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 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.     

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.     

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.     

Quantitative analysis of ascorbic acid and dehydroascorbic acid by high-performance liquid chromatography

High-performance liquid chromatography with spectrophotometric detection has been used to separate and quantitate ascorbic acid and dehydroascorbic acid. These components of vitamin C are resolved on a Lichrosorb-NH₂ column. The technique is capable of quantitatively following oxidation of ascorbic acid to dehydroascorbic acid and the reverse reduction. The technique is demonstrated to be suitable for assay of vitamin C in biological samples, foods, and pharmaceutical vitamin preparations.     

Improved method for simultaneous determination of ascorbic acid and dehydroascorbic acid,isoascorbic acid and dehydroisoascorbic acid in food and biological samples

The total vitamin C amount in different food and plasma samples was determined by a dual detection system, after HPLC separation, with direct detection of ascorbic acid and indirect fluorimetric detection of dehydroascorbic acid after a post-column O-phenyldiamine derivatisation. The two active forms of vitamin C and their d-isomers were separated within 10 min. The repeatability was determined by measurement of several fruits and vegetables and ranged from 0.3 to 1.9% (relative standard deviation) for vitamin C. The reproducibility, based on double determinations, ranged from 1.9 to 3.6% for vitamin C, depending on the matrix. The reproducibility, based on several determinations of reference materials, ranged from 2.4 to 3.7% for ascorbic acid and from 4.3 to 5.8% for dehydroascorbic acid, again depending on the matrix.     

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.     

Vitamin C transporters

Vitamin C is a wide spectrum antioxidant essential for humans, which are unable to synthesize the vitamin and must obtain it from dietary sources. There are two biologically important forms of vitamin C, the reduced form, ascorbic acid, and the oxidized form, dehydroascorbic acid. Vitamin C exerts most of its biological functions intracellularly and is acquired by cells with the participation of specific membrane transporters. This is a central issue because even in those species capable of synthesizing vitamin C, synthesis is restricted to the liver (and pancreas) from which is distributed to the organism. Most cells express two different transproter systems for vitamin C; a transporter system with absolute specificity for ascorbic acid and a second system that shows absolute specificity for dehydroascorbic acid. The dehydroascorbic acid transporters are members of the GLUT family of facilitative glucose transporters, of which at least three isoforms, GLUT1, GLUT3 and GLUT4, are dehydroascorbic acid transporters. Ascorbic acid is transported by the SVCT family of sodium-coupled transporters, with two isoforms molecularly cloned, the transporters SVCT1 y SVCT2, that show different functional properties and differential cell and tissue expression. In humans, the maintenance of a low daily requirement of vitamin C is attained through an efficient system for the recycling of the vitamin involving the two families of vitamin C transporters.     

High-performance liquid chromatographic separation of ascorbic acid,erythorbic acid,dehydroascorbic acid,dehydroerythorbic acid,diketogulonic acid,and diketogluconic acid

High-performance liquid chromatography on a Zorbax NH₂ analytical column, with acetonitrile: 0.05 m KH₂PO₄ (75:25, $\text{w}\text{w}$) used as eluant, has allowed the separation, in less than 14 min, of ascorbic acid, erythorbic acid, dehydroascorbic acid, dehydroerythorbic acid, diketogulonic acid, and diketogluconic acid. Ultraviolet monitoring at 268 nm allows ascorbic acid and erythorbic acid to be detected at the 25-ng level, while refractive index detection monitors the elution of all six compounds. Tyrosine is a good internal standard, being well separated from the other compounds and having an adequate ultraviolet absorption at 268 nm. We have found dithiothreitol to be effective in rapidly reducing dehydroascorbic acid to ascorbic acid, providing the basis for indirectly determining dehydroascorbic acid after its reduction. The potential of this high-performance liquid chromatographic procedure for evaluating the levels of these compounds in orange juice and urine is demonstrated.     

Simultaneous high-performance liquid chromatographic determination of ascorbic acid and dehydroascorbic acid in biological samples

The ascorbic acid (AA)—dehydroascorbic acid redox couple is an important component of many biological systems, and various physiological roles have been described for this vitamin. Simultaneous measurement of both AA and dehydroascorbate using high-performance liquid chromatography (HPLC) has proven difficult owing to detection problems. A simple, single-step HPLC assay for the simultaneous detection of both AA and dehydroascorbate was developed without the burden of derivatization of either compounds. This has proven to be a reliable technique and should be applicable to a wide variety of biological samples.     

