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PubMed2000

Dehydroascorbic acid irreversibly inhibits hexokinase activity.

The oxidized form of vitamin C (dehydroascorbic acid, DHA) completely and irreversibly inactivates recombinant human hexokinase type I, in a pseudo-first order fashion. The inactivation reaction occurs without saturation, indicating that DHA does not form a reversible complex with hexokinase. Further characterization of this response revealed that the inactivation does not require oxygen and that dithiothreitol, while able to prevent the DHA-mediated loss of enzyme activity, failed to restore the activity of the DHA-inhibited enzyme. Inactivation was not associated with cleavage of the peptide chain or cross-linking. The decay in enzymatic activity was however both dependent on deprotonation of a residue with an alkaline pKa and associated with covalent binding of DHA to the protein. In addition, inactivation of hexokinase decreased or increased, respectively, in the presence of the substrates glucose or MgATP. Finally, amino acid analysis of the DHA-modified hexokinase revealed a decrease of cysteine residues. Taken together, the above results are consistent with the possibility that covalent binding of the reagent with a thiol group of cysteine is a critical event for the DHA-mediated loss of hexokinase activity.

Amino Acids↗
PubMed1993

Mammalian facilitative hexose transporters mediate the transport of dehydroascorbic acid.

Although vitamin C is critical to human physiology, it is not clear how it is taken up into cells. The kinetics of cell and tissue accumulation of ascorbic acid in vitro indicate that the process is mediated by specific transporters at the cell membrane. Some experimental observations have linked the transport of ascorbic acid with hexose transport systems in mammalian cells, although no clear information is available regarding the specific role(s) of these transporters, if any, in this process. Here we use the Xenopus laevis oocyte expression system to show that the mammalian facilitative hexose transporters are efficient transporters of the oxidized form of vitamin C (dehydroascorbic acid). Two transport pathways, one with low affinity and one with high affinity for dehydroascorbic acid, were found in oocytes expressing the mammalian transporters, and these oocytes accumulated vitamin C against a concentration gradient when supplied with dehydroascorbic acid. We obtained similar results in experiments using normal human neutrophils. These observations indicate that mammalian facilitative hexose transporters are a physiologically significant pathway for the uptake and accumulation of vitamin C by cells, and suggest a mechanism for the accumulation of ascorbic acid against a concentration gradient.

Animals↗
PubMed1996

Genistein is a natural inhibitor of hexose and dehydroascorbic acid transport through the glucose transporter, GLUT1.

Genistein is a dietary-derived plant product that inhibits the activity of protein-tyrosine kinases. We show here that it is a potent inhibitor of the mammalian facilitative hexose transporter GLUT1. In human HL-60 cells, which express GLUT1, genistein inhibited the transport of dehydroascorbic acid, deoxyglucose, and methylglucose in a dose-dependent manner. Transport was not affected by daidzein, an inactive genistein analog that does not inhibit protein-tyrosine kinase activity, or by the general protein kinase inhibitor staurosporine. Genistein inhibited the uptake of deoxyglucose and dehydroascorbic acid in Chinese hamster ovary (CHO) cells overexpressing GLUT1 in a similar dose-dependent manner. Genistein also inhibited the uptake of deoxyglucose in human erythrocytes indicating that its effect on glucose transporter function is cell-independent. The inhibitory action of genistein on transport was instantaneous, with no additional effect observed in cells preincubated with it for various periods of time. Genistein did not alter the uptake of leucine by HL-60 cells, indicating that its inhibitory effect was specific for the glucose transporters. The inhibitory effect of genistein was of the competitive type, with a Ki of approximately 12 microM for inhibition of the transport of both methylglucose and deoxyglucose. Binding studies showed that genistein inhibited glucose-displaceable binding of cytochalasin B to GLUT1 in erythrocyte ghosts in a competitive manner, with a Ki of 7 microM. These data indicate that genistein inhibits the transport of dehydroascorbic acid and hexoses by directly interacting with the hexose transporter GLUT1 and interfering with its transport activity, rather than as a consequence of its known ability to inhibit protein-tyrosine kinases. These observations indicate that some of the many effects of genistein on cellular physiology may be related to its ability to disrupt the normal cellular flux of substrates through GLUT1, a hexose transporter universally expressed in cells, and is responsible for the basal uptake of glucose.

Amino Acids↗
PubMed1986

Uptake of dehydroascorbic acid and ascorbic acid to isolated nerve terminals and secretory granules from ox neurohypophyses.

