Dehydroascorbic acid

Determination of Ascorbic Acid, Total Ascorbic Acid, and Dehydroascorbic Acid in Bee Pollen Using Hydrophilic Interaction Liquid Chromatography-Ultraviolet Detection.[Pubmed:33287160]

Molecules. 2020 Dec 3;25(23). pii: molecules25235696.

Ascorbic acid (AA) is one of the essential nutrients in bee pollen, however, it is unstable and likely to be oxidized. Generally, the oxidation form (Dehydroascorbic acid (DHA)) is considered to have equivalent biological activity as the reduction form. Thus, determination of the total content of AA and DHA would be more accurate for the nutritional analysis of bee pollen. Here we present a simple, sensitive, and reliable method for the determination of AA, total ascorbic acids (TAA), and DHA in rape (Brassica campestris), lotus (Nelumbo nucifera), and camellia (Camellia japonica) bee pollen, which is based on ultrasonic extraction in metaphosphoric acid solution, and analysis using hydrophilic interaction liquid chromatography (HILIC)-ultraviolet detection. Analytical performance of the method was evaluated and validated, then the proposed method was successfully applied in twenty-one bee pollen samples. Results indicated that contents of AA were in the range of 17.54 to 94.01 microg/g, 66.01 to 111.66 microg/g, and 90.04 to 313.02 microg/g for rape, lotus, and camellia bee pollen, respectively. In addition, percentages of DHA in TAA showed good intra-species consistency, with values of 13.7%, 16.5%, and 7.6% in rape, lotus, and camellia bee pollen, respectively. This is the first report on the discriminative determination between AA and DHA in bee pollen matrices. The proposed method would be valuable for the nutritional analysis of bee pollen.

The Safe Soluble Compound Dehydroascorbic Acid Inhibits Various Upstream and Downstream Effectors of PI3K and KRAS Signaling Pathways in Undruggable PIK3CA/KRAS-Mutant Colorectal Cancer Stem-Like Cells.[Pubmed:33283545]

Nutr Cancer. 2020 Dec 7:1-16.

Efforts to develop effective drugs targeting PI3K and KRAS signaling pathways in PIK3CA/KRAS-mutant colorectal cancer stem cells (CRCSCs) remain challenging. Finding safe compounds that can easily enter CRCSCs with the ability to target metastasis-driver gene CXCR4 and pluripotency network genes as key upstream and downstream effectors of both PI3K and KRAS signaling pathways may provide promising results. PIK3CA/KRAS-mutant CRCSCs display high expression of glucose transporters (GLUTs) on their cell membrane and a glycolytic phenotype providing an opportunity to deliver antiglycolytic compounds into these cells via the GLUTs. CRC patients with low levels of vitamin C in their plasma show a shorter survival suggesting the ability of this vitamin at the physiologic levels for caspase-3 activation and apoptosis in CRCSCs. Vitamin C in an oxidized form (L-Dehydroascorbic acid; L-DHA) with antiglycolytic activity can be taken up into CRC cells via the GLUTs. This may provide selective toxicity on CRCSCs and affect CXCR4 and stemness markers genes expression in these cells. To this end, we treated PIK3CA/KRAS-mutant LS174T cells with high glycolytic activity as an attractive model for CRCSCs with L-DHA equal to the pharmacological levels of vitamin C in human plasma, after which cell numbers, metabolic activity, proliferation-rate, CXCR4 and pluripotency network genes expression, caspase-3 activity with apoptosis were evaluated. 48 h post-treatment with 100- to 1000 microM L-DHA, cell numbers were decreased and measured to be 70-47% control. L-DHA with selective toxicity on LS174T cells diminished metabolic activity and cell proliferation-rate to 1.4-0.8 (Control OD = 1.5) and 92-54.5% respectively with no toxicity on PBMCs. L-DHA decreased CXCR4, Bmi-1, Sox-2 and Oct-4 expression to 45%, 85%, 45% and 48% control respectively followed by caspase-3 reactivation by 2.5 to 4.9-fold increases and induction of apoptosis ranging from 0.5% to 58.3% for 100- to 1000 microM L-DHA. According to our data, CRC stem-like cells were highly sensitive to L-DHA in in-vitro. L-DHA selectively targeted LS174T cells and successfully reactivated caspase-3 and apoptosis in these cells. CXCR4, stemness marker genes and metabolic activity appear to be promising targets of L-DHA. Our results may provide a new therapeutic approach to target selectively GLUT-overexpressing PIK3CA/KRAS-mutant CRCSCs using L-DHA with no toxicity on normal cells.