6-Bromo-6-deoxy-L-ascorbic acid: an ascorbate analog specific for Na+-dependent vitamin C transporter but not glucose transporter pathways

Vitamin C intracellular accumulation is mediated by Na(+)-dependent vitamin C transporters SVCT1 and -2 and dehydroascorbic acid transporters GLUT1 and -3. It is unclear which pathways dominate in vivo. As a new step to resolve this issue, we identified and tested 6-bromo-6-deoxy-L-ascorbic acid as a specific candidate for SVCTs. In high performance liquid chromatography and electron paramagnetic resonance analyses, the reduced compounds ascorbic acid and 6-bromo-6-deoxy-L-ascorbic acid were similar. The oxidized products 6-bromo-6-deoxy dehydroascorbic acid (BrDHA) and dehydroascorbic acid (DHA) had comparable stabilities, based on reduction recoveries. Upon expression of GLUT1 or GLUT3 in Xenopus oocytes, BrDHA was neither transported nor bound, in contrast to robust transport of DHA. The findings were not explained by differences in the oocyte reduction of DHA and BrDHA because lysed oocytes reduced both compounds equally. Further, there was no transport of the reduced compound, 6-bromo-6-deoxy-L-ascorbic acid, by GLUT1 or GLUT3. As a prerequisite for investigating 6-bromo-6-deoxy-L-ascorbic acid transported by SVCTs, SVCT2 transport activity in oocytes was enhanced 14-fold by construction and use of a vector that added a fixed poly(A) tail to the 3' end of cRNA. For SVCT1 and SVCT2 expressed in oocytes, similar K(m) and V(max) values were observed for ascorbic acid and 6-bromo-6-deoxy-L-ascorbic acid. In human fibroblasts, predicted to have SVCT-mediated ascorbate accumulation, K(m) and V(max) values were again comparable for ascorbic acid and 6-bromo-6-deoxy-L-ascorbic acid. Using activated human neutrophils, predicted to have ascorbate accumulation mediated predominantly by DHA and GLUT transporters, 6-bromo-6-deoxy-L-ascorbic acid accumulation was <1% of accumulation when compared with ascorbic acid. We conclude that 6-bromo-6-deoxy-L-ascorbic acid is the first transport substrate identified as completely specific for SVCTs, but not GLUTs, and provide a new strategy to determine the contribution of each pathway to ascorbate accumulation.     

Simultaneous determination of ascorbic acid and dehydroascorbic acid in cultures of C3H/10T1/2 cells

Summary A reproducible method is described for the separation and quantification of ascorbic acid and dehydroascorbic acid by ion-pairing reverse-phase high performance liquid chromatography and detection by absorbance at 232 nm. Lowest detectable concentrations with a linear response of detection were 5 nmol for ascorbic acid and 50 nmol for dehydroascorbic acid. This method was applied to the analysis of C3H/10T1/2 cells and culture medium after influx or efflux experiments and single or multiple treatments with ascorbic acid. Subsequent measurement of the radioactivity in the eluted fractions increased the detectability of both ascorbic acid and dehydroascorbic acid to 10 to 20 pmol. This research was supported by grant CA 09320 and CA 31574 from the National Cancer Institute, Bethesda, MD, and grant BC441 from The American Cancer Society.     

Mechanism of vitamin C inhibition of cell death induced by oxidative stress in glutathione-depleted HL-60 cells

Vitamin C is a well known antioxidant whose precise role in protecting cells from oxidative challenge is uncertain. In vitro results have been confounded by pro-oxidant effects of ascorbic acid and an overlapping role of glutathione. We used HL-60 cells as a model to determine the precise and independent role of vitamin C in cellular protection against cell death induced by oxidative stress. HL-60 cells do not depend on glutathione to transport or reduce dehydroascorbic acid. Depletion of glutathione rendered the HL-60 cells highly sensitive to cell death induced by H2O2, an effect that was not mediated by changes in the activities of glutathione reductase, glutathione peroxidase, catalase, or superoxide dismutase. The increased sensitivity to oxidative stress was largely reversed when glutathione-depleted cells were preloaded with ascorbic acid by exposure to dehydroascorbic acid. Resistance to H2O2 treatment in cells loaded with vitamin C was accompanied by intracellular consumption of ascorbic acid, generation of dehydroascorbic acid, and a decrease in the cellular content of reactive oxygen species. Some of the dehydroascorbic acid generated was exported out of the cells via the glucose transporters. Our data indicate that vitamin C is an important independent antioxidant in protecting cells against death from oxidative stress.     

