ascorbic-acid and 10-10--dimethyl-9-9--biacridinium

Vitamin C (VC) has a strong antioxidant function evident as its ability to scavenge superoxide radicals in vitro. We verified that this property actually exists in vivo by using a real-time imaging system in which Lucigenin is the chemiluminescent probe for detecting superoxide in senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which cannot synthesize VC in vivo. SMP30/GNL KO mice were given 1.5 g/L VC [VC(+)] for 2, 4, or 8 weeks or denied VC [VC(-)]. At 4 and 8 weeks, VC levels in brains from VC(-) KO mice were <6% of that in VC(+) KO mice. Accordingly, superoxide-dependent chemiluminescence levels determined by ischemia-reperfusion at the 4- and 8 weeks test intervals were 3.0-fold and 2.1-fold higher, respectively, in VC(-) KO mice than in VC(+) KO mice. However, total superoxide dismutase activity and protein levels were not altered. Thus, VC depletion specifically increased superoxide generation in a model of the living brain.

Topics: Acridines; Animals; Antioxidants; Ascorbic Acid; Body Weight; Brain; Calcium-Binding Proteins; Carboxylic Ester Hydrolases; Intracellular Signaling Peptides and Proteins; Luminescent Agents; Mice; Mice, Knockout; Superoxides

The goal of this research was to measure in vitro the inhibitory constants of the antioxidants ascorbic and uric acid in urine, with lucigenin enhanced chemiluminescence (CL) in Fenton's system. Maximum CL emission is registered in urine containing H2O2 (5.10(-4) M), Fe2+ (5.10(-5) M), EDTA (5.10(-5) M), and chemical enhancer lucigenin (10(-4) M) at pH 5.5 and 36 degrees C. Ascorbic acid exhibits up to 4-fold stronger antioxidant effect than uric acid. The constants of antioxidant inhibition in urine were measured at concentrations 10(-3) and 10(-4) M: for ascorbic acid, 5.92 +/- 0.04 and 24.05 +/- 1.82 micromol.sec(-1); for uric acid, 1.60 +/- 0.02 and 21.45 +/- 0.97 micromol.sec(-1), respectively. Three phases of CL kinetics of urine are well observed: spontaneous CL (0-10 sec), fast flash of CL (10-50 sec), and latent period (50-300 sec). The antioxidant efficiency of ascorbic and uric acids in the final stage of catabolic processes in the body is discussed.

Topics: Acridines; Antioxidants; Ascorbic Acid; Humans; In Vitro Techniques; Kinetics; Lipid Peroxidation; Luminescent Measurements; Uric Acid

Seminal plasma protects spermatozoa from the detrimental effects of reactive oxygen species such as hydrogen peroxide. We investigated the lucigenin-dependent chemiluminescence in cell-free seminal plasma from andrological patients. The seminal plasma was separated from cells by centrifugation. In all seminal plasmas studied lucigenin-dependent chemiluminescence (LCL) was detected. The LCL showed a strong pH-dependence. The signal was stable if samples were stored at +4 degrees C for up to 4 days or up to 8 days at -80 degrees C. Filtration of the samples (0.45 and 0.22 microm pore size) did not lower their luminescence. The addition of superoxide dismutase (SOD) and ascorbic acid oxidase (AAO) lowered LCL nearly to baseline values while trolox and desferal showed moderate effect, whereas allopurinol had no effect. Electron paramagnetic resonance spectroscopy demonstrated ascorbyl radicals in seminal plasma. Physiological concentrations of ascorbic acid yielded SOD-inhibitable lucigenin-chemiluminescence. The nitroblue-tetrazolium assay showed that ascorbic acid in buffer solution produced formazan. Superoxide-anion radicals were not detected in seminal plasma by the spin-trap DEPMPO due to their low steady state concentration. It is concluded that in seminal plasma ascorbate reacts with molecular oxygen yielding ascorbyl radicals and superoxide anion. If lucigenin is added to seminal plasma, reducing substances present, such as ascorbate, reduce lucigenin to the corresponding radical; this radical reacts with molecular oxygen and also forms O2-. So LCL in human seminal plasma results from the autoxidation of ascorbate and the oxidation of the reduced lucigenin. While the physiological relevance of the former mechanism is unknown, the latter is an artifact.