Ascorbate Oxidase Induces Systemic Resistance in Sugar Beet Against Cyst Nematode Heterodera schachtii.[Pubmed:33193547]

Front Plant Sci. 2020 Oct 22;11:591715.

Ascorbate oxidase (AO) is an enzyme involved in catalyzing the oxidation of apoplastic ascorbic acid (AA) to Dehydroascorbic acid (DHA). In this research, the potential of AO spraying to induce systemic resistance was demonstrated in the interaction between sugar beet root and cyst nematode Heterodera schachtii and the mechanism was elucidated. Plant bioassays showed that roots of AO-sprayed plants were infested by a significantly lower number of females and cysts when compared with mock-sprayed control plants. Hormone measurements showed an elevated level of jasmonic acid (JA) salicylic acid (SA) and ethylene (ET) in the roots of AO-sprayed plants, with a dynamic temporal pattern of activation. Experiments with chemical inhibitors showed that AO-induced systemic resistance is partially dependent on the JA, ET and SA pathways. Biochemical analyses revealed a primed accumulation of hydrogen peroxide (H2O2), and phenylalanine ammonia lyase (PAL) activity in the roots of AO-sprayed plants upon infection by cyst nematodes. In conclusion, our data shows that AO works as an effective systemic defense priming agent in sugar beet against cyst nematode infection, through activation of multiple basal plant defense pathways.

Redox reaction-modulated fluorescence biosensor for ascorbic acid oxidase assay by using MoS2 quantum dots as fluorescence probe.[Pubmed:33167232]

Talanta. 2021 Jan 15;222:121522.

Herein, a sensitive fluorescence (FL) biosensor for the assay of ascorbic acid oxidase (AAO) was established based on the fluorescence resonance energy transfer (FRET) between MoS2 quantum dots (MQDs) and CoOOH nanoflakes. CoOOH nanoflakes as effective FL quencher could quench the FL signal of MQDs on the basis of FRET. When ascorbic acid (AA) was added to the MQDs/CoOOH nanoflakes system, the FL signal was restored due to the redox reaction between CoOOH nanoflakes and AA, in which CoOOH nanoflakes were reduced to Co(2+) by AA. In the presence of AAO, the recovered FL signal of MQDs was quenched again because of the enzymatic catalytically reaction between AAO and AA, in which AA was oxidized to Dehydroascorbic acid (DHA) and then prevented the decomposition of CoOOH nanoflakes. Under the optimal experimental conditions, this developed fluorescence method exhibited good linear ranges from 2 to 10 mU mL(-1) and 10-40 mU mL(-1) with a low detection of limit of 0.8 mU mL(-1) for AAO detection. And the limit of quantification (LOQ) of 2.6 mU mL(-1) was obtained. The proposed biosensor showed high sensitivity and selectivity, and was successfully applied for AAO determination in human serum samples.

Positive feedback between retinoic acid and 2-phospho-L-ascorbic acid trisodium salt during somatic cell reprogramming.[Pubmed:33000315]

Cell Regen. 2020 Oct 1;9(1):17.

