The Membrane Electrical Potential and Intracellular pH as Factors Influencing Intracellular Ascorbate Concentration and Their Role in Cancer Treatment

Ascorbate is an important element of a variety of cellular processes including the control of reactive oxygen species levels. Since reactive oxygen species are implicated as a key factor in tumorigenesis and antitumor therapy, the injection of a large amount of ascorbate is considered beneficial in cancer therapy. Recent studies have shown that ascorbate can cross the plasma membrane through passive diffusion. In contrast to absorption by active transport, which is facilitated by transport proteins (SVCT1 and SVCT2). The passive diffusion of a weak acid across membranes depends on the electrostatic potential and the pH gradients. This has been used to construct a new theoretical model capable of providing steady-state ascorbate concentration in the intracellular space and evaluating the time needed to reach it. The main conclusion of the analysis is that the steady-state intracellular ascorbate concentration weakly depends on its serum concentration but requires days of exposure to saturate. Based on these findings, it can be hypothesized that extended oral ascorbate delivery is possibly more effective than a short intravenous infusion of high ascorbate quantities.

Grafting Enhances Pepper Water Stress Tolerance by Improving Photosynthesis and Antioxidant Defense Systems

Currently, limited water supply is a major problem in many parts of the world. Grafting peppers onto adequate rootstocks is a sustainable technique used to cope with water scarcity in plants. For 1 month, this work compared grafted peppers by employing two rootstocks (H92 and H90), with different sensitivities to water stress, and ungrafted plants in biomass, photosynthesis, and antioxidant response terms to identify physiological–antioxidant pathways of water stress tolerance. Water stress significantly stunted growth in all the plant types, although tolerant grafted plants (variety grafted onto H92, Var/H92) had higher leaf area and fresh weight values. Var/H92 showed photosynthesis and stomata conductance maintenance, compared to sensitive grafted plants (Var/H90) and ungrafted plants under water stress, linked with greater instantaneous water use efficiency. The antioxidant system was effective in removing reactive oxygen species (ROS) that could damage photosynthesis; a significant positive and negative linear correlation was observed between the rate of CO2 uptake and ascorbic acid (AsA)/total AsA (AsAt) and proline, respectively. Moreover, in Var/H92 under water stress, both higher proline and ascorbate concentration were observed. Consequently, less membrane lipid peroxidation was quantified in Var/H92.

Effect of Rice GDP-L-Galactose Phosphorylase Constitutive Overexpression on Ascorbate Concentration, Stress Tolerance, and Iron Bioavailability in Rice

Ascorbate (vitamin C) is an essential multifunctional molecule for both plants and mammals. In plants, ascorbate is the most abundant water-soluble antioxidant that supports stress tolerance. In humans, ascorbate is an essential micronutrient and promotes iron (Fe) absorption in the gut. Engineering crops with increased ascorbate levels have the potential to improve both crop stress tolerance and human health. Here, rice (Oryza sativa L.) plants were engineered to constitutively overexpress the rice GDP-L-galactose phosphorylase coding sequence (35S-OsGGP), which encodes the rate-limiting enzymatic step of the L-galactose pathway. Ascorbate concentrations were negligible in both null segregant (NS) and 35S-OsGGP brown rice (BR, unpolished grain), but significantly increased in 35S-OsGGP germinated brown rice (GBR) relative to NS. Foliar ascorbate concentrations were significantly increased in 35S-OsGGP plants in the vegetative growth phase relative to NS, but significantly reduced at the reproductive growth phase and were associated with reduced OsGGP transcript levels. The 35S-OsGGP plants did not display altered salt tolerance at the vegetative growth phase despite having elevated ascorbate concentrations. Ascorbate concentrations were positively correlated with ferritin concentrations in Caco-2 cells – an accurate predictor of Fe bioavailability in human digestion – exposed to in vitro digests of NS and 35S-OsGGP BR and GBR samples.

