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PubMed1991

Semidehydroascorbic acid as an intermediate in norepinephrine biosynthesis in chromaffin granules.

We investigated whether semidehydroascorbic acid was an intermediate in norepinephrine synthesis in chromaffin granules and in electron transfer across the chromaffin granule membrane. Semidehydroascorbic acid was measured in intact granules by electron spin resonance. In the presence of intragranular but not extragranular ascorbic acid, semidehydroascorbic acid was formed within granules in direct relationship to dopamine beta-monooxygenase activity. However, semidehydroascorbic acid was not generated when granules were incubated with epinephrine instead of the substrate dopamine, with dopamine beta-monooxygenase inhibitors, without oxygen, and when intragranular ascorbic acid was depleted. Experiments using the impermeant paramagnetic broadening agents [K3 [Cr(C2O4)3].3H2O] and Ni(en)3(NO3)2 provided further evidence that semidehydroascorbic acid was generated only within granules. We also investigated semidehydroascorbic acid formation in the presence of intragranular and extragranular ascorbic acid. Under these conditions, semidehydroascorbic acid was formed on both sides of the granule membrane, and formation was coupled to dopamine beta-monooxygenase activity. These data indicate that dopamine beta-monooxygenase is reduced by single electron transfer from intragranular ascorbic acid, that transmembrane electron transfer occurs by single electron transfer, and that transmembrane electron transfer is directly coupled to formation of intragranular semidehydroascorbic acid via dopamine beta-monooxygenase activity.

Adrenal Medulla↗
PubMed2005

Cloning, sequencing, and characterization of alternatively spliced glutaredoxin 1 cDNA and its genomic gene: chromosomal localization, mrna stability, and origin of pseudogenes.

Alternatively spliced human glutaredoxin (Grx1(as)) cDNA was isolated from a neutrophil cDNA library, using a (32)P-labeled human glutaredoxin (Grx1) cDNA probe under non-stringent conditions. The sequence of Grx1(as) cDNA indicated that the open reading frame of the gene was identical to the open reading frame of the previously reported first human glutaredoxin (Grx1) cDNA, but the 3'-untranslated region of Grx1(as) was not homologous to Grx1 cDNA. Northern blot and RT-PCR analyses showed Grx1(as) mRNA was expressed in normal human neutrophils and transformed cells including U937, HL-60, THP, and Jurkat cells. Cloning and sequencing of the genomic gene corresponding to Grx1(as) cDNA showed that two different glutaredoxin cDNAs (Grx1(as) and Grx1) were generated from the same genomic gene via alternative splicing. Origination of Grx1(as) and Grx1 from the same gene was confirmed by chromosomal localization of the Grx1(as) gene to chromosome 5q13, the same location where the Grx1 gene was localized previously. During screening of the Grx1(as) genomic gene, two additional glutaredoxin pseudogenes were also isolated. Surprisingly, these pseudogenes contained 3'-untranslated regions that were nearly identical to the 3'-untranslated regions of Grx1(as,) not Grx1, cDNA. Because 3'-untranslated regions may be important in stabilizing mRNAs, the effect of the two 3'-untranslated regions of Grx1 and Grx1(as) on mRNA stability was investigated using luciferase reporter vectors with the 3'-untranslated regions. Luciferase activity was 2.6-fold greater in cells transfected with the reporter vector containing the 3'-untranslated region of Grx1(as) cDNA compared with the 3'-untranslated region of Grx1 cDNA. These data indicate that Grx1(as) cDNA is an alternatively spliced human Grx1 cDNA and that the Grx1(as) 3'-untranslated region may have a role in stabilizing mRNA.

3' Untranslated Regions↗
PubMed1993

Analysis of the transformation products of dehydro-L-ascorbic acid by ion-pairing high-performance liquid chromatography.

