Evaluation of total antioxidant potential (TRAP) and total antioxidant reactivity from luminol-enhanced chemiluminescence measurements.

The effect of antioxidants and biological fluids on the intensity of luminol induced chemiluminescence by radicals derived from the thermolysis of 2,2'-azo-bis(2-amidinopropane) has been employed to monitor TRAP and TOTAL ANTIOXIDANT REACTIVITY (TAR) levels. The latter parameter, which considers not only the quantity of oxidants but also their reactivity, is considered a potentially more useful index of the antioxidant status of a biological fluid. The results obtained employing human blood plasma and human urine show that the detected antioxidant capacity of both fluids is mainly related to the uric acid concentration of the samples.

Spontaneous urinary visible luminescence: characteristics and modification by oxidative stress-related clinical conditions.

Urinary visible luminescence is the result of the excretion of oxidized biomolecules and, as such, could provide a valuable index of systemic oxidative stress. The characteristics of the urinary luminescence that support this proposal are reviewed and the data obtained for patients with hyperthyroidism and children with Duchenne muscular dystrophy are also discussed. Enhanced urinary chemiluminescence was observed in both pathologies. A similar enhancement was obtained when the urinary luminescence of smokers was compared to that of non-smokers. The possibilities and limitations of this noninvasive methodology for the evaluation of systemic oxidative stress is critically evaluated.

Spontaneous urinary visible luminescence: characteristics and modification by oxidative stress-related clinical conditions

Urinary visible luminescence is the result of the excretion of oxidized biomolecules and, as such, could provide a valuable index of systemic oxidative stress. The characteristics of the urinary luminescence that support this proposal are reviewed and the data obtained for patients with hyperthyroidism and children with Duchenne muscular dystrophy are also discussed. Enhanced urinary chemiluminescence was observed in both pathologies. A similar enhancement was obtained when the urinary luminescence of smokers was compared to that of non-smokers. The possibilities and limitations of this noninvasive methodology for the evaluation of systemic oxidative stress is critically evaluated.

Enhanced urinary spontaneous luminescence in Duchenne muscular dystrophy.

Urinary spontaneous visible luminescence is enhanced in children with Duchenne Muscular Dystrophy (DMD). This result is indicative of systemic oxidative stress in DMD patients. It is proposed that measurement of the urinary luminescence could be employed to follow the progress of the disease, as well as the response of the patients to antioxidant therapy.

Inactivation of yeast alcohol dehydrogenase by alkylperoxyl radicals. Characteristics and influence of nicotinamide-adenine dinucleotides.

The study of the interaction of alkylperoxyl radicals generated by the aerobic thermolysis of 2,2'-azobis(2-amidinopropane) (AAP) with yeast alcohol dehydrogenase (YADH) revealed a high reactivity of the enzyme, with an average of about 20 radicals per added YADH tetramer being needed to elicit its total inactivation. NAD+ enhanced YADH inactivation at NAD+/YADH molar ratios from 0.25 to 1, decreasing the rate of the process when added in excess to the enzyme concentration. At NADH/YADH molar ratios greater than 1, NADH exhibited a protective effect characterized by a poorly defined induction time and lower inactivation rates, which progressively increased during the reaction period. These changes occurred concomitantly with the oxidation of NADH into NAD+, which might counteract the protective effect of NADH. Under similar conditions, NADP+ did not modify AAP-induced YADH inactivation, while NADPH exhibited a modest protection at NADPH/YADH molar ratios greater than 1. It is concluded that YADH inactivation by alkylperoxyl radicals is strongly dependent on the redox state of the NADH-NAD+ couple, as the rates of the process at different time intervals inversely correlate with the respective NADH/NAD+ ratios.

Is spontaneous urinary visible chemiluminescence a reflection of in vivo oxidative stress?

Based on the characteristics of the visible spontaneous luminiscence of human urine, we propose that this luminescence is due to the dark decomposition of long-lived luminescent intermediates produced by oxidations at the cellular level, and that it might reflect the development of oxidative stress conditions in vivo. This hypothesis is consistent with the higher emission levels monitored in the urine of hyperthyroid patients and the correlation observed between urinary thiobarbituric acid reactive substances (TBARS) levels and urinary chemiluminescence.

2,2'-Azo-bis-amidinopropane as a radical source for lipid peroxidation and enzyme inactivation studies.

