Investigating protein thiol chemistry associated with dehydroascorbate, homocysteine and glutathione using mass spectrometry.

RATIONALE: Oxidative stress is an imbalance between reactive free radical oxygen species and antioxidant defenses. Its consequences can lead to numerous pathologies. Regulating oxidative stress is the complex interplay between antioxidant recycling and thiol-containing regulatory proteins. Understanding these regulatory mechanisms is important for preventing onset of oxidative stress. The aim of this study was to investigae S-thiol protein chemistry associated with oxidized vitamin C (dehydroascorbate, DHA), homocysteine (HcySH) and glutathione (GSH) using mass spectrometry. METHODS: Glutaredoxin-1 (Grx-1) was incubated with DHA, with and without GSH and HcySH. Disulfide formation was followed by electrospray ionization mass spectrometry (ESI-MS) of intact proteins and by LC/ESI-MS/MS of peptides from protein tryptic digestions. The mechanism of DHA-mediated S-thiolation was investigated using two synthetic peptides: AcFHACAAK and AcFHACE. Three proteins, i.e. human hemoglobin (HHb), recombinant peroxiredoxin 2 (Prdx2) and Grx-1, were S-homocysteinylated followed by S-transthiolyation with GSH and investigated by ESI-MS and ESI-MS/MS. RESULTS: ESI-MS analysis reveals that DHA mediates disulfide formation and S-thiolation by HcySH as well as GSH of Grx-1. LC/ESI-MS/MS analysis allows identification of Grx-1 S-thiolated cysteine adducts. The mechanism by which DHA mediates S-thiolation of heptapeptide AcFHACAAK is shown to be via initial formation of a thiohemiketal adduct. In addition, ESI-MS of intact proteins shows that GSH can S-transthiolate S-homocysteinylated Grx-1_ HHb and Prdx2. The GS-S-protein adducts over time dominate the ESI-MS spectrum profile. CONCLUSIONS: Mass spectrometry is a unique analytical technique for probing complex reaction mechanisms associated with oxidative stress. Using model proteins, ESI-MS reveals the mechanism of DHA-facilitated S-thiolation, which consists of thiohemiketal formation, disulfide formation or S-thiolation. Furthermore, protein S-thiolation by HcySH can be reversed by reversible GSH thiol exchange. The use of mass spectrometry with in vitro models of protein S-thiolation in oxidative stress may provide significant insight into possible mechanisms of action occurring in vivo.

Development of a Physiologically Relevant Online Chemical Assay To Quantify Aerosol Oxidative Potential.

Large-scale epidemiological studies have shown a close correlation between adverse human health effects and exposure to ambient particulate matter (PM). The oxidative potential (OP) of ambient PM has been implicated in inducing toxic effects associated with PM exposure. In particular, reactive oxygen species (ROS), either bound to PM or generated by particulate components in vivo, substantially contribute to the OP and therefore toxicity of PM by lowering antioxidant concentrations in the lung, which can subsequently lead to oxidative stress, inflammation, and disease. Traditional methods for measuring aerosol OP are labor intensive and have poor time resolution, with significant delays between aerosol collection and ROS analysis. These methods may underestimate ROS concentrations in PM because of the potentially short lifetime of some ROS species; therefore, continuous online, highly time-resolved measurement of ROS components in PM is highly advantageous. In this work, we develop a novel online method for measuring aerosol OP based on ascorbic acid chemistry, an antioxidant prevalent in the lung, thus combining the advantages of continuous online measurement with a physiologically relevant assay. The method limit of detection is estimated for a range of atmospherically important chemical components such as Cu(II) 0.22 ± 0.03 µg m-3, Fe(II) 47.8 ± 5.5 µg m-3, Fe(III) 0.63 ± 0.05 µg m-3, and secondary organic aerosol 41.2 ± 6.9 µg m-3, demonstrating that even at this early stage of development, the online method is capable of measuring the OP of PM in polluted urban environments and smog chamber studies.

