Vitamin C Recycling Regulates Neurite Growth in Neurospheres Differentiated in Vitro

2020 ◽  
Author(s):  
Francisca Espinoza ◽  
Rocio Magdalena ◽  
Natalia Saldivia ◽  
Nery Jara ◽  
Fernando Martínez ◽  
...  
Keyword(s):  
Vitamin C ◽  
Bystander Effect ◽  
Dehydroascorbic Acid ◽  
Neurite Growth ◽  
Protein Carbonylation ◽  
Reduced Form ◽  
Ros Generation ◽  
Lysine Production ◽  
Neuritic Growth

Abstract Background: The reduced form of vitamin C, ascorbic acid (AA), has been related to antioxidant defense as well as gene expression and cell differentiation in the cerebral cortex. In neurons, AA is mainly oxidized to dehydroascorbic acid (DHA); however, DHA cannot accumulate intracellularly because it induces metabolic changes and cell death. In this context, it has been proposed that vitamin C recycling via neuron-astrocyte coupling maintains AA levels and prevents DHA parenchymal accumulation. To date, the role of this mechanism during the outgrowth of neurites is unknown.Methods: To stimulate neuronal differentiation, adhered neurospheres treated with AA and retinoic acid (RA) were used. Neuritic growth was analyzed by confocal microscopy, and the effect of vitamin C recycling (bystander effect) in vitro was studied using different cells (astrocytes, HL60 and U87). Reactive oxygen species (ROS) generation was also analyzed by flow cytometry and protein carbonylation / carboximetil-lysine production.Results: AA stimulates neuritic growth more efficiently than RA. However, AA is oxidized to DHA in long incubation periods, generating a loss in the formation of neurites. Surprisingly, neurite growth is maintained over time following co-incubation of neurospheres with cells (HL60, U87, or astrocytes) that efficiently capture DHA (Bystander effect). In this sense, astrocytes have high capacity to recycle DHA and stimulate the maintenance of neurites. Finally, our data indicate that DHA induces ROS generation, a condition that results in protein carbonylation and carboximetil-lysine production. Conclusions: We have demonstrated that vitamin C recycling in vitro regulates the morphology of immature neurons during the differentiation and maturation processes.

scholarly journals Vitamin C Recycling Regulates Neurite Growth in Neurospheres Differentiated In Vitro

Antioxidants ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1276
Author(s):  
Francisca Espinoza ◽  
Rocío Magdalena ◽  
Natalia Saldivia ◽  
Nery Jara ◽  
Fernando Martínez ◽  
...  
Keyword(s):  
Vitamin C ◽  
Bystander Effect ◽  
High Capacity ◽  
Dehydroascorbic Acid ◽  
Neurite Growth ◽  
Reduced Form ◽  
Incubation Periods ◽  
Neuritic Growth

The reduced form of vitamin C, ascorbic acid (AA), has been related with gene expression and cell differentiation in the cerebral cortex. In neurons, AA is mainly oxidized to dehydroascorbic acid (DHA); however, DHA cannot accumulate intracellularly because it induces metabolic changes and cell death. In this context, it has been proposed that vitamin C recycling via neuron–astrocyte coupling maintains AA levels and prevents DHA parenchymal accumulation. To date, the role of this mechanism during the outgrowth of neurites is unknown. To stimulate neuronal differentiation, adhered neurospheres treated with AA and retinoic acid (RA) were used. Neuritic growth was analyzed by confocal microscopy, and the effect of vitamin C recycling (bystander effect) in vitro was studied using different cells. AA stimulates neuritic growth more efficiently than RA. However, AA is oxidized to DHA in long incubation periods, generating a loss in the formation of neurites. Surprisingly, neurite growth is maintained over time following co-incubation of neurospheres with cells that efficiently capture DHA. In this sense, astrocytes have high capacity to recycle DHA and stimulate the maintenance of neurites. We demonstrated that vitamin C recycling in vitro regulates the morphology of immature neurons during the differentiation and maturation processes.

