Transglucosylation of ascorbic acid to ascorbic acid 2-glucoside by a recombinant sucrose phosphorylase from <Emphasis Type="Italic">Bifidobacterium longum</Emphasis>

A novel transglycosylation reaction from sucrose to l-ascorbic acid by a recombinant sucrose phosphorylase from Bifidobacterium longum was used to produce a stable l-ascorbic acid derivative. The major product was detected by HPLC, and confirmed to be 2-O-α-d-glucopyranosyl-l-ascorbic acid by LC-MS/MS analysis.     

High-performance liquid chromatographic determination of diadenosine 5′,5‴-p1,p4-tetraphosphate with precolumn fluorescence derivatization and its application to metabolism study in whole blood

Diadenosine 5′,5‴-p¹,p⁴-tetraphosphate (Ap4A) was converted with chloroacetaldehyde to the fluorescent di-1,N⁶-ethenoadenosine derivative within 60 min at 80°C. It was separated by reversed-phase HPLC and detected fluorimetrically (excitation and emission wavelengths of 275 and 410 nm, respectively). The detection limit of Ap4A was ca. 0.2 μg/ml in plasma when 10 μl of the sample was applied to the column. The rate of degradation of Ap4A added to whole blood (5 μg/ml) was examined using this method. Half-lives (means ± S.E., n = 3) were 0.88 ± 0.30 min (in rat blood), 13.7 ± 3.6 min (in dog blood and 17.2 ± 1.4 min (in human blood). A marked species difference in the degradation rate of Ap4A in blood was observed.     

The lipophilic vitamin C derivative, 6-o-palmitoylascorbate,protects human lymphocytes,preferentially over ascorbate,against X-ray-induced DNA damage,lipid peroxidation,and protein carbonylation

The aim of this study was to investigate protective effects of the lipophilic vitamin C derivative, 6-o-palmitoylascorbate (PlmtVC), against X-ray radiation-induced damages including cell death, DNA double-strand breaks (DSBs), lipid peroxidation, and protein carbonylation in human lymphocytes HEV0082, and the stability of PlmtVC under cell-cultured or cell-free condition. Irradiation with X-ray (1.5 Gy) diminished the cell viability and induced apoptosis, both of which were protected by pre-irradiational administration with PlmtVC. Gamma-H2A.X foci as a hallmark of DSBs were markedly enhanced in the irradiated cells. PlmtVC prevented X-ray-induced DSBs more appreciably than l-ascorbic acid (l-AA). Intracellular ROS production, lipid peroxidation, and protein carbonylation in HEV0082 cells were increased by X-ray at 1.5 Gy, all of which were significantly repressed by PlmtVC. PlmtVC also elevated endogenous reduced glutathione (GSH) in HEV0082 cells, and prevented X-ray-induced GSH depletion that are more appreciably over l-AA. Thus, PlmtVC prevents X-ray-induced cell death through its antioxidative activity. Stability tests showed that after being kept under physiological conditions (pH 7.4, 37 °C) for 14 days, vitamin C residual rates in PlmtVC solutions (62.2–82.0 %) were significantly higher than those in l-AA solutions (20.5–28.7 %). When PlmtVC or l-AA was added to HEV0082 lymphocytes, intracellular vitamin C in l-AA-treated cells was not detectable after 24 h, whereas PlmtVC-treated cells could keep a high level of intracellular vitamin C, suggesting an excellent stability of PlmtVC. Thus, X-ray-induced diverse harmful effects could be prevented by PlmtVC, which was suggested to ensue intrinsically from the persistent enrichment of intracellular vitamin C, resulting in relief to X-ray-caused oxidative stress.     

