Translational control of the ascorbic acid transporter SVCT2 in human platelets

Reactive oxygen species (ROS) and redox state have emerged as physiological mediators, controlling blood coagulation and thrombosis. The redox balance is obviously linked to the presence of antioxidants; in particular, vitamin C appears to be a key modulator of platelet oxidative state, since these cells physiologically accumulate ascorbic acid and, moreover, platelet ascorbate plays a role during aggregation. Here, we showed that platelets could compensate for fluctuations in ascorbate levels by modulating the expression of the Na+-dependent transporter SVCT2. Furthermore, the use of anucleated cells demonstrated, for the first time, that SVCT2 expression could be regulated at the translational level. The control of ascorbic acid uptake, through regulation of its carrier, was not only related to substrate availability, but it also occurred during platelet activation, which was accompanied by vitamin C deprivation and alteration in the redox state. Finally, we showed that changes in intracellular ascorbic acid content had physiological relevance, since they modulate the surface sulfhydryl content and the thrombus viscoelastic properties. Beside its role during aggregation, vitamin C may also have important effects during postaggregatory events.     

Rapid high-performance liquid chromatographic method for Vitamin C determination in human milk versus an enzymatic method

Vitamin C is an antioxidant that can be considered a possible biomarker of oxidative stability in human milk. A high-performance liquid chromatographic method was developed and validated for determining the total Vitamin C (ascorbic acid and dehydroascorbic acid) and ascorbic acid levels in human milk. This method was then compared with an enzymatic method (a Colorimetric technique) for quantifying ascorbic acid levels. Repeatability and reproducibility were acceptable for all methods. However, the high-performance liquid chromatography (HPLC) technique provided more satisfactory results than the enzymatic method due to this last method detected 37% less ascorbic acid and does not determine the total Vitamin C because of the enzymatic method cannot reduce the dehydroascorbic acid (DHA) to ascorbic acid. Furthermore, the HPLC method has the added advantages that it requires less reagents and material, and is simpler and less time consuming than the enzymatic method. In conclusion, the drawbacks of this enzymatic method would justify its substitution for a HPLC method.     

Ascorbic acid-independent synthesis of collagen in mice

The mouse has become the most important model organism for the study of human physiology and disease. However, until the recent generation of mice lacking the enzyme gulanolactone oxidase (Gulo), the final enzyme in the ascorbic acid biosynthesis pathway, examination of the role of ascorbic acid in various biochemical processes using this model organism has not been possible. In the mouse, similar to most mammals but unlike humans who carry a mutant copy of this gene, Gulo produces ascorbic acid from glucose. We report here that, although ascorbic acid is essential for survival, its absence does not lead to measurable changes in proline hydroxylation. Vitamin C deficiency had no significant effect on the hydroxylation of proline and collagen production during tumor growth or in angiogenesis associated with tumor or mammary gland growth. This suggests that factors other than ascorbic acid can support proline hydroxylation and collagen synthesis in vivo. Furthermore, the failure of Gulo-/- mice to thrive on a vitamin C-deficient diet therefore suggests that ascorbic acid plays a critical role in survival other than the maintenance of the vasculature.     

Coronary endothelial dysfunction is not rapidly reversible with ascorbic acid

In humans with cardiovascular risk factors, increased vascular production of superoxide anion may contribute to endothelial dysfunction by its reacting with nitric oxide and reducing its biological activity. High concentrations of ascorbic acid scavenge superoxide anion and restore normal endothelium-dependent vasodilation in humans with cardiovascular risk factors. To investigate the contribution of increased superoxide anion to endothelial dysfunction in atherosclerotic coronary arteries, we examined the effect of sequential infusions of ascorbic acid (final concentration 0.1, 1.0, and 10 mmol/L) or placebo on coronary endothelial function in 26 subjects referred for cardiac catheterization to evaluate coronary artery disease. Coronary vasomotor function was evaluated using intracoronary agonist infusion, quantitative angiography, and intracoronary Doppler measurements. At baseline, endothelium-dependent vasodilation of epicardial arteries and coronary microvessels was impaired to an equivalent extent in the ascorbic acid and placebo groups. Sequential ascorbic acid infusions had no effect on the acetylcholine-induced change in coronary artery diameter (-11+/-8, -12+/-10, and -9+/-9%) compared with the effect of placebo (-14+/-13, -16+/-10, and -13+/-9%) infusions (p=0.98). Similarly, the changes in coronary blood flow during acetylcholine infusions were equivalent during ascorbic acid (51+/-44, 67+/-66, and 62+/-52%) and placebo (61+/-104, 55+/-93, and 50+/-69%) infusions (p=0.63). Ascorbic acid also had no effect on the dilator response to intracoronary nitroglycerin (p=0.19). These data argue against an important role for superoxide-mediated "inactivation" of nitric oxide or another rapidly reversible form of oxidative stress as a mechanism of coronary endothelial dysfunction in patients with coronary atherosclerosis.     

