Effect of ascorbic acid treatment on conduit vessel endothelial dysfunction in patients with hypertension

Hypertension is associated with low plasma ascorbic acid levels and impaired endothelial function. Recent evidence suggests that increased vascular oxidative stress contributes to the pathophysiology of endothelial dysfunction and hypertension. We recently showed that chronic oral ascorbic acid therapy lowers blood pressure in hypertensive patients. We hypothesized that it would also improve endothelial vasomotor function. In a randomized, double-blind, placebo-controlled study, we examined the effect of acute (2 g po) and chronic (500 mg/day for 1 mo) ascorbic acid treatment on brachial artery flow-mediated dilation in 39 patients with hypertension. Compared with 82 age- and gender-matched normotensive controls, these patients had impaired endothelium-dependent, flow-mediated dilation of the brachial artery [8.9 +/- 6.1 vs. 11.2 +/- 5.7% (SD), P < 0.04]. After therapy, plasma ascorbic acid concentrations increased acutely from 50 +/- 12 to 149 +/- 51 micromol/l and were maintained at 99 +/- 33 micromol/l with chronic treatment (both P < 0.001). As previously reported, chronic ascorbic acid therapy reduced systolic and mean blood pressure in these patients. However, acute or chronic ascorbic acid treatment had no effect on brachial artery endothelium-dependent, flow-mediated dilation or on endothelium-independent, nitroglycerin-mediated dilation. These results demonstrate that conduit vessel endothelial dysfunction secondary to hypertension is not reversed by acute or chronic treatment with oral ascorbic acid. The effects of this treatment on resistance vessel vasomotor function warrant further investigation.     

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.     

Effect of Tetracycline on Leucocyte Ascorbic Acid Levels

Fourteen men aged 73-94 showed a fall in leucocyte ascorbic acid levels during five days'' treatment with 1 g of tetracycline daily. A control group of nine men aged 74-90 showed no fall in leucocyte ascorbic acid levels. Of the control group, three were treated with phenobarbitone 60 mg thrice daily, three with phenylbutazone 200 mg thrice daily, and three with aloxiprin 1,200 mg four times a day. A further two men aged 87 and 90 showed increased urinary excretion of ascorbic acid while receiving 1 g of tetracycline daily.     

Production of ascorbic acid glucoside by alginate-entrapped mycelia of <Emphasis Type="Italic">Aspergillus niger</Emphasis>

The mycelia of Aspergillus niger, cultivated in a medium containing 45 g l⁻¹ maltose, 66 g l⁻¹ yeast extract, and 5 g l⁻¹ K₂HPO₄ at 30°C and 200 rpm, were used as a biocatalyst in the glucosylation of ascorbic acid. Free mycelia from 3-day-old culture, when used in a 6-h reaction with maltose as the acyl donor, gave 16.07 g l⁻¹ ascorbic acid glucoside corresponding to a volumetric productivity of 2.68 g l⁻¹ h⁻¹ and a conversion of 67%. Mycelia from 3-day-old cultures were entrapped in calcium alginate beads and used as a catalyst in the glucosylation of ascorbic acid. An ascorbic acid-to-maltose molar ratio of 1:9 was found to be optimum, and the conversion reached 75% after 12 h. The concentration of ascorbic acid glucoside produced at this molar ratio was 17.95 g l⁻¹, and the productivity was 1.5 g l⁻¹ h⁻¹. The biocatalyst was repeatedly used in a fixed bed bioreactor for the synthesis of ascorbic acid glucoside and approximately 17 g l⁻¹ of ascorbic acid glucoside corresponding to a volumetric productivity of 1.42 g l⁻¹ h⁻¹ was produced in each use. The conversion was retained at 70% in each use. The entrapped mycelia also exhibited exceptionally high reusability and storage stability. The product was purified to 85% by anion exchange and gel permeation chromatography with a final yield of 75%.     

Changes in hydrophilic antioxidant activity in Avena sativa and Triticum aestivum leaves of different age during de-etiolation and high-light treatment

The steady-state of reactive oxygen species (ROS) in plant cells is controlled by ROS-producing and scavenging agents. A large cellular pool of antioxidant metabolites is involved in their control. Variations in this antioxidant pool may be monitored by measuring changes in hydrophilic antioxidant activity (free radical-quenching activity of water-soluble components) and ascorbic acid levels. The de-etiolation process and induction of light stress in Avena sativa and Triticum aestivum leaves were used as physiological models to study the antioxidant status at different ages. The data showed that five-day-old green plants and de-etiolated plants of the same age have similar hydrophilic antioxidant activity (8 mol ASC equivalents g FW^–1), which increases during the de-etiolation process. In oat and wheat, young leaves (five days old) had higher antioxidant status (hydrophilic antioxidant activity and ascorbic acid level) than old leaves (10 and 20 days old). High-light treatment caused a decrease in antioxidant status, especially in young leaves. Hydrophilic antioxidant activity and ascorbic acid levels recovered totally or partially after 30 or 60 min in the dark. This capacity also depends on age and species. The ascorbic acid/hydrophilic antioxidant activity ratio is presented as an indicator of antioxidant variations in response to stress, but taking into account the absolute levels of antioxidants.     