SPECTROPHOTOMETRIC DETERMINATION OF ASCORBIC ACID BY HORSERADISH PEROXIDASE

L-ascorbic acid is one of the essential nutrients and most common food supplements, fortificants, and preservatives. It is commercially available as solutions, drops, tablets, capsules, crystal powder, beverage mixtures, multivitamin formulations, and multi antioxidant formulations. The usual daily dose is from 25 mg to 1.5 g. Ascorbic acid is a distinctly reducing agent with low redox potential (0.18 and 0.08 V at pH 4.5 and 6.4, respectively). Based on ascorbate property, numerous methods for its quantitative determination are developed, from titrimetric, electrochemical, and chromatographic methods, to fluorometric and kinetic ones. Enzyme peroxidase is interfered with by ascorbic acid, which decreases the oxidation speed of its co-substrates during hydrogen peroxide decomposition by peroxidase. Absorbance changes at the wavelength of corresponding reagents are in correlation with ascorbate concentration. During this study, benzidine and o-tolidine have been used as chromogenic reagents. Reaction conditions were optimized for various buffer systems, calibration curves were constructed, and limits of detection (0.04 μmol/L) and quantification (0.12 μmol/L) were calculated. Using calibration charts, it was possible to detect ascorbic acid within limits from 0.4 to 10 μmol/L. The optimized method was applied for the determination of ascorbic acid in pharmaceutical products. The method was characterized by exceptional sensitivity and accuracy, but only for preparations not containing substances that affect enzyme peroxidase.

Organization and control of the ascorbate biosynthesis pathway in plants

ABSTRACTThe enzymatic steps involved in l-ascorbate biosynthesis in photosynthetic organisms (the Smirnoff-Wheeler, SW pathway) has been well established and here we comprehensively analyze the subcellular localization, potential physical interactions of SW pathway enzymes and assess their role in control of ascorbate synthesis. Transient expression of GFP-fusions in Nicotiana benthamiana and Arabidopsis (Arabidopsis thaliana) mutants complemented with genomic constructs showed that while GME is cytosolic, VTC1, VTC2, VTC4, and l-GalDH have cytosolic and nuclear localization. While transgenic lines GME-GFP, VTC4-GFP and l-GalDH-GFP driven by their endogenous promoters accumulated the fusion proteins, the functional VTC2-GFP protein is detected at low level using immunoblot in a complemented vtc2 null mutant. This low amount of VTC2 protein and the extensive analyses using multiple combinations of SW enzymes in N. benthamiana supported the role of VTC2 as the main control point of the pathway on ascorbate biosynthesis. Interaction analysis of SW enzymes using yeast two hybrid did not detect the formation of heterodimers, although VTC1, GME and VTC4 formed homodimers. Further coimmunoprecipitation (CoIP) analysis indicted that consecutive SW enzymes, as well as the first and last enzymes (VTC1 and l-GalDH), associate thereby adding a new layer of complexity to ascorbate biosynthesis. Finally, metabolic control analysis incorporating known kinetic characteristics, showed that previously reported feedback repression at the VTC2 step confers a high flux control coefficient and rationalizes why manipulation of other enzymes has little effect on ascorbate concentration.One sentence summaryMetabolic engineering, genetic analysis and functional mutant complementation identify GDP-l-galactose phosphorylase as the main control point in ascorbate biosynthesis in green tissues.

Spectrophotometric determination of ascorbic acid by horseradish peroxidase

L-ascorbic acid is one of the essential nutrients and most common food supplements, fortificants, and preservatives. It is commercially available as solutions, drops, tablets, capsules, crystal powder, beverage mixtures, multivitamin formulations, and multi antioxidant formulations. The usual daily dose is from 25 mg to 1.5 g. Ascorbic acid is a distinctly reducing agent with low redox potential (0.18 and 0.08 V at pH 4.5 and 6.4, respectively). Based on ascorbate property, numerous methods for its quantitative determination are developed, from titrimetric, electrochemical, and chromatographic methods, to fluorometric and kinetic ones. Enzyme peroxidase is interfered with by ascorbic acid, which decreases the oxidation speed of its co-substrates during hydrogen peroxide decomposition by peroxidase. Absorbance changes at the wavelength of corresponding reagents are in correlation with ascorbate concentration. During this study, benzidine and o-tolidine have been used as chromogenic reagents. Reaction conditions were optimized for various buffer systems, calibration curves were constructed, and limits of detection (0.04 mmol/L) and quantification (0.12 mmol/L) were calculated. Using calibration charts, it was possible to detect ascorbic acid within limits from 0.4 to 10 mmol/L. The optimized method was applied for the determination of ascorbic acid in pharmaceutical products. The method was characterized by exceptional sensitivity and accuracy, but only for preparations not containing substances that affect enzyme peroxidase.

