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PubMed2001

Dehydroascorbic acid and oxidative stress in haemodialysis patients.

BACKGROUND: The amount of dehydroascorbic acid contained within total ascorbic acid (oxidized as well as non-oxidized forms) in plasma, hereafter referred to as the dehydroascorbic acid fraction, may be a measure of oxidative stress during haemodialysis. In the present study, we determined this fraction in chronic haemodialysis patients. METHODS: Using high performance liquid chromatography, dehydroascorbic acid and total ascorbic acid levels were measured in 80 maintenance haemodialysis patients for a period of > 2 years as well as in 49 controls, to examine a possible association of these compounds with clinical parameters and/or drugs taken by the patients. RESULTS: Dialysis patients who had an increased plasma urate level (P < 0.05) and had been taking allopurinol (P < 0.05) or NSAID (non-steroid anti-inflammatory drugs) (P < 0.01), and dialysis patients who were younger (< or = 55 years), as compared with older dialysis patients (P < 0.01), were found to have a lower dehydroascorbic acid fraction by multivariate analysis. Mean plasma dehydroascorbic acid levels and dehydroascorbic acid fractions were significantly lower in the younger haemodialysis patients (4.8 +/- 0.7 micromol/l and 28.4 +/- 3.9%) than in healthy younger controls (13.3 +/- 1.1 micromol/l and 41.1 +/- 1.8%) (P < 0.0001 and P < 0.01, respectively). Moreover, a correlation was found between plasma dehydroascorbic acid fraction and plasma lipid peroxide (r = 0.66, P < 0.01) in patients who had not been taking allopurinol and/or NSAID. CONCLUSION: We found that dehydroascorbic acid fraction was related to patients' age, plasma urate level and to taking allopurinol or NSAID. Dehydroascorbic acid fraction may be another indirect index of oxidative stress.

Aging↗

PubMed1983

Reinvestigation of the diabetogenic effect of dehydroascorbic acid.

To investigate the diabetogenic effect of pure dehydroascorbic acid, male Wister- and Sprague-Dawley rats received i.v. injections of the substance. No hyperglycemia and no decreased glucose tolerance were found. I.v. administration of the hydrolysis products of dehydroascorbic acid and of a solution containing monodehydroascorbate likewise did not increase blood glucose values. It is concluded that in previously performed experiments not dehydroascorbic acid itself but one or several impurities might have produced hyperglycemia in the rat. The electron transfer proteins tested (ascorbate:ferricytochrome b5 oxidoreductase, cytochrome b5, NADH:ferricytochrome b5 oxidoreductase, NADH:monodehydroascorbate oxidoreductase), which might participate in the reduction of dehydroascorbic acid, could not be induced in liver microsomes from Wistar rats by the injection of dehydroascorbic acid, its hydrolysis products, or monodehydroascorbate.

Animals↗

PubMed2000

Enzymatically validated liquid chromatographic method for the determination of ascorbic and dehydroascorbic acids in fruit and vegetables.

A liquid chromatographic method has been described for the determination of total vitamin C, ascorbic acid (AA) and dehydroascorbic acid (DHAA) in fruits and vegetables. The complete separation of AA and DHAA could be achieved on a C18 column using 0.2 M KH2PO4 (pH adjusted to 2.4 with H3PO4) as the mobile phase at a flow-rate of 0.5 ml/min. Since the detection sensitivity was poor for DHAA even at 210 nm, it was estimated as the difference between the total AA after DHAA reduction and AA content of the original sample, using dithiothreitol (DTT) as the precolumn reductant. The reaction times for the complete conversion of DHAA to AA at room temperatures were 150, 120, 90 and 75 min for 1, 2, 4 and 8 mmol DTT per mmol of DHAA, respectively. The percentage recovery ranged from 81.7 to 105.9. AA contents of some selected fruits and vegetables were analyzed comparatively by liquid chromatography and enzymatic assay to validate the method.

Ascorbic Acid↗

PubMed1996

Enzymatic method to determine dehydroascorbic acid in biological samples and in bread dough at various stages of mixing.

