Claude Tallineau

Plasmalogen degradation by oxidative stress: production and disappearance of specific fatty aldehydes and fatty α-hydroxyaldehydes

Plasmalogens are often considered as antioxidant molecules that protect cells from oxidative stress. Their vinyl ether bond could indeed be among the first targets for newly formed radicals. However, the long chain aldehydes released from plasmalogens were seldom studied and possible injurious or harmless effects were poorly examined. Thus, the sensitivity of the vinyl ether bond of plasmalogens was investigated in a cerebral cortex homogenate under UV irradiation- or Fe2+/ascorbate-induced peroxidation. Kinetics of aldehyde production was followed by gas chromatography/mass spectrometry. This confirmed that plasmalogens were highly sensitive to oxidative stress (70% cleavage after 90 min UV irradiation and 30% after 30 min of Fe2+/ascorbate). The aldehydes corresponding to sn-1 position 16:0, 18:0, or 18:1 were poorly detected. Conversely, oxidation of plasmalogens yielded preferentially 15:0, 17:0, and 17:1 aldehydes under UV and the alpha-hydroxyaldehydes 16:0-OH and 18:0-OH following a Fe2+/ascorbate oxidation. Kinetics showed that free aldehydes and above all free alpha-hydroxyaldehydes disappeared from the medium as soon as produced. Consequently, the behavior of these released aldehydes in the tissues has to be investigated in order to ascertain the protective effect of plasmalogens against oxidation.

Autoxidation of Rat Brain Homogenate: Evidence for Spontaneous Lipid Peroxidation. Comparison with the Characteristics of Fe2+- and Ascorbic Acid-Stimulated Lipid Peroxidation

Aerobically-incubated brain homogenates are known to undergo autoxidation characterized by spontaneous TBARS production, presumably as a result of lipid peroxidation. However, TBARS measurement alone, because of its lack of specificity, is not sufficient to demonstrate the occurrence of lipid peroxidation in complex biological systems. This study, undertaken to determine whether or not spontaneous oxidation of rat brain homogenate is due to lipid peroxidation, measured different specific markers of this process (fatty acids, lipid aldehydes and the formation of fluorescence products) and studied changes in alpha-tocopherol. Incubation of rat brain homogenates at 37 degrees C under air led to spontaneous TBARS formation, which was accompanied by lipid aldehydes and lipid fluorescence products as well as polyunsaturated fatty acid (PUFA) degradation. Alpha-tocopherol was also consumed. On the whole, these results demonstrate that autoxidation of brain homogenate is a spontaneous lipid peroxidation process. When homogenates were exposed to Fe2+ and ascorbic acid-induced oxidative stress, lipid peroxidation was enhanced. However, spontaneous and stimulated peroxidation showed similar patterns not characteristic of classical lipid peroxidation, i.e. without the lag and accelerating phases typical of a propagating chain reaction. PUFA degradation was limited despite stimulation of peroxidation.

Copper-induced lipid peroxidation and hemolysis in whole blood: evidence for a lack of correlation

In order to establish a possible relationship between hemolytic and peroxidant activities of copper ions, lipid peroxidation was studied in plasma and whole blood incubated for 24 h with different concentrations of copper. The lipid peroxidation was investigated by the determination of thiobarbituric acid-reactive species, conjugated dienes and fluorescent lipid chromophores. The copper-induced lipoperoxidation was clearly demonstrated in plasma incubated with high concentrations of copper (12.10(-4) and 20.10(-4) M); in whole blood, all the lipoperoxidation products were increased in the plasma, while the fluorescent lipid chromophores remained unchanged in red cells. With a copper concentration similar to that found in acute copper intoxication (4.10(-4) M) no lipoperoxidation was observed and yet hemolysis occurred, reduced glutathione (GSH) decreased dramatically and methemoglobin (MetHb) increased. From these results, we assume that, despite its prooxidant activity and its capacity to produce lipoperoxidation, it has not been proven that copper ions at pathophysiological concentrations induce hemolysis by an oxidative mechanism.

Evidence for the involvement of (Cu-ATP)2− in the inhibition of human erythrocyte (Ca2+ + Mg2+-ATPase by copper

The effects of copper on the activity of erythrocyte (Ca2+ + Mg2+)-ATPase have been tested on membranes stripped of endogenous calmodulin or recombined with purified calmodulin. The interactions of copper with Ca2+, calmodulin and (Mg-ATP)2- were determined by kinetic studies. The most striking result is the potent competitive inhibition exerted by (Cu-ATP)2- against (Mg-ATP)2- (Ki = 2.8 microM), while free copper gives no characteristic inhibition. Our results also demonstrate that copper does not compete with calcium either on the enzyme or on calmodulin. The fixation of calmodulin on the enzyme is not altered in the presence of copper as shown by the fact that the dissociation constant remains unaffected. It may be speculated that (Cu-ATP)2- is the active form of copper, which could plausibly be at the origin of some of the pathological features of erythrocytes observed in conditions associated with excess copper.

