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International audience; No data are reported on changes in mitochondrial membrane phospholipids in non-alcoholic fatty liver disease. We determined the content of mitochondrial membrane phospholipids from rats with non alcoholic liver steatosis, with a particular attention for cardiolipin (CL) content and its fatty acid composition, and their relation with the activity of the mitochondrial respiratory chain complexes. Different dietary fatty acid patterns leading to steatosis were explored.With high-fat diet, moderate macrosteatosis was observed and the liver mitochondrial phospholipid class distribution and CL fatty acids composition were modified. Indeed, both CL content and its C18:2n-6 content were increased with liver steatosis. Moreover, mitochondrial ATP synthase activity was positively correlated to the total CL content in liver phospholipid and to CL C18:2n-6 content while other complexes activity were negatively correlated to total CL content and/or CL C18:2n-6 content of liver mitochondria. The lard-rich diet increased liver CL synthase gene expression while the fish oil-rich diet increased the (n-3) polyunsaturated fatty acids content in CL. Thus, the diet may be a significant determinant of both the phospholipid class content and the fatty acid composition of liver mitochondrial membrane, and the activities of some of the respiratory chain complex enzymes may be influenced by dietary lipid amount in particular via modification of the CL content and fatty acid composition in phospholipid.

Antioxidant activities of polyphenolic compounds extracted (PPEs) from ripe fruits of oil palms are investigated by studying their in vitro effects on human low-density lipoprotein (LDL) oxidation. Four oil palm species ( Elaeis guineensis ) are issued from the National Centre of Agronomic Research of Côte d'Ivoire, of which two are parental varieties (HP1 and HP2), while the other two are crossing varieties (HP3 and HP4). The main identified compounds were rutin (HP3 and HP4) and caffeic and chlorogenic (5-caffeoyl quinic) acids (HP1, HP3, and HP4). The highest total phenolic content was found for HP4, while it was significantly lower for HP2. Antioxidative effects were monitored by Cu(2+)- or 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH)-induced generation of conjugated dienes (lag time and oxidation rate). The highest PPE specific antioxidant activity (SAA) values were obtained with crossing varieties (HP3 and HP4) in the copper-oxidation assay. In the AAPH-oxidation assay, SAA values were comparable for all four varieties. PPEs were effective at preventing LDL-vitamin E depletion in vitro. They could exert direct beneficial antioxidant effects on vitamin E and other antioxidants contained in food and beverages in vivo, within the gastrointestinal (GI) tract. These data could also be of particular importance for a healthier nutrition or the management of chronic diseases by a polyphenol-rich diet.

Dietary lipids are known to affect the composition of the biological membrane and functions that are involved in cell death and survival. The mitochondrial respiratory chain enzymes are membrane protein complexes whose function depends on the composition and fluidity of the mitochondrial membrane lipid. The present study aimed at investigating the impact of different nutritional patterns of dietary lipids on liver mitochondrial functions. A total of forty-eight Wistar male rats were divided into six groups and fed for 12 weeks with a basal diet, lard diet or fish oil diet, containing either 50 or 300 g lipid/kg. The 30 % lipid intake increased liver NEFA, TAG and cholesterol levels, increased mitochondrial NEFA and TAG, and decreased phospholipid (PL) levels. SFA, PUFA and unsaturation index (UI) increased, whereas MUFA andtrans-fatty acids (FA) decreased in the mitochondrial membrane PL in 30 % fat diet-fed rats compared with 5 % lipid diet-fed rats. PL UI increased with fish oil dietv.basal and lard-rich diets, and PLtrans-FA increased with lard dietv.basal and fish oil diets. The 30 % lipid diet intake increased mitochondrial membrane potential, membrane fluidity, mitochondrial respiration and complex V activity, and decreased complex III and IV activities. With regard to lipid quality effects, β-oxidation decreased with the intake of basal or fish oil diets compared with that of the lard diet. The intake of a fish oil diet decreased complex III and IV activities compared with both the basal and lard diets. In conclusion, the characteristics and mitochondrial functions of the rat liver mitochondrial membrane are more profoundly altered by the quantity of dietary lipid than by its quality, which may have profound impacts on the pathogenesis and development of non-alcoholic fatty liver disease.

We have recently shown that low density lipoprotein (LDL) was able to denitrate albumin-bound 3-NO(2)-Tyr residues and to concomitantly release NO(3)(-) through a Ca(2+)-dependent process that has been ascribed to a specific protein structure. A lipophilic food component (gamma-tocopherol), which is easily loaded into LDL has been found to totally inhibit denitrating activity. We presently found that ellagic acid (EA) and its methylated derivatives, 4,4'O-methyl- and 3,3'O-methyl-ellagic acids (MeEA1 and MeEA2, respectively), amphipathic phenolic components of certain fruits and beverages, were also able to inhibit this activity, with a total inhibition for EA and a 60% inhibition for MeEA1 and MeEA2. EA exhibited the highest affinity for protein plasma, whereas a higher affinity of MeEA1 and MeEA2 (with MeEA1 > MeEA2) than EA was found for lipoprotein fractions, suggesting that the inhibition-driving property is protein affinity. As a result of this nitratase-inhibition property EA and its natural metabolite MeEA2 may have a beneficial role in special physiopathological conditions.