An enzymatic method is described for measuring L-dehydroascorbic acid in perchloric acid extracts of biological samples. The enzyme used in the assay was glutathione dehydrogenase (glutathione:dehydroascorbate oxidoreductase), which was purified from wheat flour using three column chromatography steps. The enzyme catalyzes the reduction of dehydroascorbic acid by glutathione, and the ascorbic acid product is measured spectrophotometrically at 265 nm. The assay is a fast (about 2 min) and simple two-step procedure. First, a mixture of extract and buffer is set to zero absorbance against air in a spectrophotometer. Second, a solution of glutathione dehydrogenase and glutathione is added and the absorbance after 2 min is used in a formula to calculate nmol dehydroascorbic acid/g or ml sample [513.8(Abs-0.013) x factor for dilution of extract]. The formula was derived from a calibration graph using pure L-dehydroascorbic acid standards. Pure L-dehydroascorbic acid was prepared from pure L-ascorbic acid, and the ascorbate oxidase used to catalyze the reaction was removed by ultrafiltration. Commercial L-dehydroascorbic acid (Aldrich) was unsuitable for use as a standard because the purity was only 56-67% in comparison to laboratory-prepared L-dehydroascorbic acid. The sensitivity of the assay was such that when dehydroascorbic acid was added to healthy human blood plasma during extraction with perchloric acid at the level of 7.5 nmol/ml plasma the dehydroascorbic acid could be measured with complete recovery. Low levels of dehydroascorbic acid were detected in fresh fruits and vegetables. When samples were ground for several minutes before extraction with perchloric acid, the dehydroascorbic acid levels increased more than 10-fold. Dehydroascorbic acid increased rapidly during mixing of bread dough containing added L-ascorbic acid.

Ascorbic Acid↗

PubMed1987

Ascorbic and dehydroascorbic acids simultaneously quantified in biological fluids by liquid chromatography with fluorescence detection, and comparison with a colorimetric assay.

We describe a "high-performance" liquid-chromatographic method for separating and quantifying ascorbic acid (AA) and dehydroascorbic acid (DHA) in plasma and urine. We used a reversed-phase C18 column with an ion-pair reagent and detected the analytes by post-column reaction with 4,5-dimethyl-o-phenylenediamine to form a fluorescent derivative (measured at excitation and emission wavelengths of 365 and 440 nm, respectively). Isoascorbic acid (IA) is the internal standard. Retention times for DHA, AA, and IA are 5.6, 15.5, and 19.9 min, respectively. Between-day CVs for AA in plasma in concentrations of 8 and 20 mg/L were 9% and 7%, respectively. The limit of detection is 10 and 4 ng for AA and DHA, respectively. Results by the present method and the methoxyaniline colorimetric method for AA are comparably accurate.

Ascorbic Acid↗

PubMed1987

A highly sensitive high-performance liquid chromatography method for the estimation of ascorbic and dehydroascorbic acid in tissues, biological fluids, and foods.

A highly sensitive procedure for determining ascorbic acid (AA) and dehydroascorbic acid (DHAA) by high-performance liquid chromatography with electrochemical detection in biological fluids, tissues, and foods is described. AA is separated in a C18 reverse-phase column after extraction from the sample with metaphosphoric acid. An aliquot of 20 microliter of diluted extract is injected into the column for the estimation of AA. DHAA is indirectly estimated by converting it to AA after reduction with DL-homocysteine at pH 7.0-7.2 for 30 min at 25 degrees C. After dilution, a 20-microliter aliquot is injected into the column to obtain total vitamin C (AA + DHAA). The concentration of DHAA is calculated by subtraction. AA can be reproducibly quantified at concentrations as low as 50 pg/20 microliter of sample extract. The method described here used a specially designed mobile phase, gave greater stability and a noiseless baseline, and increased substantially the sensitivity and precision. The procedure is rapid, analysis being completed within 10 min after sample preparation, and has been successfully applied to biological fluids, tissues, and foods.

Animals↗

PubMed1994

Spectrophotometric determination of dehydroascorbic acid in biological samples.

We describe a method for accurately and precisely measuring dehydroascorbic acid in perchloric acid extracts prepared from human plasma, lymphocytes, and mammalian cells. Samples were assayed by spectrophotometrically monitoring the kinetics of the concentration-dependent absorbance changes of dehydroascorbic acid with phosphate-methanol-containing buffers. The lowest detectable dehydroascorbate concentration using this assay is estimated to be below 0.1 mumol/liter. Total analysis time is less than 10 min and allows the simultaneous measurement of numerous samples. The calibration curve is linear (r > 0.995) over the range 0-200 mumol/liter. The dehydroascorbic acid concentrations measured in supplemented samples agree with known concentrations. Interference of ascorbic acid and 2,3-diketogulonic acid with this assay was excluded. The correlation with a highly specific chromatographic procedure gave comparable results over the range of physiologically relevant concentrations. The procedure avoids the most commonly applied method of measuring the native ascorbic acid, then reducing the dehydroascorbic acid, and finally measuring the total ascorbic acid and determining dehydroascorbic acid by the difference. Stabilization of ascorbic acid during assay was achieved by addition of desferrioxamine.

3T3 Cells↗

PubMed2000

Simple in-line postcolumn oxidation and derivatization for the simultaneous analysis of ascorbic and dehydroascorbic acids in foods.

