Reactions of 1-methyl-2-phenylindole with malondialdehyde and 4-hydroxyalkenals. Analytical applications to a colorimetric assay of lipid peroxidation.

Under acidic and mild-temperature conditions, 1-methyl-2-phenylindole was found to react with malondialdehyde (MDA) and 4-hydroxyalkenals to yield a stable chromophore with intense maximal absorbance at 586 nm. The use of methanesulfonic acid results in optimal yields of chromophore produced from MDA as well as from 4-hydroxynonenal. By contrast, the use of hydrochloric acid results in an optimal yield of chromophore produced from MDA and a negligible reaction of 4-hydroxynonenal. Taking advantage of such chromogenic reactions, we developed a new colorimetric assay of lipid peroxidation. Using a methanesulfonic acid-based medium, MDA and 4-hydroxyalkenals can be measured at the 586 nm wavelength. However, the presence of endogenous inhibitors of the reaction with 4-hydroxyalkenals is common, and this means that the latter may be underestimated in some biological samples. The assay performed in a hydrochloric acid-based medium enables the specific measurement of MDA in the presence of 4-hydroxyalkenals. Upon hydrolysis of Schiff bases in hydrochloric acid (pH 1.5), either assay can be used to specifically measure the amount of total MDA in biological samples because 4-hydroxyalkenals undergo an irreversible cyclization reaction under the hydrochloric acid-based conditions of hydrolysis. The two assays were applied to the determination of the amount of MDA alone and of MDA and 4-hydroxyalkenals in an in vitro model of lipid peroxidation. This methodology was also used to clarify complex patterns of tissue-specific MDA production in vivo, following hydrolysis of Schiff bases, in rodents treated with doxorubicin.

Reactions of N-methyl-2-phenylindole with malondialdehyde and 4-hydroxyalkenals. Mechanistic aspects of the colorimetric assay of lipid peroxidation.

Under specific acidic conditions, both malondialdehyde (1, MDA) and 4-hydroxynonenal (2, 4-HNE) react with N-methyl-2-phenylindole (3) to give the same chromophoric cyanine 4 with maximal absorbance at 586 nm. Under such conditions, the reaction of 3 with 4-HNE (2) as well as with alkanals yields a second chromophoric cyanine 10 with maximal absorbance at 505 nm. The influence of different acids, iron(III), and oxygen on the reaction of 3 with such aldehydes was studied in detail. Under anaerobic conditions, the acid-induced reaction of 4-HNE with 3 afforded three rapidly interconverting intermediates, 5-7. Their subsequent fragmentation to 4 and hexanal in the presence of iron(III) and oxygen is consistent with the tandem beta-fragmentation of an indolyl radical cation. 1-Indolylalkenes were identified as essential intermediates in the acid-induced reaction of 3 with alkanals. A very mild iron(III)-catalyzed fragmentation of these intermediates afforded the corresponding 3-formylindole 11 as the direct precursor of the 505 nm chromophore 10. Such reactions were markedly influenced by the nature of the acid. Contrary to the rapid chromogenic reaction of 4-HNE which was observed in the presence of methanesulfonic acid, the HCl-induced reaction of 4-HNE with 3 did not afford the 586 nm chromophore. Furthermore, hexanal did not yield the 505 nm chromophore 10 upon reaction with 3 in the presence of HCl, again in contrast with the rapid chromogenic reaction which was observed in the presence of methanesulfonic acid. Comparison of the reaction mixtures under the two assay conditions confirmed that the same intermediates were formed. We conclude that the nature of the acid plays a crucial role in the oxidative fragmentation of intermediates into chromophores, allowing the selective assay of MDA in the presence of 4-HNE, using HCl acidic conditions.

[Metabolism and antioxidant function of glutathione]. / Métabolisme et fonction antioxydante du glutathion.

Glutathione (gamma-glutamyl-cysteinyl-glycine or GSH) is a cysteine-containing tripeptide with reducing and nucleophilic properties which play an important role in cellular protection from oxidative damage of lipids, proteins and nucleic acids. GSH regulates the metabolism of proteins and their activities by means of thiol-disulfide exchange. During oxidative stress, GSH plays a key role of protection and detoxification as a cofactor of glutathione peroxidases and glutathione-S-transferases. There are synergistic interactions between GSH and other components of the antioxidant defense system such as vitamin C, vitamin E and superoxide dismutases.

Partial prevention of glutathione depletion in rats following acute intoxication with diethylmaleate.

To assess the ability of L-2-oxothiazolidine-4-carboxylate (OTC) to stimulate the biosynthesis of glutathione (GSH) in non-fasted male rats, the time-courses of GSH and cysteine contents were studied in liver, kidney, heart and brain, following a single intraperitoneal injection of OTC (5 mmol/kg), with or without co-administration of the GSH depletor diethylmaleate (3 mmol/kg). In the absence of diethylmaleate, OTC did not change the GSH or cysteine content of heart and kidney. The liver was the only organ where systemic administration of OTC resulted in a fast and quasi-linear increase in GSH as a function of time, with no appreciable lag-time. A maximal, i.e. 2.1-fold increase in liver GSH was induced by OTC at the times corresponding to the low GSH values of the diurnal cycle observed in control rats. A smaller, i.e. 1.4-fold increase in brain GSH was observed after 6 hours. A marked increase in cysteine always preceded that of GSH in liver and brain. In the liver, the OTC-mediated stimulation of GSH biosynthesis was optimal when cysteine delivery was achieved at the onset of the cysteine decrease that was observed in the diurnal cycle of control rats. These results support the view that cysteine is a limiting factor in the biosynthesis of GSH. Following an acute dose of diethylmaleate (3 mmol/kg), OTC afforded a general and significant protection of rat tissues against GSH depletion.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutathione and cysteine depletion in rats and mice following acute intoxication with diethylmaleate.

We examined the dose-dependent glutathione (GSH) depletion in liver, kidney, heart and brain of rats and mice, and cysteine depletion in rat kidney, following i.p. administration of diethylmaleate (DEM). In either rodent, the fall in total GSH concentration in liver and heart reached an upper value of 90 and 80% with 3 and 4 mmol DEM/kg respectively, which did not increase with higher doses. This study suggests that the residual level of GSH corresponds to the mitochondrial pool, in which case DEM might serve as a tool for the measurement of mitochondrial GSH ex vivo. In further experiments, we studied the time course of GSH and cysteine after administration of 3 mmol DEM/kg in rat tissues. Maximal depletion was reached approximately 1 hr after the i.p. injection. Subsequent GSH repletion was fast in liver and kidney, whereas it was slow in heart and brain, with a return to control values by 8-12 and by 48 hr after intoxication, respectively. This study provides new data for cardiac GSH and renal cysteine decrease after intoxication with DEM and should help to optimize GSH depletion models for further pharmacological investigations, especially when the use of inhibitors of glutathione metabolic turnover is undesirable and when side-effects other than GSH depletion must be avoided.