Polarized spectroscopic elucidation of N-acetyl-l-cysteine, l-cysteine, l-cystine, l-ascorbic acid and a tool for their determination in solid mixtures

Method of linear polarized vibrational (both IR- and Raman) spectroscopy of oriented colloids in nematic host is applied on N-acetyl-l-cysteine, l-cysteine, l-cystine and l-ascorbic acid with a view to obtain experimental bands assignment and local structural elucidation in solid-state. Structural results are compared with available crystallographic data for all of the systems studied. Scopes and limitations of the polarized method are shown. Discussion on the correlation between polarized spectroscopic data and the space group type as well as the number of the molecules in the unit cell (Z) is performed. Compounds with monoclinic space group P2₁, containing Z = 1 (N-acetyl-l-cysteine) and 2 (l-cysteine and l-ascorbic acid) are elucidated. One of the rare for organic molecules, hexagonal P6₁22 space group and Z = 6 (l-cystine) is also elucidated. Experimental assignment of the characteristics frequencies is obtained, explaining the typical for the crystals Fermi-resonance, Fermy–Davydov and Davydov splitting effects. For first time in the literature we are reported the orientation of the solid-mixture in nematic host, using the trade product ACC (Hexal, Germany), containing mainly N-acetyl-l-cysteine and l-ascorbic acid. Quantitative IR-spectroscopic approach for determination of solid mixtures is presented as well. The intensity ratio between 1,716 cm^?1 (characteristic for N-acetyl-l-cysteine) and 990 cm^?1, (attributed N-acethyl-cysteine and vitamin C) is used. Linear regression analysis between content and the peak ratio data for ten solid-binary mixtures, leads to straight-line plot y = 1.08₂ (±0.04₉) + (?0.11₄ ± 0.01₁)x, where x = 1/X _i . Factor r of 0.9641 and a reliability of 98.85% are obtained. The analysis of ACC 200 (Hexal, Germany) show that the IR measurements leads to standard deviation of 0.010 and 0.011 at P about 0.0500 for the systems and a confidence of >98.77₁%.     

Short-term parenteral application of α-tocopherol leads to increased concentration in plasma and tissues of the rat

Numerous studies suggest that supplemental vitamin E prior to or during vast surgeries might diminish or even prevent ischemia/reperfusion-induced injuries. In the present placebo-controlled study male Sprague-Dawley rats were supplemented parenterally or orally with α-tocopherol for three consecutive days. The applied amount of α-tocopherol was 2.3 μmol per day for oral and 1.2 μmol per day for parenteral supplementation. The enrichment of vitamin E concentrations in plasma and tissue samples (aortic endothelium, liver, and lung) was determined by HPLC. The vitamin E level was elevated following intravenous supplementation in plasma (21.4±1.9 μmol/L vs. 10.2±1.7 μmol/L in parenteral control group), in aortic endothelium (1.1±0.2 pmol/mm² vs. 0.5±0.1 pmol/mm²) and in liver and lung (41.3±7.5 pmol/mg vs. 22.9±6.5 pmol/mg and 75.6±13.6 pmol/mg vs. 51.7±5.9 pmol/mg, respectively). Oral supplementation for three days also led to an increased level in liver (38.2±7.7 pmol/mg vs. 22.9±6.6 pmol/mg in oral control group) and in lung (67.8±5.7 pmol/mg vs. 51.7±9.3 pmol/mg) but not in aortic endothelium or plasma (0.8±0.3 pmol/mm² vs. 0.6±0.3 pmol/mm² and 12.0±2.2 μmol/L vs. 10.7±2.6 μol/L.)     

Determination Method for l-Ascorbic Acid in Foods with Immobilized Ascorbate Oxidase

Pumpkin (Cucurbita moschata) ascorbate oxidase was entrapped within 6% (w/v) Ca-alginate gel beads, and then the beads were treated with 1% (w/v) glutaraldehyde for 20 hr at 4°C. The immobilized ascorbate oxidase was much more stable than the free form. Under the optimum conditions, the immobilized enzyme remained fully active for 3 months and after 50 assays. A linear relationship was found between immobilized ascorbate oxidase activity and l-ascorbic acid concentration in the range of 2 ~ 20 μg/ml. The immobilized preparation could be employed for the simple and rapid determination of l-ascorbic acid in foods.     

Influence of citrate and EDTA anticoagulants on plasma malondialdehyde concentrations estimated by high-performance liquid chromatography

Estimation of lipid peroxidation through MDA formation measured by assaying thiobarbituric acid (TBA) reactive products separated by HPLC remains the method of choice to study the development of oxidative stress in blood plasma. In this report we describe the influence of citrate and EDTA anticoagulants used for blood collection on estimation of MDA concentrations using HPLC analysis of MDA-TBA adducts. We analyzed a group of 40 blood donors (21 men and 19 women), median age 27 years, range 19–48 years. The mean MDA concentration in citrate plasma was 1.43±0.51 μmol/l (range: 0.61–2.57 μmol/l) and in EDTA plasma 0.36±0.10 μmol/l (range: 0.13–0.63 μmol/l). There was a significant difference in MDA mean concentration that we attribute to different antioxidant properties of anticoagulants used for blood collection. Consistency in the choice of anticoagulant is clearly extremely important.     

Synthesis of l-(+)-Tartaric Acid from l-Ascorbic Acid via 5-Keto-d-Gluconic Acid in Grapes

5-Keto-l-idionic acid (5-keto-d-gluconic acid, d-xylo-5-hexulosonic acid) was found as a metabolic product of l-ascorbic acid in slices of immature grapes, Vitis labrusca L. cv `Delaware'. Specifically labeled compounds, recognized as metabolic products of l-ascorbic acid in grapes, were fed to young grape tissues to investigate the metabolic pathway from l-ascorbic acid to l-(+)-tartaric acid.     

Over-expression of l-galactono-γ-lactone dehydrogenase increases vitamin C, total phenolics and antioxidant activity in lettuce through bio-fortification

Epidemiological studies have shown that regular consumption of fruits and vegetables is associated with reduced risk of chronic diseases. Vegetables can provide vitamins, phenolics, flavonoids, minerals and dietary fibers for optimal health benefits. However, some nutrients contained in many fruits and vegetables cannot meet of the complete nutrition need in the human body. Biotechnology has the potential to improve the nutritional value of crops. Considering the high consumption of romaine lettuce in human diet worldwide, the objective of study is to enhance the contents of vitamin C, phenolics and antioxidant activity in lettuce leaves by genetic engineering techniques. The gene expression level, vitamin C content, total phenolics, as well as total and cellular antioxidant activities were analyzed by real-time PCR, HPLC, Folin–Ciocalteu, Hydro-PSC and CAA methods, respectively. The bio-fortification of genetically engineered lettuce increased vitamin C up to 48.94 ± 1.34 mg/100 g FW following the increased over-expression of _At GLDH. This is almost a 3.2-fold increase as the content when compared with wild type lettuce (p < 0.05). In addition, phenolic compounds in transgenic lettuce contained 120.4 ± 1.62 mg GA equiv./100 g FW, almost double the phenolic content of the wild type. Total antioxidant activities were 735.4 ± 47.7 μmol vitamin C equiv./100 g FW, cellular antioxidant activities were 7.33 ± 0.86 μmol quercetin equiv./100 g FW (PBS wash) and 18.14 ± 0.68 μmol quercetin equiv./100 g FW (No PBS wash) in transgenic lettuce, respectively, 1.5, 4 and twofold increases when compared with the wild type. This study suggests that bio-fortification by genetic engineering has great potential to improve vitamin C, phenolic contents and antioxidant activity in lettuce.     

The Biosynthesis of (+)-Tartaric Acid in Pelargonium crispum

Metabolic conversion of l-galactono-1, 4-lactone and l-ascorbic acid to (+)-tartaric acid and oxalic acid has been studied in Pelargonium crispum, cv. Prince Rupert. Experiments with specifically labeled substrates suggest a path of conversion involving cleavage of l-ascorbic acid, or a metabolic product of l-ascorbic acid, between C₂ and C₃, such that oxalic acid arises from the two carbon fragment and (+)-tartaric acid from the four carbon fragment.     