Ascorbic acid may not be involved in cryptochrome-based magnetoreception

Seventeen years after it was originally suggested, the photoreceptor protein cryptochrome remains the most probable host for the radical pair intermediates that are thought to be the sensors in the avian magnetic compass. Although evidence in favour of this hypothesis is accumulating, the intracellular interaction partners of the sensory protein are still unknown. It has been suggested that ascorbate ions could interact with surface-exposed tryptophan radicals in photoactivated cryptochromes, and so lead to the formation of a radical pair comprised of the reduced form of the flavin adenine dinucleotide cofactor, FAD • − , and the ascorbate radical, Asc • − . This species could provide a more sensitive compass than a FAD–tryptophan radical pair. In this study of Drosophila melanogaster cryptochrome and Erithacus rubecula (European robin) cryptochrome 1a, we use molecular dynamics simulations to characterize the transient encounters of ascorbate ions with tryptophan radicals in cryptochrome in order to assess the likelihood of the [FAD • − Asc • − ]-pathway. It is shown that ascorbate ions are expected to bind near the tryptophan radicals for periods of a few nanoseconds. The rate at which these encounters happen is low, and it is therefore concluded that ascorbate ions are unlikely to be involved in magnetoreception if the ascorbate concentration is only of the order of 1 mM or less.

Ascorbate concentration in Arabidopsis thaliana and expression of ascorbate related genes using RNAseq in response to light and the diurnal cycle

We explore where transcriptional regulation of ascorbate concentration lies in plants. Is it in biosynthesis,recycling, regulation or consumption? Arabidopsis thaliana plants were grown under controlled environment at four photon flux density levels (PFD). Rosettes from plants were harvested at the four PFD levels and over a diurnal cycle and after a step change in PFD and analysed for ascorbate concentration and transcript levels measured by RNAseq. Ascorbate concentrations and expression of genes in the L-galactose ascorbate biosynthesis, recycling,consumption pathways and regulation are presented to provide a full analysis of the control of ascorbate by environmentally modulated gene expression. Ascorbate concentration responded to PFD levels but not to time of day and showed only a small response to change of PFD after 2 days. Of the L-galactose pathway genes, only GDP galactose phosphorylase (GGP) showed a significant response in to different PFDs, time of day and to change in PFD. Other genes also showed limited responses. This study compares gene expression of a range of ascorbate related genes to changes in environment in a unified way and supports the concept that GGP is the key regulatory gene in ascorbate biosynthesis and that post transcriptional regulation is also important.HighlightIn a comprehensive study of expression of all ascorbate related genes the data is consistent with the control of leaf ascorbate concentration by transcription being through the expression of GDP galactose phosphorylase.

Ascorbate metabolism in vegetative and reproductive organs of “cherry” tomato

Ascorbate metabolism is an essential procedure for all plant cells that plays important roles in several physiological processes such as plant development and reactive oxygen species detoxification. To shed more light on ascorbate metabolism in certain organs of tomato plants, we performed a detailed compartmentalized analysis of ascorbate concentration, ascorbate peroxidase/dehydroascorbate reductase enzyme activities and transcript accumulation of genes related to ascorbate metabolism. Our results showed higher level of ascorbate concentration and ascorbate peroxidase and dehydroascorbate reductase activities in young leaves and shoot tips, while min. ascorbate concentration was recorded in root tips. The study of the expression of the genes involved in ascorbate metabolism revealed that several genesfollowed similar patterns. However, APX3 gene expression was considerably higher in reproductive organs, while plastidialAPX6 and DHAR2genes transcripts were barely detectable in root tips. Organ-specific expression of genes involved in ascorbate metabolism suggests that different isoenzymes have a specific role in regulation of the redox status of some of the organs in tomato plants.