Alterations in the ganglioside composition of rat cortical brain slices during experimental lactic acidosis: implication of an enzymatic process independent of the oxidative stress

Several in vitro studies have shown that lactic acidosis plays a role in brain damage by enhancing free radical formation and lipid peroxidation. The purpose of this study was to determine whether gangliosides are affected by lactic acid-induced oxidation in rat brain tissues. Cortical brain slices were incubated at 37 degrees C for 5 or 17 h in Krebs-Ringer buffer containing 20 mM lactic acid (final pH 5.5) previously equilibrated with 100% O2. Damage from lipid peroxidation was estimated by measurement of thiobarbituric acid-reactive substances (TBARS) and analysis of polyunsaturated fatty acids (PUFAs). Gangliosides were studied by high-performance thin-layer chromatography. Incubation with lactic acid induced overproduction of TBARS, whereas PUFAs were only slightly degraded, even after 17 h of incubation. However, the major modifications in the ganglioside profile occurred after 17 h of incubation. Gangliosides GD1a and GT1b decreased in conjunction with a substantial increase in the GM1 percentage. The addition of butylated-hydroxytoluene and desferrioxamine in the incubation medium, or incubation under 100% nitrogen, abolished TBARS production but not the ganglioside modifications, indicating that the change in ganglioside distribution was not related to oxidative stress induced by lactic acid. To investigate the possibility of an enzymatic process activated by the pH shift, slices were incubated with lactic acid in presence of 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, a specific inhibitor of sialidase. In these conditions, no change in gangliosides profile occurred. These results demonstrate that sialidase is responsible for the alterations in the gangliosides composition of rat cortical brain slices during lactic acidosis.

Gentamicin-induced kidney damage and lipid peroxidation in rats

Although it has been reported that injections of gentamicin induces lipid peroxidation in rat renal cortex (Ramsammy et al. (1985) Biochem. Pharmacol. 34, 3895-3900), our results showed no modification of thiobarbituric-reagent substances (TBARS) or in analysis of the polyunsaturated fatty acid profile. Moreover, endogenous vitamin E and glutathione were not consumed. In in vitro systems, gentamicin incubated with microsomes, homogenates and kidney slices from the normal rat failed to induce lipid peroxidation. We show that the increase in TBARS in vivo detected by Ramsammy et al. was wrongly attributed to the oxidant power of gentamicin. As this antibiotic does react positively to thiobarbituric acid in the presence of a system generating free radicals, it is possible that these authors accidentally introduced such a system into their experiments.

MPTP toxicity in rat striatal slices: dopamine uptake alteration does not appear to be related to lipid peroxidation

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which is used to create experimental models of parkinsonism, induces both dopaminergic neurotoxicity and peroxidation reactions. The present work investigated the interaction between the dopamine (DA) uptake system, lipid peroxidation and MPTP in a rat striatum slice model. [3H]DA uptake was decreased and the concentration of thiobarbituric acid reactive substances (TBARS) increased after a plain preincubation in Krebs-Ringer bicarbonate buffer for 150 min. The decrease in [3H]DA uptake and the increase in TBARS were suppressed by the iron-chelating agent desferrioxamine. Inhibition of [3H]DA uptake was intensified, [3H]GBR 12 935 binding to DA uptake sites was decreased and TBARS production was inhibited in slices after preincubation with MPTP. MPTP effects were inhibited by L-deprenyl, a MAO-B inhibitor. These results suggest that the spontaneous decrease in DA uptake during simple preincubation in pure Krebs-Ringer solution was related to spontaneous TBARS generation. During MPTP preincubation, alteration of the DA uptake mechanism was not due to additional lipid peroxidation since TBARS production was decreased. MPTP effects could have resulted from other events which are discussed.

Lack of correlation between TBARS production and PUFA degradation during incubation of membrane erythrocytes in an OH. (Fe2+/H2O2) generator system.

We investigated the effects of an OH⋅ (Fe2+/H2O2) generator system of erythrocyte membrane, particularly the time-course of lipid peroxidation as estimated by measurement of conjugated dienes, thiobarbituric reactive substances (TBARS), lipofuscin-like pigments, and α-tocopherol. Polyunsaturated fatty acids (PUFAs), especially arachidonic acid (20∶4 ω 6) and docosahexenoic acid (22∶6 ω 3), were also measured. Erythrocyte membranes were suspended in phosphate buffer containing Fe2+ (200 μM) and H2O2 (1.42 mM), and incubated in a shaking water bath at 37°C. Initially, there was an increase in TBARS and lipofuscin-like pigments, two well-known end products of PUFA oxidative degradation, whereas PUFAs remained unchanged (incubation time: 1 h). After two or more hours of incubation, marked lipid peroxidation was noted, with the appearance of conjugated dienes and a decrease of PUFAs, indicating that lipid peroxidation had occurred after a lag phase during which TBARS were not produced from PUFAs. This suggests that another OH⋅ target was involved.

Evidence of lipoperoxidation induced by lactic acid on kidney homogenates

Sawas and Gilbert (Arch. Int. Pharmacodyn. Ther., 276 (1985) 301-312) reported that the commercial solution of haloperidol induces lipoperoxidation of kidney homogenates from Sprague-Dawley rats. However, it would appear that this effect is attributable to the excipient, lactic acid, rather than to haloperidol itself. Lactic acid enhances susceptibility to lipoperoxidation of kidney homogenates in a dose- and time-dependent manner by increasing production of thiobarbituric acid-reactive substances and slightly decreasing polyunsaturated fatty acids such as arachidonic acid and docosahexaenoic acid. This stimulation of lipoperoxidation may be attributed to a mechanism less dependent on enzymatic action than on Fe2+ and Fe3+. Lactic acid may facilitate iron release and formation of iron complexes, factors which increase susceptibility to oxidative stress.