Determination of cellular vitamin C dynamics by HPLC-DAD.

A redox-sensitive inter-conversion between ascorbic acid (ASC) and its oxidized form dehydroascorbic acid (DHA) in the intracellular environment has been of exceptional interest to recent metabolomics and pharmaceutical research. We developed a chromatographic protocol to instantly determine these vitamers with each identity from cellular extracts, without any labeling and pretreatments. Owing to its simplicity, one can readily continue the assay for hours, an otherwise difficult to cover timescale at which the intracellular DHA-ASC conversion comes into play. The method was validated for the analysis of pancreatic cancer cells, to our knowledge the first-ever study on a nucleated cell type, to trace in detail their kinetics of glucose transporter-dependent DHA uptake and, simultaneously, that for the intracellular ASC conversion. The simplest of all the relevant techniques and yet with the unique ability to provide each vitamer identity on a high-throughput basis, this method should offer the most practical option for VC-involved physiological and pharmaceutical studies including high-dose VC cancer therapy.

[Resveratrol alleviates oxidative injury from high glucose-induced human lens epithelial cells and its possible mechanism].

OBJECTIVE: To investigate the effects and its mechanism of resveratrol against human lens epithelial cells (LEC) apoptosis mediated by high glucose-induced oxidative injury. METHODS: An experimental study. LEC were cultured in different concentrations (5.5, 15.0, 25.0, 35.0, 45.0 mmol/L) of glucose medium or 25.0 mmol/L glucose medium at different time (0, 6, 12, 24, 48, 72 h), and established an interventional models of (5.0, 15.0, 25.0, 35.0 mg/L) resveratrol. Av-FITC-PI (annexin V-fluorescein isothiocyanate-propidium iodium) was used to detect apoptosis. The amount of ROS was calculated by flow cytometry. The expression of apoptosis of the protein Bcl-2 and Bax, iNOS, NF-κB, IκB and MnSOD were showed by Western blotting and the amount of oxidative damage marker MDA was explored by Spectrometers and Analytical Photometers. Differences between the two groups were evaluated by One-way ANOVA. RESULTS: The apoptosis of LEC induced by high glucose was time-and dose-dependent obviously. As the glucose concentration increased and duration prolonged, the expression of anti-apoptotic protein Bcl-2 was decreased and pro-apoptotic protein Bax was increased.Intracellular ROS and MDA induced by high glucose were increased significantly with dose-and time-dependence. Compared with 5.5 mmol/L group, ROS generation increased significantly in the concentration of 15.0 mmol/L (F = 14.06, P = 0.035), 25.0 mmol/L (F = 17.46, P = 0.000), 35.0 mmol/L (F = 16.58, P = 0.001), 45.0 mmol/L (F = 12.88, P = 0.000) and were statistically significant. Compared with 5.5 mmol/L glucose cultured group, ROS generation increased significantly at 6 h (F = 6.778, P = 0.014), 12 h (F = 6.551, P = 0.001), 24 h (F = 7.327, P = 0.001), 48 h (F = 10.84, P = 0.000), 72 h (F = 13.36, P = 0.000) in LEC cultured group by 25.0 mmol/L glucose and were statistically significant. Compared with 5.5 mmol/L group, the content of MDA were significantly increased in 15.0 mmol/L (F = 1.177, P = 0.035), 25.0 mmol/L (F = 1.704, P = 0.000), 35.0 mmol/L (F = 2.412, P = 0.001) and 45.0 mmol/L (F = 2.347, P = 0.000) glucose medium and were statistically significant. Compared with 5.5 mmol/L cultured group, the content of MDA were significantly increased at 6 h (F = 1.704, P = 0.014), 12 h (F = 5.676, P = 0.001), 24 h (F = 3.325, P = 0.001), 48 h (F = 6.669, P = 0.000), 72 h (F = 3.011, P = 0.000) in LEC cultured group by 25.0 mmol/L high glucose and were statistically significant. When resveratrol (5.0, 15.0, 25.0, 35.0 mg/L) was added to 25.0 mmol/L glucose medium, respectively, the apoptotic cells were decreased, the expression of pro-apoptotic protein Bax was decreased and anti-apoptotic protein Bcl-2 was increased.Intracellular ROS (compared with the basic concentration of 5.5 mmol/L, F values were 14.76, 7.018, 13.96, 4.733, 1.921, P values were 0.000, 0.000, 0.003, 0.086, 0.100 respectively) and MDA (compared with the basic concentration of 5.5 mmol/L, F values were 2.454, 1.108, 1.630, 1.563, 2.250, P values were 0.000, 0.001, 0.026, 0.068, 0.183 respectively) were decreased. MnSOD expression was increased, iNOS and NF-κB activation were inhibited. CONCLUSION: Resveratrol could alleviates oxidative injury from high glucose-induced LEC, and inhibited of iNOS-mediated oxidative damage through inhibiting the activities of NF-κB could be the mechanism of this effect.