Changes in ascorbic acid content and ascorbate peroxidase activity during the development of Acetabularia mediterranea

Abstract. The level of peroxidase activity utilizing ascorbic acid changes during the development of the green alga, Acetabularia mediterranea. During development almost parallel levels of peroxidase activity and ascorbic acid content are detectable: both steadily decrease as algae progress from very young, slowly growing cells to the rapid growth stage and then to cells exhibiting differentiation into primordium and cap. Changes in the levels of the enzyme and its substrate in the cytoplasm and periplasm were demonstrated using biochemical and cytochemical procedures. Concomitant with these developmental changes, we also observed changes in the stage-specific patterns of ascorbic acid concentration: growing algae exhibit a pronounced negative apicobasal gradient of ascorbic acid. Acetabularia cultivated at 1,200 lux (the normal intensity in a 12-h-light/12-h-dark cycle) and at 700 lux (intensity at which growth is reduced, and cap formation is delayed) were also compared. The higher light intensity induced a moderate decrease in the ascorbic acid content without noticeable changes in the compartmental distribution in the cytoplasm and periplasm, and an increase in the level of periplasmic peroxidase activity with little change in the total peroxidase activity. Catalase was found to be present at very low levels and is unlikely to play a role in H₂O₂ catabolism. Possible roles for ascorbic acid and peroxidase in the development of Acetabularia are discussed.     

The determination of ascorbic acid and uric acid in human seminal plasma using an HPLC with UV detection

Oxidative stress has been proposed as one of the potential causes for infertility in men. Ascorbic acid and uric acid play important role in protection of spermatozoa against free radicals. A method for the simultaneous determination of ascorbic acid and uric acid in human seminal plasma using HPLC with UV detection and investigation their clinical significance as antioxidants protecting male germ cells against oxidative damage are described. Semen samples were obtained from consecutive male partners of couples presenting for a fertility evaluation. After liquefaction, the samples were centrifuged and the supernatants were diluted with dithiothreitol solution and after a filtration injected onto an analytical column. For the separation, a reverse-phase column MAG 1, 250 mm × 4.6 mm, Labiospher PSI 100 C18, 5 μm, was used. The mixture of ethanol and 25 mmol/L sodium dihydrogenphosphate (2.5:97.5, v/v), pH 4.70 was used as a mobile phase. Analytical performance of this method is satisfactory for both ascorbic acid and uric acid: the intra-assay and inter-assay coefficients of variation were below 10%. Quantitative recoveries from spiked seminal plasma were between 92.1 and 102.1%. We have found no significant differences in both ascorbic acid and uric acid concentration between the smokers and non-smokers (351.0 ± 237.9 μmol/L and 323.7 ± 99.5 μmol/L vs. 444.8 ± 245.5 μmol/L and 316.6 ± 108.9 μmol/L, p>0.05). This assay is a simple and reproducible HPLC method for the simultaneous measurement of ascorbic acid and uric acid in human seminal plasma.     

Ascorbate is particularly effective against LDL oxidation in the presence of iron(III) and homocysteine/cystine at acidic pH

Metal-catalyzed LDL oxidation is enhanced by the presence of homocysteine. In this study, the effectiveness of ascorbic acid against low-density lipoprotein (LDL) oxidation by iron(III) and copper(II) in the presence of homocysteine and the main plasma disulfide cystine was investigated. Relative to the degree of LDL oxidation reached in the absence of antioxidants, ascorbic acid was particularly effective against iron-catalyzed LDL oxidation at pH 6.0. This can be explained from its stability under acidic conditions and is likely to be important in ischemia, in inflammation and exhausting exercise. At pH 7.4, an ascorbic acid concentration at least as high as the concentration of homocysteine might be necessary to efficiently inhibit LDL oxidation by iron(III) and copper(II) in the presence of homocysteine and cystine. Histidine increased the efficiency of ascorbic acid as an antioxidant against copper-mediated oxidation in this system. The capacity of homocysteine to regenerate ascorbic acid from dehydroascorbic acid appeared to play a minor role in inhibition of ascorbic acid oxidation by copper as compared to copper chelation by homocysteine.     