Concerning the presence and formation of ascorbic acid in yeasts

The selective, sensitive method of analysis of ascorbic acid by high performance liquid chromatography with electrochemical detection (HPLC/EC) has been used to determine the ascorbic acid content of cell extracts from yeasts grown in glucose-free medium, 0.3 M D-glucose, and 0.112 M L-galactono-1,4-lactone. Saccharomyces cerevisiae (strain G-25 and its tetraploid) and a commercial baker's yeast contained less than 2 μg ascorbic acid g^?1 wet wt. of cells when grown for 22 h in glucose-free medium. In 0.3 M D-glucose, only the commercial baker's yeast gave a slight increase (2–50 μg g^?1 wet wt. in 22 h). In 0.112 M L-galactono-1,4-lactone, all three strains produced ascorbic acid (372–587 μg g^?1 wet wt. in 22 h). Lypomyces starkeyi, a species previously reported to contain a significant amount of ascorbic acid (Heick et al., Can. J. Biochem., 47 (1972) 752), was essentially devoid of ascorbic acid under all three conditions of incubation although it did contain an HPLC/EC reactive peak (R_T = 0.87 relative to ascorbic acid) that was readily oxidized by charcoal in the presence of oxygen. The identity of this new compound remains to be determined.     

Effect of oxygen on ascorbic acid uptake and concentration in embryonic chick brain

The effects of oxygen on ascorbic acid concentration and transport were studied in chick embryo (Gallus gallus domesticus). During normoxic incubations, plasma ascorbic acid concentration peaked on fetal day 12 and then fell, before increasing again on day 20 when pulmonary respiration began. In contrast, cerebral ascorbic acid concentration rose after day 6, was maintained at a relatively high level during days 8–18, and then fell significantly by day 20. Exposure of day 16 embryos for 48 h to 42% ambient O₂ concentration decreased ascorbic acid concentration by four-fifths in plasma and by one-half in brain, compared to values in normoxic (21% O₂) or hypoxic (15% O₂) controls. Hyperoxic preincubation of embryos also inhibited ascorbic acid transport, as evidenced by decreased initial rates of saturable and Na⁺-dependent [¹⁴C]ascorbic acid uptake into isolated brain cells. It may be concluded that changes in ascorbic acid concentration occur in response to oxidative stress, consistent with a role for the vitamin in the detoxification of oxygen radicals in fetal tissues. However, changing O₂ levels have less effect on ascorbic acid concentration in brain than in plasma, indicating regulation of the vitamin by brain cells. Furthermore, the effect of hyperoxia on cerebral vitamin C may result, in part, from inhibition of cellular ascorbic acid transport.     

A comparative study between a brain Na+,K(+)-ATPase inhibitor (endobain E) and ascorbic acid

In the search of Na⁺,K⁺-ATPase modulators, we have reported the isolation by gel filtration and HPLC of a brain fraction, termed endobain E, which highly inhibits Na⁺,K⁺-ATPase activity. In the present study we compared some properties of endobain E with those of ascorbic acid. Kinetic experiments assaying synaptosomal membrane K⁺-p-nitrophenylphosphatase (K⁺-p-NPPase) activity in the presence of endobain E or ascorbic acid showed that in neither case did enzyme inhibition prove competitive in nature versus K⁺ or p-NPP concentration. At pH 5.0, endobain E and ascorbic acid maximal UV absorbance was 266 and 258 nm, respectively; alkalinization to pH 14.0 led to absorption drop and shift for endobain E but to absorbance disappearance for ascorbic acid. After cysteine treatment, endobain E absorbance decreased, whereas that of ascorbic acid remained unaltered; iodine treatment led to absorbance drop and shift for endobain E but to absorbance disappearance for ascorbic acid. HPLC analysis of endobain E disclosed the presence of two components: one eluting with retention time and UV spectrum indistinguishable from those of ascorbic acid and a second, as yet unidentified, both exerting Na⁺,K⁺-ATPase inhibition.     