Determination of ascorbic and dehydroascorbic acids in blood plasma and serum by liquid chromatography

A liquid-chromatography (LC) method with ultraviolet detection for measuring ascorbic (AA) and dehydroascorbic acid (DHA) in human blood and serum was studied. The method used an ODS reversed-phase column and cetyltrimethylammonium bromide as an ion-pairing agent. AA was measured before and after the reduction of DHA with dithiothreitol. The absene of interferences resulting from hemolysis products was verified and also the stability of the ascorbic acid in metaphosphoric acid extracts. The analytical parameters, linearity (1–80 μg/ml), accuracy (recovery, 96.7–100.7%) and precision (C.V.=3.1%), show that the method is reliable and adequate for measuring the total vitamin C content in serum and plasma.     

Vitamin C homeostasis in skeletal muscle cells

In skeletal muscle, vitamin C not only enhances carnitine biosynthesis but also protects cells against ROS generation induced by physical exercise. The ability to take up both ascorbic and dehydroascorbic acid from the extracellular environment, together with the ability to recycle the intracellular vitamin, maintains high cellular stores of ascorbate. In this study, we examined vitamin C transport and recycling, by using the mouse C2C12 and rat L6C5 muscle cell lines, which exhibit different sensitivity to oxidative stress and GSH metabolism. We found that: (1) both cell lines express SVCT2, whereas SVCT1 is expressed at very low levels only in proliferating L6C5 cells; furthermore L6C5 myoblasts are more efficient in ascorbic acid transport than C2C12 myoblasts; (2) C2C12 cells are more efficient in dehydroascorbic acid transport and ascorbyl free radical/dehydroascorbic acid reduction; (3) differentiation is paralleled by decreased ascorbic acid and dehydroascorbic acid transport and reduction and increased ascorbyl free radical reduction; (4) differentiated cells are more responsive to oxidative stress induced by glutathione depletion; indeed, myotubes showed increased SVCT2 expression and thioredoxin reductase-mediated dehydroascorbic acid reduction. From our data, SVCT2 and NADPH-thioredoxin-dependent DHA reduction appears to belong to an inducible system activated in response to oxidative stress.     

In vitro oxidation of ascorbic acid and its prevention by GSH

The interaction of glutathione (GSH) with ascorbic acid and dehydroascorbic acid was examined in in-vitro experiments in order to examine the role of GSH in protecting against the autoxidation of ascorbic acid and in regenerating ascorbic acid by reaction with dehydroascorbic acid. If a buffered solution (pH 7.4) containing 1.0 mM ascorbic acid was incubated at 37 degrees C, there was a rapid loss of ascorbic acid in the presence of oxygen. When GSH was added to this solution, ascorbic acid did not disappear. Maximum protection against ascorbic acid autoxidation was achieved with as little as 0.1 mM GSH. Cupric ions (0.01 mM) greatly accelerated the rate of autoxidation of ascorbic acid, an effect that was inhibited by 0.1 mM GSH. Other experiments showed that GSH complexes with cupric ions, resulting in in a lowering of the amount of GSH in solution as measured in GSH standard curves. These results suggest that the inhibition of ascorbic acid autoxidation by GSH involves complexation with cupric ions that catalyze the reaction. When ascorbic acid was allowed to autoxidize at 37 degrees C the subsequent addition of GSH (up to 10 mM) did not lead to the regeneration of ascorbic acid. This failure to detect a direct reaction between GSH and the dehydroascorbic acid formed by oxidation of ascorbic acid under this condition was presumably due to the rapid hydrolysis of dehydroascorbic acid. When conditions were chosen, i.e., low temperature, that promote stability of dehydroascorbic acid, the direct reaction between GSH and dehydroascorbic acid to form ascorbic acid was readily detected. The marked instability of dehydroascorbic acid at 37 degrees C raises questions regarding the efficiency of the redox couple between GSH and dehydroascorbic acid in maintaining the concentration of ascorbic acid in mammalian cells exposed to an oxidative challenge.     

