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

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.

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Vitamin C Recycling Regulates Neurite Growth in Neurospheres Differentiated in Vitro

10.21203/rs.3.rs-51679/v1 ◽

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.

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

Biochemical Journal ◽

10.1042/0264-6021:3420049 ◽

1999 ◽

Vol 342 (1) ◽

pp. 49 ◽

Cited By ~ 17

Author(s):

Joanne M. UPSTON ◽

Ari KARJALAINEN ◽

Fyfe L. BYGRAVE ◽

Roland STOCKER

Keyword(s):

Vitamin C ◽

Plasma Vitamin ◽

Potential Contributor

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

Blood ◽

10.1182/blood.v84.5.1628.bloodjournal8451628 ◽

1994 ◽

Vol 84 (5) ◽

pp. 1628-1634 ◽

Cited By ~ 3

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.

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Cellular and intracellular transport of vitamin C. The physiologic aspects

Orvosi Hetilap ◽

10.1556/oh.2013.29712 ◽

2013 ◽

Vol 154 (42) ◽

pp. 1651-1656 ◽

Cited By ~ 6

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.

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

Blood ◽

10.1182/blood.v84.5.1628.1628 ◽

1994 ◽

Vol 84 (5) ◽

pp. 1628-1634 ◽

Cited By ~ 84

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.

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Chromatographic and Extraction Techniques for Vitamin C Assay

Journal of AOAC INTERNATIONAL ◽

10.1093/jaoac/48.5.985 ◽

1965 ◽

Vol 48 (5) ◽

pp. 985-991

Author(s):

Elmer De Ritter

Keyword(s):

Ascorbic Acid ◽

Vitamin C ◽

Thin Layer ◽

Dehydroascorbic Acid ◽

Reduced Form ◽

Extraction Techniques ◽

Total Vitamin ◽

High Efficient ◽

Efficient Extraction ◽

Layer Chromatography

Abstract A review is presented of chromatographic procedures used in the assay of vitamin C. Paper, thin layer, and column chromatography have been used to advantage for separating interfering substances. Even the closely related erythorbic acid, which has no vitamin C activity, can be separated from ascorbic acid by various techniques on paper. Total vitamin C can be determined chromatographically after reduction of dehydroascorbic acid with H2S or after oxidation of the reduced form to dehydroascorbic acid. A combination of column and thin layer chromatography on silica gel of the dinitrosazone formed by reaction of dehydroascorbic acid with 2,4-dinitrophenylhydraiine is recommended as an effective method for achieving specificity in vitamin C assays of foods and feeds where the level of interference is high. Efficient extraction procedures are described for determining added ascorbic acid in feeds and mineral premixes.

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Role of monosaccharide transporter in vitamin C uptake by placental membrane vesicles

AJP Cell Physiology ◽

10.1152/ajpcell.1986.250.4.c637 ◽

1986 ◽

Vol 250 (4) ◽

pp. C637-C641 ◽

Cited By ~ 25

Author(s):

R. L. Ingermann ◽

L. Stankova ◽

R. H. Bigley

Keyword(s):

Vitamin C ◽

Concentration Gradient ◽

Cytochalasin B ◽

Dehydroascorbic Acid ◽

Membrane Vesicles ◽

Sodium Concentration ◽

Reduced Form ◽

High Concentration ◽

Simple Diffusion ◽

Monosaccharide Transporter

Dehydroascorbic acid (DHA), the reversibly oxidized form of vitamin C, was taken up much more rapidly than L-glucose into membrane vesicles prepared from the maternal face of the human placental syncytiotrophoblast. DHA uptake was sensitive to inhibition by cytochalasin B and was independent of a sodium concentration gradient. At equilibrium, the concentration of DHA in the vesicles did not exceed that of the medium. DHA and the D-glucose analogue, 3-O-methyl-D-glucose (3-O-MG) appeared to compete with one another for the transporter. The 3-O-MG and DHA inhibitory constants were indistinguishable. Vesicles loaded with a high concentration of 3-O-MG and suspended in low 3-O-MG displayed a marked, transitory enhancement of DHA, but not L-glucose uptake. These findings suggest that DHA is taken into the first cellular boundary of the placenta between maternal and fetal circulations by the sodium-independent monosaccharide transporter. In contrast to DHA, L-ascorbic acid, the reversibly reduced form of vitamin C, was taken into these vesicles much more slowly. This uptake was not affected by cytochalasin B nor by a sodium concentration gradient; it appeared to occur by simple diffusion.

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Vitamin C Recycling Regulates Neurite Growth in Neurospheres Differentiated In Vitro

Antioxidants ◽

10.3390/antiox9121276 ◽

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.

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