Photostability and interaction of ascorbic acid in cream formulations.

The kinetics of photolysis of ascorbic acid in cream formulations on UV irradiation has been studied using a specific spectrophotometric method with a reproducibility of ± 5%. The apparent first-order rate constants (k(obs)) for the photolysis of ascorbic acid in creams have been determined. The photoproducts formed in the cream formulations include dehydroascorbic acid and 2,3-diketogulonic acid. The photolysis of ascorbic acid appears to be affected by the concentration of active ingredient, pH, and viscosity of the medium and formulation characteristics. The study indicates that the ionized state and redox potentials of ascorbic acid are important factors in the photostability of the vitamin in cream formulations. The viscosity of the humectant present in the creams appears to influence the photostability of ascorbic acid. The results show that the physical stability of the creams is an important factor in the stabilization of the vitamin. In the cream formulations stored in the dark, ascorbic acid undergoes aerobic oxidation and the degradation is affected by similar factors as indicated in the photolysis reactions. The rate of oxidative degradation in the dark is about seventy times slower than that observed in the presence of light.

Influence of fruit ripening stage and harvest period on the antioxidant content of sweet pepper cultivars.

Pepper (Capsicum annuum L.) fruits are highly appreciated by producers and consumers for their economical and nutritional value. Four different cultivars of coloured peppers in immature and mature stages were harvested throughout the spring and examined for their content of phenolic compounds, ascorbic acid and total antioxidant capacity (TAA) as well as for lipid peroxidation and carbonyl proteins as index of oxidative stress. Ripening and harvest period influenced the antioxidants and the development of oxidative processes in the cultivars differently: lipid peroxidation increased in mature peppers except in one cultivar (Y1075), while no changes in protein oxidation or in TAA were produced, except in Y1075 in which both parameters increased. Each cultivar presented differences in antioxidant compounds depending on the harvest period, but we could recommend May as the optimal if all cultivars have to be harvested at the same time, when levels of ascorbate, phenols and TAA were not decreased, fresh weight and proteins were elevated, and levels of oxidation were not as high as in June (except for Y1075). A previous study of the response of each cultivar to different environmental conditions results essential to establish a good program of selection of cultivars with high quality and productivity.

Determination of dehydroascorbic acid in mouse tissues and plasma by using tris(2-carboxyethyl)phosphine hydrochloride as reductant in metaphosphoric acid/ethylenediaminetetraacetic acid solution.