Retinoic acid (RA) and 2-phospho-L-ascorbic acid trisodium salt (AscPNa) promote the reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells. In the current studies, the lower abilities of RA and AscPNa to promote reprogramming in the presence of each other suggested that they may share downstream pathways at least partially. The hypothesis was further supported by the RNA-seq analysis which demonstrated a high-level overlap between RA-activated and AscPNa activated genes during reprogramming. In addition, RA upregulated Glut1/3, facilitated the membrane transportation of Dehydroascorbic acid, the oxidized form of L-ascorbic acid, and subsequently maintained intracellular L-ascorbic acid at higher level and for longer time. On the other hand, AscPNa facilitated the mesenchymal-epithelial transition during reprogramming, downregulated key mesenchymal transcriptional factors like Zeb1 and Twist1, subsequently suppressed the expression of Cyp26a1/b1 which mediates the metabolism of RA, and sustained the intracellular level of RA. Furthermore, the different abilities of RA and AscPNa to induce mesenchymal-epithelial transition, pluripotency, and neuronal differentiation explain their complex contribution to reprogramming when used individually or in combination. Therefore, the current studies identified a positive feedback between RA and AscPNa, or possibility between vitamin A and C, and further explored their contributions to reprogramming.

Silencing of ascorbate oxidase results in reduced growth, altered ascorbic acid levels and ripening pattern in melon fruit.[Pubmed:32987259]

Plant Physiol Biochem. 2020 Nov;156:291-303.

Ascorbate oxidase (AO, EC 1.10.3.3) is a copper-containing enzyme localized at the apoplast, where it catalyzes the oxidation of ascorbic acid (AA) to Dehydroascorbic acid (DHA) via monoDehydroascorbic acid (MDHA) intermediate. Despite it has been extensively studied, no biological roles have been definitively ascribed. To understand the role of AO in plant metabolism, fruit growth and physiology, we suppressed AO expression in melon (Cucumis melo L.) fruit. Reduction of AO activity increased AA content in melon fruit, which is the result of repression of AA oxidation and simultaneous induction of certain biosynthetic and recycling genes. As a consequence, ascorbate redox state was altered in the apoplast. Interestingly, transgenic melon fruit displayed increased ethylene production rate coincided with elevated levels of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO, EC 1.14.17.4) activity and gene expression, which might contribute to earlier ripening. Moreover, AO suppressed transgenic melon fruit exhibited a dramatic arrest in fruit growth, due to a simultaneous decrease in fruit cell size and in plasmalemma (PM) ATPase activity. All the above, support for the first time, the in vivo AO participation in the rapid fruit growth of Cucurbitaceae and further suggest an alternative route for AA increase in ripening fruit.

A kinetic study and mechanisms of reduction of N, N'-phenylenebis(salicyalideneiminato)cobalt(III) by L-ascorbic acid in DMSO-water medium.[Pubmed:32939409]

Heliyon. 2020 Sep 1;6(9):e04621.

The kinetics of reduction of N, N (1) -phenylenebis-(salicylideneiminato)cobalt (III), referred to as [Co(Salophen)](+) by L-ascorbic acid (H2A) was studied in mixed aqueous medium (DMSO:H2O; 1:4 v/v) under pseudo-first-order conditions at 33 +/- 1 degrees C, mu = 0.1 mol dm(-3) (NaCl) and lambda max = 470 nm. L-ascorbic acid was oxidized to Dehydroascorbic acid with kinetics that was first order in both the [H2A] and [Co(Salophen)(+)] and second-order overall. The reaction involves two parallel reaction pathways; an acid-dependent and the inverse acid-dependent pathways. The inverse acid pathway shows that there is a pre-equilibrium step before the rate determining-step in which a proton is lost. The kinetics followed negative Bronsted-Debye salt effect. Evidence was obtained for the presence of free radicals but none to support the formation of an intermediate complex of significant stability during the reaction. Overall, the data obtained suggest an outer-sphere mechanism for the reaction. A plausible mechanism is proposed.

Metabolomics Fingerprint Induced by the Intranigral Inoculation of Exogenous Human Alpha-Synuclein Oligomers in a Rat Model of Parkinson's Disease.[Pubmed:32937957]

Int J Mol Sci. 2020 Sep 14;21(18). pii: ijms21186745.

