Spontaneous N epsilon-methylation and N epsilon-formylation reactions between L-lysine and formaldehyde inhibited by L-ascorbic acid.

For the inhibition of spontaneous N epsilon-methylation and N epsilon-formylation reactions between L-lysine and formaldehyde, L-ascorbic acid proved to be most suitable. The inhibition was not complete unless the molar concentration of ascorbic acid exceeded that of formaldehyde. T.l.c., potentiometric titration, n.m.r. spectroscopy and radiometric analysis were applied in the study of the inhibition process. Formaldehyde was reduced by L-ascorbic acid to ethylene glycol.     

Effects of Ascorbic Acid and Analogs on the Activity of Testicular Hyaluronidase and Hyaluronan Lyase on Hyaluronan

We have evaluated the inhibition of testicular hyaluronidase and hyaluronan lyase by L-ascorbic acid and chemical analogs. We observed that L-ascorbic acid, D-isoascorbic acid and dehydroascorbic acid inhibited both types of enzymes, but showed stronger effects towards hyaluronan lyase. But these compounds were observed to degrade the substrate, hyaluronan, by themselves. Of the other ascorbic acid analogs tested, saccharic acid inhibited hyaluronan lyase, while not affecting the enzymatic activity of testicular hyaluronidase, nor affecting the physic-chemical stability of hyaluronan. This is the first compound, to our knowledge, to be shown to possess such selective inhibition. Therefore, we propose that saccharic acid could serve as a lead compound for the development of potent and selective inhibitors of bacterial hyaluronan lyase or of polysaccharide lyase enzymes in general as we observed this compound to be capable of inhibiting chondroitinase ABC in addition to hyaluronan lyase.     

Effects of ascorbic acid and analogs on the activity of testicular hyaluronidase and hyaluronan lyase on hyaluronan

We have evaluated the inhibition of testicular hyaluronidase and hyaluronan lyase by L-ascorbic acid and chemical analogs. We observed that L-ascorbic acid, D-isoascorbic acid and dehydroascorbic acid inhibited both types of enzymes, but showed stronger effects towards hyaluronan lyase. But these compounds were observed to degrade the substrate, hyaluronan, by themselves. Of the other ascorbic acid analogs tested, saccharic acid inhibited hyaluronan lyase, while not affecting the enzymatic activity of testicular hyaluronidase, nor affecting the physic-chemical stability of hyaluronan. This is the first compound, to our knowledge, to be shown to possess such selective inhibition. Therefore, we propose that saccharic acid could serve as a lead compound for the development of potent and selective inhibitors of bacterial hyaluronan lyase or of polysaccharide lyase enzymes in general as we observed this compound to be capable of inhibiting chondroitinase ABC in addition to hyaluronan lyase.     

The interaction of L-ascorbic acid with the active center of myrosinase.

Only L-ascorbic acid activated plant myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1), whereas ascorbic acid analogs did not. The enzyme protein was conformationally changed by the addition of L-ascorbic acid to the spectrophotometric analysis, approx. 1.5 amino residues appeared on the surface of the enzyme and about 2.3 tryptophan residues were buried in the molecule when 1 mM L-ascorbic acid was added. Optimum temperature for the myrosinase activity was approx. 55 degrees C without L-ascorbic acid, but with L-ascorbic acid it was about 35 degrees C; that for beta-glucosidase activity was the same (55 degrees C) with or without L-ascorbic acid. The effect of chemical modification of the functional groups of myrosinase on the interaction of L-ascorbic acid was investigated and the interaction of L-ascorbic acid with the active center of the enzyme is proposed.     