Ascorbic acid enables reversible dopamine receptor 3H-agonist binding

The effects of ascorbic acid on dopaminergic ³H-agonist receptor binding were studied in membrane homogenates of bovine anterior pituitary and caudate, and rat striatum. In all tissues virtually no stereospecific binding (defined using luM (+)butaclamol) of the ³H-agonists N-propylnorapomorphine (NPA), apomorphine, or dopamine could be demonstrated in the absence of ascorbic acid. Although levels of total ³H-agonist binding were three to five times greater in the absence than in the presence of 0.1% ascorbic acid, the increased binding was entirely non-stereospecific. Greater amounts of dopamine-inhibitable ³H-NPA binding could be demonstrated in the absence of 0.1% ascorbic acid, but this measure of “specific binding” was demonstrated not to represent dopamine receptor binding since several other catecholamines and catechol were equipotent with dopamine and more potent than the dopamine agonist (±)amino-6,7-dihydroxy-1,2,3,4-tetrahydronapthalene (ADTN) in inhibiting this binding. High levels of dopamine-displaceable ³H-agonist binding were detected in fresh and boiled homogenates of cerebellum, an area of brain which receives no dopaminergic innervation, further demonstrating the non-specific nature of ³H-agonist binding in the absence of ascorbic acid. These studies emphasize that under typical assay conditions ascorbic acid is required in order to demonstrate reversible and specific ³H-agonist binding to dopamine receptors.     

Direct biosynthesis of ascorbic acid from glucose by Xanthomonas campestris through induced free-radicals

Ascorbic acid (20.4 g l^-1 in 50 h) was synthesized directly from glucose by Xanthomonas campestris as an adaptive response to induced free-radicals through HOCl treatment. Identity of ascorbic acid was confirmed through IR and NMR spectroscopy.     

Accumulation of Ascorbic Acid in the Cotyledons of Morning Glory (Pharbitis nil) Seedlings during the Induction of Flowering by Low-Temperature Treatment and the Effect of Prior Exposure to High-Intensity Light

Seedlings of Pharbitis nil strain ‘Violet’ werecultured at a low temperature, which induces their floweringeven in continuous light, with or without prior exposure tohigh-intensity light, which enhances the flower-inducing effectof the exposure to low temperature. Analysis by HPLC of extractsof cotyledons showed that the level of an unstable compoundincreased during these treatments, in addition to the increasein levels of phenylpropanoids reported previously. The compoundwas identified as ascorbic acid from the spectroscopic data.The change in the concentration of ascorbic acid at low temperaturewas correlated with the increase in the induction of floweringand the increase in levels of the phenylpropanoids. The rapidincrease in level of ascorbic acid after exposure to high-intensitylight reflected the promotive effect of high-intensity lighton the induction of flowering at low temperature. However, levelsof ascorbic acid also increased in seedlings of P. nil strain‘Kidachi’ that were cultured in high-intensity light,a treatment that does not induce flowering in this strain. Thus,ascorbic acid cannot be associated with the induction of floweringby high-intensity light alone. Ascorbic acid increased the rateof formation of caffeic acid from p-coumaric acid in vitro,a result that suggests that ascorbic acid might be involvedin the increases in levels of phenylpropanoids in the seedlings. (Received April 17, 1995; Accepted August 1, 1995)     

Chromium and chronic ascorbic acid depletion effects on tissue ascorbate,manganese, and14C retention from14C-ascorbate in guinea pigs

Chromium (Cr) potentiates the effects of insulin and a role for insulin in ascorbic acid transport has been reported. Therefore, the effects of Cr and ascorbate depletion on tissue ascorbic acid and¹⁴C distribution and excretion after a¹⁴C ascorbate dose were investigated in guinea pigs. As utilization of dietary Cr is affected by interaction with other minerals, tissue manganese (Mn), zinc (Zn), copper (Cu), and iron (Fe) were examined. For 20 wk, 40 weanling animals were fed either a Cr-deficient (<0.06 μg Cr/g diet, ?Cr) or a Cr-adequate (2 μg Cr from CrCl₃/g diet, +Cr) casein-based diet and were given 1 mg ascorbate/d (?C) or 10 mg ascorbate/d (+C) for 20 wk. Animals fed the Cr-depleted diet had decreased weight at 20 wk (p<0.01). Six hours before necropsy, animals were dosed by micropipette with 1.8 μCi ofl-[carboxyl-¹⁴C] ascorbic acid and placed in metabolic cages. Ascorbate supplementation increased Fe concentrations in most analyzed tissues, hepatic¹⁴C, tissue ascorbate and Mn concentration in the adrenal and testes, but decreased the concentrations of Cu in the kidney and Mn in the spleen. Liver Mn concentration was higher and kidney Mn concentration was lower in +Cr animals. Interactions between Cr and ascorbic acid affected Mn concentrations in bone and brain. These results indicate that ascorbate and Cr may affect Mn distribution. Chromium supplementation decreased plasma cortisol, brain¹⁴C and the amount of¹⁴C expired as carbon dioxide. These findings suggest that dietary Cr may affect ascorbic acid metabolism and the metabolic response to stress.     

