2,2'-Azo-bis(2-amidinopropane) thermolysis induces luminol luminescence. The luminescence intensity is quenched by SOD, catalase, Trolox and human blood serum. However, the time course of the light intensity profile is different for the different additives. In particular, the quenching efficiency of Trolox and human blood serum decreases with time after addition. Double quenching experiments show that SOD and Trolox are not competitive quenchers, while a simple competition can be established between Trolox and human blood serum in trapping a common intermediate. From the kinetic analysis of the data it is concluded that, at least at low additive concentrations, Trolox scavenges a luminol derived radical. Higher concentrations of Trolox or human blood serum produce induction times that are proportional to the additives concentrations. The possibility of employing luminol luminescence in the evaluation of TRAP levels and the capacity of biological samples to scavenge free radicals is discussed.

The kinetics of hydrolysis of 2-naphthyl acetate (2-NA) catalyzed by α-chymotrypsin (α-CT), in reverse micellar solutions formed by glycerol (GY)−water (38% v/v) mixture/sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/n-heptane has been determined by spectroscopic measurements. To compare the efficiency of this reaction with that observed in micelles with water in the core, as well as in the corresponding homogeneous media, the reaction was also studied in water/AOT/n-heptane reverse micellar solutions and in both homogeneous media (water and GY−water, 38% v/v mixture). In every media, α-CT was characterized by the absorption and emission spectra, the fluorescence lifetimes, and the fluorescence anisotropy of its tryptophan residues. The effect of AOT concentration on the kinetic parameters obtained in the micellar systems was determined, at a constant molar ratio of the inner polar solvent and surfactant. Moreover, the data obtained allowed the evaluation of the 2-NA partition constant between the organic and the micellar pseudophase. It is shown that the addition of GY to the micelle interior results in an increase in the catalytic properties of α-CT. The fluorescence anisotropy studies in the different media show that the addition of GY increases the viscosity as compared with the aqueous systems. It seems that the GY addition to the reverse micellar aggregates results in a decrease of the conformational mobility of α-CT, which leads to an increase of the enzyme stability and activity.

Although the importance of seminal plasma in the protection of spermatozoa against reactive oxygen species is well known, only a few studies have investigated its antioxidative properties and the possible relationship between infertility and plasmatic antioxidant defences. The aim of the present study was to assess the status of the total non-enzymatic antioxidant defences of human seminal plasma. Semen samples were obtained from 101 patients consulting for infertility and 15 fertile donors. A total reactive antioxidant potential (TRAP) in seminal plasma was determined by luminol-chemiluminescence. The relationship of seminal TRAP values to lipid peroxidation was also evaluated. Our results show that semen samples from fertile controls show total antioxidant capacity at higher frequency and value than equivalent samples from suspected subfertile men. This fact as well as the inverse relationship observed between antioxidant capacity and lipid peroxidation potential strongly suggest that impaired antioxidant defences may play a role in infertile disorders.

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We recorded potassium currents from single skeletal muscle channels incorporated into bilayers or using macropatches of Xenopus laevis oocytes membranes expressing the human Slowpoke (hSlo) ␣-subunit. Exposure of the intracellular side of KV,Ca channels to H2O2 (4-23 mM) leads to a time-dependent decrease of the open probability (Po) without affecting the unitary conductance. H2O2 did not affect channel activity when added to the extracellular side. These results provide evidence for an intracellular site(s) of H 2O2 action. Desferrioxamine (60 M) and cysteine (1 mM) completely inhibited the effect of H2O2, indicating that the decrease in Po was mediated by hydroxyl radicals. The reducing agent dithiothreitol (DTT) could not fully reverse the effect of H2O2. However, DTT did completely reverse the decrease in Po induced by the oxidizing agent 5,5Ј-dithio-bis-(2-nitrobenzoic acid). The incomplete recovery of KV,Ca channel activity promoted by DTT suggests that H2O2 treatment must be modifying other amino acid residues, e.g., as methionine or tryptophan, besides cysteine. Noise analysis of macroscopic currents in Xenopus oocytes expressing hSlo channels showed that H2O2 induced a decrease in current mediated by a decrease both in the number of active channels and Po.

Maple syrup urine disease (MSUD) is an inherited neurometabolic disorder caused by deficiency of branched-chain alpha-keto acid dehydrogenase complex activity which leads to tissue accumulation of the branched-chain alpha-keto acids (BCKAs) alpha-ketoisocaproic acid (KIC), alpha-ketoisovaleric acid (KIV) and alpha-keto-beta-methylvaleric acid (KMV) and their respective amino acids. Neuropathologic findings characteristic of the disease are cerebral edema and atrophy, whose pathophysiology is poorly known. In the present study, we investigated the in vitro effect of BCKAs on various parameters of oxidative stress, namely chemiluminescence (CL), thiobarbituric acid-reactive substances (TBA-RS), total radical-trapping antioxidant potential (TRAP), total antioxidant reactivity (TAR), and the activities of the antioxidant enzymes catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD) in cerebral cortex of 30-day-old rats. The major effects observed were with KIC, which significantly increased CL and TBA-RS measurements, decreased TRAP and TAR values, and markedly inhibited GPx activity. KMV and KIV increased CL and decreased TRAP and TAR values. In contrast, these compounds did not affect CAT and SOD activities. Taken together, it was shown that: the BCKAs studied stimulated lipid peroxidation and reduced the brain antioxidant defences, suggesting an increased production of free radicals. In case the in vitro effects here detected also occur in vivo in MSUD, it can be presumed that oxidative stress might contribute, at least in part, to the brain damage found in the affected patients.

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