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.

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.

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.

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.

Influence of vitamin C supplementation on oxidative stress and neutrophil inflammatory response in acute and regular exercise.

Exercise induces a multitude of physiological and biochemical changes in blood affecting its redox status. Tissue damage resulting from exercise induces activation of inflammatory cells followed by the increased activity of myeloperoxidase (MPO) in circulation. Vitamin C readily scavenges free radicals and may thereby prevent oxidative damage of important biological macromolecules. The aim of this study was to examine the effect of vitamin C supplementation on oxidative stress and neutrophil inflammatory response induced by acute and regular exercise. Experiment was conducted on acute exercise group (performing Bruce Treadmill Protocol (BTP)) and regular training group. Markers of lipid peroxidation, malondialdehyde (MDA), MPO activity, and vitamin C status were estimated at rest and after BTP (acute exercise group) and before and after vitamin C supplementation in both groups. Our results showed increased postexercise Asc in serum independently of vitamin supplementation. They also showed that vitamin C can significantly decrease postexercise MDA level in both experimental groups. Increased postexercise MPO activity has been found in both groups and was not affected by vitamin C supplementation. We concluded that vitamin C supplementation can suppress lipid peroxidation process during exercise but cannot affect neutrophil inflammatory response in either exercise group.

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.

Deficit irrigation strategies combined with controlled atmosphere preserve quality in early peaches.

Due to the water scarcity in the Mediterranean countries, irrigation must be optimized while keeping fruit quality. The effect of deficit irrigation strategies on changes in quality parameters of the early "Flordastar" peaches was studied. The deficit irrigation was programmed according to signal intensity of the maximum daily trunk shrinkage; deficit irrigation plants were irrigated to maintain maximum daily trunk shrinkage signal intensity values close to 1.4 or 1.3 in the case of DI1 or DI2 plants, respectively. Results were compared to a control watered at 150% crop evapotranspiration. Fruits were stored up to 14 days at 0 ℃ and 95% Relative Humidity (RH) in air or in controlled atmosphere (controlled atmosphere; 3-4 kPa O2 and 12-14 kPa CO2), followed by a retail sale period of 4 days at 15 ℃ and 90-95% Relative Humidity in air. Weight losses were lower in controlled atmosphere stored peaches from deficit irrigation. Air-stored fruits developed a more intense red color due to a faster ripening, which was not affected by the type of watering. At harvest, deficit irrigation peaches showed higher soluble solids content, which provided a better sensory evaluation. The soluble phenolic content was initially higher (55.26 ± 0.18 mg gallic acid equivalents/100 g fresh weight) and more stable throughout postharvest life in DI1 fruits than in those from the other irrigation treatments. Concerning vitamin C, control fruits at harvest showed higher ascorbic acid than dehydroascorbic acid content (5.43 versus 2.43 mg/100 g fresh weight, respectively), while water stressed peaches showed the opposite results. The combination of DI2 and controlled atmosphere storage allowed saving a significant amount of water and provided peaches with good overall quality, maintaining the bioactive compounds analyzed.

[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.

Plant adaptation to extreme environments: the example of Cistus salviifolius of an active geothermal alteration field.

Cistus salviifolius is able to colonise one of the most extreme active geothermal alteration fields in terms of both soil acidity and hot temperatures. The analyses of morpho-functional and physiological characters, investigated in leaves of plants growing around fumaroles (G leaves) and in leaves developed by the same plants after transfer into growth chamber under controlled conditions (C leaves) evidenced the main adaptive traits developed by this pioneer plant in a stressful environment. These traits involved leaf shape and thickness, mesophyll compactness, stomatal and trichome densities, chloroplast size. Changes of functional and physiological traits concerned dry matter content, peroxide and lipid peroxidation, leaf area, relative water and pigment contents. A higher reducing power and antioxidant enzymatic activity were typical of G leaves. Though the high levels of stress parameters, G leaves showed stress-induced specific morphogenic and physiological responses putatively involved in their surviving in active geothermal habitats.

New evaluation of fetal oxidative stress: measurement of the umbilical cord blood dimethyl sulfate-induced ascorbyl free radical by an electron spin resonance method.