Vitamin C enhancement of brood rearing by caged honeybees fed a chemically defined diet

Pollen collected by bees was sampled during a 3-h period once a week from April to October 1983 and analyzed for vitamin C (L-ascorbic acid and dehydroascorbic acid). The levels were highly variable and ranged from a low of 136 μg/g pollen (April) to a high of 1943 μg/g pollen (May). Overall, caged honeybees fed diets containing 1,000 and 2,000 μg/g L-ascorbic acid reared significantly more bees to the sealed stage than bees fed diets with 500 μg/g ascorbic acid or control bees. The levels of vitamin C in prepupae reared by bees ranged from 64.5 to 103.5 μg/g body mass. Vitamin C is either synthesized from simple precursors or from symbiotic microorganisms in the gut since honeybees fed the ascorbic acid-free control had equivalent levels of ascorbic acid to those fed the enriched diets. The total diet consumption by bees during the 10-week study showed that the four diets were equally attractive.     

Effects of ascorbic acid on the uptake of serotonin in differentiated neuroblastoma cells

Ascorbic acid causes concentration-dependent and time-dependent effects on [³H]-serotonin (³H-5HT) uptake into differentiated neuroblastoma N-2a cells. Preincubation of cells with ascorbic acid inhibits both passive diffusion and active transport of ³H-5HT (0.1 μM). The kinetic characteristics of the active uptake process change with ascorbic acid treatment, resulting in an increase in the Km from 0.27 μM to 3.0 μM and in the Vmax from 453 to 2369 fmol/min/10⁶ cells. This inhibitory effect of ascorbic acid appears to be due to its reducing properties.     

Dietary ascorbic acid deficiency in guinea pigs: No effect on ethanol preference,spiroperidol binding,or monoamine oxidase activity

Ascorbic acid levels are commonly reported to be decreased in alcoholics. Although this deficiency could be due to dietary factors, there is evidence that ascorbic acid may be involved in the metabolism and acute effects of ethanol, possibly related to the pathogenesis of alcoholism. Therefore, we examined ethanol preference in guinea pigs receiving an ascorbate deficient $\text{vs}$ a normal diet. Brain and spleen ascorbic acid levels were dramatically decreased, but ethanol preference was not altered by the acute dietary deficiency of this vitamin. In addition, an acute stressor (cold water swim), alone or in combination with ascorbate deficiency, had no effect on ethanol preference. At termination of the experiment, two measures of brain aminergic function (MAO activity and ³H-spiroperidol binding), purportedly altered by ethanol or ascorbic acid or both, were not associated with tissue ascorbate levels.     

Dehydroascorbate reduction

Dehydroascorbic acid is generated in plants and animal cells by oxidation of ascorbic acid. The reaction is believed to occur by the one-electron oxidation of ascorbic acid to semidehydroascorbate radical followed by disproportionation to dehydroascorbic acid and ascorbic acid. Semidehydroascorbic acid may recycle to ascorbic acid catalyzed by membrane-bound NADH-semidehydroscorbate reductase. However, disproportionation of the free radical occurs at a rapid rate, 10⁵ M^–1 s^–1, accounting for measurable cellular levels of dehydroascorbate. Dehydroascorbate reductase, studied earlier and more extensively in plants, is now recognized as the intrinsic activity of thioltransferases (glutaredoxins) and protein disulfide isomerase in animal cells. These enzymes catalyze the glutathione-dependent two-electron regeneration of ascorbic acid. The importance of the latter route of ascorbic acid renewal was seen in studies of GSH-deficient rodents (Meister, A. (1992)Biochem. Pharmacol. 44 1905–1915). GSH deficiency in newborn animals resulted in decreased tissue ascorbic acid and increased dehydroascorbate-to-ascorbate ratios. Administration of ascorbic acid daily to GSH-deficient animals decreased animal mortality and cell damage from oxygen stress. A cellular role is proposed for dehydroascorbate in the oxidation of nascent protein dithiols to disulfides catalyzed in the endoplasmic reticulum compartment by protein disulfide isomerase.     