Ascorbic acid (AA) has a strong anti-oxidant function evident as its ability to scavenge superoxide radicals in vitro. Moreover, AA is an essential ingredient for post-translational proline hydroxylation of collagen molecules. Dehydroascorbic acid (DHA), the oxidized form of AA, is generated from these reactions. In this study, we describe an improved method for assessing DHA in biological samples. The use of 35 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) as a reductant completely reduced DHA to AA after 2 h on ice in a 5% solution of metaphosphoric acid containing 1 mM ethylenediaminetetraacetic acid (EDTA) at pH 1.5. This method enabled us to measure the DHA content in multiple tissues and plasma of 6-weeks-old mice. The percentages of DHA per total AA differed markedly among these tissues, i.e., from 0.8 to 19.5%. The lung, heart, spleen and plasma had the highest levels at more than 10% of DHA per total AA content, whereas the cerebrum, cerebellum, liver, kidney and small intestine had less than 5% of DHA per total AA content. This difference in DHA content may indicate an important disparity of oxidative stress levels among physiologic sites. Therefore, this improved method provides a useful standard for all DHA determinations.

Rapid determination of ascorbic acid, dehydroascorbic acid, and total vitamin C by electrochemiluminescence with a thin-layer electrochemical cell.

This paper reports on a rapid and sensitive method for the simultaneous determination of ascorbic acid (H(2)A), dehydroascorbic acid (DHA), and total vitamin C by electrochemiluminescence (ECL) using a thin-layer electrochemical cell. Significant ECL signals can be generated by the anodic oxidation of Ru(bpy)(3) (2+) in the presence of H(2)A or DHA in pH 8.8 phosphate buffer solution. Because of the extremely small dead volume of the thin-layer cell (approximately 1.5 microL), almost all amount of H(2)A is assumed to be completely oxidized to DHA with a short pre-electrolysis step. As a result, it is possible to determine the reductive vitamin C (H(2)A) by square wave voltammetry before the pre-electrolysis step, while total vitamin C (sum of H(2)A and DHA) is able to be determined at a subsequent ECL step. The method was employed for the determination of vitamin C in commercial beverages with the analytical results in good agreement with the certified values.

Vitamin C in camu-camu [Myrciaria dubia (H.B.K.) McVaugh]: evaluation of extraction and analytical methods.

Camu-camu, a typical Amazonian fruit, is known for the high vitamin C content of the peel and pulp. As vitamin C is widely used in the food, pharmaceutical, and cosmetics industries, it is of interest to study new sources, extraction techniques, and analytical methods for the identification and quantification of this compound. Here, evaluation was made of extraction and quantification methods, as well as the differences in vitamin C content according to the origin and part of the camu-camu fruit. The extraction techniques studied were pressurized liquid extraction (PLE), acid extraction, and maceration. The analytical methods evaluated were titrimetry and chromatography. Camu-camu samples were obtained from different regions, and the peel and pulp were studied separately. Acid extraction using sulfuric acid as solvent provided the highest vitamin C yields, while PLE, as a completely clean technique, proved to be a promising alternative for the recovery of ascorbic acid (L-AA). The application of an ultra-high performance liquid chromatography methodology (UHPLC-DAD) enabled the fast identification and quantification of L-AA and dehydroascorbic acid (DHAA), with high resolution, sensitivity, and specificity. The results obtained using the chromatographic and titration methods were not significantly different (p < 0.05), indicating that titrimetry is useful for routine analyses. L-AA and DHAA were found in the peel, but only L-AA was found in the pulp. The variation of vitamin C content among the lots could be explained by the edaphoclimatic conditions. The combination of a clean extraction technique and a fast analytical method is a promising approach for the determination of vitamin C in food products.

Development and validation of a liquid chromatographic method for the determination of ascorbic acid, dehydroascorbic acid and acetaminophen in pharmaceuticals.

A reversed-phase ion pair liquid chromatographic method (RP-LC) for the determination of dehydroascorbic acid (DHA) and ascorbic acid (AA) and also acetaminophen, which is combined in pharmaceuticals, is proposed and validated. AA and acetaminophen were analyzed directly, while DHA was determined after pre-column derivatization with 4,5-dimethyl-1,2-phenylenediamine (DMPD). The derivatization reaction was carried out under mild conditions (10min at ambient temperature) in the dark in sodium acetate buffer (80mM; pH 3.7) solution containing EDTA as metal scavenger. The chromatographic separations were performed on a Phenomenex Synergi 4u hydro-RP (150mmx4.6mm) under isocratic elution conditions, using cetyltrimethylammonium bromide (CTAB) as ion-pairing reagent in the mobile phase. Linear responses were observed for each compound. The intra-day precision (R.S.D.) was < or =1.40% and there was no significant difference between intra- and inter-day data. Recovery studies showed good results for all compounds (99.7-101.8%) with R.S.D. ranging from 0.56 to 1.82%. The limits of quantitation were about 40, 50 and 140pmol for acetaminophen, AA and DHA, respectively. The DHA impurity values found in dosage forms were < or =0.2% of AA.

Fatty acid profile of human milk of Portuguese lactating women: prospective study from the 1st to the 16th week of lactation.

BACKGROUND/AIMS: Fatty acid (FA) composition varies over the course of the day and during lactation. The aim of this study was to evaluate FA composition and its compositional stability in human milk, from day 7 to week 16 of lactation. METHODS: Human milk was collected from all feedings over 24 h at day 7 and weeks 4, 8, 12 and 16 of lactation in 31 lactating women. FAs were analyzed through gas chromatography. Comparisons were made with analysis of variance. RESULTS: Total monounsaturated FAs decreased from 33.04 +/- 2.58% wt/wt at day 7 to 31.48 +/- 3.32% wt/wt at week 16 of lactation, much at the expenses of the decrease in the major monounsaturated FA found in human milk, oleic acid. Main polyunsaturated FAs n-6 and n-3 showed fluctuations from day 7 up to week 16 of lactation, but with no statistical significance. Arachidonic acid significantly decreased from transitional to mature milk. CONCLUSIONS: The FA profile obtained throughout the study time points presented very low levels of oleic acid and very high linoleic acid/alpha-linoleic acid ratios which reflect recent changes in Portuguese women's food patterns. Despite this, the arachidonic acid/docosahexaenoic acid ratio [corrected] remained constant during the study, suggesting a protective metabolic mechanism.

Development of a quantitative method for the analysis of total L-ascorbic acid in foods by high-performance liquid chromatography.

A new method has been developed for the determination of total vitamin C in foods. The method requires less time than the traditional methodologies and uses a radical oxidation of L-ascorbic acid (AA) to obtain dehydro-L-ascorbic acid (DHAA) by means of a peroxyl radical generated in situ by thermal decomposition of an azo-compound, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). The dehydro-L-ascorbic acid is condensed with benzene-1,2-diamine (o-phenylenediamine, OPDA) to form its highly fluorescent quinoxaline derivative, 3-(1,2-dihydroxyethyl)furo[3,4-b]quinoxaline-1-one (DFQ), which is then separated on a C(18) column eluted with a mobile phase of 80 mM phosphate buffer and methanol at pH=7.8 and detected fluorometrically at lambda(ex)=355 nm and lambda(em)=425 nm. The reaction conditions for the complete conversion of AA to DFQ were 56 degrees C, 36 min and a mumol AAPH/AA ratio of 60. The sample, extracted with an aqueous metaphosphoric acid solution, was analyzed after being filtered through a 0.45 microm membrane. The method has shown good repeatability, sensitivity and accuracy compared to the results obtained with the reference method. The response of the detection system was linear within a range of 0.5-8.0 microg/mL with a correlation coefficient of 0.9997. The limit of detection was 0.27 microg/mL and the limit of quantification was 0.83 microg/mL. The AA contents of some selected foods were analyzed.

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.

Leaf extracellular ascorbate in relation to O(3) tolerance of two soybean cultivars.

