ascorbic-acid and 2-4-dinitrophenylhydrazine

Purpose of this study was to examine the dose dependant effects of sesame seed powder as a dietary supplement on hypercholesteraemic and oxidative stress conditions in male albino rats. Sesame seed (Sesamum indicum) powder was administered at 5% and 10% dose levels along with either normal or hypercholesteraemic diet for duration of four weeks. Administration of sesame seed powder to hypercholesteraemic rats resulted in a significant decline in plasma, hepatic total lipid and cholesterol levels and, plasma LDL-cholesterol levels with an increase in plasma HDL-cholesterol levels. Further, these animals also showed increased fecal excretion of cholesterol, neutral sterol and bile acid along with increases in hepatic HMG-CoA reductase activity and bile acid content. Additionally sesame seed feeding improved the hepatic antioxidant status (catalase and SOD enzyme activities) with a reduction in lipid peroxidation. No significant changes in lipid and antioxidant profiles occurred in the normocholesteraemic rats administered with sesame seed powder. These beneficial effects of sesame seed on hypercholesteraemic rats appeared to be due to its fiber, sterol, polyphenol and flavonoid content, enhancing the fecal cholesterol excretion and bile acid production and as well as increasing the antioxidant enzyme activities.

Topics: Animals; Anticholesteremic Agents; Antioxidants; Ascorbic Acid; Bile Acids and Salts; Body Weight; Cholesterol; Diet; Eating; Feces; Hydroxymethylglutaryl CoA Reductases; Indicators and Reagents; Lipid Metabolism; Lipid Peroxidation; Lipids; Liver; Male; Organ Size; Phenylhydrazines; Phytosterols; Rats; Seeds; Sesamum; Sterols

This study investigates the oxidative damage of biomolecules in livers of mice treated with morphine intraperitoneally. The oxidative damage of DNA as measured by single cell electrophoresis and high-performance liquid chromatography equipped with electrochemical and UV detection, the protein carbonyl content was measured by 2,4-dinitrophenylhydrazine method, and the malondialdehyde content was measured by the HPLC method. The activities of antioxidative enzymes, superoxide dismutase, catalase and glutathione peroxidase, and the activity of alanine aminotransferase were assayed by spectrophotometer method. Glutathione and oxidized glutathione were detected by fluorescence spectrophotometer method. All the indexes of oxidative damage, such as 8-OHdG, protein carbonyl group and malondialdehyde content, and the activity of alanine aminotransferase (n=27) increased significantly compared to those of control (n=27) (P<0.01) in livers of morphine-administered alone mice, while the indexes related with the in vivo antioxidative capacity, such as the ratio of glutathione and oxidized glutathione, activities of superoxide dismutase, catalase and glutathione peroxidase significantly decreased (P<0.01). When mice were treated with morphine combined with exogenous antioxidants, glutathione and ascorbic acid, all the indexes of oxidative damage and the activity of alanine aminotransferase showed no changes as compared to those of control (P>0.05), i.e., both glutathione and ascorbic acid completely abolished the damage of morphine on the hepatocyte. These results implied that morphine caused a seriously oxidative stress in mice livers and hence caused hepatotoxicity, while exogenous antioxidants were able to prevent the oxidative damage of biomolecules and hepatotoxicity caused by morphine. Thus, blocking oxidative damage may be a useful strategy for the development of a new therapy for opiate abuse.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Alanine Transaminase; Animals; Antioxidants; Ascorbic Acid; Catalase; Chemical and Drug Induced Liver Injury; China; Chromatography, High Pressure Liquid; Deoxyguanosine; DNA Damage; Drug Administration Schedule; Drug Therapy, Combination; Electrophoresis; Glutathione; Glutathione Peroxidase; Injections, Intraperitoneal; Liver; Malondialdehyde; Mice; Morphine; Organic Chemicals; Oxidation-Reduction; Oxidative Stress; Phenylhydrazines; Proteins; Superoxide Dismutase