Anemia associated with changes in iron and iron-59 utilization in copper deficient rats fed high levels of dietary ascorbic acid and iron

The influence of dietary copper, iron, and ascorbic acid on iron utilization was examined in a 2×2×2 factorial experiment. Male Sprague-Dawley weanling rats were fed copper-deficient (Cu-, 0.42 μg Cu/g) or copper-adequate (Cu+, 5.74 μg Cu/g) diets that contained one of two levels of iron (38 or 191μg Fe/g) and ascorbic acid (0 or 1% of the diet). These eight diets were fed for 20 d, and rats received an oral dose of 4 μCi iron-59 on d 15. Compared to Cu+ rats, the Cu− rats had 27% lower hemoglobin levels with 45, 59, and 65% lower cytochrome c oxidase (CCO) activities in the liver, heart, and bone marrow, respectively (p<0.0001). High dietary iron or ascorbic acid did not alter hemoglobin in Cu+ rats. However, hemoglobin was 23% lower in Cu− rats fed the highest, rather than the lowest levels of iron and ascorbic acid. Liver CCO was decreased (p<0.02) in Cu− rats fed high iron. Among Cu− rats, ascorbic acid did not influence CCO but decreased hemoglobin by 17% (p<0.001), reduced the percentage of absorbed iron-59 in the erythrocytes by 91% (p<0.05) and depressed the percentage apparent absorption of iron (p<0.05). These results suggest that the effects of elevated dietary iron and ascorbic acid on iron utilization are influenced by copper status.     

Stimulation of transepithelial sodium and chloride transport by ascorbic acid. Induction of Na+ channels is inhibited by amiloride

Ascorbic acid increases the short circuit current (I_sc) across the amphibian cornea when it is present at either surface of this epithelium. These effects were additive. The effect was greater when it was on the tear side. The response returned to baseline levels when the ascorbic acid was washed from the bathing media. The effect of ascorbic acid on I_sc when it was on the aqueous humor side of the cornea could be blocked by bumetanide but that due to the vitamin's presence on the tear side was unchanged. The ascorbic acid could enter the tissue and crossed the cornea at similar rates in either direction. When the cornea was bathed by a Cl^?-free solution or exposed to bumetanide, the rise in I_sc observed with ascorbic acid on the tear side was equivalent to an increased Na⁺ flux from the tear to the aqueous humor side. In normal (Cl^? present) Conway solution the rise in the I_sc seen with ascorbic acid on the aqueous humor side was equal to an increased flux of Cl^? from the aqueous to the tear surface. However, when ascorbic acid was present on the opposite, tear, side the increased I_sc reflected a rise in both Cl^? and Na⁺ transport, aqueous-to-tear side, and tear-to-aqueous side, respectively. Thiol reagents (tear side), including reduced glutathione (10^?5 M), blocked the effect of ascorbic acid (10^?3 M) providing they were added to the bathing solution prior to the vitamin. However, they had no effect once the response had been established. The effect of the reduced glutathione appeared to be of a non-competitive nature. Oxidized glutatione (10^?4 M) (and cystamine) blocked the effect of ascorbic acid (10^?3 M) when present on the tear side prior to the vitamin. However, they also increased the rate of decline of the response when added subsequently to the ascorbic acid. Amiloride (as low as 5·10^?9 M), on the tear side but not the aqueous humor side, prevented the response to ascorbic acid but could not reverse it, once it was established. The possible nature of the effect of ascorbic acid is discussed in relation to its pharmacological interactions with thiol and disulfide reagents and amiloride.     

The stability of ascorbic acid in Artemia urmiana following enrichment and subsequent starvation

The effects of vitamin enrichment on ascorbic acid (AA) levels in Artemia urmiana were studied by applying an emulsion containing ascorbyl palmitate (AP) as a vitamin C source. Nauplii were kept at 28°C in incubators containing the enrichment medium (cod liver oil, AP, sodium polysurbate, α‐tocopherol and tap water) for 0, 12, 18 and 24 h and then starved at 5°C for 0, 12, 18 and 24 h. AA was determined using a reversed phase high performance liquid chromatography coupled to an electrochemical detector. The results showed that nauplii of A. urmiana had high levels of ascorbic acid in their body tissues (1534 ± 166 μg g^?1 dry weight) and that the AA concentration increased following enrichment. The maximum enrichment level was reached by hour 18, declining by hour 24. There was a significant difference in AA levels between enriched and non‐enriched artemia (P < 0.05). Although AA contents were enhanced in all groups during starvation in cold conditions, the increases were not considerable. However, a clear correlation could be observed between duration of starving and rise in AA levels in non‐enriched and 12 and 18 h enriched groups.