L-ascorbic acid microencapsulated with polyacylglycerol monostearate for milk fortification

Efficiency was examined of microencapsulating L-ascorbic acid by polyglycerol monostearate (PGMS), and changes in the chemical and sensorial aspects of L-ascorbic acid and/or iron-fortified milk during storage were evaluated. The selected core materials were ferric ammonium sulfate and L-ascorbic acid. The highest efficiency (94.2%) of microencapsulation was found with the ratio of 5:1 as the coating to core material. The release of ascorbic acid from the microcapsules increased sharply from 1.6 to 6.7% up to 5 d of storage. The TBA value was the lowest in the milk sample with added encapsulated iron and unencapsulated L-ascorbic acid up to 5 d of storage in comparison with the other treated samples. A sensory analysis showed that most aspects were not significantly different between the control and fortified samples encapsulated with ascorbic acid after 5 d of storage. The results indicate that L-ascorbic acid microencapsulated with PGMS can be applied to fortify milk and acceptable milk products can be prepared with microencapsulated L-ascorbic acid and iron.     

Action of ascorbic acid on a myosin molecule derived from carp

The influence of L-ascorbic acid at 40 degrees C incubation on the subfragment-1 and rod regions, prepared by chymotryptic digestion of myosin, and myosin was investigated by SDS-polyacrylamide gel electrophoresis and transmission electron microscopy respectively. It was observed that L-ascorbic acid acted more readily on the subfragment-1 region of myosin. Further, circular dichroism measurement indicated that L-ascorbic acid did not affect the structure of myosin. These results suggest that L-ascorbic acid acts more readily on the myosin subfragment-1 region and promotes the gelation of myosin without producing a conformational change in this protein.     

Biosynthesis of L-ascorbic acid in plants: new pathways for an old antioxidant

The biosynthetic pathway of L-ascorbic acid (vitamin C) in plants has been established for several years. However, recent reports describe alternative pathways, revealing a more complex picture of L-ascorbic acid biosynthesis than had been expected. GDP-L-gulose and myo-inositol are proposed as new intermediates in L-ascorbic acid biosynthesis, indicating that part of the animal pathway might also be operating in plants. Enzymatic studies on the GDP-mannose- 3',5'-epimerase and L-galactono-1,4-lactone dehydrogenase suggest that they are important regulatory steps for L-ascorbic acid biosynthesis.     

Degradation of L-Ascorbic Acid and Mechanism of Non-enzymic Browning Reaction

In the presence of oxygen, L-ascorbic acid sol ution (0.05 M) browned more intense1 y than dehydro-L-ascorbic acid solution (0.05 M) during storage for longer period.

The mixed solution of L-ascorbic acid (ASA) and dehydro-L-ascorbic acid (DHA) with the ratio of 1:1 or 1:3 in concentration gave more intense browning than DHA solution during storage at 38°C for about 3 weeks. Essentially the same type of browning was observed in case of the mixture of ASA and DHA with D-glucose. Browning of partially oxidized ASA solution also showed substantially the same results as those mentioned above.     

Antioxidant alpha-keto-carboxylate pyruvate protects low-density lipoprotein and atherogenic macrophages

Oxidative modification of low-density lipoprotein (oxLDL) plays a pathogenic role in atherogenesis. Classical antioxidants such as L-ascorbic acid can inhibit formation of oxLDL. Alpha-Keto-carboxylates such as pyruvate and congeners also display antioxidant properties in some cell-free and intact cell systems. We tested the hypothesis that pyruvate or alpha-keto-glutarate may function as antioxidants with respect to LDL incubated with 5 or 10 microM Cu2+ alone or in combination with THP-1-derived macrophages. alpha-Hydroxy-carboxylates (L-lactate), linear aliphatic monocarboxylates (acetate/caprylate) and L-ascorbic acid served as controls. The oxLDL formation was ascertained by electrophoretic mobility and oxLDL cytotoxicity was judged by macrophage viability and thiobarbituric acid reactive substances (TBARS) formation. Cu2+ alone was not cytotoxic but increased electrophoretic mobility of cell-free LDL, stimulating TBARS. Millimolar pyruvate, alpha-ketoglutarate, or micromolar L-ascorbic acid partially inhibited oxLDL formation, while alpha-hydroxy-carboxylate or the aliphatic mono-carboxylates had no measurable antioxidant properties in cell-free LDL. Co-culture of LDL with macrophages and Cu2+ augmented TBARS release and resulted in 95% macrophage death. Pyruvate improved macrophage viability with 5 microM Cu2+ up to 60%. L-Ascorbic acid (> or = 100 microM) protected macrophages up to 80%. When > or = 100 microM L-ascorbic acid was combined with pyruvate, oxLDL formation and macrophage death were fully prevented. Thus, alpha-keto-carboxylates, but not physiological alpha-hydroxy-carboxylates or aliphatic monocarboxylates qualify as antioxidants in LDL systems. Since alpha-keto-carboxylates enhanced the antioxidant power of L-ascorbic acid, our findings may have implications for strategies attenuating atherosclerosis.     