The effect of beta-carotene on protein oxidation was examined under different oxygen (O(2)) tensions and with other antioxidants: alpha-tocopherol, ascorbic acid, and mixtures of antioxidants. Human serum albumin (HSA) was incubated with 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) to induce protein oxidation (carbonyl formation), under 15, 150, and 760 torr of O(2) tension. Antioxidant activity was related to O(2) tension, antioxidant concentrations and interaction between mixtures of antioxidants: (1) Under 15 torr of O(2), incubating HSA with AAPH, 1. 6 microM beta-carotene, 80 microM alpha-tocopherol, 160 microM ascorbic acid, and mixtures (0.1 microM beta-carotene, 5.0 microM alpha-tocopherol and 10 microM ascorbic acid) resulted in 24, 29, 39, and 44% reduction of carbonyl formation, respectively. (2) Under 150 torr of O(2) tension, the antioxidant effect of beta-carotene was decreased by 4% but increasing O(2) tension did not diminish the antioxidant effects of alpha-tocopherol, ascorbic acid, or antioxidant mixtures. (3). Under 760 torr of O(2) tension, adding 1. 6 microM beta-carotene resulted in 26% more carbonyl formation. (4) Under 760 torr of O(2) tension, the antioxidant effect of ascorbic acid was decreased 32% compared to what was observed at 150 torr of O(2) tension. Changes in O(2) tension had no effect on the antioxidant effect of alpha-tocopherol. The mixture of antioxidants inhibited carbonyl formation by 37% and was 7% less effective than that of 15 and 150 torr of O(2) tension. High concentration of beta-carotene produces more protein oxidation in the presence of high O(2) tension by a prooxidant mechanism. Mixtures of beta-carotene, alpha-tocopherol, and ascorbic acid provided better protective effects on protein oxidation than any single compound.

Topics: Amidines; Antioxidants; Ascorbic Acid; beta Carotene; Enzyme-Linked Immunosorbent Assay; Humans; Oxidants; Oxidation-Reduction; Oxygen; Phenylhydrazines; Serum Albumin; Vitamin E

Degradation of collagen by oxidant species may play an important role in the progression of rheumatoid arthritis. Whilst the overall effects of this process are reasonably well defined, little is known about the sites of attack, the nature of the intermediates, or the mechanism(s) of degradation. In this study electron paramagnetic resonance spectroscopy with spin trapping has been used to identify radicals formed on collagen and related materials by metal ion-H2O2 mixtures. Attack of the hydroxyl radical, from a Fe(II)-H2O2 redox couple, on collagen peptides gave signals from both side chain (.CHR'R"), and alpha-carbon[.C(R)(NH-)CO-,R = side-chain]radicals. Reaction with collagen gave both broad anisotropic signals, from high-molecular-weight protein-derived radicals, and isotropic signals from mobile species. The latter may be low-molecular-weight fragments, or mobile side-chain species; these signals are similar to those from the alpha-carbon site of peptides and the side-chain of lysine. Enzymatic digestion of the large, protein-derived, species releases similar low-molecular-weight adducts. The metal ion employed has a dramatic effect on the species observed. With Cu(I)-H2O2 or Cu(II)-H2O2 instead of Fe(II)-H2O2, evidence has been obtained for: i) altered sites of attack and fragmentation, ii) C-terminal decarboxylation, and iii) hydrogen abstraction at N-terminal alpha-carbon sites. This altered behaviour is believed to be due to the binding of copper ions to some substrates and hence site-specific damage. This has been confirmed in some cases by electron paramagnetic resonance studies of the Cu(II) ions.

Topics: Amino Acids; Ascorbic Acid; Benzenesulfonates; Collagen; Collagenases; Copper; Electron Spin Resonance Spectroscopy; Hydrogen Peroxide; Hydroxyl Radical; Iron; Metals; Nitroso Compounds; Oxidants; Oxidation-Reduction; Peptides; Phenylhydrazines; Protein Binding; Spin Trapping

We demonstrate that total ascorbic acid (TAA, the sum of ascorbic acid and dehydroascorbic acid) in properly prepared human plasma is stable at -70 degrees C for at least 6 years when preserved with dithiothreitol. TAA in human plasma or serum preserved with metaphosphoric acid degrades slowly, at the rate of no more than 1% per year. As assessed from our stability data and from data obtained from 23 laboratories over a period of > 2 years, the intralaboratory repeatability of TAA measurement is approximately 2 mumol/L, irrespective of TAA concentration. Nonchromatographic analytical methods involving dinitrophenylhydrazine and 0-phenylenediamine yield biased results relative to chromatographic methods. Within groups of laboratories that use roughly similar analytical methods, the interlaboratory measurement reproducibility CV for TAA is 15%.