Dehydro-L-ascorbic acid, the oxidation form of L-ascorbic acid, converts nonenzymatically to a variety of transformation products depending upon the incubating conditions. These products, called the ascorbate cascade, possess different absorption spectra and reducing activities. Ion-pairing high-performance liquid chromatography with multiwavelength uv absorption and amperometric electrochemical detection has proven to be a useful technique for the analysis of these products. Separation and characterization in the present investigations reveal three kinds of transformation products: the polymerized intermediates of browning reaction, the reductant possessing an absorption maximum at 345 nm as the six-carbon compound, and erythro-L-ascorbic acid, 2-furoic acid, 3-hydroxy-2-pyrone, 5-methyl-3,4-dihydroxytetrone, reductic acid, and furfural as the five-carbon compound. These products possess an absorption maximum above 240 nm, and most of them are electrochemically active reducing compounds which are derived from nonreducing dehydro-L-ascorbic acid through the processes of delactonization, decarboxylation, and intramolecular rearrangement.

Chromatography, High Pressure Liquid↗
PubMedDate not supplied

Ascorbate transport in pig coronary artery smooth muscle: Na(+) removal and oxidative stress increase loss of accumulated cellular ascorbate.

Pig deendothelialized coronary artery rings and smooth muscle cells cultured from them accumulated ascorbate from medium containing Na(+). The accumulated material was determined to be ascorbate using high-performance liquid chromatography. We further characterized ascorbate uptake in the cultured cells. The data fitted best with a Hill coefficient of 1 for ascorbate (K(asc) = 22 +/- 2 microM) and 2 for Na(+) (K(Na) = 84 +/- 10 mM). The anion transport inhibitors sulfinpyrazone and 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) inhibited the uptake. Transferring cultured cells loaded with (14)C-ascorbate into an ascorbate-free solution resulted in a biphasic loss of radioactivity - an initial sulfinpyrazone-insensitive faster phase and a late sulfinpyrazone-sensitive slower phase. Transferring loaded cells into a Na(+)-free medium increased the loss in the initial phase in a sulfinpyrazone-sensitive manner, suggesting that the ascorbate transporter is bidirectional. Including peroxide or superoxide in the solution increased the loss of radioactivity. Thus, ascorbate accumulated in coronary artery smooth muscle cells by a Na(+)-dependent transporter was lost in an ascorbate-free solution, and the loss was increased by removing Na(+) from the medium or by oxidative stress.

Actins↗
PubMedDate not supplied

Peroxynitrous acid--where is the hydroxyl radical?

Peroxynitrite is an inorganic toxin of physiological interest, formed from the diffusion-controlled reaction of superoxide and nitrogen monoxide with a rate constant of (1.6 +/- 0.3) x 10(10) M(-1) s(-1). On the basis of three experiments we conclude that homolysis of the O-O bond in peroxynitrous acid is unlikely: (1) the yield of nitrite from the decomposition of peroxynitrite shows a dependence on the peroxynitrite concentration and is lower than expected for homolysis; (2) the yield of [15N]nitrate from the reaction of [15N]nitrite with peroxynitrous acid predicted by homolysis does not correspond to that found experimentally, and (3) the reaction of peroxynitrous acid with monohydroascorbate does not yield ascorbyl radicals. Activation volumes determined from high-pressure kinetic studies are inconclusive.

Dehydroascorbic Acid↗
PubMed1995

Ascorbic acid and cell survival of adriamycin resistant and sensitive MCF-7 breast tumor cells.

The ability of human cells to regenerate ascorbic acid from dehydroascorbate is partially dependent on the glutathione redox status of the cell and the relative activity of dehydroascorbate reductases. Mammalian dehydroascorbate reductase activity is associated with two proteins known as thioltransferase (glutaredoxin) and protein disulfide isomerase. We compared the specific activity of thioltransferase, protein disulfide isomerase, and other GSH-related enzymes in Adriamycin-resistant human breast tumor cells, MCF-7 ADRR, and Adriamycin-sensitive, MCF-7 WT, tumor cells. MCF-7 ADRR cells had higher activities of glutathione peroxidase (34.7 fold), nonseleno-glutathione peroxidase (glutathione S-transferases; 5.3 fold), thioredoxin (2.3 fold), and thioltransferase (4.0 fold) compared with the WT Adriamycin-sensitive cell line. Thioltransferase was detected in Western blots in extracts of ADRR MCF-7 cells but not in WT MCF-7 cells. alpha-Tocopherol in the membrane and cytosolic fractions was 2.8 and 3.0 fold higher, respectively, in Adriamycin-resistant compared with Adriamycin-sensitive cells. Supplementation of MCF-7 cells with L-ascorbic acid 2-phosphate (2 and 10 mM) had no effect on WT cell viability after 5 days incubation with up to 0.33 microM Adriamycin. In contrast, supplementation of ADRR MCF-7 cells with L-ascorbic acid 2-phosphate resulted in enhanced resistance up to 3.4 microM Adriamycin over a 5-day incubation. Both lines of MCF-7 cells demonstrated the ability to utilize ascorbic acid as the 2-phosphate derivative. After 48 h incubation with 8.6 microM Adriamycin, the resistant cells maintained normal viability and ascorbate-dehydroascorbate levels, whereas drug-sensitive cells had significantly lower ascorbate with a higher percent dehydroascorbate and increased cell death as judged by cell protein levels (52% of controls).