When uptake of L-[14C]ascorbic acid ([14C]AA) to various organs in guinea-pigs was studied after intracardiac injection, the adenohypophysis, pars intermedia, and the neurohypophysis had an uptake per milligramme protein which was about half of the uptake to the adrenals. Adrenal uptake was 20 +/- 2.8 pmol mg-1 protein microCi-1 injected. The uptake to the different parts of the hypophysis was considerably higher than the uptake to pancreas, liver, kidney, spleen and other organs. When isolated nerve endings (neurosecretosomes) from ox neurohypophyses were incubated with a medium containing labelled dehydroascorbic acid ([14C]DHA), the uptake was much slower than when the medium contained labelled ascorbic acid. The uptake of [14C]DHA showed a linear dependence on concentration, and was not influenced by addition of Mg2+ and ATP. Addition of Mg2+ + ATP, omission of Ca2+ and Mg2+ or exchange of Na+ in the medium with K+ had no effect on the uptake of ascorbic acid. When isolated secretory granules from ox neurohypophyses were incubated with a medium containing [14C]DHA, uptake was considerably faster than the uptake when they were incubated in a medium containing [14C]AA. The uptake of dehydroascorbic acid was linear with the concentration in the medium and was not changed by addition of Mg2+ ATP. Addition of 10 mM NH4Cl or exchange of 120 mM K+ in the incubation medium with Na+ did not change the uptake of dehydroascorbic acid. The contents of copper, zinc, iron and cobalt were determined in isolated nerve endings (A) and membranes (B) as well as in lysate (C) from isolated neurosecretory granules. The results (in nmol mg-1 protein) were for Cu: (A): 0.25 +/- 0.01 (SEM), (B): 0.67 +/- 0.16, (C): 0.22 +/- 0.06; for Zn: (A): 0.53 +/- 0.13, (B): 6.97 +/- 0.75, (C): 1.8 +/- 0.53; and for Fe: (A): 15.6 +/- 1.9, (B): 6.92 +/- 0.32, (C): 3.15 +/- 0.43. In all preparations the cobalt content was below the detection limit (less than 5 pmol mg-1 protein).

Animals↗
PubMed2000

Dehydroascorbic acid transport by GLUT4 in Xenopus oocytes and isolated rat adipocytes.

Dehydroascorbic acid (DHA), the first stable oxidation product of vitamin C, was transported by GLUT1 and GLUT3 in Xenopus laevis oocytes with transport rates similar to that of 2-deoxyglucose (2-DG), but due to inherent difficulties with GLUT4 expression in oocytes it was uncertain whether GLUT4 transported DHA (Rumsey, S. C. , Kwon, O., Xu, G. W., Burant, C. F., Simpson, I., and Levine, M. (1997) J. Biol. Chem. 272, 18982-18989). We therefore studied DHA and 2-DG transport in rat adipocytes, which express GLUT4. Without insulin, rat adipocytes transported 2-DG 2-3-fold faster than DHA. Preincubation with insulin (0.67 micrometer) increased transport of each substrate similarly: 7-10-fold for 2-DG and 6-8-fold for DHA. Because intracellular reduction of DHA in adipocytes was complete before and after insulin stimulation, increased transport of DHA was not explained by increased internal reduction of DHA to ascorbate. To determine apparent transport kinetics of GLUT4 for DHA, GLUT4 expression in Xenopus oocytes was reexamined. Preincubation of oocytes for >4 h with insulin (1 micrometer) augmented GLUT4 transport of 2-DG and DHA by up to 5-fold. Transport of both substrates was inhibited by cytochalasin B and displayed saturable kinetics. GLUT4 had a higher apparent transport affinity (K(m) of 0.98 versus 5.2 mm) and lower maximal transport rate (V(max) of 66 versus 880 pmol/oocyte/10 min) for DHA compared with 2-DG. The lower transport rate for DHA could not be explained by binding differences at the outer membrane face, as shown by inhibition with ethylidene glucose, or by transporter trans-activation and therefore was probably due to substrate-specific differences in transporter/substrate translocation or release. These novel data indicate that the insulin-sensitive transporter GLUT4 transports DHA in both rat adipocytes and Xenopus oocytes. Alterations of this mechanism in diabetes could have clinical implications for ascorbate utilization.

Adipocytes↗
PubMed1998

Dehydroascorbic acid prevents apoptosis induced by oxidized low-density lipoprotein in human monocyte-derived macrophages.