OBJECTIVE: The aim is to evaluate intrapartum fetal oxidative stress in real-time by umbilical cord blood dimethyl sulfate (DMSO)-induced ascorbyl free radical (AFR) measured by an electron spin resonance (ESR) method. METHODS: 75 mothers delivering at gestational age after 37 weeks were recruited. They were divided into three groups: spontaneous vaginal birth (n = 27), elective cesarean section (CS) (n = 34), and emergency CS due to non-reassuring fetal status (n = 14). Umbilical artery (UA) and venous (UV) cord blood gas analysis was performed. Serum levels of DMSO-induced AFR (AFR/DMSO) that reflect vitamin C concentrations, was measured by ESR spectroscopy. RESULTS: Blood gas analysis showed no significant differences among the groups. UA-AFR/DMSO level of elective CS group was significantly lower compared with spontaneous delivery group (0.32 ± 0.12 vs. 0.46 ± 0.14, p < 0.005). Emergency CS group showed significantly lower levels of UA-AFR/DMSO compared with elective CS group (0.25 ± 0.11 vs. 0.32 ± 0.12, p < 0.005). UV-AFR/DMSO levels had no significant difference among the groups. CONCLUSIONS: It is suggested that fetal cord blood AFR/DMSO is a sensitive marker to assess fetal oxidative stress during delivery.

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.

Vitamina C, carotenoides, fenólicos totais e atividade antioxidante de goiaba, manga e mamão procedentes da ceasa do estado de Minas Gerais / Vitamin C, carotenoids, phenolic compounds and antioxidant activity of guava, mango and papaya from ceasa of Minas Gerais state

Este estudo teve como objetivo analisar o conteúdo de compostos antioxidantes (ácido ascórbico - AA, ácido desidroascórbico - ADA, vitamina C total, licopeno, β-caroteno, β-criptoxantina e estimativa de compostos fenólicos) e avaliar a atividade antioxidante, em goiaba, manga e mamão. A análise de carotenoides e vitamina C foi realizada por Cromatografia Líquida de Alta Eficiência (CLAE). O teor de fenólicos totais foi determinado utilizando o reagente de Folin-Ciocalteu e leitura espectrofotométrica. A atividade antioxidante foi avaliada pelo Teste do 2,2-diphenil-2-picril-hidrazil (DPPH•) e do Poder Redutor. A Anova (α= 0,05) foi utilizada para a análise dos dados. Os teores dos constituintes antioxidantes diferiram entre as três frutas, mas a goiaba foi a fruta que apresentou teores mais elevados de compostos fenólicos, vitamina C total, ADA e licopeno, além dos maiores valores para atividade antioxidante. Foi constatada forte correlação entre os testes que avaliaram a atividade antioxidante e o teor de fenólicos totais, demonstrando serem estes os principais compostos antioxidantes a contribuírem para a atividade antioxidante das frutas analisadas, em ambos os testes. É importante incentivar a utilização das frutas avaliadas neste estudo, tanto em nível doméstico quanto em estabelecimentos de alimentação coletiva para aumentar o consumo de antioxidantes naturais pela população.

This study aimed to analyze the content of antioxidant compounds (ascorbic acid - AA, dehydroascorbic acid - DHA, total vitamin C, lycopene, β-carotene, β-cryptoxanthin and phenolic compounds) and to evaluate the antioxidant activity in guava, mango and papaya. The analysis of carotenoids and vitamin C was performed by high performance liquid chromatography (HPLC). The content of phenolic compounds was determined using the Folin-Ciocalteu reagent and spectrophotometric reading. Antioxidant activity was evaluated by testing the 2.2-diphenyl-2-picryl-hydrazil (DPPH•) and reducing power. ANOVA was used for data analysis (α= 0.05). The levels of antioxidant constituents differed among the three fruits; guava was the fruit that had the highest levels of phenolic compounds, total vitamin C, lycopene and DHA, and the highest values for antioxidant activity. There was a strong correlation between tests that evaluated antioxidant activity and the content of phenolic compounds, demonstrating that these are the main antioxidant compounds to contribute to antioxidant activity of fruits examined in both tests. It is important to encourage the use of fruits evaluated in this study, both at home and in food service establishments, in order to increase the intake of natural antioxidants by the population.

Thermal stability of ascorbic acid and ascorbic acid oxidase in african cowpea leaves ( Vigna unguiculata ) of different maturities.

Cowpea, an African leafy vegetable ( Vigna unguiculata ), contains a high level of vitamin C. The leaves harvested at 4-9 weeks are highly prone to vitamin C losses during handling and processing. Therefore, the purpose of this research was to study the effect of thermal treatment on the stability of ascorbic acid oxidase (AAO), total vitamin C content (l-ascorbic acid, l-AA), and dehydroascorbic acid (DHAA) and l-AA/DHAA ratio in cowpea leaves harvested at different maturities (4, 6, and 8 weeks old). The results showed that AAO activity, total vitamin C content, and l-AA/DHAA ratio in cowpea leaves increased with increasing maturity (up to 8 weeks). Eight-week-old leaves were the best source of total vitamin C and showed a high ratio of l-AA/DHAA (4:1). Thermal inactivation of AAO followed first-order reaction kinetics. Heating at temperatures above 90 °C for short times resulted in a complete AAO inactivation, resulting in a protective effect of l-AA toward enzyme-catalyzed oxidation. Total vitamin C in young leaves (harvested at 4 and 6 weeks) was predominantly in the form of DHAA, and therefore temperature treatment at 30-90 °C for 10 min decreased the total vitamin C content, whereas total vitamin C in 8-week-old cowpea leaves was more than 80% in the form of l-AA, so that a high retention of the total vitamin C can be obtained even after heating and/or reheating (30-90 °C for 10 min) before consumption. The results indicated that the stability of total vitamin C in situ was strongly dependent on the plant maturity stage and the processing conditions applied.