Animals↗
PubMedDate not supplied

Paramagnetic changes in pulmonary tumors.

Electron spin resonance studies on healthy and tumorous human lung samples have been conducted in order to determine possible differences in free radical concentration and shaped of the spectra between the different sections of the lung. It could be shown that in healthy lung tissue the signal caused by the semidehydroascorbate (SDA) radical is not prominent because of the prevailing high partial oxygen pressure. On formation of a tumor, the spin concentration increases, possibly due to the higher metabolic rate; here, the SDA peak is also more pronounced which indicates alterations in the interaction between cell constituents and ascorbic acid. Within the tumor, the spin concentration is considerably reduced which is probably caused by a still higher concentration of ascorbic acid. Addition of ascorbic acid to the different lung specimens enhanced the just described effect while oxidizing substances, such as H2O2, reversed it.

Dehydroascorbic Acid↗
PubMed1982

Interference by ascorbic acid in test systems involving peroxidase. I. Reversible indicators and the effects of copper, iron, and mercury.

I describe the mechanism whereby ascorbic acid can hamper test systems involving a peroxide-generating system, peroxidase, and a benzidine-type indicator. In test media such as urines, abnormally high concentrations of ascorbic acid may give rise to false negatives in the determination of analytes such as glucose. Absorbance measurements in solution or reflectance measurements on commercially available paper reagent strips demonstrate either inhibition of visible activity by ascorbic acid or a lag time in the development of oxidized indicator color. The duration of the lag time is proportional to the ascorbic acid concentration, is inversely proportional to the enzyme concentration, and is also affected by concentrations of hydrogen peroxide and o-tolidine indicator. The same results were seen in both citrate buffer pH 5 and phosphate buffer pH 7. Because the complete system (o-tolidine indicator, hydrogen peroxide, and peroxidase) must be present if the ascorbate is to be oxidized rapidly, this indicates that ascorbic acid inhibits color development by re-reducing oxidized indicator as fast as it is formed; the o-tolidine then acts catalytically in oxidizing ascorbic acid. Added Cu2+ and Fe3+, both known to react with ascorbic acid, had measurable but small effects on the system. In contrast, Hg2+ abolished the ascorbic acid-elicited lag time, even when present in near-stoichiometric concentration. Hg2+ showed little inhibitory effect on the activity of either glucose oxidase or peroxidase. Presumably it rapidly oxidizes ascorbic acid to dehydroascorbate. The reaction of mercuric ion with ascorbate was measured by reflectance measurements of paper reagent strips in addition to absorbance measurements of solution assays; equivalent results were obtained. Incorporation of Hg2+ into reagent strips can thus render both strips and solution diagnostic tests insensitive to interfering substances such as ascorbic acid.

Ascorbic Acid↗
PubMedDate not supplied

[Relation between ascorbic acid metabolism and the body' supply of vitamin K].

Experiments on rats were made to study the content of ascorbic (AA), Dehydroascorbic (DAA) and diketogulonic (DKGA) acids in the blood serum, daily urine, liver, adrenal, kidney, spleen and lung tissues under varying body supply with vitamin K. The rats with vitamin K deficiency manifested the decreased content of AA, DAA and the elevated content of DKGA in the blood serum, daily urine and test tissues. The percentage of AA absorption by blood proteins was found to be increased upon vitamin K deficiency in the body. The changes in AA metabolism correlated with the reduced capillary resistance. Administration of AA to rats with vitamin K deficiency led to an increase in the content of AA, DAA and to a lowering of DKGA in the blood serum and tissues promoting the normalization of capillary resistance. The data obtained attest to the dependence of AA metabolism on the body supply with vitamin K, pointing to the necessity of exercising the control over AA metabolism in different forms of vitamin K deficiency.