High-dose vitamin C supplementation increases skeletal muscle vitamin C concentration and SVCT2 transporter expression but does not alter redox status in healthy males

Antioxidant vitamin C (VC) supplementation is of potential clinical benefit to individuals with skeletal muscle oxidative stress. However, there is a paucity of data reporting on the bioavailability of high-dose oral VC in human skeletal muscle. We aimed to establish the time course of accumulation of VC in skeletal muscle and plasma during high-dose VC supplementation in healthy individuals. Concurrently we investigated the effects of VC supplementation on expression levels of the key skeletal muscle VC transporter sodium-dependent vitamin C transporter 2 (SVCT2) and intramuscular redox and mitochondrial measures. Eight healthy males completed a randomized placebo-controlled, crossover trial involving supplementation with ascorbic acid (2×500 mg/day) over 42 days. Participants underwent muscle and blood sampling on days 0, 1, 7, and 42 during each treatment. VC supplementation significantly increased skeletal muscle VC concentration after 7 days, which was maintained at 42 days (VC 3.0±0.2 (mean±SEM) to 3.9±0.4 mg/100 g wet weight (ww) versus placebo 3.1±0.3 to 2.9±0.2 mg/100 g ww, p=0.001). Plasma VC increased after 1 day, which was maintained at 42 days (VC 61.0±6.1 to 111.5±10.4 µmol/L versus placebo 60.7±5.3 to 59.2±4.8 µmol/L, p<0.001). VC supplementation significantly increased skeletal muscle SVCT2 protein expression (main treatment effect p=0.006) but did not alter skeletal muscle redox measures or citrate synthase activity. A main finding of our study was that 7 days of high-dose VC supplementation was required to significantly increase skeletal muscle vitamin C concentration in healthy males. Our findings implicate regular high-dose vitamin C supplementation as a means to safely increase skeletal muscle vitamin C concentration without impairing intramuscular ascorbic acid transport, antioxidant concentrations, or citrate synthase activity.     

Nδ-acetylornithine and S-methylcysteine in blood plasma

N^δ-Acetylornithine and S-methylcysteine have been identified as minor components of deproteinized blood plasma of human and bovine blood. Human blood plasma contains a variable amount of acetylornithine, averaging 1.1 ± 0.4 μmol/l (range 0.8–0.2 μmol/l). Urine contains a very small amount of acetylornithine, approximately 1 nmol/mg creatinine (1 μmol/day). Human blood plasma contains 3.9 ± 1.9 μmol/l (range 1.4–6.5 μmol/l) of S-methylcysteine. Urine contains approximately 5 nmol/mg creatinine; after acid hydrolysis the amount is increased to 20 nmol/mg creatinine.     

Biosynthesis of (+)-Tartaric Acid from l-[4-C]Ascorbic Acid in Grape and Geranium

The metabolic fate of l-[4-¹⁴C]ascorbic acid has been examined in the grape (Vitis labrusca L.) and lemon geranium (Pelargonium crispum L. L'Hér. cv. Prince Rupert) under conditions comparable to data from l-[1-¹⁴C]ascorbic acid and l-[6-¹⁴C]ascorbic acid experiments. In detached grape leaves and immature berries, l-[4-¹⁴C]ascorbic acid and l-[1-¹⁴C]ascorbic acid were equivalent precursors to carboxyl labeled (+)-tartaric acid. In geranium apices, l-[4-¹⁴C]ascorbic acid yielded internal labeled (+)-tartaric acid while l-[6-¹⁴C]ascorbic acid gave an equivalent conversion to carboxyl labeled (+)-tartaric acid. These findings clearly show that two distinct processes for the synthesis of (+)-tartaric acid from l-ascorbic acid exist in plants identified as (+)-tartaric acid accumulators. In grape leaves and immature berries, (+)-tartaric acid synthesis proceeds via preservation of a four-carbon fragment derived from carbons 1 through 4 of l-ascorbic acid while carbons 3 through 6 yield (+)-tartaric acid in geranium apices.     