Inhibitory effect of an ellagic acid-rich pomegranate extract on tyrosinase activity and ultraviolet-induced pigmentation

A pomegranate extract (PE) from the rind containing 90% ellagic acid was tested for its skin-whitening effect. PE showed inhibitory activity against mushroom tyrosinase in vitro, and the inhibition by the extract was comparable to that of arbutin, which is a known whitening agent. PE, when administered orally, also inhibited UV-induced skin pigmentation on the back of brownish guinea pigs. The intensity of the skin-whitening effect was similar between guinea pigs fed with PE and those fed with L-ascorbic acid. PE reduced the number of DOPA-positive melanocytes in the epidermis of UV-irradiated guinea pigs, but L-ascorbic acid did not. These results suggest that the skin-whitening effect of PE was probably due to inhibition of the proliferation of melanocytes and melanin synthesis by tyrosinase in melanocytes. PE, when taken orally, may be used as an effective whitening agent for the skin.     

Transport model of the human Na+-coupled L-ascorbic acid (vitamin C) transporter SVCT1

Vitamin C (L-ascorbic acid) is an essential micronutrient that serves as an antioxidant and as a cofactor in many enzymatic reactions. Intestinal absorption and renal reabsorption of the vitamin is mediated by the epithelial apical L-ascorbic acid cotransporter SVCT1 (SLC23A1). We explored the molecular mechanisms of SVCT1-mediated L-ascorbic acid transport using radiotracer and voltage-clamp techniques in RNA-injected Xenopus oocytes. L-ascorbic acid transport was saturable (K(0.5) approximately 70 microM), temperature dependent (Q(10) approximately 5), and energized by the Na(+) electrochemical potential gradient. We obtained a Na(+)-L-ascorbic acid coupling ratio of 2:1 from simultaneous measurement of currents and fluxes. L-ascorbic acid and Na(+) saturation kinetics as a function of cosubstrate concentrations revealed a simultaneous transport mechanism in which binding is ordered Na(+), L-ascorbic acid, Na(+). In the absence of L-ascorbic acid, SVCT1 mediated pre-steady-state currents that decayed with time constants 3-15 ms. Transients were described by single Boltzmann distributions. At 100 mM Na(+), maximal charge translocation (Q(max)) was approximately 25 nC, around a midpoint (V(0.5)) at -9 mV, and with apparent valence approximately -1. Q(max) was conserved upon progressive removal of Na(+), whereas V(0.5) shifted to more hyperpolarized potentials. Model simulation predicted that the pre-steady-state current predominantly results from an ion-well effect on binding of the first Na(+) partway within the membrane electric field. We present a transport model for SVCT1 that will provide a framework for investigating the impact of specific mutations and polymorphisms in SLC23A1 and help us better understand the contribution of SVCT1 to vitamin C metabolism in health and disease.     