Soybean [Glycine max (L.) Merr.] cultivars Essex and Forrest that exhibit differences in ozone (O(3)) sensitivity were used in greenhouse experiments to investigate the role of leaf extracellular antioxidants in O(3) injury responses. Charcoal-filtered air and elevated O(3) conditions were used to assess genetic, leaf age, and O(3) effects. In both cultivars, the extracellular ascorbate pool consisted of 80-98% dehydroascorbic acid, the oxidized form of ascorbic acid (AA) that is not an antioxidant. For all combinations of genotype and O(3) treatments, extracellular AA levels were low (1-30nmolg(-1) FW) and represented 3-30% of the total antioxidant capacity. Total extracellular antioxidant capacity was twofold greater in Essex compared with Forrest, consistent with greater O(3) tolerance of Essex. The results suggest that extracellular antioxidant metabolites in addition to ascorbate contribute to detoxification of O(3) in soybean leaves and possibly affect plant sensitivity to O(3) injury.

Role of Zucchini and Its Distinctive Components in the Modulation of Degenerative Processes: Genotoxicity, Anti-Genotoxicity, Cytotoxicity and Apoptotic Effects.

Zucchini (Cucurbita pepo subsp. pepo) is a seasonal vegetable with high nutritional and medical values. Many useful properties of this fruit are attributed to bioactive compounds. Zucchini fruits ("Yellow" and "Light Green" varieties) and four distinctive components (lutein, ß-carotene, zeaxanthin and dehydroascorbic acid) were selected. Firstly, the lutein, ß-carotene, zeaxanthin and dehydroascorbic acid contents were determined in these fruits. Then, in order to evaluate the safety and suitability of their use, different assays were carried out: (i) genotoxicity and anti-genotoxicity tests to determine the safety and DNA-protection against hydrogen peroxide; (ii) cytotoxicity; and (iii) DNA fragmentation and Annexin V/PI (Propidium Iodide) assays to evaluate the pro-apoptotic effect. Results showed that: (i) all the substances were non-genotoxic; (ii) all the substances were anti-genotoxic except the highest concentration of lutein; (iii) "Yellow" zucchini epicarp and mesocarp exhibited the highest cytotoxic activity (IC50 > 0.1 mg/mL and 0.2 mg/mL, respectively); and (iv) "Light Green" zucchini skin induced internucleosomal DNA fragmentation, ß-carotene being the possible molecule responsible for its pro-apoptotic activity. To sum up, zucchini fruit could play a positive role in human health and nutrition due to this fruit and its components were safe, able to inhibit significantly the H2O2-induced damage and exhibit anti-proliferative and pro-apoptotic activities toward HL60 (human promyelocytic leukemia cells) tumor cells. The information generated from this research should be considered when selecting potential accessions for breeding program purposes.

Degradation kinetics of the antioxidant additive ascorbic acid in packed table olives during storage at different temperatures.

The kinetics of ascorbic acid (AA) loss during storage of packed table olives with two different levels of added AA was investigated. Three selected storage temperatures were assayed: 10 degrees C, ambient (20-24 degrees C), and 40 degrees C. The study was carried out in both pasteurized and unpasteurized product. The effect of pasteurization treatment alone on added AA was not significant. In the pasteurized product, in general AA degraded following a first-order kinetics. The activation energy calculated by using the Arrhenius model averaged 9 kcal/mol. For each storage temperature, the increase in initial AA concentration significantly decreased the AA degradation rate. In the unpasteurized product, AA was not detected after 20 days in samples stored at room temperature and AA degradation followed zero-order kinetics at 10 degrees C, whereas at 40 degrees C a second-order reaction showed the best fit. In both pasteurized and unpasteurized product, the low level of initial dehydroascorbic acid disappeared during storage. Furfural appeared to be formed during storage, mainly at 40 degrees C, following zero-order kinetics.

Spectrophotometric method for fast quantification of ascorbic acid and dehydroascorbic acid in simple matrix for kinetics measurements.