scholarly journals Bioaccessibility of Antioxidants in Blackcurrant Juice After Treatment Using Supercritical Carbon Dioxide

2021 ◽  
Author(s):  
Urszula Trych ◽  
Magdalena Buniowska ◽  
Sylwia Skąpska ◽  
Ireneusz Kapusta ◽  
Krystian Marszałek
Keyword(s):  
Carbon Dioxide ◽  
Supercritical Carbon Dioxide ◽  
Vitamin C ◽  
Model Experiment ◽  
Dehydroascorbic Acid ◽  
Total Anthocyanins ◽  
The Stability ◽  
Supercritical Carbon ◽  
Thermally Treated

Blackcurrant juice (Ribes nigrum L.) was subjected to supercritical carbon dioxide (SCCD) at 10, 30 and 60 MPa for 10 min at 45°C as well as thermally treated at 45°C and 85°C for 10 min to determine the stability, antioxidant capacity (AC) and bioaccessibility (BAc) of vitamin C, total anthocyanins and their individual monomers. An in vitro gastrointestinal digestion model completed with dialysis was used to assess BAc. The use of SCCD at each of the pressures applied improved the stability of vitamin C, total anthocyanins, and AC before in vitro digestion. As a result of digestion, L-ascorbic acid was oxidized to L-dehydroascorbic acid, and finally, the total content of vitamin C, anthocyanins, and AC decreased. SCCD did not significantly improve the BAc of vitamin C and total anthocyanins. The highest BAc of vitamin C was noted in fresh juice (FJ) (40%) and after mild heat treatment at 45°C (T45) (46%). The highest BAc of total anthocyanins was also noted in the FJ (4.4%). The positive effect of the application of SCCD on the BAc of the delphinidin-3-O-glycosides was observed compared to T45 and thermal pasteurization at 85°C (T85). Moreover, cyanidins were generally more bioaccessible than delphinidins in all samples. AC after digestion was higher in SCCD samples compared to thermally treated measured using ABTS+• and DPPH• assays, whereas in dialysate similar trends were observed only for AC measured using the ABTS+• assay. This phenomenon was justified by the formation of individual metabolites detected by UPLC-PDA-MS / MS in the model experiment with delphinidin-3-O-rutinoside. The protocatechuic acid which is well known as a strong antioxidant was detected in the model experiment after digestion. Further research is needed to better understand the metabolic pathway of anthocyanins and the possible uses of SCCD to improve the health properties of fruit products.

scholarly journals Human HL-60 myeloid leukemia cells transport dehydroascorbic acid via the glucose transporters and accumulate reduced ascorbic acid

Blood ◽  
1994 ◽  
Vol 84 (5) ◽  
pp. 1628-1634 ◽  
Author(s):  
JC Vera ◽  
CI Rivas ◽  
RH Zhang ◽  
CM Farber ◽  
DW Golde
Keyword(s):  
Ascorbic Acid ◽  
Vitamin C ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Dehydroascorbic Acid ◽  
Glucose Transporters ◽  
Leukemic Cells ◽  
Leukemia Cell Line ◽  
Reduced Form ◽  
A Cell

The cellular accumulation of vitamin C, a substance critical to human physiology, is mediated by transporters located at the cell membrane, and is regulated in a cell-specific manner. Neoplastic cells may have special needs for vitamin C. Therefore, we investigated the transport of vitamin C in a human myeloid leukemia cell line (HL-60). The HL-60 cells lacked the capacity to transport the reduced form of vitamin C, ascorbic acid, but they showed a remarkable ability to transport the oxidized form of vitamin C, dehydroascorbic acid (DHA). Uptake- accumulation studies indicated that the HL-60 cells accumulated ascorbic acid when provided with DHA. Kinetic analysis showed the presence of two functional activities involved in the uptake of DHA, one with low affinity and one with high affinity. Cytochalasin B and phloretin, which inhibit the passage of glucose through the facilitative glucose transporters, also inhibited the transport of DHA by HL-60 cells. Transport of DHA was completed by D- but not L-hexoses, and was sensitive to D-hexose-dependent counter transport acceleration. These data support the concept that HL-60 myeloid leukemic cells transport DHA through the facilitative hexose transporters (glucose transporters) and accumulate the reduced form of ascorbic acid.

