Catherine Cordelet

Oxyphytosterols are present in plasma of healthy human subjects

The oxidised derivatives of phytosterols (oxyphytosterols) were identified in plasma samples from thirteen healthy human volunteers, using MS. All the samples contained noticeable quantities of (24R)-5beta,6beta-epoxy-24-ethylcholestan-3beta-ol (beta-epoxysitostanol) and (24R)-ethylcholestan-3beta,5alpha,6beta-triol (sitostanetriol) and also trace levels of (24R)-5alpha,6alpha-epoxy-24-ethylcholestan-3beta-ol (alpha-epoxysitostanol), (24R)-methylcholestan-3beta,5alpha,6beta-triol (campestanetriol) and (24R)-ethylch olest-5-en-3beta-ol-7-one(7-ketositosterol). The amounts of these oxyphytosterols in plasma varied from 4.8 to 57.2 ng/ml. There are two possibilities concerning the origin of these compounds. First, they could come from the small amounts of oxyphytosterols in food. Second, they could originate from the in vivo oxidation of phytosterols in plasma. Very few data actually exist concerning these compounds. Their identification in human samples suggests that further research is necessary in this field.

Induction of similar features of apoptosis in human and bovine vascular endothelial cells treated by 7-ketocholesterol

Cholesterol oxides have numerous cytotoxic effects and those oxidized in the C7 position have been shown to induce apoptosis in bovine aortic endothelial cells (BAEC). The aim of the present study was to determine whether apoptosis also occurs in human vascular endothelial cells (HUVEC) treated with 7‐ketocholesterol. To this end, cultured BAEC and HUVEC were incubated for 48 h with 7‐ketocholesterol (concentration range 5–80 μg/ml) and the characteristics of cell death were assessed by various methods: counting of adherent and non‐adherent cells; analysis of DNA fragmentation pattern; and morphological study by light, fluorescence, and electron microscopy. The 7‐ketocholesterol treatment was accompanied by a decrease in the number of adherent cells and an increase in the number of non‐adherent cells. Apoptotic cells, recognized by fragmented and/or condensed nuclei after staining with Hoechst 33342 or Giemsa, were mainly detected among non‐adherent cells, and agarose gel electrophoresis revealed a typical internucleosomal DNA fragmentation among 7‐ketocholesterol‐treated cells. The DNA fragmentation was no longer detected when HUVEC and BAEC were simultaneously incubated with 0·5 mmol/l zinc chloride, which is known to inhibit Ca2+/Mg2+‐dependent endonucleases. Finally, the ultrastructural abnormalities observed by electron microscopy in both 7‐ketocholesterol‐treated HUVEC and BAEC were remarkably similar and were mainly characterized by condensed chromatin, altered mitochondria, disturbed organization of the cytoskeleton, and vacuoles containing myelin figures and/or cell debris; apoptotic bodies were also frequently detected. It is concluded that 7‐ketocholesterol constitutes a potent inducer of apoptosis in endothelial vascular cells of both bovine and human origin, suggesting that cholesterol oxides may be involved in the early steps of the atherosclerotic process in humans.

Oxidative injury of isolated cardiomyocytes: dependence on free radical species

The contribution of lipid peroxidation to myocardial injury by free radicals (FR) is still unclear. Consequently, we examined the functional damages inflicted on cultured rat cardiomyocytes (CM) during FR stress provoked by the xanthine/xanthine oxidase system (X/XO) or by a hydroperoxidized fatty acid ((9 Z, 11 E, 13 (S), 15 Z)-13-hydroperoxyocta-decatrienoic acid; 13-HpOTrE), in order to simulate in vitro the initial phase and the propagation phase of the FR attack, respectively. Transmembrane potentials were recorded with glass microelectrodes and contractions were monitored photometrically. The EPR spectroscopy showed that X/XO produced superoxide and hydroxyl radicals during 10 min. The X/XO system altered sharply and irreversibly the spontaneous electrical and mechanical activities of the CM. However, the gas chromatographic analysis showed that these drastic functional damages were associated with comparatively moderate membrane PUFA degradation. Moreover, the EPR analysis did not reveal the production of lipid-derived FR. 13-HpOTrE induced a moderate and reversible decrease in electrical parameters, with no change in CM contractions. These results indicate that the functional consequences of FR attack are dependent on the radical species present and do not support the idea that the membrane lipid breakdown is a major factor of myocardial oxidant dysfunction.

Cross-influence of membrane polyunsaturated fatty acids and hypoxia-reoxygenation on α- and β-adrenergic function of rat cardiomyocytes

The purpose of the present investigation was to determine whether the beneficial effects of polyunsaturated fatty acids (PUFA) may influence ischemia-reperfusion-induced alterations of myocardial α- and β-adrenoceptor (α-AR, β-AR) responsiveness. This study was carried out using monolayer cultures of neonatal rat ventricular myocytes in a substrate-free, hypoxia-reoxygenation model of ischemia. The cardiomyocytes (CM) were incubated during 4 days in media enriched either with n−6 PUFA (arachidonic acid, AA) or with n−3 PUFA (eicosapentaenoic acid, EPA, and docosahexaenoic acid, DHA). The n−6/n−3 ratio in n−3 CM was close to 1.2, compared to 20.1 in n−6 CM. The contractile parameters of n−6 CM and n−3 CM were similar in basal conditions as well as during hypoxia and reoxygenation. In basal conditions, the phospholipid (PL) enrichment with long chain n−3 PUFA resulted in an increased chronotropic response to isoproterenol (ISO) and to phenylephrine (PHE). After posthypoxic reoxygenation, the chronotropic response to β-AR activation in n−6 CM was significantly enhanced as compared with the control response in normoxia. In opposition, the ISO-induced rise in frequency in n−3 CM in control normoxia and after reoxygenation was similar. In these n−3 CM, the changes in contractile parameters, which accompanied the chronotropic response, were also similar in reoxygenation and in normoxic periods, although the rise in shortening velocity was slightly increased after reoxygenation. In response to PHE addition, only the chronotropic effect of n−6 CM appeared significantly enhanced after hypoxic treatment. These results suggested that increasing n−3 PUFA in PL reduced the increase in α- and β-AR functional responses observed after hypoxia-reoxygenation. This effect may partly account for the assumed cardiac protective effect of n−3 PUFA, through the attenuation of the functional response to catecholamines in the ischemic myocardium.