Parkinson's disease (PD) is considered a synucleinopathy because of the intraneuronal accumulation of aggregated alpha-synuclein (alphaSyn). Recent evidence points to soluble alphaSyn-oligomers (alphaSynO) as the main cytotoxic species responsible for cell death. Given the pivotal role of alphaSyn in PD, alphaSyn-based models are crucial for the investigation of toxic mechanisms and the identification of new therapeutic targets in PD. By using a metabolomics approach, we evaluated the metabolic profile of brain and serum samples of rats infused unilaterally with preformed human alphaSynOs (HalphaSynOs), or vehicle, into the substantia nigra pars compacta (SNpc). Three months postinfusion, the striatum was dissected for striatal dopamine (DA) measurements via High Pressure Liquid Chromatography (HPLC) analysis and mesencephalon and serum samples were collected for the evaluation of metabolite content via gas chromatography mass spectrometry analysis. Multivariate, univariate and correlation statistics were applied. A 40% decrease of DA content was measured in the HalphaSynO-infused striatum as compared to the contralateral and the vehicle-infused striata. Decreased levels of Dehydroascorbic acid, myo-inositol, and glycine, and increased levels of threonine, were found in the mesencephalon, while increased contents of fructose and mannose, and a decrease in glycine and urea, were found in the serum of HalphaSynO-infused rats. The significant correlation between DA and metabolite content indicated that metabolic variations reflected the nigrostriatal degeneration. Collectively, the metabolomic fingerprint of HalphaSynO-infused rats points to an increase of oxidative stress markers, in line with PD neuropathology, and provides hints for potential biomarkers of PD.

Total Vitamin C, Ascorbic Acid, Dehydroascorbic Acid, Antioxidant Properties, and Iron Content of Underutilized and Commonly Consumed Fruits in Sri Lanka.[Pubmed:32908860]

Int J Food Sci. 2020 Aug 20;2020:4783029.

Sri Lanka is rich in a wide diversity of fruits, but many are underutilized by the people in Sri Lanka despite their nutritional value. This is mainly due to little awareness of the palatability of many fruits and hence low popularity in the market. The present study aimed at providing comparative data on the main biochemical and nutritional parameters of thirty-seven (37) species of fruits grown in Sri Lanka, including 22 underutilized fruits and 15 commonly consumed fruits. The main parameters of the comparison were the contents of ascorbic acid (AA), total vitamin C (TVC), total phenolic content (TPC), total flavonoid content (TFC), total iron (Fe), and antioxidant capacities (ACs). The mean AA, TVC, TPC, TFC, and Fe contents in 100 g of fresh edible portions of fruits ranged from 2.0 to 185.0 mg, 8.1 to 529.6 mg, 12.9 to 2701.7 mg gallic acid equivalent, 0.2 to 117.5 mg quercetin equivalents, and 0.1 to 1.1 mg, respectively. The IC50 values in a DPPH assay varied between 0.8 to 1856.7 mg/mL and FRAP values in a FRAP assay ranged from 4.2 to 2070 mumol FeSO4/g in the studied fruits. Fruits were ranked based on the levels of the abovementioned biochemical properties. Using this ranking, 12 of the top 15 fruits were underutilized. Phyllanthus emblica (Indian gooseberry) is at the top of these underutilized fruits, and Psidium guajava (guava) is the best among commonly consumed fruits. These results indicate that underutilized fruits in Sri Lanka can be recommended as high quality and low-cost alternatives for securing nutritional requirements. Hence, underutilized fruits can be promoted as healthy additional fruits in Sri Lanka.

Dehydroascorbic Acid Affects the Stability of Catechins by Forming Conjunctions.[Pubmed:32906587]

Molecules. 2020 Sep 7;25(18). pii: molecules25184076.