scholarly journals Efflux of hepatic ascorbate: a potential contributor to the maintenance of plasma vitamin C

1999 ◽  
Vol 342 (1) ◽  
pp. 49-56 ◽  
Author(s):  
Joanne M. UPSTON ◽  
Ari KARJALAINEN ◽  
Fyfe L. BYGRAVE ◽  
Roland STOCKER
Keyword(s):  
Vitamin C ◽  
Dehydroascorbic Acid ◽  
Liver Cells ◽  
Reduced Form ◽  
Radical Scavenger ◽  
Plasma Vitamin ◽  
Temperature Sensitive ◽  
Isolated Liver Cells ◽  
Potential Contributor ◽  
Whole Blood Cells

Ascorbate (AH, the reduced form of vitamin C) is an important radical scavenger and antioxidant in human plasma; the resulting ascorbyl radical can disproportionate to AH and dehydroascorbic acid (DHA). Here we address potential maintenance mechanism(s) for extracellular AH by examining the ability of cells to convert extracellularly presented DHA to AH. DHA was rapidly transported into human liver (HepG2), endothelial and whole blood cells invitro by plasma membrane glucose transporters and reduced intracellularly. Liver cells displayed the highest capacity to release the intracellularly accumulated AH. The proteins responsible for DHA uptake and AH release could be distinguished by inhibitor studies. Thus, unlike DHA uptake, AH efflux was largely insensitive to cytochalasin B and thiol-reactive agents but was inhibited by phloretin, 4,4′-di-isothiocyanostilbene-2,2′-disulphonate and isoascorbate. Efflux of AH from cells was temperature-sensitive and saturable with a low affinity (millimolar, intracellular) for AH. In addition to isolated liver cells, perfusion of intact rat and guinea-pig liver with DHA resulted in AH in the circulating perfusate. Our results show that hepatocytes take up and reduce DHA and subsequently release part of the AH formed, probably via a membrane transporter. By converting extracellular DHA to extracellular AH, the liver might contribute to the maintenance of plasma AH, a process that could be important under conditions of oxidative stress.

scholarly journals Vitamin C Is a Kinase Inhibitor: Dehydroascorbic Acid Inhibits IκBα Kinase β

2004 ◽  
Vol 24 (15) ◽  
pp. 6645-6652 ◽  
Author(s):  
Juan M. Cárcamo ◽  
Alicia Pedraza ◽  
Oriana Bórquez-Ojeda ◽  
Bing Zhang ◽  
Roberto Sanchez ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Vitamin C ◽  
Enzymatic Activity ◽  
Redox State ◽  
Kinase Inhibitor ◽  
Dehydroascorbic Acid ◽  
Molecular Action ◽  
Cellular Signal ◽  
Direct Linkage

ABSTRACT Reactive oxygen species (ROS) are key intermediates in cellular signal transduction pathways whose function may be counterbalanced by antioxidants. Acting as an antioxidant, ascorbic acid (AA) donates two electrons and becomes oxidized to dehydroascorbic acid (DHA). We discovered that DHA directly inhibits IκBα kinase β (IKKβ) and IKKα enzymatic activity in vitro, whereas AA did not have this effect. When cells were loaded with AA and induced to generate DHA by oxidative stress in cells expressing a constitutive active IKKβ, NF-κB activation was inhibited. Our results identify a dual molecular action of vitamin C in signal transduction and provide a direct linkage between the redox state of vitamin C and NF-κB signaling events. AA quenches ROS intermediates involved in the activation of NF-κB and is oxidized to DHA, which directly inhibits IKKβ and IKKα enzymatic activity. These findings define a function for vitamin C in signal transduction other than as an antioxidant and mechanistically illuminate how vitamin C down-modulates NF-κB signaling.