Temporal effects of alpha-tocopherol and L-ascorbic acid on in vitro fertilized porcine embryo development

The susceptibility of embryos to reactive oxygen species (ROS) varies in different stages of embryo development. The present study evaluated temporal effects of alpha-tocopherol and L-ascorbic acid on the porcine embryo development, and investigated whether a single or twice supplements of these two antioxidants at a divided concentrations favors the embryo development. In order to determine temporal effects of alpha-tocopherol and/or L-ascorbic acid, 100 microM alpha-tocopherol or 200 microM L-ascorbic acid were supplemented to the North Carolina State University (NCSU)-23 embryo culture media at 0, 48, 96 and 120 h of culture. In another set of experiments, the concentration was divided into two equal halves, i.e., 50 microM alpha-tocopherol and 100 microM L-ascorbic acid, and supplemented twice at 0 and 48, 0 and 96, or 48 and 96 h of culture. Supplementing culture media with 100 microM alpha-tocopherol for the entire culture period of 168 h or starting from the 48 h of culture yielded higher blastocyst percentage compared with the control or starting from the 96 or 120 h of culture. L-Ascorbic acid (200 microM) alone or together with alpha-tocopherol (100 microM) with a single supplement did not affect the frequency of blastocyst formation or number of cells in blastocyst. L-ascorbic acid with a divided supplements yielded higher blastocyst percentage compared with the control. No synergistic effect was observed on embryo development at a single supplement of these antioxidants. Although, at divided supplements higher blastocyst percentage was observed compared with control group, no further beneficial effect was observed compared with alpha-tocopherol or L-ascorbic acid alone. Our results demonstrated that the embryotrophic effects of alpha-tocopherol and/or L-ascorbic acid, in terms of frequency of blastocyst formation and number of cells in blastocyst, depends on the concentration and supplementation timing.     

Effect of glucose on carbohydrate synthesis from alanine or lactate in hepatocytes from starved rats.

Euglena gracilis was found to contain a peroxidase that specifically require L-ascorbic acid as the natural electron donor in the cytosol. The presence of an oxidation-reduction system metabolizing L-ascorbic acid was demonstrated in Euglena cells. Oxidation of L-ascorbic acid by the peroxidase, and the absence of ascorbic acid oxidase activity, suggests that the system functions to remove H2O2 in E. gracilis, which lacks catalase.     

Effect of flavonoids on vitamin C activity of D-isoascorbic acid.

Large peroral doses of D-isoascorbic acid, a vitamin C stereoisomer (50 mg per animal per day), were retained in the guinea-pig organism to a smaller extent than the same doses of L-ascorbic acid (vitamin C). Simultaneous administration of the flavonoids rutin and epicatechin increased the amount of D-isoascorbic acid retained in the liver, brain and wall of the small intestine by up to 100%, but four weeks after its extraction from the food the amount of L-ascorbic acid left in the guinea-pig organism still exceeded D-isoascorbic acid reserves. This difference, which was found in all the organs studied, was the largest in the groups simultaneously given flavonoids. In guinea-pigs which, like man, are dependent on an exogenous vitamin C supply, D-isoascorbic acid was metabolized at a manifestly higher rate than L-ascorbic acid, irrespective of whether flavonoids were administered or not. In liver, brain and small intestine wall homogenates, the oxidized forms of both stereoisomers were reduced in the presence of reduced glutathione, but the reduction rate of D-isodehydroascorbic acid was higher and it was stimulated by the two flavonoids more strongly than the reduction of L-dehydroascorbic acid. The stuterospecific.     