Amelioration of age-dependent increase in protein carbonyls of cerebral hemispheres of mice by melatonin and ascorbic acid

Melatonin secreted by the pineal gland acts as a free radical scavenger besides its role as a hormonal signaling agent. It detoxifies a variety of free radicals and reactive oxygen intermediates including hydroxyl radical, peroxynitrite anion and singlet oxygen. Ascorbic acid (Vitamin C), a water soluble vitamin, is a naturally occurring antioxidant and cofactor in various enzymes. Protein carbonyls are formed as a consequence of the oxidative modification of proteins by reactive oxygen species. Oxidative modification alters the function of protein and is thought to play an important role in the decline of cellular functions during aging. In the present study, the effect of melatonin and ascorbic acid on age-related carbonyl content of cerebral hemispheres in mice was investigated. Protein carbonyls of cerebral hemispheres have been found to be significantly higher in 18-month-old mice as compared to 1-month old mice. Administration of a single dose of melatonin (10 mg/kg body weight) and ascorbic acid (10 mg/kg body weight) intraperitoneally for three consecutive days decreases the carbonyl content in 1- and 18-month-old mice significantly. The present study thus suggests that the formation of protein carbonyls in the cerebral hemispheres of the aging mice can be prevented by the antioxidative effects of melatonin and ascorbic acid that could in turn be beneficial in having health benefits from age-related neurodegenerative diseases.     

Determination of ascorbic acid and dehydroascorbic acid in biological samples by high-performance liquid chromatography using subtraction methods: reliable reduction with tris[2-carboxyethyl]phosphine hydrochloride

Determination of dehydroascorbic acid in biological samples most commonly involves indirect measurement. The concentration is calculated by subtraction of the measured ascorbic acid concentration from that of total ascorbic acid analyzed after reduction of the dehydroascorbic acid present; a methodology also referred to as subtraction methods. Consequently, successful determination of dehydroascorbic acid is dependent on proper sample handling, quantitative reduction of the compound, and accurate quantification of both ascorbic acid and total ascorbic acid. In this paper, the recently introduced reductant tris[2-carboxyethyl]phosphine (TCEP) is evaluated as a reliable alternative to the commonly used reducing agent dithiothreitol (DTT). The results show that TCEP offers a more efficient reduction of dehydroascorbic acid at low pH compared to that of DTT. Moreover, while DTT maintains a reducing sample environment for less than 24 h, TCEP show complete protection from oxidation of ascorbic acid for at least 96 h following sample preparation. Removal of TCEP prior to analysis is unnecessary. A revised HPLC-EC method incorporating TCEP as reductant as well as the coanalysis of isoascorbic acid and uric acid is presented. The within- and between-day coefficients of variation for the complete assay are less than 1.5 and 3.5% for all analytes. As a whole, the method presented here is simpler and more reliable than existing methods.     

Collagen biosynthesis; tissue culture experiments to ascertain the role of ascorbic acid in collagen formation

Quantitative studies of collagen formation by chick embryonic lung tissue grown in media deficient in, or completely lacking, ascorbic acid have been carried out. Cell growth and collagen formation in such cultures can proceed almost normally in media lacking ascorbic acid. Ascorbic acid in combination with whole embryo extract, dialyzed media, or synthetic mixture number 703 was found to have no appreciable effect on cell growth or total collagen formation. This is in marked contrast to the almost total failure of collagen formation in scorbutic animals and suggests that for slow collagen biosynthesis as distinct from more prolific collagen-producing systems, ascorbic acid plays an indirect role.     

Ascorbic acid analysis using high-performance liquid chromatography with coulometric electrochemical detection

A method for the detection of ascorbic acid using high-performance liquid chromatography with coulometric electrochemical detection and a technique for stabilization of the vitamin are described. Since less than 1 pmol of ascorbic acid can be detected, this assay provides significantly greater sensitivity than nearly all of the currently available procedures. Stabilization of 10 pmol or less of ascorbic acid at room temperature for up to 4 h and for several weeks at -70 degrees C facilitates storage of a large number of samples and measurement of ascorbic acid using an automated sampling device. This method was used to quantitate the amounts of ascorbic acid in human polymorphonuclear leukocytes and bovine adrenomedullary chromaffin granules. The calculated concentrations found for human neutrophils (1.35 mM) and bovine chromaffin granules (10.0 mM) are in agreement with previously published data. The assay is suitable for the determination of ascorbic acid in biological samples where only a small amount of tissue is available or very low amounts of ascorbic acid are found. This method is the first application of coulometric electrochemical detection to ascorbic acid HPLC analysis.     