A new analytical procedure for the simultaneous determination of L-ascorbic acid (AA), isoascorbic acid (IAA), L-dehydroascorbic acid (DHAA), and isodehydroascorbic acid (IDHAA) in food by high-performance liquid chromatography (HPLC) is developed. After separation on an HPLC column, an in-line oxidation of AA and IAA to DHAA and IDHAA, respectively, is performed on a short column of activated charcoal. The dehydroascorbic acids are derivatized with a 1,2-phenylenediamine solution in a heated capillary Tefzel reactor into fluorescent quinoxaline compounds and monitored fluorometrically. The chromatographic method provides good separation of LAA, LDHAA, and their diastereoisomers in a relatively short time (-10 min). After optimization of postcolumn derivatization conditions, calibration runs and recovery tests are performed. The fluorescent response in terms of peak area is highly proportional to the concentration of all derivatives examined over a range of 0.1 to 100 microg/mL solution for LAA, LDHAA, IAA, and IDHAA. Recoveries were in the range of 97 to 103%. The detection limit is 0.1 mg of each ascorbic acid derivative per 100 g food. A wide variety of foods (fruits, fruit juices, vegetables, vegetable products, milk, liver, and sausage) are analyzed by the developed procedure. The Vitamin C (LAA and LDHA) contents determined according to the present analytical method are in the same order of magnitude as the result of precolumn derivatization and the fluorometric methods. The described method is a highly specific procedure for determining Vitamin C in food. It is simple to handle, only slightly susceptible to disturbance, perfectly suitable for serial determinations, and yields reproducible results.

Ascorbic Acid↗

PubMed1983

A specific high-performance liquid chromatography assay for dehydroascorbic acid shows an increased content in CLL lymphocytes.

A method for the assay of dehydroascorbic acid using high-performance liquid chromatography with uv detection is described. The dehydroascorbic acid is separated from ascorbic acid and reduced with dithiothreitol, and is then quantitated as ascorbic acid following rechromatography. Since as little as 22 pmol can be detected, sensitivity is at least 40-fold greater than that of other currently available procedures. This method was used to measure the level of dehydroascorbic acid in normal and chronic lymphocytic leukemia lymphocytes. A significantly higher concentration of dehydroascorbic acid was found in leukemic (21.80 +/- 3.55 nmol/10(8) cells, mean +/- SE) than in normal lymphocytes (9.32 +/- 1.15 nmol/10(8) cells) (P less than 0.03). Analysis of extracts from normal B cell lymphocytes revealed comparable dehydroascorbic acid levels to unfractionated lymphocytes, indicating that the elevated level in chronic lymphocytic leukemia was not simply a reflection of the increased percentage of B lymphocytes in this disorder. These studies illustrate that the technique can be used to measure the dehydroascorbic acid content from sources where only scanty material is available or low levels are found.

Ascorbic Acid↗

PubMed1995

Accumulation of vitamin C (ascorbate) and its oxidized metabolite dehydroascorbic acid occurs by separate mechanisms.

It is unknown whether ascorbate alone (vitamin C), its oxidized metabolite dehydroascorbic acid alone, or both species are transported into human cells. This problem was addressed using specific assays for each compound, freshly synthesized pure dehydroascorbic acid, the specially synthesized analog 6-chloroascorbate, and a new assay for 6-chloroascorbate. Ascorbate and dehydroascorbic acid were transported and accumulated distinctly; neither competed with the other. Ascorbate was accumulated as ascorbate by sodium-dependent carrier-mediated active transport. Dehydroascorbic acid transport and accumulation as ascorbate was at least 10-fold faster than ascorbate transport and was sodium-independent. Once transported, dehydroascorbic acid was immediately reduced intracellularly to ascorbate. The analog 6-chloroascorbate had no effect on dehydroascorbic acid transport but was a competitive inhibitor of ascorbate transport. The Ki for 6-chloroascorbate (2.9-4.4 microM) was similar to the Km for ascorbate transport (9.8-12.6 microM). 6-Chloroascorbate was itself transported and accumulated in fibroblasts by a sodium-dependent transporter. These data provide new information that ascorbate and dehydroascorbic acid are transported into human neutrophils and fibroblasts by two distinct mechanisms and that the compound available for intracellular utilization is ascorbate.

Ascorbic Acid↗

PubMed2002

Accumulation of intracellular ascorbate from dehydroascorbic acid by astrocytes is decreased after oxidative stress and restored by propofol.