The methanol method for the quantification of ascorbic acid and dehydroascorbic acid in biological samples

We present a fast to perform spectrophotometric method for the quantification of ascorbic acid and its oxidized form dehydroascorbic acid in biological samples. The assay detects a chromophore formed during the reaction of dehydroascorbic acid with methanol in phosphate/citrate buffer. This reaction can also be employed for the determination of ascorbate (vitamin C) in the presence of ascorbate oxidase. The major advantage of the developed protocol for the determination of both forms of vitamin C is a simple spectrophotometrical single end point determination. It is demonstrated that the methanol method is an improvement compared with a commercially available test kit for the determination of vitamin C. Using the methanol method, a dose-dependent increase in intracellular ascorbic acid was determined upon incubation of L-929 cells and RAW 264.7 macrophages with increasing concentrations of extracellular ascorbate. In blood serum, vitamin C was determined at concentrations between 46 and 97 microM. Supplementation with different amounts of ascorbate showed satisfying recovery. In L-929 cells, even unphysiologically high amounts of reactive nitrogen species were unable to completely oxidize intracellular vitamin C.     

Reduction of dehydroascorbic acid at low pH

Ascorbic acid and dehydroascorbic acid are unstable in aqueous solution in the presence of copper and iron ions, causing problems in the routine analysis of vitamin C. Their stability can be improved by lowering the pH below 2, preferably with metaphosphoric acid. Dehydroascorbic acid, an oxidised form of vitamin C, gives a relatively low response on the majority of chromatographic detectors, and is therefore routinely determined as the increase of ascorbic acid formed after reduction. The reduction step is routinely performed at a pH that is suboptimal for the stability of both forms. In this paper, the reduction of dehydroascorbic acid with tris-[2-carboxyethyl] phosphine (TCEP) at pH below 2 is evaluated. Dehydroascorbic acid is fully reduced with TCEP in metaphosphoric acid in less than 20 min, and yields of ascorbic acid are the same as at higher pH. TCEP and ascorbic acid formed by reduction, are more stable in metaphosphoric acid than in acetate or citrate buffers at pH 5, in the presence of redox active copper ions. The simple experimental procedure and low probability of artefacts are major benefits of this method, over those currently applied in a routine assay of vitamin C, performed on large number of samples.     

Superoxide dictates the mode of U937 cell ascorbic acid uptake and prevents the enhancing effects of the vitamin to otherwise nontoxic levels of reactive oxygen/nitrogen species

Exposure of U937 cells to low micromolar levels of ascorbic acid or dehydroascorbic acid, while resulting in identical ascorbic acid accumulation, is unexpectedly associated with remarkably different responses to exogenous oxidants. We observed that otherwise nontoxic levels of hydrogen peroxide, tert-butylhydroperoxide or peroxynitrite promote toxicity in cells preloaded with ascorbic acid, whereas hardly any effect was detected in cells pretreated with dehydroascorbic acid. Further experiments performed with peroxynitrite in cells preloaded with ascorbic acid provided evidence for a very rapid nonapoptotic death, preceded by early Bax mitochondrial translocation and by mitochondrial permeability transition. The notion that conversion of extracellular ascorbic acid to dehydroascorbic acid prevents the enhancing effects on oxidant toxicity and nevertheless preserves the net amount of vitamin C accumulated was also established using ascorbate oxidase as well as various sources of superoxide, namely, xanthine/xanthine oxidase or ATP-driven NADPH oxidase activation. These findings suggest that superoxide-dependent conversion of extracellular ascorbic acid to dehydroascorbic acid represents an important component of the overall survival strategy of some cell types to reactive oxygen/nitrogen species.     

Reduction of dehydroascorbic acid at low pH

Ascorbic acid and dehydroascorbic acid are unstable in aqueous solution in the presence of copper and iron ions, causing problems in the routine analysis of vitamin C. Their stability can be improved by lowering the pH below 2, preferably with metaphosphoric acid. Dehydroascorbic acid, an oxidised form of vitamin C, gives a relatively low response on the majority of chromatographic detectors, and is therefore routinely determined as the increase of ascorbic acid formed after reduction. The reduction step is routinely performed at a pH that is suboptimal for the stability of both forms. In this paper, the reduction of dehydroascorbic acid with tris-[2-carboxyethyl] phosphine (TCEP) at pH below 2 is evaluated. Dehydroascorbic acid is fully reduced with TCEP in metaphosphoric acid in less than 20 min, and yields of ascorbic acid are the same as at higher pH. TCEP and ascorbic acid formed by reduction, are more stable in metaphosphoric acid than in acetate or citrate buffers at pH 5, in the presence of redox active copper ions. The simple experimental procedure and low probability of artefacts are major benefits of this method, over those currently applied in a routine assay of vitamin C, performed on large number of samples.