Topics: Ascorbic Acid; Chromatography; Dehydroascorbic Acid; Dithiothreitol; Drug Stability; Freezing; Humans; Kinetics; Laboratories; Phenylenediamines; Phenylhydrazines; Phosphorous Acids; Plasma; Reproducibility of Results; Specimen Handling; Stereoisomerism; Time Factors

An HPLC micro-method with fluorescence detection has been developed to determine total vitamin C (vit C) and dehydroascorbic acid (DHA) concentrations in human plasma samples. This method is based on the rapid, specific reaction of DHA with dimethyl-o-phenylenediamine (DMPD) to form a fluorescent quinoxaline derivative that is quantified by HPLC in less than 5 minutes. The method was assessed with reference to the direct 2,4-dinitrophenylhydrazine (DNPH) colorimetric method. They were well correlated (r3 = 0.879), but the DMPD-HPLC method had the limit of detection 6 times lower than the standard method and the relative error for a vitamin standard was 10 times better than that of the standard method. The plasma DHA to total vit C ratio varied from 10 to 60%, depending on sample processing. Plasma that were immediately analysed contained 10% DHA whatever the subject's age; frozen deproteinized samples kept 1 week (-67 degrees C) had 20%, and blood samples kept for one hour at room temperature before treatment had up to 60% DHA. The ratio in capillary samples taken from the finger was 11-42%. This rapid, specific and very sensitive micro-method is well suited to routine measurements of plasma vit C.

Topics: Adult; Aged; Aged, 80 and over; Ascorbic Acid; Chromatography, High Pressure Liquid; Colorimetry; Dehydroascorbic Acid; Fluorometry; Humans; Linear Models; Middle Aged; Oxidation-Reduction; Phenylenediamines; Phenylhydrazines; Reference Standards; Reproducibility of Results; Sensitivity and Specificity; Statistics, Nonparametric; Temperature; Time Factors

A reverse-phase high pressure liquid chromatographic technique (RP-HPLC) was developed for the analysis of ascorbic acid in bass liver. Ascorbic acid was extracted from bass liver and simultaneously assayed by RP-HPLC. The results were compared to the Dinitrophenylhydrazine (DNPH) method now in use. Recovery studies showed about 97% by the HPLC method compared to about 96% by the DNPH method. There was no statistically significant difference found in the values obtained from the two methods. The HPLC method described here is considered the preferred method both in terms of a shorter analysis time and greater sensitivity.

Topics: Animals; Aquaculture; Ascorbic Acid; Bass; Chromatography, High Pressure Liquid; Liver; Phenylhydrazines; Spectrophotometry

1. Concentrations of ascorbic acid (ascorbic and dehydro-ascorbic; A + D; measured by the 2,4-dinitrophenylhydrazine method) of nearly three times those of plasma are present in gastric juice samples from patients with normal gastric histology. 2. A significant reduction in gastric juice ascorbic acid (A + D) was observed in patients with chronic gastritis. This reduction in concentration was independent of the grade of gastritis. 3. Concentrations of ascorbic acid (A + D) in gastric biopsy specimens were consistently higher in the antrum than in the body of the stomach. 4. These data demonstrate that considerable quantities of ascorbic acid (A + D) are normally 'secreted' into the stomach. 5. Ascorbic acid (ascorbic only; A; measured by h.p.l.c.) was present predominantly in its biologically active form in the patients with normal gastric histology. However, in patients with gastritis, independent of grade, ascorbic acid was present predominantly in its oxidized, biologically inactive form.

Topics: Adult; Aged; Ascorbic Acid; Chromatography, High Pressure Liquid; Chronic Disease; Female; Gastric Juice; Gastritis; Humans; Hydrogen-Ion Concentration; Leukocytes; Male; Middle Aged; Phenylhydrazines

Topics: Ascorbic Acid; Autoanalysis; Humans; Phenylhydrazines; Phosphotungstic Acid; Spectrophotometry

In leukocytes a dynamic relationship between the reduced form of ascorbic acid (AA) and its oxidized product dehydro-AA has been described. It is therefore important to know which form of the vitamin predominates when choosing a methodology. The purpose of this study was to find out if the majority of ascorbate in human leukocytes isolated by centrifugation through Percoll is in the reduced AA form by measuring reduced AA by HPLC and comparing the values to those obtained by using the 2,4-dinitrophenylhydrazine (DNPH) method which measures total ascorbate, and quantifying the reduced and oxidized forms of the vitamin in leukocytes using a modification of the DNPH method. There was no significant difference (P greater than .05) between the HPLC and DNPH values for 12 individuals and 87% of the AA was found to be in the reduced form. These results support the assumption that the majority of AA found in a mixed leukocyte population isolated through Percoll is in the reduced form and that both methods can be used for AA measurements.