Human low-density lipoprotein (LDL) oxidized with Cu2+ or the radical generator 2,2'-azobis(2-methyl-propionamidine) hydrochloride (AAPH) induces apoptosis in mature human monocyte-derived macrophages as assessed by staining with fluorescein-isothiocyanate-labeled annexin V, by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, and by staining of the 7A6 mitochondrial antigen. Oxidized LDL-induced apoptosis was dose and time dependent and clearly distinct from apoptosis induced by serum deprivation. Human autologous serum and lipoprotein-deficient human serum prevented apoptosis induced by oxidized LDL. Supplementation of serum-free culture medium with 25 microM ascorbic or isoascorbic acid only partially protected macrophages from apoptosis, whereas dehydroascorbic acid (DHAA) completely inhibited apoptosis induced by either Cu2+- or AAPH-oxidized LDL. Apoptosis was also inhibited by the structural analogue alloxan. Both cyclic multiketones dose-dependently inhibited oxidized LDL-induced apoptosis with IC50 in the submicromolar range. Prior loading of macrophages with ascorbic acid did not prevent the induction of apoptosis. Apoptosis was reduced by more than 90% after treatment of oxidized LDL with DHAA, whereas after incubation with either ascorbic or isoascorbic acid there was no such reduction. Removal of free DHAA by gel filtration did not reverse the inactivation. Parameters of LDL oxidation such as electrophoretic mobility, alpha-tocopherol content, thiobarbituric-acid-reactive subtances and lipid peroxide levels did not correlate to apoptotic activity. Also, binding and uptake of Texas-red-labeled oxidized LDL was not prevented by DHAA. Dithiothreitol-treatment of oxidized LDL, however, reduced the apoptotic activity by 76%. Our results suggest that oxidized thiols on apoB may be essential for the induction of apoptosis by oxidized LDL in human macrophages.

Alloxan↗
PubMed1989

Liquid chromatographic and fluorescent derivative aerobic degradation studies of dehydroascorbic acid in aqueous solution at elevated temperatures.

The aqueous degradation of dehydroascorbic acid (DHA) has been studied in the temperature range 52-90 degrees C. The DHA was determined by reversed-phase liquid chromatography and by derivatisation of DHA with o-phenylenediamine to form the fluorescent quinoxaline. The pseudo-first-order degradation of DHA has been verified and rate constants for the process are presented. The role of DHA in the degradation of ascorbic acid and previous DHA solution stability studies are discussed.

Ascorbic Acid↗
PubMed1979

[The amount of L-dehydroascorbic acid on total-vitamin C of vegetable products (author's transl)].

Spectrophotometric determinations of vitamin C were carried out on different kinds of vegetable products in raw state, after steam-cooking and freezedrying. Indicators used were in part 2,6-dichlorophenol-indophenol (DIP) and in part N-bromosuccinimide (NBS) and starch iodide, which at the same time allows a determination of other reducing interfering substances. The amount of L-dehydroascorbic acid (DAS), which together with L-ascorbic acid (AS) becomes vitamin C or total-vitamin C (VC), proved to be of special interest. During homogenisation of the vegetable samples in oxalic oxid solution AS was not oxidized. Other precautions like cooling or on oxygen-free atmosphere were not required. DAS was not found in raw vegetable products or after cooking. A high proportion of interfering substances especially when estimating VC was considered the cause for the normally measured amount of DAS. In freeze-dried samples of vegetable products a small amount DAS could be identified depending on the freezing process.

Ascorbic Acid↗
PubMed1986

Dehydroascorbic acid and ascorbic acid transport systems in the guinea pig ileum.

The transport properties of dehydroascorbic acid and ascorbic acid in membrane vesicle preparations of guinea pig ileum were evaluated. Na-dependent transport of ascorbic acid in the brush-border membrane was confirmed, and an Na-independent mechanism was found in the basolateral membrane. The electrically neutral oxidized form of vitamin C is transported by an Na-independent mechanism at both cell surfaces. Transport of each substrate is saturable and exhibits cis-inhibition and trans-stimulation in the presence of structural analogues. Additional studies on ascorbate metabolism will be necessary to support a comprehensive model of intestinal handling of vitamin C.

Animals↗
PubMed1981

Interaction of mammalian hemoglobins with dehydroascorbic acid.

Reactive sulfhydryl groups of major hemoglobins from guinea-pig, rat and cat reduced dehydroascorbic acid to ascorbic acid leading to formation of intrachain disulfide bonds. Hybridization experiments indicated that the reduction was carried out by the alpha chain of cat hemoglobin.