Topics: Acridines; Adult; Allopurinol; Antioxidants; Ascorbate Oxidase; Ascorbic Acid; Chromans; Deferoxamine; Electron Spin Resonance Spectroscopy; Free Radicals; Humans; Hydrogen-Ion Concentration; Infertility, Male; Luminescent Measurements; Male; Oxidants; Oxidation-Reduction; Oxygen; Oxygen Consumption; Reactive Oxygen Species; Semen; Semen Preservation; Superoxide Dismutase; Temperature

Lucigenin has been frequently used for the chemiluminescent detection of superoxide (*O-2) in intact tissue. More recent studies, however, revealed that lucigenin per se causes formation of *O-2 raising doubt about this probe to detect reliably *O-2. We therefore tested a more recently described chemiluminescence probe (2-methyl-6-phenyl-3,7-dihydroimidazol[1,2-alpha]pyrazine-3-one (CLA)) to estimate the ability of vascular tissue to generate *O-2 as an alternative to lucigenin. In a cell free system as well as in vascular tissue, CLA-enhanced chemiluminescence was dose dependently inhibited by superoxide dismutase (SOD), vitamin C and sodium nitroprusside (SNP). Electron spin resonance studies revealed that lucigenin (250 microM) but not CLA (1 microM) caused extra *O-2 production in vascular tissue. Stimulation of vessels with NADH (200 microM) increased CLA enhanced chemiluminescence, which was inhibited by low concentrations of superoxide dismutase (20U/ml). Endothelial removal as well as the nitric oxidase-synthase inhibitor increased CLA chemiluminescence in vessels. We conclude that CLA is a sensitive and specific chemiluminescence probe to detect *O-2 production in intact vascular tissue.

Topics: Acridines; Animals; Aorta; Ascorbic Acid; Cell Extracts; Electron Spin Resonance Spectroscopy; Luminescent Measurements; Muscle, Smooth, Vascular; omega-N-Methylarginine; Pyrazines; Rabbits; Superoxide Dismutase; Superoxides

For the investigation of luminol (LM)-and lucigenin (LC)-amplified chemiluminescence (CL) in rat liver microsomes using both a liquid-scintillation counter (LKB/Wallac 1219 Rackbeta) and a Berthold luminometer (AutoLumat LB 953) optimal incubation mixtures and conditions and basic kinetics have been established. Whereas calibration curves for both LM- and LC-CL are performed with hydrogenperoxide (LC quantum yield is 6.25 fold higher as that of LM), distinct differences were revealed with microsomes, indicating that different reactive oxygen species (ROS) are determined: Both LM- and LC-CL follow the kinetics of enzymatic reactions in terms of dependence on protein and NADPH or NADH concentration, time course, temperature etc., but with differences. LM-CL does not work without addition of Fe2+, whereas LC-CL does. Both copper ions and copper bound in a complex abolish CL, LC-CL being much more sensitive. Isolated cytochrome P-450 (P450) and NADPH P450 reductase from liver of pheno-barbital treated rats alone proved to be inactive in LM-and LC-CL production, whereas te combination 1:1 without and with addition of lipid was highly active in both LM-and LC-CL. Ascorbic acid and glutathione as scavengers diminish both LM- and LC-CL in concentrations higher then 10(5). Dimethyl-sulfoxide (DMSO) was ineffective in LM-CL up to concentrations of 0.2 M, the very high concentration of 2 M diminished LM-CL only to 1/3. LC-CL was diminished starting at concentrations of 100 mM and at 2 M only 10% of maximum LC-CL was observed. The trap substance N-t-butyl-a-phenylnitrone (BNP) also diminished LC-CL more effectively than LM-CL. Clearcut differences were revealed by the addition of catalase and superoxide dismutase: both enzymes diminished LM-CL only, without any influence on LC-CL. Hexobarbital, a potent uncoupler of P450, enhances LM-CL fivefold, whereas LC-CL is barely influenced. Aniline (without uncoupling capability) decreased both LM-and LC-CL increasingly with increasing concentrations. Therefore the conclusion is drawn that LM-CL measures in liver microsomes predominantly superoxide anion radicals, whereas LC-CL is mainly a measure for microsomal hydroxyl radical formation or of reactive organic radicals. With microsomes of phenobarbital and beta-naphthoflavone treated rats CL was much higher but in principle the same kinetic characteristics could be shown. All results on microsomes were obtained uniformly with the liquid scintillation counter and the Berthold