Primary rat astrocyte cultures absorbed dehydroascorbic acid from the medium and reduced it to intracellular ascorbate. Uptake of dehydroascorbic acid (5-200 microM) was inhibited only partially by glucose (10 mM). The remaining glucose-insensitive component of dehydroascorbic acid uptake was inhibited reversibly by sulfinpyrazone (IC(50) = 80 microM). Dehydroascorbic acid uptake was not mediated by Na(+)-ascorbate cotransporters or volume-sensitive anion channels because it was neither Na(+)-dependent nor blocked by the channel antagonist, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Oxidative stress, induced in astrocytes by the lipophilic radical generator tert-butyl hydroperoxide, decreased intracellular glutathione concentration and inhibited accumulation of intracellular ascorbate from dehydroascorbic acid. Subsequent administration of either the native antioxidant alpha-tocopherol (200 microM) or anesthetic concentrations of the antioxidant sedative propofol (1-8 microM, administered 30 min after tert-butyl hydroperoxide), did not change glutathione concentration but restored the ability of astrocytes to accumulate intracellular ascorbate from dehydroascorbic acid. These results are consistent with a novel mechanism of astrocytic ascorbate accumulation that is inhibited by lipophilic radicals and protected by lipophilic antioxidants such as propofol.

Animals↗

PubMed1986

Glucose-independent transport of dehydroascorbic acid in human erythrocytes.

It has been previously reported that glucose and its structural analogs inhibit dehydroascorbic acid (DHA) transport across the membranes of nonpolar cells, which led to the suggestion that the hexose transporter mediates dehydroascorbic acid transport. The present study examines the role of the erythrocyte hexose transport system in dehydroascorbic acid uptake. We have confirmed that dehydroascorbic acid may be a ligand of the hexose transport system under certain experimental conditions. However, there is an additional pathway of dehydroascorbic acid transport that is uninfluenced by external glucose. This pathway is one of facilitated diffusion, demonstrating saturation kinetics of transport, cis-inhibition, and trans-stimulation. The Km for the system is 412 microM. It is suggested that this previously undescribed sugar-independent transporter is the physiologically important route of DHA uptake in erythrocytes.

Adult↗

PubMed2002

Very low vitamin C activity of orally administered L-dehydroascorbic acid.

The biological activity of L-dehydroascorbic acid (DHA), which is easily formed from L-ascorbic acid (ASC) during storage and cooking processes, has been considered to be equivalent to that of ASC on the basis of studies made several decades ago, when a specific method to determine ASC was not available. The nutritional activity of orally ingested DHA has now been evaluated by comparing ASC concentrations in 12 tissues of rats administered four different doses of ASC. Determinations were made by using the specific and sensitive method, which had been developed by us. Here it is shown that the efficiency of DHA was almost 10% of that of ASC on a molar basis, based on animal experiments using the inherently scorbutic ODS rat, which is a convenient human model animal to investigate the metabolism of vitamin C. On the basis of these findings, it is proposed that it is necessary to reevaluate the nutritional requirement of vitamin C based on both ASC and DHA contents of foods.

Administration, Oral↗

PubMed1997

Increased facilitated transport of dehydroascorbic acid without changes in sodium-dependent ascorbate transport in human melanoma cells.

Many cell types transport vitamin C solely in its oxidized form, dehydroascorbic acid, through facilitative glucose transporters. These cells accumulate large intracellular concentrations of vitamin C by reducing dehydroascorbic acid to ascorbate, a form that is trapped intracellularly. Certain specialized cells can transport vitamin C in its reduced form, ascorbate, through a sodium-dependent cotransporter. We found that normal human melanocytes and human malignant melanoma cells are able to transport vitamin C using both mechanisms. Melanoma cell lines transported dehydroascorbic acid at a rate that was at least 10 times greater than the rate of transport by melanocytes, whereas both melanoma cells and melanocytes transported ascorbate with similar efficiency. Dehydroascorbic acid transport was inhibited by deoxyglucose and cytochalasin B, indicating the direct participation of facilitative glucose transporters in the transport of oxidized vitamin C. Melanoma cells accumulated intracellular vitamin C concentrations that were up to 100 times greater than the corresponding extracellular dehydroascorbic acid concentrations, whereas intracellular accumulation of vitamin C by melanocytes never exceeded the extracellular level of dehydroascorbic acid. Melanoma cells transported dehydroascorbic acid through at least two different transporters, each with a distinct K(m), a finding that agreed well with the presence of several glucose transporter isoforms in these cells. Only one kinetic component of ascorbate uptake was identified in both melanocytes and melanoma cells, and ascorbate transport was sodium dependent and inhibited by ouabain. Both cell types were able to accumulate intracellular concentrations of vitamin C that were greater than the extracellular ascorbate concentrations. The data indicate that melanoma cells and normal melanocytes transport vitamin C using two different transport systems. The transport of dehydroascorbic acid is mediated by a facilitated mechanism via glucose transporters, whereas transport of ascorbic acid involves a sodium-ascorbate cotransporter. The differential capacity of melanoma cells to transport the oxidized form of vitamin C reflects the increased expression of facilitative transporters associated with the malignant phenotype.

Ascorbic Acid↗

PubMed1953