Animals↗
PubMed1998

Substrates of hexokinase, glucose-6-phosphate dehydrogenase, and glyceraldehyde-3-phosphate dehydrogenase prevent the inhibitory response induced by ascorbic acid/iron and dehydroascorbic acid in rabbit erythrocytes.

Exposure of intact rabbit erythrocytes or erythrocyte lysates to ascorbic acid/FeCl3 in a glucose-free saline promoted a rapid decline in reduced glutathione and this response was paralleled by inactivation of hexokinase. Under the same conditions, the activity of the enzymes glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase did not show appreciablevariations in intact cells, but was severely inhibited in the cell-free system. Similar results were obtained by replacing ascorbic acid/FeCl3 with dehydroascorbic acid. In addition, both treatments effectively inhibited the activity of purified hexokinase as well as those of glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. Further studies using the cell-free system indicated that the inhibition of enzyme activities elicited by either of the two treatments was effectively counteracted by the specific substrates of these enzymes. The fact that the hexokinase substrate glucose freely permeates the plasma membrane, unlike the substrates of glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphatedehydrogenase, explains the selective inhibition of hexokinase observed in intact cells. The above results also indicate that dehydroascorbic acid is an inhibitor of these enzymes and strongly suggest that it is at least in part responsible for the effects mediated by the cocktail ascorbic acid/FeCl3.

Animals↗
PubMed1976

Effects of tetraacetyl-bis-dehydroascorbic acid, a derivative of ascorbic acid, on Ehrlich cells and HeLa cells (human carcinoma cells).

Derivatives of ascorbic acid were synthesized, and the studies were made on their effects in Ehrlich ascites carcinoma cells, in regard to the inhibition and the prolongation of survival time as well as on the morphological degeneration in HeLa cells. In a model infection study carried out by using tetraacetyl-bis-dehydroascorbic acid in dd mice infected with Ehrlich cells, it was proved that the prolongation of survival time was nearly double in comparison to the control group mice. Also, it was noted that hypertrophy due to abdominal dropsy and body weight were reduced much more than in the control group. From these results, the inhibiting effect of tetraacetyl-bis-dehydroascorbic acid was confirmed. While in the case of DHA and other derivatives, almost no inhibition and prolongation of survival time were observed. As for HeLa cells in a tissue culture, tetraacetyl-bis-DHA, in a dosage of 125-250 mug/ml, demonstrated definitely its morphological degeration. After 125 mug/ml of tetraacetyl-bis-DHA was added to a tissue culture solution of HeLa cells, the cells were washed and recultured. No growth of the cells was observed. Consequently, this substance was confirmed to be anti-HeLa substance with a low toxicity.

Animals↗
PubMedDate not supplied

[Cause of dehydroascorbic acid accumulation in the blood of patients with insulin-dependent diabetes mellitus].

It is known that dehydroascorbic acid (DHAA) produces a diabetogenic effect and its content in the blood increases in diabetes mellitus. It was previously established that the generation of reducing equivalents (RE) in the course of hexosemonophosphate shunt, CO2 production and SH-glutathione regeneration in erythrocytes with and without moderate and maximum oxidation load in vitro were not disturbed in diabetes. The authors have proposed a procedure to study blood and erythrocyte DHAA reductase activity in suspension in health and in insulin-dependent diabetes mellitus by means of redoxstatometry using a device of original design. A significant acceleration of RE transfer through the erythrocyte membrane was detected in diabetes. A lowered participation in this process of the AA in equilibrium DHAA "shuttle" system was recorded in the blood of patients with diabetes mellitus what was mostly expressed under the conditions of acidosis in vitro. Probably "shuttle" function in diabetes was provided by some other redox system which might be located in the plasma. The predominant functioning of this redox system and a decrease of DHAA reductase activity in diabetes resulted in the accumulation of DHAA in the blood of patients with type I diabetes mellitus.

Adult↗
PubMed1985

Mitogenic inhibition and effect on survival of mice bearing L1210 leukemia using a combination of dehydroascorbic acid and hydroxycobalamin.

The present study was designed to test the effect of a combination of dehydroascorbic acid (DHA) and hydroxycobalamin (vitamin B12) on the survival of mice bearing L1210 leukemia. Results showed a significant increase in survival of treated mice compared with controls (p less than or equal to 0.0001) (Student's t-test). This positive effect was significantly lost when DHA was substituted by ascorbic acid (AA) in the same experimental conditions. In vitro findings also revealed that the DHA-B12 combination specifically inhibited mitoses of L1210 cells while non-neoplastic L929 cells were not affected.

Animals↗