Although tea catechins in green tea and green tea beverages must be stable to deliver good sensory quality and healthy benefits, they are always unstable during processing and storage. Ascorbic acid (AA) is often used to protect catechins in green tea beverages, and AA is easily oxidized to form Dehydroascorbic acid (DHAA). However, the function of DHAA on the stability of catechins is not clear. The objective of this study was to determine the effects of DHAA on the stability of catechins and clarify the mechanism of effects by conducting a series of experiments that incubate DHAA with epigallocatechin gallate (EGCG) or catechins. Results showed that DHAA had a dual function on EGCG stability, protecting its stability by inhibiting hydrolysis and promoting EGCG consumption by forming ascorbyl adducts. DHAA also reacted with (-)-epicatechin (EC), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin (EGC) to form ascorbyl adducts, which destabilized them. After 9 h of reaction with DHAA, the depletion rates of EGCG, ECG, EC, and EGC were 30.08%, 22.78%, 21.45%, and 13.55%, respectively. The ability of DHAA to promote catechins depletion went from high to low: EGCG, ECG, EGC, and EC. The results are important for the processing and storage of tea and tea beverages, as well as the general exploration of synergistic functions of AA and catechins.

Glutathione and Glutathione-Like Sequences of Opioid and Aminergic Receptors Bind Ascorbic Acid, Adrenergic and Opioid Drugs Mediating Antioxidant Function: Relevance for Anesthesia and Abuse.[Pubmed:32872204]

Int J Mol Sci. 2020 Aug 28;21(17). pii: ijms21176230.

Opioids and their antagonists alter vitamin C metabolism. Morphine binds to glutathione (l-gamma-glutamyl-l-cysteinyl-glycine), an intracellular ascorbic acid recycling molecule with a wide range of additional activities. The morphine metabolite morphinone reacts with glutathione to form a covalent adduct that is then excreted in urine. Morphine also binds to adrenergic and histaminergic receptors in their extracellular loop regions, enhancing aminergic agonist activity. The first and second extracellular loops of adrenergic and histaminergic receptors are, like glutathione, characterized by the presence of cysteines and/or methionines, and recycle ascorbic acid with similar efficiency. Conversely, adrenergic drugs bind to extracellular loops of opioid receptors, enhancing their activity. These observations suggest functional interactions among opioids and amines, their receptors, and glutathione. We therefore explored the relative binding affinities of ascorbic acid, Dehydroascorbic acid, opioid and adrenergic compounds, as well as various control compounds, to glutathione and glutathione-like peptides derived from the extracellular loop regions of the human beta 2-adrenergic, dopamine D1, histamine H1, and mu opioid receptors, as well as controls. Some cysteine-containing peptides derived from these receptors do bind ascorbic acid and/or Dehydroascorbic acid and the same peptides generally bind opioid compounds. Glutathione binds not only morphine but also naloxone, methadone, and methionine enkephalin. Some adrenergic drugs also bind to glutathione and glutathione-like receptor regions. These sets of interactions provide a novel basis for understanding some ways that adrenergic, opioid and antioxidant systems interact during anesthesia and drug abuse and may have utility for understanding drug interactions.

Facile Preparation of Homogeneous Copper Nanoclusters Exhibiting Excellent Tetraenzyme Mimetic Activities for Colorimetric Glutathione Sensing and Fluorimetric Ascorbic Acid Sensing.[Pubmed:32844641]

ACS Appl Mater Interfaces. 2020 Sep 23;12(38):42521-42530.