Changes in ascorbic acid levels in apoplastic fluid during growth of pine hypocotyls. Effect on peroxidase activities associated with cell walls

The apoplastic fluid of pine ( Pinus pinaster Aiton) hypocotyls contains ascorbic acid (AA) and dehydroascorbic acid (DHA). The amounts of ascorbic and dehydroascorbic acids were in the nmol (g fresh weight)⁻¹ range and decreased with the hypocotyl age as well as along the hypocotyl axis. The ratio AA/(AA+DHA) also decreased with the hypocotyl age and along the hypocotyl. Both ascorbic oxidase and peroxidase activity against ascorbic acid showed very low activity not only in the apoplastic fluid but also in the fractions ionically and covalently bound to the cell walls. However, the peroxidase activity in the three abovementioned fractions was strongly increased in the presence of ferulic acid. That stimulation effect increased with the hypocotyl age and from the apical towards the basal region of the hypocotyls of 10-day-old seedlings. Furthermore, the oxidation of ferulic acid by apoplastic and ionically- and covalently-bound peroxidases was inhibited by ascorbic acid as long as ascorbate was available. A regulatory role of apoplastic ascorbic acid levels in the formation of dehydrodiferulic bridges between wall polysaccharides catalysed by cell wall peroxidases and thus in the cell wall stiffening during plant growth is proposed.     

Sodium-dependent vitamin C transporter SVCT2: Expression and function in bone marrow stromal cells and in osteogenesis

Ascorbic acid (Vitamin C) has a critical role in bone formation and osteoblast differentiation, but very little is known about the molecular mechanisms of ascorbic acid entry into bone marrow stromal cells (BMSCs). To address this gap in knowledge, we investigated the identity of the transport system that is responsible for the uptake of ascorbic acid into bone marrow stromal cells (BMSCs). First, we examined the expression of the two known isoforms of the sodium-coupled ascorbic acid transporter, namely SVCT1 and SVCT2, in BMSCs (Lin ? ve Sca1 + ve) and bone at the mRNA level. Only SVCT2 mRNA was detected in BMSCs and bone. Uptake of ascorbic acid in BMSCs was Na⁺-dependent and saturable. In order to define the role of SVCT2 in BMSC differentiation into osteoblasts, BMSCs were stimulated with osteogenic media for different time intervals, and the activity of SVCT2 was monitored by ascorbic acid uptake. SVCT2 expression was up-regulated during the osteogenic differentiation of BMSCs; the expression was maximal at the earliest phase of differentiation. Subsequently, osteogenesis was inhibited in BMSCs upon knock-down of SVCT2 by lentivirus shRNA. We also found that the expression of the SVCT2 could be negatively or positively modulated by the presence of oxidant (Sin-1) or antioxidant (Ascorbic acid) compounds, respectively, in BMSCs. Furthermore, we found that this transporter is also regulated with age in mouse bone. These data show that SVCT2 plays a vital role in the osteogenic differentiation of BMSCs and that its expression is altered under conditions associated with redox reaction. Our findings could be relevant to bone tissue engineering and bone related diseases such as osteoporosis in which oxidative stress and aging plays important role.     

A newly established strain of spontaneously hypertensive rat with a defect of ascorbic acid biosynthesis.

To investigate the effects of ascorbic acid deficiency on the pathogenesis of hypertension and/or its complications, we established a rat strain with both genetic hypertension and a defect of ascorbic acid biosynthesis. The od gene (L-gulono-gamma-lactone oxidase gene) of the ODS (Osteogenic Disorder Shionogi) rat, which is a rat mutant unable to synthesize ascorbic acid, was introduced into spontaneously hypertensive rats (SHR), and a novel congenic strain, SHR-od, was established. SHR-od showed scurvy when fed an ascorbic acid-free diet. Systolic blood pressure of male SHR-od began to increase at 9 weeks of age and reached 190-200 mmHg at 20 weeks of age. In 25-week-old SHR-od, ascorbic acid deficiency when fed an ascorbic acid-free diet for 6 weeks caused a remarkable reduction of blood pressure to lower than 110 mmHg. The wall to lumen ratio of the testicular artery in ascorbic acid-deficient SHR-od was lower than that of the control rats. When rats were fed a diet supplemented with ascorbic acid (300 mg/kg), ascorbic acid concentration in SHR-od was lower in the serum and liver than that in ODS rats. These results indicate that ascorbic acid could be closely related to the development of hypertension in SHR-od. We believe that SHR-od will be a useful model for experimental studies on hypertension and its complications, since all of them suffer from hypertension spontaneously and the level of ascorbic acid deficiency in these rats could be controlled at will both in concentration and duration.     

Liquid chromatographic behavior of ascorbate on amine columns

Quantitation of ascorbate at concentrations normally found in biological samples and foods has previously been shown to be possible by HPLC analysis. Prefilled amine columns from three manufacturers were presently used to evaluate their potential for separating low concentrations of [14C]ascorbic acid from its degradation products, [14C]dehydroascorbic acid and [14C]diketogulonic acid. A successful separation was achieved on some columns with as little as 200 cpm (30 pmol) of total ascorbate injected. On other columns, injection of 30-500 pmol of ascorbate resulted in as much as 80% of [14C]ascorbic acid eluting with an unpredictable retention time. In these instances the inclusion of nonlabeled ascorbic acid (0.5 mg/ml) to the sample resulted in most of the [14C]ascorbic acid activity eluting at the expected retention time of ascorbic acid. The inclusion of ascorbic acid in samples injected onto the column also resulted in a more discrete peak in the elution of dehydroascorbic acid, and more complete recovery of the total [14C]activity (ascorbic acid, dehydroascorbic acid, and diketogulonic acid) injected onto the column.     

COLLAGEN BIOSYNTHESIS : TISSUE CULTURE EXPERIMENTS TO ASCERTAIN THE ROLE OF ASCORBIC ACID IN COLLAGEN FORMATION

Quantitative studies of collagen formation by chick embryonic lung tissue grown in media deficient in, or completely lacking, ascorbic acid have been carried out. Cell growth and collagen formation in such cultures can proceed almost normally in media lacking ascorbic acid. Ascorbic acid in combination with whole embryo extract, dialyzed media, or synthetic mixture number 703 was found to have no appreciable effect on cell growth or total collagen formation. This is in marked contrast to the almost total failure of collagen formation in scorbutic animals and suggests that for slow collagen biosynthesis as distinct from more prolific collagen-producing systems, ascorbic acid plays an indirect role.     

Influence of cultural and physiochemical factors on ascorbate stability in plant tissue culture media

Ascorbic acid rapidly decays in plant tissue culture media. Within 50 min to 3 h after preparing 100 mM solutions, ascorbic acid was destroyed. Autoclaving, shaking flasks, high light intensity and increasing pH over a range from 4.5–7 accelerated decay. Ascorbic acid was oxidized to dehydroascorbic acid which also underwent decay. Within 11 h and 15 min after adding ascorbic acid both ascorbic acid and its oxidation product, dehydroascorbic acid, disappeared from medium. Since ascorbic acid is rapidly destroyed in plant tissue culture media it may not exert its effect as an intact molecule. Instead its antioxidant/antibrowning role in plant cell, tissue and organ cultures may be mediated by some product of further oxidation.     

Role of ascorbic acid in photosynthesis

Experimental data concerning the role of ascorbic acid in both the maintenance of photosynthesis and in the protection of the photosynthetic apparatus against reactive oxygen species and photoinhibition are reviewed. The function of ascorbic acid as an electron donor in the “Krasnovsky reaction”, as well as its physiological role as a donor to components of the photosynthetic electron transport chain, which was first studied by A. A. Krasnovsky in the 1980s, is discussed. Data on the content and transport of ascorbic acid in plant cells and chloroplasts are presented.     

Source of electron energy for animal metabolism

Summary The histochemical localization of ascorbic acid was carried out in the pectoral muscles of various birds using a modified method. — The greater deposition of silver granules within the red fibres as well as in their nuclei than in those of the white ones is correlated with higher metabolic activity of the former. — The possible role of ascorbic acid as a participant in energy transfer mechanisms in the muscle fibres; in lipid synthesis and the importance of AA and its free radical in protein metabolism are discussed.     

抗坏血酸在高粘度聚乙烯醇胶体溶液中分散均一性考察

目的:考察了采用实验室常用的磁力搅拌法和改进的机械搅拌法后,聚乙烯醇胶体溶液中抗坏血酸的分布均一度。接着,在机械搅拌法的基础上,研究了抗坏血酸粉末与抗坏血酸溶液在胶中分散的均一性。方法:分散混合方法:机械搅拌法,磁力搅拌法;均一性检验方法:以单位质量胶载药量RSD值作为指标;统计学分析方法:t检验。结果:采用磁力搅拌法不能够较好地混合抗坏血酸与PVA胶体溶液;而机械搅拌法RSD值控制在10%以内,说明胶中抗坏血酸均一分布,符合生产要求。T检验表明两种方法具有显著性差异。药物溶于水后分散更为均一。结论:解决药物在高粘度溶液介质中均匀分散的问题。