2,3-Diketogulonic Acid↗
PubMed1992

Short term effects of oxidized ascorbic acid on bovine corneal endothelium and human placenta.

Studies on the toxic effects of dehydro-L-ascorbic acid (DHAA) have been extended to include evaluations over time periods up to 3 hr. and to test for specific effects on a membrane transport protein, a membrane-bound enzyme and a soluble intracellular enzyme. In studies on cultured corneal endothelial cells, DHAA concentrations of 1, 2, and 5 mM over 3 hr. had an inhibitory effect on subsequent uptake of DHAA present at a tracer level. Surviving fragments of human placenta and alkaline phosphatase activity of the placental brush-border membrane were susceptible to the effect of DHAA at a high concentration (10 mM). Because intracellular metabolism of DHAA was not affected, and an increase in membrane permeability was not detected, it is concluded that a specific membrane transport protein might be the site of DHAA-induced damage. These studies support the concept that the oxidized form of ascorbic acid (vitamin C) has potential toxic effects on biological systems and suggests that proteins that mediate transport and metabolism may be sites where DHAA causes damage.

Alkaline Phosphatase↗
PubMed2003

Sugar hydrazone-metal complexes: transition- and non-transition metal complexes of monosaccharide S-alkylhydrazonecarbodithioates and dehydro-L-ascorbic acid bis(S-alkylhydrazonecarbodithioates).

Copper(II), nickel(II) and palladium(II) complexes with aldehydo-D-arabinose-, L-arabinose-, D-galactose-, D-glucose- and D-mannose- S-methyl- and S-benzylhydrazonecarbodithioates were synthesized and characterized by elemental analyses, infrared and UV-Vis. In these complexes the aldehydo sugar hydrazone acts as a mononegative NS bidentate ligand. The reaction of Cu(II) chloride, however, proceeded with reduction, and copper(I) complexes were isolated. The hydrazone molecule in these Cu(I) complexes acts as neutral NS bidentate ligand. Dehydro-L-ascorbic acid bis(S-methylhydrazinecarbodithioate) and bis(hydrazinecarbothioamide), as well as their corresponding Cu(II), Ni(II), zinc(II) and Pd(II) complexes were prepared and characterized. Electrospray (ES) and field desorption (FD) mass spectra suggest that the Cu(II), Ni(II), and Pd(II) complexes are monomeric (square planar), whereas the Zn(II) are dimeric and pentacoordinate.

Aldehydes↗
PubMed2001

Spectrophotometric determination of ascorbic acid with iron(III) and p-carboxyphenylfluorone in a cationic surfactant micellar medium.

A simple and highly sensitive spectrophotometric method for the determination of ascorbic acid (AA) was established by using iron(III) and p-carboxyphenylfluorone (PCPF) in a cationic surfactant micellar medium. The apparent molar absorptivity of the proposed method, which does not require an extraction procedure, was 2.05 x 10(6) dm3 mol-1 cm-1 at 655 nm. Beer's law was obeyed in the concentration range of 0.02-0.12 microgram/cm3 for AA. The procedure was successfully applied to assays of AA in pharmaceutical preparations. It is suggested that the method is based on a coupled redox-complexation reaction in which the first step is the oxidation of AA by iron(III), and the second step includes the formations of the iron(II)-PCPF (1:2) complex and the dehydroascorbic acid-iron(III)-PCPF (1:1:2) complex.

Ascorbic Acid↗
PubMed1992

Mode of formation of quinoxaline versus 2[1H]-quinoxalinone rings from dehydro-D-erythorbic acid.