1. 2,2'-Azo-bis-amidinopropane (ABAP) thermal decomposition produces free radicals that initiate the lipid peroxidation of erythrocyte ghost membranes. 2. Addition of 6-n-propyl-2-thiouracil decreases the rate of the process, both by decreasing consumption of the natural antioxidants of the membranes and by direct interaction with the free radicals involved in the lipid peroxidation. 3. Peroxyl radicals produced in ABAP thermal decomposition inactivate lysozyme, horseradish peroxidase (HRP) and glucose oxidase, in that order. The number of enzyme molecules inactivated per radical introduced into the system increases with enzyme concentration. 4. Competitive studies employing mixtures of enzymes show that the order of reactivity of these enzymes towards the peroxyl radicals is the opposite to that obtained for the rate of enzyme inactivation. It is concluded that inactivation efficiency is determined mainly by the average number of free radicals that must react with an enzyme molecule to produce its inactivation, and that this number is directly related to the molecular weight of the enzyme.

A light-induced tryptophan-riboflavin binding: biological implications.

We review here the covalent photo-binding induced by visible light between the essential amino acid tryptophan and the vitamin riboflavin. We discuss the biological implications of this photoadduct in relation to the hepatotoxic and cytotoxic effect associated to parenteral nutrients and to culture media exposed to the action of light, respectively. We also analyze the formation of a photo-binding between riboflavin and the residues of tryptophan present in the proteins of the eye lens, a tissue which is permanently exposed to visible light.

Free radical scavenging activity of carnosine.

The capacity of carnosine to decrease free radical-induced damage was evaluated using the oxidation of brain homogenates, the 2,2'-azobis-2-amidino propane-induced oxidation of erythrocyte ghost membranes, the radiation induced inactivation of horseradish peroxidase and the 2,2'-azobis-2-amidino propane-induced inactivation of lysozyme. Carnosine addition up to 17 mM did not produce any significant protection in either lipid peroxidation system, as assayed by the oxygen uptake rate. Carnosine addition reduces the intensity of the visible luminescence emitted, apparently due to a dark decomposition of the luminescent intermediates. Carnosine addition protects horseradish peroxidase and lysozyme from free radical mediated inactivation. The mean carnosine concentrations required to inhibit the inactivation rates by 50% were 0.13 mM and 0.6 mM for horseradish peroxidase and lysozyme, respectively.

Alpha-oxidation of alpha-hydroxyfatty acids in rat brain. Possible involvement of an alpha-peroxylactone.

Combined--but not individual--microsomal and supernatant fractions obtained from rat brains not only consume oxygen but also provoke emission from added chlorophyll. These results are consistent with literature data (Levis and Mead, J. Biol. Chem. 239, 77 [1964]) for trapping of radioactive 14CO2 following addition of alpha-hydroxy-[1-14C]stearic acid. The most plausible explanation for emission is the interaction of chlorophyll with an alpha-peroxylactone. An intermediary alpha-peroxylactone in alpha-oxidation is consistent with other available data (Salim-Hanna, Campa and Cilento, Photochem. Photobiol. 45, 849 [1987]; Campa, Salim-Hanna and Cilento, Photochem. Photobiol. 49, 349 [1989]) and, on chemical grounds, provides a feasible route to the final products.

Toxic effect of a photo-induced tryptophan-riboflavin adduct on F9 teratocarcinoma cells and preimplantation mouse embryos.

Solutions containing L-tryptophan and riboflavin exposed to visible light, under N2 atmosphere, yield a tryptophan-riboflavin adduct, able to inhibit the growth of cultured F9 teratocarcinoma cells. This same effect was found in the presence of a mixture of the tryptophan photooxidation products and the adduct, when using solutions previously irradiated with visible light in an O2 atmosphere. A cytotoxic effect was also observed with embryos incubated in the presence of a tryptophan-riboflavin adduct, in the latter case necrosis and embryo development arrest occurred.

Riboflavin status and photo-induced riboflavin binding to the proteins of the rat ocular lens.

The presence of radioactivity in lenses of rats injected intraperitoneally with a solution of tryptophan and 14C-riboflavin, irradiated during 48 hours and under N2 atmosphere, was detected. The excitation fluorescence spectrum (lambda emission = 520 nm) of the soluble lens fraction corresponds to the riboflavin decomposition product of lumichrome type. When 14C-riboflavin enriched lenses were exposed to visible light, a photo-induced binding between riboflavin and a water insoluble protein fraction of the lenses was observed. This finding may help to clarify the lens modifications during aging and in cataractogenesis.

Obtention of a photo-induced adduct between a vitamin and an essential aminoacid. Binding of riboflavin to tryptophan.

Studies in animals have suggested that the products of the irradiation of tryptophan in the presence of riboflavin may play a role in the development of hepatic dysfunction during parenteral nutrition. In this paper we describe the formation of an adduct between tryptophan and riboflavin obtained as a consequence of an anaerobic irradiation of these compounds. Through the use of molecular sieves and of an ion-exchange resin it was possible to separate the photo-adduct from the dimer riboflavin and other reaction products. The various fractions were characterized on the basis of their absorption and emission spectra. Also used were measures of anisotropy of fluorescence emission in order to characterize the derived adduct.