Thermal stability of L-ascorbic acid and ascorbic acid oxidase in broccoli (Brassica oleracea var. italica).

UNLABELLED: The thermal stability of vitamin C (including l-ascorbic acid [l-AA] and dehydroascorbic acid [DHAA]) in crushed broccoli was evaluated in the temperature range of 30 to 90 degrees C whereas that of ascorbic acid oxidase (AAO) was evaluated in the temperature range of 20 to 95 degrees C. Thermal treatments (for 15 min) of crushed broccoli at 30 to 60 degrees C resulted in conversion of l-AA to DHAA whereas treatments at 70 to 90 degrees C retained vitamin C as l-AA. These observations indicated that enzymes (for example, AAO) could play a major role in the initial phase (that is, oxidation of l-AA to DHAA) of vitamin C degradation in broccoli. Consequently, a study to evaluate the temperature-time conditions that could result in AAO inactivation in broccoli was carried out. In this study, higher AAO activity was observed in broccoli florets than stalks. During thermal treatments for 10 min, AAO in broccoli florets and stalks was stable until around 50 degrees C. A 10-min thermal treatment at 80 degrees C almost completely inactivated AAO in broccoli. AAO inactivation followed 1st order kinetics in the temperature range of 55 to 65 degrees C. Based on this study, a thermal treatment above 70 degrees C is recommended for crushed vegetable products to prevent oxidation of l-AA to DHAA, the onset of vitamin C degradation. PRACTICAL APPLICATION: The results reported in this study are applicable for both domestic and industrial processing of vegetables into products such as juices, soups, and purees. In this report, we have demonstrated that processing crushed broccoli in a temperature range of 30 to 60 degrees C could result in the conversion of l-ascorbic acid to dehydroascorbic (DHAA), a very important reaction in regard to vitamin C degradation because DHAA could be easily converted to other compounds that do not have the biological activity of vitamin C.

Determination of furan precursors and some thermal damage markers in baby foods: ascorbic acid, dehydroascorbic acid, hydroxymethylfurfural and furfural.

The presence of ascorbic acid (AA), vitamin C (AA + dehydroascorbic acid (DHAA)) and furfural as potential precursors of furan in commercial fruit and vegetable jarred baby food was studied. Hydroxymethylfurfural (HMF) was also determined and used, together with furfural levels, as markers of thermal damage. AA, calculated DHAA and vitamin C values ranged between 22.4 and 103, 2.9 and 13.8, and 32.1 and 113.2 mg/100 g, respectively, in fruit-based baby food. However, no trace of AA was found in the vegetable-based baby food samples tested, probably because these samples are not enriched in vitamin C and the content of this vitamin in fresh vegetables is destroyed during processing. Furfural values ranged from not detected to 236 microg/100 g, being higher in vegetable samples than in fruit samples possibly because of greater AA degradation favored by a higher pH in the vegetable samples. HMF values (range: not detected-959 microg/100 g), however, were higher in the fruit samples, probably due to greater carbohydrate content degradation and as a consequence of the Maillard reaction, favored by a lower pH in these samples. According to these results, HMF would be the optimum indicator of thermal treatment for fruits, and furfural for vegetables. The higher furfural content of vegetable baby food could be considered an index of greater AA degradation and, therefore, the furan content might be higher in this kind of sample than in fruit-based baby food.

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.

Monitoring of vitamin C species in aqueous solution by flow injection analysis coupled with an on-line separation with reversed-phase column.

Two vitamin C species of ascorbic acid and dehydroascorbic acid in aqueous solution were monitored by flow injection analysis. Ascorbic acid and dehydroascorbic acid were resolved by a reversed-phase column, and dehydroascorbic acid was reduced to ascorbic acid by an on-line post-column reaction with dithiothreitol. Both natural and reduced ascorbic acids were photometrically detected at 260 nm, and the two vitamin C species were simultaneously determined. The determination range was from 0 to 8 x 10(-5)M with a limit of detection of 1.7 x 10(-6)M. The proposed method was applied to the conversion monitoring of ascorbic acid and dehydroascorbic acid in weakly acidic to weakly alkaline aqueous solutions, as well as to the determination of the vitamin C in some beverage samples.