The mode of formation of the quinoxaline versus 2[1H]-quinoxalinone rings by the reaction of o-diamines with dehydro-D-erythorbic acid has been investigated. The study was carried out by using one and two molar equivalents of 1,2-diamino-4,5-dimethylbenzene (3b) to give 6,7-dimethyl-3-(1-oxo-D-erythro-2,3,4-trihydroxybutyl)-2[1H]-quino xalinone (4b) and 2-(2-amino-4,5-dimethylphenylcarbamoyl)-3-(D-erythro-glycerol-1-yl )- 6,7-dimethylquinoxaline (6), respectively. The former product exists predominantly as the two furanosyl anomers. Sequential reaction of 4a with 3b has been studied, and the location of each diamine in the product was deduced by using 1H-n.m.r. spectroscopy. A mechanism for the reaction is proposed. Acetate and acetal derivatives of the compound are prepared.

Dehydroascorbic Acid↗
PubMed1990

Reaction of ascorbate with lysine and protein under autoxidizing conditions: formation of N epsilon-(carboxymethyl)lysine by reaction between lysine and products of autoxidation of ascorbate.

N epsilon-(Carboxymethyl)lysine (CML) has been identified as a product of oxidation of glucose adducts to protein in vitro and has been detected in human tissue proteins and urine [Ahmed, M. U., Thorpe, S. R., & Baynes, J. W. (1986) J. Biol. Chem. 261, 4889-4894; Dunn, J. A., Patrick, J. S., Thorpe, S. R., & Baynes, J. W. (1989) Biochemistry 28, 9464-9468]. In the present study we show that CML is also formed in reactions between ascorbate and lysine residues in model compounds and protein in vitro. The formation of CML from ascorbate and lysine proceeds spontaneously at physiological pH and temperature under air. Kinetic studies indicate that oxidation of ascorbic acid to dehydroascorbate is required. Threose and N epsilon-threuloselysine, the Amadori adduct of threose to lysine, were identified in the ascorbate reaction mixtures, suggesting that CML was formed by oxidative cleavage of N epsilon-threuloselysine. Support for this mechanism was obtained by identifying CML as a product of reaction between threose and lysine and by analysis of the relative rates of formation of threuloselysine and CML in reactions of ascorbate or threose with lysine. The detection of CML as a product of reaction of ascorbate and threose with lysine suggests that other sugars, in addition to glucose, may be sources of CML in proteins in vivo. The proposed mechanism for formation of CML from ascorbate is an example of autoxidative glycosylation of protein and suggests that CML may also be an indicator of autoxidative glycosylation of proteins in vivo.

Ascorbic Acid↗
PubMed2000

The reactive oxygen species--and Michael acceptor-inducible human aldo-keto reductase AKR1C1 reduces the alpha,beta-unsaturated aldehyde 4-hydroxy-2-nonenal to 1,4-dihydroxy-2-nonene.

The human aldo-keto reductase AKR1C1 (20alpha(3alpha)-hydroxysteroid dehydrogenase) is induced by electrophilic Michael acceptors and reactive oxygen species (ROS) via a presumptive antioxidant response element (Burczynski, M. E., Lin, H. K., and Penning, T. M. (1999) Cancer Res. 59, 607-614). Physiologically, AKR1C1 regulates progesterone action by converting the hormone into its inactive metabolite 20alpha-hydroxyprogesterone, and toxicologically this enzyme activates polycyclic aromatic hydrocarbon trans-dihydrodiols to redox-cycling o-quinones. However, the significance of its potent induction by Michael acceptors and oxidative stress is unknown. 4-Hydroxy-2-nonenal (HNE) and other alpha,beta-unsaturated aldehydes produced during lipid peroxidation were reduced by AKR1C1 with high catalytic efficiency. Kinetic studies revealed that AKR1C1 reduced HNE (K(m) = 34 microm, k(cat) = 8.8 min(-1)) with a k(cat)/K(m) similar to that for 20alpha-hydroxysteroids. Six other homogeneous recombinant AKRs were examined for their ability to reduce HNE. Of these, AKR1C1 possessed one of the highest specific activities and was the only isoform induced by oxidative stress and by agents that deplete glutathione (ethacrynic acid). Several hydroxysteroid dehydrogenases of the AKR1C subfamily catalyzed the reduction of HNE with higher activity than aldehyde reductase (AKR1A1). NMR spectroscopy identified the product of the NADPH-dependent reduction of HNE as 1,4-dihydroxy-2-nonene. The K(m) of recombinant AKR1C1 for nicotinamide cofactors (K(m) NADPH approximately 6 microm, K(m)(app) NADH >6 mm) suggested that it is primed for reductive metabolism of HNE. Isoform-specific reverse transcription-polymerase chain reaction showed that exposure of HepG2 cells to HNE resulted in elevated levels of AKR1C1 mRNA. Thus, HNE induces its own metabolism via AKR1C1, and this enzyme may play a hitherto unrecognized role in a response mounted to counter oxidative stress. AKRs represent alternative GSH-independent/NADPH-dependent routes for the reductive elimination of HNE. Of these, AKR1C1 provides an inducible cytosolic barrier to HNE following ROS exposure.