Topics: Ascorbic Acid; Chromatography, High Pressure Liquid; Female; Humans; Leukocytes; Male; Oxidation-Reduction; Phenylhydrazines

Rabbit muscle phosphoglucomutase was irreversibly inactivated upon preincubation with vitamin C (Vit C). Fe(III), NADH.NADH oxidase.Fe(III), or ferritin.Vit C. Substrate, glucose 1-phosphate and Mg2+ afforded partial protection. No altered amino acid could be detected in the inactive enzyme. Enzyme so inactivated was more susceptible to trypsin. More importantly, during inactivation, the enzyme lost up to 70% of its enzyme-bound phosphate; the completely inactivated enzyme retained the remainder of the bound phosphate which was isolatable as phosphoserine residing in the 22-amino acid long tryptic peptide. Free phosphoserine as well as those in phosphorylase alpha and phosphocasein were resistant to the oxidizing system, suggesting that the phosphoserine of phosphoglucomutase is uniquely vulnerable to these treatments. Alternatively, a fraction of the total 1 mol of phosphate in the phosphoform of phosphoglucomutase may not be associated with phosphoserine. Phosphoglyceromutase, which has phosphohistidine at its active site, was also inactivated by the oxidizing system. However, it did not release any of the bound phosphate.

Topics: Amino Acids; Animals; Ascorbic Acid; Catalase; Ferric Compounds; Ferritins; Histidine; Iron; Magnesium; Multienzyme Complexes; Muscles; NADH, NADPH Oxidoreductases; Phenylhydrazines; Phosphates; Phosphoglucomutase; Phosphorylase a; Phosphoserine; Rabbits; Sulfhydryl Compounds; Trypsin

Intracellular proteolytic degradation of glutamine synthetase occurs in two distinct steps in Escherichia coli (Levine, R. L., Oliver, C. N., Fulks, R. M., and Stadtman, E. R. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2120-2124). In the first step, a mixed function oxidation modifies the glutamine synthetase. The modified enzyme, which is catalytically inactive, becomes susceptible to proteolytic attack. In the second step, a protease specific for the modified enzyme catalyzes the actual proteolytic degradation. The oxidatively modified glutamine synthetase was studied to determine the chemical differences between it and the native enzyme. Only a single alteration was found; one of sixteen histidine residues/subunit was altered by the oxidative modification. The modification introduced a carbonyl group into the protein, permitting isolation of a stable dinitrophenylhydrazone. No other differences were detected between the native and modified proteins. Specifically, the cysteine, methionine, phenylalanine, tyrosine, and tryptophan contents were not altered. A number of other prokaryotic and eukaryotic enzymes are also susceptible to oxidative modification. This covalent modification may be important in intracellular proteolysis, in mammalian host defense systems, in prevention of autolysis, in aging processes, and in oxygen toxicity.

Topics: Amino Acids; Ascorbic Acid; Escherichia coli; Glutamate-Ammonia Ligase; Histidine; Kinetics; Macromolecular Substances; Oxidation-Reduction; Phenylhydrazines

Topics: 2,6-Dichloroindophenol; Animals; Ascorbic Acid; Biological Assay; Bronchi; Male; Oxidation-Reduction; Phenylhydrazines; Pulmonary Alveoli; Rats; Rats, Inbred Strains; Specific Pathogen-Free Organisms; Therapeutic Irrigation; Trichloroacetic Acid

Topics: Ascorbic Acid; Beer; Chromatography, High Pressure Liquid; Phenylhydrazines

Topics: Animals; Ascorbic Acid; Chemical Phenomena; Chemistry; Ferrozine; Guinea Pigs; Humans; Kinetics; Male; Phenylhydrazines; Sulfhydryl Compounds; Tissue Distribution; Triazines

Measurements of ascorbic acid concentration in leukocytes by "high-performance" liquid chromatography (HPLC) provides better nutritional assessment, leading to better management, particularly of presymptomatic and critically ill patients. This procedure includes a simple, reproducible cell-separation technique that requires no more than 2 mL of whole blood. Cell populations are separable with greater than 95% purity and greater than 99% viability. Ascorbic acid is assayed by HPLC. The vitamin can be reproducibly quantified in concentrations as low as 0.1 microgram/mL of cell extract. The chromatographic procedure is very rapid, analysis being completed within 15 min after specimen preparation. The assay is suitable also for urine and protein-free filtrates of plasma and of other biological materials. Reference intervals for plasma, mononuclear leukocytes, and polymorphonuclear leukocytes were established. A preliminary clinical evaluation revealed that hospital patients were at a greater risk of ascorbic acid deficiency than expected.