Antimetabolic activity of L-ascorbic acid in human and animal tumors

1. The effect of high concentrations of L-ascorbic acid on the growth of some human and animal transformed and non-transformed cell lines has been investigated. Directly implemented into culture of transformed cell lines it decreased [3H]thymidine, [3H]uridine and [3H]leucine incorporation into cells. Vitamin C inhibited DNA synthesis by transformed cells 3-4 times more efficiently than by normal cells. 2. In vivo treatment of athymic nude mice bearing human mammary carcinoma with 500 mg/kg L-ascorbic acid for the first 15 days markedly inhibited the growth of tumor cells. 3. As determined by alkaline elution, both DNA strand breaks and DNA cross links were observed in mammary carcinoma cells treated with vitamin C. DNA-DNA and DNA-protein cross links in cells treated with L-ascorbic acid were revealed by the proteinase K assay. Removal of vitamin C caused an immediate onset of spontaneous repair of single or double stranded DNA breaks. If, however, vitamin was reintroduced into cell culture, this spontaneous repair was reversed. 4. Our results indicate an antimetabolic activity of L-ascorbic acid in human and animal transformed cells, probably due to lethal damages in DNA.     

Antioxidant activity of L-ascorbic acid in wild-type and superoxide dismutase deficient strains of Saccharomyces cerevisiae

Much has been published on the non-enzymatic antioxidant L-ascorbic acid (vitamin C), but even so its interaction with endogenous cellular defense systems has not yet been fully elucidated. Our study investigated the antioxidant activity of L-ascorbic acid in wild-type strain EG103 (SOD) Saccharomyces cerevisiae and isogenic mutant strains deficient in cytosolic superoxide dismutase (sod1delta), mitochondrial superoxide dismutase (sod2delta) or both (sod1delta sod2delta), metabolizing aerobically or anaerobically with and without the stressing agent paraquat. The results show that during both aerobic and anaerobic metabolism there was a significant increase in the survival of both wild-type S. cerevisiae cells and the mutant cells (sod1delta, sod2delta and sod1delta sod2delta) when pretreated with L-ascorbic acid before exposure to paraquat. Exposure to paraquat resulted in higher catalase activity but this significantly decreased when the cells were pre-treated with L-ascorbic acid. These results demonstrate that due to the damage caused by paraquat, the antioxidant protection of L-ascorbic acid seems to be mediated by catalase levels in yeast cells.     

Effect of ascorbic acid on release of acetylcholine from synaptic vesicles prepared from different species of animals and release of noradrenaline from synaptic vesicles of rat brain.

Low concentrations of L-ascorbic acid caused release of acetylcholine from isolated synaptic vesicles (rat, guinea-pig and rabbit) in the presence of 2mM ATP, 2 mM MgCl₂ and 10^?5 M CaCl. The half maximum effect was obtained with about 2 to 2.5 ωM L-ascorbic acid, and the effect was inhibited by addition of 1mM EGTA. The release of noradrenaline from rat synaptic vesicles was also enhanced by L-ascorbic acid, but the concentration for half maximal stimulation was about 20 ωM, indicating that noradrenaline release was less sensitive to L-ascorbic acid than acetylcholine release. The physiological function of L-ascorbic acid in the brain is discussed in relation to release of transmitters.     

The challenge of increasing vitamin C content in plant foods

The term "vitamin" is used to define a number of organic compounds that have to be obtained from different foods because the organism itself cannot synthesize them in the quantities needed to sustain life. Vitamin C is the common name for L-ascorbic acid. In humans, the principal role of this molecule is to scavenge reactive oxygen species, due to its antioxidant capacity, and to serve as cofactor for many enzymes. A deficiency of L-ascorbic acid is traditionally linked to human diseases such as scurvy. Plant foods are the principal source of L-ascorbic acid for humans. There is a high variability of L-ascorbic acid content in the various plant organs that are used for human consumption. This diversity is related to the specific functions played by L-ascorbic acid in the different plant tissues. The net content of L-ascorbic acid in plants is determined through a balance of the activities of different biosynthetic, recycling, and catabolic pathways. Here we review the importance of L-ascorbic acid for human health, the current knowledge on its metabolism and function in plants, and the efforts that have already been made by genetic modification to improve its content in plant organs used for human food. We provide a current and forward looking perspective of how plant science can contribute to improving the L-ascorbic acid content in crop species using gene transformation, quantitative trait loci and association mapping-based approaches.