20-Hydroxysteroid Dehydrogenases↗
PubMed2000

Chemical characteristics of dehydro-L-ascorbic acid.

Dehydro-L-ascorbic acid (DAA) exists mainly in its C2 hydrated bicyclic form (5) in an aqueous solution, and monocyclic DAA (3), which is the expected reaction product immediately after the oxidation of AA, has not been observed by NMR spectroscopy. The formation mechanism for 5 from 3 and the stability of 5 were examined by the semi-empirical molecular orbital method (MOPAC). It was indicated that the protonation reaction was the key step in the formation of 5, therefore, the formation of 5 is thought to be more difficult under physiological conditions which mostly involve in the neutral or slightly alkaline state. However, by NMR, it was confirmed that, even in a neutral or slightly alkaline state very close to physiological conditions, the predominant form of DAA existing in an aqueous solution immediately after the enzymatic oxidation of AA was confirmed to be 5, although the possible existence of other forms of DAA at very low concentrations could not be completely excluded.

Dehydroascorbic Acid↗
PubMed2004

Oxidative stress in limpets exposed to different environmental conditions in the Beagle Channel.

The aim of this work was to study the oxidative profile of digestive glands of two limpets species (Nacella (Patinigera) magellanica and Nacella (Patinigera) deaurata) exposed to different environmental conditions. The intertidal population of N. (P.) magellanica is subjected to a wide variety of stresses not experienced by N. (P.) deaurata. Although a typical electron paramagnetic resonance (EPR) spectrum of ascorbyl radical in digestive gland from both limpets was observed, neither ascorbyl radical content nor the ascorbyl radical content/ascorbate content ratio was significantly different, suggesting that the difference in the environmental conditions did not appear to be responsible for developing alterations in the oxidative status of both organisms at the hydrophilic level (e.g. cytosol). Lipid peroxidation in the digestive glands was estimated, both as the content of thiobarbituric acid reactive substances (TBARS) and as the content of lipid radicals assessed by EPR, in both organisms. TBARS and lipid radical content were 34.8 and 36.5%, respectively, lower in N. (P.) magellanica as compared to N. (P.) deaurata. On the other hand, total iron content and the rate of generation of superoxide anion were 47.9 and 51.4%, respectively, lower in N. (P.) magellanica as compared to N. (P.) deaurata. The activity of catalase and superoxide dismutase (SOD) was 35.3 and 128.6% higher in N. (P.) magellanica as compared to N. (P.) deaurata, respectively. No significant differences were determined between the digestive glands of both molluscs regarding the content of total thiols. alpha-Tocopherol and beta-carotene content were significantly lower in N. (P.) magellanica as compared to N. (P.) deaurata. A distinctive EPR signal for the adduct Fe--MGD--NO (g = 2.03 and a(N) = 12.5 G) was detected in the homogenates of digestive glands of both limpets. A significant difference in the content of the Fe-MGD-NO adduct in digestive glands from N. (P.) magellanica and N. (P.) deaurata (491 +/- 137 and 839 +/- 63 pmol/g FW, respectively) was observed. Taken as a whole, the data presented here indicated that coping with environmental stressing conditions requires a complex adjustment of the physiological metabolic pathways to ensure survival by minimizing intracellular damage. It is likely that N. (P.) magellanica has a particular evolutionary adaptation to extreme environmental conditions by keeping iron content low and antioxidant activities high.

Analysis of Variance↗