Topics: Ascorbic Acid; Cell Separation; Chromatography, High Pressure Liquid; Humans; Leukocytes; Monocytes; Neutrophils; Phenylhydrazines; Reference Values

The evaluation of a recently published colorimetric method for plasma ascorbic acid determination, using phosphotungstic acid (PTA), was performed by comparison with the largely employed 2,4-dinitrophenylhydrazine (DNPH) procedure. The method has been evaluated according to International Federation of Clinical Chemistry (I.F.C.C.) recommendations. In particular, calibration procedures have been performed and precision, accuracy, linearity, specificity and sensitivity have been studied in biological samples. Linear regression analysis indicates that the two methods do not correlate completely. The PTA method shows a better recovery. The PTA method shares with the DNPH procedure a poor precision at low concentrations of vitamin C in plasma such as to make results less reliable at the clinically significative levels.

Topics: Ascorbic Acid; Colorimetry; Humans; Phenylhydrazines; Phosphotungstic Acid

Gadusol, C8H12O6, has been isolated from roes of the cod (Gadus morhua L.), i.e., ovaries that contain ripe eggs just before spawning. The concentration is about 4 g/kg dry wt. It has been identified as 1,4,5-trihydroxy-5-hydroxymethyl-2-methoxycyclo-hex-1-en-3-one and this structure was confirmed by synthesis of the anhydro tetra-acetate derivative from methyl 3,5-diacetoxy-4-methoxybenzoate. Concentrations of gadusol in the roes of other marine teleost fish examined are of the same order as in cod roes. Gadusol has some properties similar to ascorbic acid and both compounds, after oxidation, react with 2,4-dinitrophenylhydrazine in the commonly-used assay procedure for ascorbic acid. Specific assays showed that the concentrations of gadusol in the roes of marine fish are severalfold greater than those of ascorbic acid. Gadusol is structurally related to the mycosporines previously reported from a number of different organisms.

Topics: Animals; Ascorbic Acid; Chemical Phenomena; Chemistry; Cyclohexanols; Female; Fishes; Ovary; Ovum; Phenylhydrazines

Application ot the alpha,alpha'- dipyridyl method for determination of ascorbic acid in urine is described. The urine sample was acidified with trichloracetic acid and shaken with activated carbon to remove interfering substances. The acid filtrate was first neutralized (pH 7.0) by adding Na2HPO4. The dehydroascorbic acid was then reduced back to ascorbic acid by incubation with dithiothreitol. After removal of the excess dithiothreitol with N-ethylmaleimide, ascorbic acid was determined by measuring the reduction of ferric ion. The ferrous ion produced was coupled to alpha,alpha'-dipyridyl in the presence of H3PO4. Ferrous ion in urine samples, which theoretically interferes with the method, was removed by a combination of Na2HPO4 and H3PO4.

Topics: 2,2'-Dipyridyl; Ascorbic Acid; Chlorides; Dehydroascorbic Acid; Ferric Compounds; Oxidation-Reduction; Phenylhydrazines; Pyridines; Specific Gravity; Spectrophotometry; Urine

Topics: Ascorbic Acid; Hydrazines; Phenylhydrazines; Temperature

Topics: Ascorbic Acid; Body Fluids; Humans; Hydrazines; Phenylhydrazines; Urine; Vitamins

Topics: Ascorbic Acid; Humans; Hydrazines; Phenylhydrazines; Vitamins

Topics: Ascorbic Acid; Dehydroascorbic Acid; Humans; Hydrazines; Phenylhydrazines; Vitamins

Topics: Ascorbic Acid; Food; Humans; Hydrazines; Phenylhydrazines; Psidium

Topics: Ascorbic Acid; Hydrazines; Phenylhydrazines; Sugar Acids; Tissue Extracts

Topics: Ascorbic Acid; Hydrazines; Phenylhydrazines