scholarly journals Alternative pathways of dehydroascorbic acid degradation in vitro and in plant cell cultures: novel insights into vitamin C catabolism

2011 ◽  
Vol 440 (3) ◽  
pp. 375-385 ◽  
Author(s):  
Harriet T. Parsons ◽  
Tayyaba Yasmin ◽  
Stephen C. Fry
Keyword(s):  
Reactive Oxygen Species ◽  
Vitamin C ◽  
Dehydroascorbic Acid ◽  
Oxygen Species ◽  
Reactive Intermediate ◽  
Species Status ◽  
Alternative Pathways ◽  
Irreversible Oxidation

L-Ascorbate catabolism involves reversible oxidation to DHA (dehydroascorbic acid), then irreversible oxidation or hydrolysis. The precursor–product relationships and the identity of several major DHA breakdown products remained unclear. In the presence of added H2O2, DHA underwent little hydrolysis to DKG (2,3-dioxo-L-gulonate). Instead, it yielded OxT (oxalyl L-threonate), cOxT (cyclic oxalyl L-threonate) and free oxalate (~6:1:1), essentially simultaneously, suggesting that all three product classes independently arose from one reactive intermediate, proposed to be cyclic-2,3-O-oxalyl-L-threonolactone. Only with plant apoplastic esterases present were the esters significant precursors of free oxalate. Without added H2O2, DHA was slowly hydrolysed to DKG. Downstream of DKG was a singly ionized dicarboxy compound (suggested to be 2-carboxy-L-xylonolactone plus 2-carboxy-L-lyxonolactone), which reversibly de-lactonized to a dianionic carboxypentonate. Formation of these lactones and acid was minimized by the presence of residual unreacted ascorbate. In vivo, the putative 2-carboxy-L-pentonolactones were relatively stable. We propose that DHA is a branch-point in ascorbate catabolism, being either oxidized to oxalate and its esters or hydrolysed to DKG and downstream carboxypentonates. The oxidation/hydrolysis ratio is governed by reactive oxygen species status. In vivo, oxalyl esters are enzymatically hydrolysed, but the carboxypentonates are stable. The biological roles of these ascorbate metabolites invite future exploration.

Cellular and intracellular transport of vitamin C. The physiologic aspects

2013 ◽  
Vol 154 (42) ◽  
pp. 1651-1656 ◽  
Author(s):  
András Szarka ◽  
Tamás Lőrincz
Keyword(s):  
Ascorbic Acid ◽  
Vitamin C ◽  
Intracellular Transport ◽  
Initial Rate ◽  
Plasma Concentrations ◽  
Dehydroascorbic Acid ◽  
Reduced Form ◽  
Human Diet ◽  
Sodium Dependent ◽  
The One

Vitamin C requirement is satisfied by natural sources and vitamin C supplements in the ordinary human diet. The two major forms of vitamin C in the diet are L-ascorbic acid and L-dehydroascorbic acid. Both ascorbate and dehydroascorbate are absorbed along the entire length of the human intestine. The reduced form, L-ascorbic acid is imported by an active mechanism, requiring two sodium-dependent vitamin C transporters (SVCT1 and SVCT2). The transport of the oxidized form, dehydroascorbate is mediated by glucose transporters GLUT1, GLUT3 and possibly GLUT4. Initial rate of uptake of both ascorbate and dehydroascorbate is saturable with increasing external substrate concentration. Vitamin C plasma concentrations are tightly controlled when the vitamin is taken orally. It has two simple reasons, on the one hand, the capacity of the transporters is limited, on the other hand the two Na+-dependent transporters can be down-regulated by an elevated level of ascorbate. Orv. Hetil., 154 (42), 1651–1656.