Increased oxidative stress and altered serum macro-minerals and trace elements levels are associated with coronary artery disease

BackgroundThis study was designed to evaluate the serum malondialdehyde (MDA), non-enzymatic antioxidants (vitamin A and C), macro-minerals (magnesium and calcium), and trace elements (zinc, copper, and iron) levels in patients with coronary artery disease (CAD) and to explore their role in disease progression.MethodsThis prospective case-control study was comprised of 40 CAD patients and 40 healthy volunteers as cases and control subjects, respectively. The level of lipid peroxidation was assessed by measuring the serum MDA level using a UV spectrophotometer. The levels of vitamins A and C were determined by high-performance liquid chromatography (HPLC) and UV spectrophotometric method, respectively. Atomic absorption spectroscopy (AAS) was used to measure serum macro-minerals (Mg and Ca) and trace elements (Zn, Cu, and Fe) concentrations.ResultsThe mean age of CAD patients and control subjects was 53.90 ± 2.22 and 37.03 ± 1.50 years, respectively. This study revealed significantly higher concentrations of MDA (p < 0.01) and lower concentrations of vitamin A (p < 0.01), and vitamin C (p < 0.05) in the CAD patients than in control subjects. The mean values of Mg, Cu, Zn, Ca, and Fe were 11.67 ± 0.64, 1.17 ± 0.03, 0.43 ± 0.02, 107.38 ± 1.81, and 1.66 ± 0.04 μg/mL, respectively for the CAD patients and 19.38 ± 0.65, 1.07 ± 0.02, 0.87 ± 0.02, 94.29 ± 1.89, and 1.52 ± 0.05 μg/mL, respectively for the controls and the differences were significant (p < 0.05) between the patients and controls.ConclusionFrom these findings, we can suggest that there is a strong association of CAD with an elevated level of MDA, depleted levels of antioxidants, and altered macro-minerals and trace elements concentrations.     

Elicitor-enhanced production of gymnemic acid in cell suspension cultures of <Emphasis Type="Italic">Gymnema sylvestre</Emphasis> R. Br.

Cell suspension cultures of Gymnema sylvestre treated with four different elicitors, methyl jasmonate (MJ), yeast extract, chitin and pectin were studied for the production of gymnemic acid as gymnemagenin equivalent, that was analyzed by high performance liquid chromatography (HPLC). All the four tested elicitors induced gymnemic acid production in cell suspension cultures. Highest gymnemic acid content was achieved following treatment with yeast extract (100.47 ± 0.28 mg/l), this was followed by MJ (70.43 ± 0.26 mg/l), pectin (64.19 ± 0.23 mg/l) and chitin (62.72 ± 0.13 mg/l). The addition of elicitors has shown a significant influence on cell growth that affected cell growth compared to respective controls. The highest gymnemic acid production was obtained after 20 days of elicitation in cultures treated with 0.5 g l^−l yeast extract, it was 5.25-folds greater than in control. These results suggest that the addition of an elicitor to Gymnema sylvestre cell suspension cultures could stimulate and enhance gymnemic acid production. In our present study we could able to overproduce gymnemic acid up to 51.97 ± 0.26 mg l^−l (dry weight basis) in yeast extract treated cell suspension cultures.     

Photochemiluminescent detection of antiradical activity; IV: testing of lipid-soluble antioxidants

A new method for quantification of antiradical properties of pure lipid-soluble antioxidants and for measurement of integral antioxidant capacity in the lipid phase (ACL) of polycomponent systems, such as blood plasma or tissue homogenates, is developed. It is based on an antioxidant-sensitive inhibition of a photo-induced, chemiluminescence accompanied autoxidation of luminol. The sensitivity of the photochemiluminescent (PCL) assay lies within nmol quantities of substances, the measuring range for α-tocopherol is between 0.1 and 3 nmol. The interassay variability of the method is lower than 5%, the intraassay variability <2%. The antioxidant efficiency of γ-tocopherol was found to be 43% of α-tocopherol. The results of the PCL measurements on pure antioxidants and on lipid extracts from blood plasma were compared with the level of, ‘vitamin E’ (VE) determined as a sum of α- and γ-tocopherol by HPLC. Very good coincidence of both methods was observed for pure substances (r = 0.998, P<0.001). The ACL of human blood plasma was found to be 27.98 ± 0.68 μmol equivalents of α-tocopherol/l (mean ± mean error, n = 142), it is ∼ 25% more than the concentration of VE found in the same samples (22.09 ± 0.59 μmol/l). In this case, the correlation of both parameters was lower: r = 0.811, P<0.001. The animal experiments showed that synthetic antioxidants may not only increase the value of ACL of blood plasma but in the same time reduce the concentration of biological antioxidants, e.g. VE drastically. The prooxidant activity of synthetic antioxidants in vivo or the replacing of structured α-tocopherol from its position can be the cause. This important circumstance has to be considered during the testing of new antioxidants for clinical application.     