A simple, rapid and reliable method was developed for quantifying ascorbic (AA) and dehydroascorbic (DHAA) acids and validated in 20mM malate buffer (pH 3.8). It consists in a spectrophotometric measurement of AA, either directly on the solution added with metaphosphoric acid or after reduction of DHAA into AA by dithiothreitol. This method was developed with real time measurement of reactions kinetics in bulk reactors in mind, and was checked in terms of linearity, limits of detection and quantification, fidelity and accuracy. The linearity was found satisfactory on the range of 0-6.95mM with limits of detection and quantification of 0.236mM and 0.467mM, respectively. The method was found acceptable in terms of fidelity and accuracy with a coefficient of variation for repeatability and reproducibility below 6% for AA and below 15% for DHAA, and with a recovery range of 97-102% for AA and 88-112% for DHAA.

Furan precursors in food: a model study and development of a simple headspace method for determination of furan.

Furan was previously detected in foods that had undergone thermal treatment. Because furan is now classified as a possible human carcinogen, a model system was developed to investigate the origins of furan. Also, a simple, rapid isotope dilution (d4-furan) headspace method was developed to measure furan. Two pathways of furan formation have been identified in the model systems tested so far. The first is the oxidation of polyunsaturated fatty acids at elevated temperatures, and the second is linked to the decomposition of ascorbic acid derivatives. The analytical procedure, based on the use of a 50 microL injection (from the headspace of a 1.5 mL vial containing 0.5 mL water) into the split/splitless injection port of a gas chromatograph/mass spectrometer (electron ionization, selected-ion monitoring), showed linearity in the 10-1000 ng/g range with a limit of detection of 1 ng/g.

[Determination of ascorbic and dehydroascorbic acid concentration using HPLC method in smokers with stable coronary artery disease scheduled for coronary artery bypass grafting (CABG)]. / Oznaczenie stezenia kwasu askorbowego i dehydroaskorbowego metoda HPLC u palaczy ze stabilna choroba wiencowa zakwalifikowanych do operacji pomostowania tetnic wiencowych (CABG).

Ascorbic acid is a vitamin soluble in water and its deficiency in human body causes scurvy. Its symptoms in adults are gingivitis, susceptibility of blood vessels to damage and bleeding, changes in bones and cartilage and retarded wound healing. Ascorbic acid is necessary in redox processes taking place in cell. It is reversibly oxidized to dehydroascorbic acid and partially metabolized to inactive sulphide and oxalic acid, which is expelled in urine. It is well absorbed from the digestive system and easily reaches the tissues. Healthy organism contains 1.5 g of ascorbic acid and daily requirement for ascorbic acid is estimated for 30-100 mg. Ascorbic acid is not synthesized by humans, but it is an essential dietary vitamin for the species. Ascorbic acid is used in treatment deficiency in daily demand for vitamin C, caused by improper diet, poor absorption or cigarette smoking. It is used in large doses in general weakness, infectious diseases and during the recovery period. Positive results have been obtained after therapy with vitamin C of Mollera-Barlowa disease, Schonlein-Henoch disease, Werlhof disease, haemophilia and also in patients with stable coronary artery disease. Vitamin C is assumed to be a basic antioxidant, although its role in pathological conditions is controversial. However, it seems that the complexity of the oxidant-antioxidant system makes the question of participation of vitamin C (and other scavengers of free radicals) in pathogenesis of diseases still open.

Precision of dehydroascorbic acid quantitation with the use of the subtraction method--validation of HPLC-DAD method for determination of total vitamin C in food.

In food analysis, a method for determination of vitamin C should enable measuring of total content of ascorbic acid (AA) and dehydroascorbic acid (DHAA) because both chemical forms exhibit biological activity. The aim of the work was to confirm applicability of HPLC-DAD method for analysis of total content of vitamin C (TC) and ascorbic acid in various types of food by determination of validation parameters such as: selectivity, precision, accuracy, linearity and limits of detection and quantitation. The results showed that the method applied for determination of TC and AA was selective, linear and precise. Precision of DHAA determination by the subtraction method was also evaluated. It was revealed that the results of DHAA determination obtained by the subtraction method were not precise which resulted directly from the assumption of this method and the principles of uncertainty propagation. The proposed chromatographic method should be recommended for routine determinations of total vitamin C in various food.