scholarly journals Human HL-60 myeloid leukemia cells transport dehydroascorbic acid via the glucose transporters and accumulate reduced ascorbic acid

Blood ◽  
1994 ◽  
Vol 84 (5) ◽  
pp. 1628-1634 ◽  
Author(s):  
JC Vera ◽  
CI Rivas ◽  
RH Zhang ◽  
CM Farber ◽  
DW Golde
Keyword(s):  
Ascorbic Acid ◽  
Vitamin C ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Dehydroascorbic Acid ◽  
Glucose Transporters ◽  
Leukemic Cells ◽  
Leukemia Cell Line ◽  
Reduced Form ◽  
A Cell

Abstract The cellular accumulation of vitamin C, a substance critical to human physiology, is mediated by transporters located at the cell membrane, and is regulated in a cell-specific manner. Neoplastic cells may have special needs for vitamin C. Therefore, we investigated the transport of vitamin C in a human myeloid leukemia cell line (HL-60). The HL-60 cells lacked the capacity to transport the reduced form of vitamin C, ascorbic acid, but they showed a remarkable ability to transport the oxidized form of vitamin C, dehydroascorbic acid (DHA). Uptake- accumulation studies indicated that the HL-60 cells accumulated ascorbic acid when provided with DHA. Kinetic analysis showed the presence of two functional activities involved in the uptake of DHA, one with low affinity and one with high affinity. Cytochalasin B and phloretin, which inhibit the passage of glucose through the facilitative glucose transporters, also inhibited the transport of DHA by HL-60 cells. Transport of DHA was completed by D- but not L-hexoses, and was sensitive to D-hexose-dependent counter transport acceleration. These data support the concept that HL-60 myeloid leukemic cells transport DHA through the facilitative hexose transporters (glucose transporters) and accumulate the reduced form of ascorbic acid.

scholarly journals Vitamin C protects HL60 and U266 cells from arsenic toxicity

Blood ◽  
2005 ◽  
Vol 105 (10) ◽  
pp. 4004-4012 ◽  
Author(s):  
Nicos Karasavvas ◽  
Juan M. Cárcamo ◽  
George Stratis ◽  
David W. Golde
Keyword(s):  
Vitamin C ◽  
Culture Media ◽  
Dehydroascorbic Acid ◽  
Transition Metal Ions ◽  
Glutathione Depletion ◽  
Apparent Paradox ◽  
Free Transition ◽  
Rpmi 8226 ◽  
Dose Dependent

AbstractAlthough there is no compelling evidence that vitamin C has antitumor activity in humans, clinical trials are testing the hypothesis that ascorbic acid (AA) will enhance the efficacy of arsenic trioxide (As2O3) in myeloma. In vitro, AA cytotoxicity depends on its interaction with free transition metal ions in culture media leading to the generation of H2O2 and other reactive oxygen species (ROSs). Therefore, to circumvent the extracellular in vitro pro-oxidant effects of AA, we loaded HL60, U266, and RPMI-8226 cells with vitamin C by incubation with dehydroascorbic acid (DHA). Loading cells in this manner resulted in prominent, dose-dependent protection of As2O3-treated cells as measured by viability, colony formation, and apoptosis assays. Glutathione depletion enhanced cell sensitivity to the cytotoxic effects of As2O3 and vitamin C loading provided protection. AA was found to generate cytotoxic concentrations of H2O2 in culture medium without cells and copper/iron chelators inhibited this reaction. However, AA did not generate H2O2 in simple buffer or human plasma. Direct incubation with AA resulted in increased intracellular ROSs, whereas DHA incubation decreased it. These results clarify an apparent paradox and indicate that vitamin C loading in HL60, U266, and RPMI-8226 cells ameliorates As2O3 cytotoxicity.

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