Topics: Acridines; Aniline Compounds; Animals; Ascorbic Acid; Catalase; Copper; Cyclic N-Oxides; Cytochrome P-450 Enzyme System; Dimethyl Sulfoxide; Glutathione; Hexobarbital; Iron; Kinetics; Luminescent Measurements; Luminol; Male; Microsomes, Liver; NADPH Dehydrogenase; Nitrogen Oxides; Proteins; Rats; Rats, Wistar; Superoxide Dismutase; Temperature; Time Factors

NMDA, the specific agonist of glutamate gated ion channels permeable to calcium, is implicated as a causal factor in the pathogenesis of several neurobiological disorders such as stroke, seizures, ischemia, and chronic neurodegenerative diseases. On the other hand, evidence on the roles of oxidative mechanisms involved in NMDA-induced neurotoxicity is accumulating. In this study, we have used chemiluminescence measurements as an easy, rapid and sensitive assay to investigate the effects of NMDA and oxidative stress on brain cell vulnerability. Rat brain homogenates were incubated with increasing concentrations of glutamate and NMDA. Production of reactive oxygen species was followed by single photon emission measurements using the specific enhancers luminol and lucigenin. Increases in emission were observed at excitotoxic concentrations of glutamate and NMDA. Other parameters of oxidative stress such as diene conjugates, TBARS and carbonyl groups were also investigated. Our results indicated that chemiluminescence measurements may be used to study involvement of oxidative stress in neurotoxicity.

Topics: Acridines; Animals; Ascorbic Acid; Brain; Free Radicals; Frontal Lobe; Luminescent Measurements; Luminol; N-Methylaspartate; Nitroprusside; Oxidative Stress; Rats; Reactive Oxygen Species; Thiobarbituric Acid Reactive Substances

During oxidation of Fe(2+) catalyzed by tetracyclines there was recorded lucigenin, activated chemiluminescence evident of generation of the oxygen radicals. It was also observed that during the Fe(2+) oxidation by the molecular oxygen catalyzed by tetracyclines there generated hydrogen peroxide which accelerated the Fe(2+) oxidation recorded photometrically by the formation of strongly absorbing tetracycline complexes with Fe(2+). In the presence of ascorbate reducing Fe(2+) in the complexes with tetracyclines and their subsequent oxidation there generated radicals modifying the antibiotic molecules evident from a change in their absorption spectra after the respective incubation. The results offered a pattern describing the mechanism of the tetracycline toxic effect on biological objects.

Topics: Acridines; Ascorbic Acid; Catalysis; Ferrous Compounds; Luminescent Measurements; Oxidation-Reduction; Reactive Oxygen Species; Spectrophotometry; Tetracyclines

The effects of several antioxidants on the three major functions of human neutrophils--oxidative burst, secretion and leukotriene formation--were investigated with special emphasis on the lipophilicity. The most striking differences were obtained when ascorbate and the lipophilic ester ascorbyl palmitate were compared. As expected, the luminol- and lucigenin-dependent chemiluminescence was inhibited by all antioxidants to a different degree. Ascorbyl palmitate was able to block the biphasic luminol-dependent response completely with IC50 values of 10 and 25 microM for the first and second phase, respectively. In contrast, ascorbate only blocked efficiently the first phase of the response. The secretion of elastase was inhibited by ascorbyl palmitate dose-dependently with an IC50 value of around 200 microM, whereas ascorbate was completely inactive. Electron microscopy supported the assumption that inhibition was due to a block in degranulation and not to enzyme inactivation. This was further supported by a parallel, although somewhat lower, inhibition of other secretory enzymes like myeloperoxidase, beta-glucuronidase or lysozyme. Cells treated with the Ca2+-ionophore A23187 responded by LTB4-synthesis which was also inhibited by ascorbyl palmitate. A very efficient inhibition was observed in cell homogenates with an IC50 value of 1.5 microM. No inhibition by ascorbate was detected in both systems. Concomitant with the inhibition of 5-lipoxygenase the activity of 15-lipoxygenase increased. We conclude that cellular reductants may control neutrophil functions and that the inhibition by ascorbyl palmitate of the three processes relevant for inflammatory responses could be of therapeutic importance.

Topics: Acridines; Antioxidants; Arachidonate 5-Lipoxygenase; Ascorbic Acid; Cytoplasmic Granules; Dose-Response Relationship, Drug; Humans; Leukotriene B4; Luminescent Measurements; Luminol; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils