furthermore, ascorbic acid depletion in mice incapable of synthesizing ascorbic acid (Gulo-/-) dramatically restricts the in vivo growth of implanted Lewis lung carcinoma tumors. Histopathological analyses of these tumors reveal poorly formed blood vessels, extensive hemorrhagic foci, and decreased collagen and von Willebrand factor expression. Our data indicate that ascorbic acid plays an essential role in tumor angiogenesis and growth, and that restriction of ascorbic acid or pharmacological inhibition of prolyl hydroxylase may prove to be novel therapeutic approaches to the treatment of cancer.

α-Lipoic acid-enrichment partially reverses tissue ascorbic acid depletion in pacu (Piaractus mesopotamicus) fed vitamin C-devoid diets

Effects of dietary α-lipoic acid (LA) and ascorbic acid (AA) on the growth, tissue vitamin C and tocopherol (vitamin E) levels, and malondialdehyde levels were examined in the tropical fish pacu, Piaractus mesopotamicus. Pacu juveniles were fed one of four casein-gelatin-based diets for 8 weeks: with 0.05% AA and 0.1% LA (+AA+LA), with AA and without LA (+AA–LA), without AA and with LA (−AA+LA), and without AA and LA (−AA−LA). When the fish received quantities of feed equal to 1.9–2.5% of its body weight, growth was not influenced, regardless of the presence of AA or LA throughout most of the experimental period. Growth was, however, slightly but significantly lower at week 8 in the AA-deficient/LA-supplemented group. An AA-deficient diet caused a highly significant reduction in both total AA and dehydroascorbic acid content in the liver and gill tissues. This reduction of tissue AA concentrations was reversed in a significant manner by LA (antioxidant-sparing effect). The 8-week-long vitamin C deprivation was sufficient to initiate the reduction in tissue ascorbic acid; however, total ascorbate in the liver of fish in the (−)AA/(+)LA group was 127.7±54.3 nmol g−1 tissue, whereas it was 28.6±26.3 nmol g−1 in the (−)AA/(−)LA group, a 4.4-fold difference. This mitigating effect of the addition of the endogenous antioxidant LA to the diet indicates that LA exerts a vitamin C-sparing effect in teleost fish that by far exceeds the phenomena demonstrated in non-scurvy-prone mammals. There was no difference among the different diet groups for vitamin E and malondialdehyde levels in the liver. These results suggest that LA is a potent substance for the prevention of AA deficiency in cultured fishes. The optimal dietary level of LA needs to be determined in the light of the slight reduction in body weight gain after 8 weeks of feeding in the absence of AA.

Bergenin, A Nigerian Alcoholic Beverage Additive From Sacoglottis Gabonensis As An Antioxidant Protector Of Mammalian Cells Against 2,4—Dinitrophenyl Hydrazine-Induced Lipid Peroxidation

The effect of bergenin isolated from Sacoglottis gabonensis bark extract, on membrane lipid peroxidation and tissue ascorbic acid level was studied using rat as the experimental animal and 2,4-dinitrophenyl as the experimental oxidant. Pretreatment with bergenin significantly reduced but did not completely abolish DNPH-induced lipid peroxidation in the liver,brain and red blood cell as evidenced from the levels of the two intermediates, lipid hydroperoxide and aldehydes, measured. Bergenin also protected against DNPH-induced depletion of tissue ascorbic acid and had a sparing effect in untreated animals. The results suggest that bergenin may be responsible for the earlier observed anti-lipid peroxidizing effect of the bark extract.

Gentisic acid, an aspirin metabolite, inhibits oxidation of low-density lipoprotein and the formation of cholesterol ester hydroperoxides in human plasma

Gentisic acid, an aspirin metabolite, has an antioxidant effect, although its detailed mechanism remains elusive. The present study was designed to determine whether it inhibits low-density lipoprotein (LDL) oxidation and the formation of lipid hydroperoxides in human plasma. The susceptibility of LDL oxidative modification was investigated by a method using 2,2′-azobis or Cu 2+. To study the effect of gentisic acid on free radical-induced damage to plasma lipids, cholesterol ester hydroperoxides generated by incubating human fresh plasma with Cu 2+ and gentisic acid was analyzed. Gentisic acid inhibited LDL oxidation in a concentration-dependent manner. It significantly inhibited the formation of cholesterol ester hydroperoxides in plasma, and was consumed after the depletion of ascorbic acid and reduced form of coenzyme Q-10 (CoQH 2-10), whereas concentrations of other antioxidants remained unchanged. Gentisic acid had a potent free radical scavenging activity with a minimal chelating effect. The potent antioxidant property of gentisic acid may partly account for the anti-atherogenic effects of aspirin.

Ascorbic acid decreases oxidant stress in endothelial cells caused by the nitroxide tempol

Stable nitroxide radicals have been considered as therapeutic antioxidants because they can scavenge more toxic radicals in biologic systems. However, as radicals they also have the potential to increase oxidant stress in cells and tissues. We studied the extent to which this occurs in cultured EA.hy926 endothelial cells exposed to the nitroxide Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl). Tempol was rapidly reduced by the cells, as manifest by an increase in the ability of the cells to reduce extracellular ferricyanide and by disappearance of the Tempol EPR signal. Cells loaded with ascorbic acid, which directly reacts with Tempol, showed increased rates of Tempol-dependent ferricyanide reduction, and a more rapid loss of the Tempol EPR signal than cells not containing ascorbate. In this process, intracellular ascorbate was oxidized, and was depleted at lower Tempol concentrations than was GSH, another important intracellular low molecular weight antioxidant. Further evidence that Tempol concentrations of 100–1000 μM induced an oxidant stress was that it caused an increase in the oxidation of dihydrofluorescein in cells and inhibited ascorbate transport at concentrations as low as 50–100 μM. The presence of intracellular ascorbate both prevented dihydrofluorescein oxidation and spared GSH from oxidation by Tempol. Such sparing was not observed when GSH was depleted by other mechanisms, indicating that it was likely due to protection against oxidant stress. These results show that whereas Tempol may scavenge other more toxic radicals, care must be taken to ensure that it does not itself induce an oxidant stress, especially with regard to depletion of ascorbic acid.

Short-term ascorbic acid deficiency does not impair antioxidant status in lens of guinea pigs.

We examined whether short-term ascorbic acid deficiency impairs antioxidant status in the lens of guinea pigs. Male guinea pigs aged 4 wk were given a scorbutic diet (20 g/animal per day) with and without ascorbic acid (400 mg/animal per day) in drinking water for 3 wk. The ascorbic acid-deficient group showed no lens opacity. The ascorbic acid-deficient group had 14% of serum ascorbic acid concentration, 6% of aqueous humor ascorbic acid concentration, and 18% of lens ascorbic acid content in the ascorbic acid-adequate group. There were no differences in the contents of lens reduced glutathione and thiobarbituric acid reactive substances, an index of lipid peroxidation, between the ascorbic acid-deficient and adequate groups, while the deficient group had higher lens vitamin E content than the adequate group. The ascorbic acid-deficient group had higher serum vitamin E concentration than the ascorbic acid adequate group, while there were no differences in the concentrations of serum reduced glutathione and tiobarbituric acid reactive substances between the deficient and adequate groups. These results indicate that short-term ascorbic acid deficiency does not impair antioxidant status in the lens of guinea pigs despite induction of severe ascorbic acid depletion in the tissue, which may result in no cataract formation.

Control of ascorbic acid efflux in rat luteal cells: role of intracellular calcium and oxygen radicals.

In luteal cells, prostaglandin (PG)F2a mobilizes intracellular calcium concentration ([Ca]i), generates reactive oxygen species (ROS), depletes ascorbic acid (AA) levels, inhibits steroidogenesis, and ultimately induces cell death. We investigated the hypothesis that [Ca]i mobilization stimulates ROS, which results in depletion of cellular AA in rat luteal cells. We used a self-referencing AA-selective electrode that noninvasively measures AA flux at the extended boundary layer of single cells and fluorescence microscopy with fura 2 and dichlorofluorescein diacetate (DCF-DA) to measure [Ca]i and ROS, respectively. Menadione, a generator of intracellular superoxide radical (O2-), PGF2a, and calcium ionophore were shown to increase [Ca]i and stimulate intracellular ROS. With calcium ionophore and PGF2a, but not menadione, the generation of ROS was dependent on extracellular calcium influx. In unstimulated cells there was a net efflux of AA of 121.5 +/- 20.3 fmol x cm-1 x s-1 (mean +/- SE, n = 8), but in the absence of extracellular calcium the efflux was significantly reduced (10.3 +/- 4.9 fmol x cm-1 x s-1; n = 5, P < 0.05). PGF2a and menadione stimulated AA efflux, but calcium ionophore had no significant effect. These data suggest two AA regulatory mechanisms: Under basal conditions, AA efflux is calcium dependent and may represent recycling and maintenance of an antioxidant AA gradient at the plasma membrane. Under luteolytic hormone and/or oxidative stress, AA efflux is stimulated that is independent of extracellular calcium influx or generation of ROS. Although site-specific mobilization of calcium pools and ROS cannot be ruled out, the release of AA by PGF2a-stimulated luteal cells may occur through other signaling pathways.