Incorporation and metabolism of trans 20∶5 in endothelial cells. Effect on prostacyclin synthesis

To study the ability of long-chain trans fatty acids (FA) to be incorporated and metabolized into endothelial cells, bovine aortic endothelial cells were incubated with medium enriched eicosapentaenoic acid (EPA) bound to albumin (M2) or one of its geometrical isomers: 20∶5 5c,8c,11t,14c,17c(M3), 20∶5 5c,8c,11c,14c,17t(M4), or 20∶5 5c,8c,11t,14c,17t(M5). After 48 h of incubation, supernatant and cells were harvested and their lipids were analyzed, including prostacyclin synthesis. EPA and 22∶5n−3 of endothelial cells incubated with M2 were, respectively, three and two times higher than in control cells (incubated in M1, without any fatty acid added), whereas 22∶6n−3 increased only in the supernatant, suggesting its release after biosynthesis. However, 18∶2n−6 and 22∶4n−6 decreased (about 30%). Trans 20∶5 isomers represented 4.7, 3.9, and 5.2% of total phospholipid FA in endothelial cells incubated with M3, M4, and M5, respectively. They were elongated into trans 22∶5 and trans 24∶5, as revealed by gas chromatography-mass spectrometry and gas chromatography-Fourier transform infrared analysis. In cells incubated with M2, M3, M4, and M5, prostacyclin synthesis was inhibited by 49.0, 62.5, 60.5, and 72.0%, respectively. This effect may be due to less available arachidonic acid in the cells and to a competition between EPA isomers and AA at the level of cyclooxygenase pathway, as it was demonstrated that 20∶5 Δ17t was metabolized by this enzyme.

Involvement of oxygen free radicals in the respiratory uncoupling induced by free calcium and ADP-magnesium in isolated cardiac mitochondria: Comparing reoxygenation in cultured cardiomyocytes

Recently, we have observed that the simultaneous application of free calcium (fCa) and ADP-magnesium (Mg) reduced the ADP:O ratio in isolated cardiac mitochondria. The uncoupling was prevented by cyclosporin A, an inhibitor of the permeability transition pore. The purpose of this study was to know if the generation of oxygen free radicals (OFR) is involved in this phenomenon and if it occurs during reoxygenation (Reox) of cultured cardiomyocytes. Cardiac mitochondria were harvested from male Wistar rats. Respiration was assessed in two media with different fCa concentrations (0 or 0.6 μM) with palmitoylcarnitine and ADP-Mg as respiration substrates. The production of Krebs cycle intermediates (KCI) was determined. Without fCa in the medium, the mitochondria displayed a large production of citrate + isocitrate + α-ketoglutarate. fCa drastically reduced these KCI and promoted the accumulation of succinate. To know if OFR are involved in the respiratory uncoupling, the effect of 4OH-TEMPO (250 μM), a hydrosoluble scavenger of OFR, was tested. 4OH-TEMPO completely abolished the fCa- and ADP-Mg-induced uncoupling. Conversely, vitamin E contributed to further decreasing the ADP:O ratio. Since no hydrosoluble electron acceptor was added in our experiment, the oxygen free radical-induced oxidized vitamin E was confined near the mitochondrial membranes, which should reduce the ADP:O ratio by opening the permeability transition pore. The generation of OFR could result from the matrix accumulation of succinate. Taken together, these results indicate that mitochondrial Ca uptake induces a slight increase in membrane permeability. Thereafter, Mg enters the matrix and, in combination with Ca, stimulates the isocitrate and/or α-ketoglutarate dehydrogenases. Matrix succinate favors oxygen free radical generation that further increases membrane permeability and allows respiratory uncoupling through proton leakage. To determine whether the phenomenon takes place during Reox, cultured cardiomyocytes were subjected to hypoxia and Reox. 14C-palmitate was added during Reox to determine the KCI profile. Succinate had not increased during Reox. In conclusion, calcium- and ADP-Mg-induced respiratory uncoupling is due to oxygen free radical generation through excess matrix accumulation of succinate. The phenomenon does not occur during reoxygenation because of a total restoration of mitochondrial magnesium and/or ADP concentration.

Adaptation of the free bag technique to evaluate the use of the nitrogenous component of feeds in the large intestine of the pony.

ther directly or after pepsin predigestion (6 h in a pepsin solution: 2 g/l of 0.1 N HCI). It was recovered in the feces. The quantity of nitrogen found in the bag enabled us to evaluate the quantity of alimentary nitrogen really degraded in the large intestine (de Boer et al, 1987). First we compared 3 sizes of bags filled with alfalfa hay. They measured 2.5 x 3 cm with 0.35 g of substratum, 2.5 x 6 cm with 0.8 g of substratum and 3 x 11 cm with 1.7 g of substratum, respectively. The 32 measures of DM disappearance gave the following mean results: small bags: 30.0 ± 3.3; middle-sized bags: 31.4 t 5.4; large bags: 38.1 ± 2.7. We consequently chose to introduce the large bags at 5:00 pm to evaluate nitrogen digestion in the large intestine, either by introducing the bag containing the feed directly or after pepsin digestion. We studied 4 feeds: al-