Low selenium status in alcoholic cirrhosis is correlated with aminopyrine breath test

The relationship among impaired selenium status, lipid peroxidation, and liver function was examined in 19 hospitalized patients with severe alcoholic cirrhosis. Plasma selenium was found to be significantly lower (mean±SD: 54±13 μg/L) than in healthy controls (83±11 μg/L) and plasma malondialdehyde, assessed as thiobarbituric acid reactants, which reflects lipid peroxidation, was increased (2.0±1.2 μmol/L vs <1.2 μmol/L in controls). The mean¹⁴C aminopyrine breath test, an indicator of liver function, was lower than normal (2.7±1.9 vs 6.3±0.9% in controls) and found to be significantly correlated with plasma selenium (r=0.59,p<0.05). A prospective, randomized selenium supplementation trial was conducted in a group of 16 patients who received either daily 100 μg selenium as enriched yeast during 4 mo or a placebo. Among the 10 patients who completed the study, plasma selenium significantly increased in the supplemented group (n=4; before: 58±10 μg/L, and after 101±12 μg/L,p<0.01) contrary to the placebo group (n=6, before: 47±10 μg/L, after: 57±9 μg/L, n.s.),¹⁴C aminopyrine breath test improved in three out of four selenium-supplemented patients and in three out of six placebo patients, but the small number of patients did not allow statistical evaluation. These results demonstrate that low selenium status in alcoholic cirrhosis is correlated to liver function and could be improved by supplementation.     

Elevating vitamin C content via overexpression of myo-inositol oxygenase and l-gulono-1,4-lactone oxidase in Arabidopsis leads to enhanced biomass and tolerance to abiotic stresses

l-ascorbic acid (vitamin C) is an abundant metabolite in plant cells and tissues. Ascorbate functions as an antioxidant, as an enzyme cofactor, and plays essential roles in multiple physiological processes including photosynthesis, photoprotection, control of cell cycle and cell elongation, and modulation of flowering time, gene regulation, and senescence. The importance of this key molecule in regulating whole plant morphology, cell structure, and plant development has been clearly established via characterization of low vitamin C mutants of Arabidopsis, potato, tobacco, tomato, and rice. However, the consequences of elevating ascorbate content in plant growth and development are poorly understood. Here, we demonstrate that Arabidopsis lines overexpressing a myo-inositol oxygenase or an l-gulono-1,4-lactone oxidase, containing elevated ascorbate, display enhanced growth and biomass accumulation of both aerial and root tissues. To our knowledge, this is the first study demonstrating such a marked positive effect in plant growth in lines engineered to contain elevated vitamin C content. In addition, we present evidence showing that these lines are tolerant to a wide range of abiotic stresses including salt, cold, and heat. Total ascorbate content of the transgenic lines remained higher than those of controls under the abiotic stresses tested. Interestingly, exposure to pyrene, a polycyclic aromatic hydrocarbon and known inducer of oxidative stress in plants, leads to stunted growth of the aerial tissue, reduction in the number of root hairs, and inhibition of leaf expansion in wild type plants, while these symptoms are less severe in the overexpressers. Our results indicate the potential of this metabolic engineering strategy to develop crops with enhanced biomass, abiotic stress tolerance, and phytoremediation capabilities.