Nanozymes are artificial enzymes, which can substitute traditional biological enzymes for multifield applications. However, to date, it remains challenging to search novel mimic enzymes or multienzyme mimics. Herein, a facile and green method for preparing monodisperse, homogeneous copper nanoclusters (Cu NCs) with smaller size was developed, which used cysteamine as a template and hydrazine hydrate as a reductant to reduce Cu(2+). The as-prepared Cu NCs exhibited excellent tetraenzyme-like activities, including peroxidase (POD)-, catalase (CAT)-, superoxide dismutase (SOD)-, and ascorbic acid oxidase (AAO)-mimic activities. The mechanisms, kinetics, and catalytic performances of Cu NCs were systematically studied. Moreover, based on the POD-like activity of Cu NCs, sensitive and simple colorimetric sensing glutathione (GSH) was explored, with the low limit of detection of 0.89 muM GSH (S/N = 3). Additionally, a novel fluorimetric ascorbic acid (AA) sensor was developed with the linear range of 0.5-30 muM and limit of detection (LOD) of 0.144 muM, on the basis of the principle that AA is oxidized to Dehydroascorbic acid (DHAA) specifically catalyzed by the AAO-like activity of Cu NCs, while DHAA can further react with o-phenylenediamine (OPDA) to generate a highly fluorescent quinoxaline (DFQ) derivative. The as-proposed colorimetric GSH sensor and the fluorimetric AA sensor were capable of detecting GSH and AA, respectively, in real samples accurately and reproducibly. Thus, the Cu NCs-based multienzyme mimic is a promising candidate for biocatalysis and biosensing.

Dual effects of ascorbic acid on the stability of EGCG by the oxidation product dehydroascorbic acid promoting the oxidation and inhibiting the hydrolysis pathway.[Pubmed:32799157]

Food Chem. 2021 Feb 1;337:127639.

A series of incubation systems of pure (-)-Epigallocatechin gallate (EGCG), ascorbic acid (AA) and Dehydroascorbic acid (DHAA) at 80 degrees C were performed to investigated the effect and mechanism of AA on the stability of EGCG. Results shows the dual function of AA, protect action at low concentration and promoting degradation at high concentration, and the critical concentration is about 10mmol/L. The protective properties of AA due to the reversible reaction from AA to DHAA inhibiting oxidation pathway of EGCG to EGCG quinone or other activated intermediates, and both AA and DHAA can inhibit the hydrolysis of EGCG. The properties of promoting degradation is mainly due to the fact that DHAA, the oxidation product of AA, can react with EGCG to generate some ascorbyl adducts of EGCG. This result is helpful to control the stability of catechins and further clarify the complex interaction on healthy between EGCG and AA.

Improved UPLC-UV Method for the Quantification of Vitamin C in Lettuce Varieties (Lactuca sativa L.) and Crop Wild Relatives (Lactuca spp.).[Pubmed:32716366]

J Vis Exp. 2020 Jun 30;(160).

Vitamins, especially vitamin C, are important micronutrients found in fruits and vegetables. Vitamin C is also a major contributor to their antioxidant capacity. Lettuce is one of the most popular vegetables among consumers worldwide. An accurate protocol to measure vitamin C content in lettuce and other related species is crucial. We describe here a method using the ultra-high-performance liquid chromatography-ultraviolet (UPLC-UV) technique, in which sample preparation, vitamin extraction and chromatography conditions were optimized. Samples were collected to represent the entire plant, frozen at -80 degrees C and lyophilized to prevent undesirable oxidation and make their manipulation easier. The extraction of vitamin C was carried out in acidic media, which also contributed to its stability. As vitamin C can be present in two different interconvertible forms, ascorbic acid (AA) and Dehydroascorbic acid (DHAA), both compounds should be measured for accurate quantification. The DHAA was quantified indirectly after its reduction to AA because AA shows a higher absorptivity than DHAA in the UV range of the spectrum. From the same extract, two measurements were carried out, one before and one after that reduction reaction. In the first case, we were quantifying the AA content, and in the second one, we quantified the sum of AA and DHAA (TAA: total ascorbic acid) in the form of AA. Then, DHAA quantity was indirectly obtained by subtracting AA coming from the first measurement from TAA. They were determined by UPLC-UV, using a commercial AA standard to build a calibration curve and optimizing the chromatographic procedure, to obtain AA peaks that were completely resolved in a short time. This protocol could be easily extrapolated to any other plant material with slight or no changes. Its accuracy revealed statistically significant differences otherwise unperceived. Other strengths and limitations are discussed more in depth in the manuscript.