In blood, peroxynitrite (ONOO- ) and CO2 react to form NO2* and CO3* through the short-lived adduct ONOOCO2-, leading to protein-bound tyrosine nitration. ONOO(-) -modified LDL is atherogenic. Oxidatively modified LDL generally appears in the vessel wall surrounded by antioxidants. Human serum albumin (HSA) is one of them, partly associated to LDL as a LDL-albumin complex (LAC). The purpose was to study the effect of a mild nitration on LAC and whether albumin may interfere with LDL nitration. To do so, SIN-1 was used as ONOO- generator in the presence or absence of 25 mM HCO3-. The human serum albumin (HSA)-bound tyrosine nitration rate was found to be 4.4 x 10(-3) mol/mol in the presence of HCO3-. HSA decreased the LAC-tyrosine nitration rate from 2.5 x 10(-3) to 0.6 x 10(-3) mol/mol. It was concluded that HSA impaired the apoB-bound tyrosine nitration. These findings raise the question of the patho-physiological significance of these nitrations and their interactions which may potentially prevent both atheromatous plaque formation and endothelium dysfunction in particular and appear to be beneficial in terms of atherogenic risk.

The purpose of this double clinical study was (1) to evaluate the effect of one single intake (300 ml) of red wine (RW) on the plasma antioxidant capacity (pAOC) and plasma phenolics over the 24-h time period following the intake, and (2) to compare the long-term effects of daily intakes (250 ml/d) of RW, white wine (WW) and Champagne (CH) on the plasma and LDL characteristics of healthy subjects. In the first part, blood samples were collected just before and after wine consumption. In the second part, subjects received the 3 types of wine successively, only at the mealtime, over 3-week periods separated by a 3-week wash out. Blood samples were drawn in fasting condition before and after each 3-week wine consumption period. The peak of pAOC was at 3-4 h following the single intake of RW, that of catechin was at 4 h (0.13 micromol/l) and that of gallic acid and caffeic acid was earlier (or = 1.5 and 0.3 micromol/l, respectively). In plasma, the major form of gallic acid was 4-O-methylated, but a minor form (the 3-O-methyl derivative) appeared. In the long term study, no wine was able to change LDL oxidizability, but some other parameters were modified specifically: RW decreased pAOC (without changing TBARS and uric acid plasma levels), LDL lipids and total cholesterol (TC), and increased plasma apoA1, whereas CH increased plasma vitamin A. The beneficial effect of RW seems to mainly be explained by its action on lipid and lipoprotein constants, and not by its antioxidant one.

Using an approach in line with that of a previous report, we assessed the antioxidant activity of several natural, polyphenol- or tocotrienol-rich mixtures: extracts from Elaesis Guineensis oil (A) and Vitis vinifera (B), a Coffea robusta powder (C), and extracts from Olea europea mill wastewaters (D), Solanum melongena (E), and Lycopersicon esculentum (F). The copper- and 2-2'-azobis(2-amidinopropane) hydrochloride (AAPH)-oxidation systems were used in the presence of low-density lipoprotein. For comparison, antioxidant activities of chlorogenic acid and catechin, as archetypes of molecules highly efficient with the copper- and the AAPH-oxidation system, respectively, were assessed. The aim was to establish the occurrence of synergistic antioxidant actions among some of these natural mixtures. On a molar basis, the highest specific antioxidant activities (SAA) were found for B, chlorogenic acid, and C in the copper system, and for A, catechin, and B in the AAPH system. On a mass basis, the highest SAA were found, respectively, for chlorogenic acid, B, and catechin, and for catechin, chlorogenic acid, and B. These results show that large discrepancies take place in the evaluations between the two systems. B and C exhibited a synergistic antioxidant efficiency, in the presence or absence of A, but only with the copper system. This was also true for the two types of A+B+C mixture that were tested. It is thought that this association might provide an ideal combination, incorporating both the radical scavenger and the transition-metal ion chelation properties of B and C.

International audience; Red sorghum is a source of phenolic compounds (PCs), including 3-deoxyanthocyanidins that may protect against oxidative stress related disease such as atherosclerosis. HPLC was used to characterise and quantify PCs extracted from red or white sorghum whole grain flour. Antioxidant activity was measured by an oxygen radical absorbance capacity assay and against LDL-oxidisability, and further compared to that of synthesised 3-deoxyanthocyanidins (i.e., luteolinidin and apigeninidin). Phenolic content of red and white sorghums was evaluated as 3.90 ± 0.01 and 0.07 ± 0.01 mmol gallic acid equivalents L- 1, respectively. Luteolinidin and apigeninidin were mainly found in red sorghum. Red sorghum had almost 3 and 10 times greater specific antioxidant activity compared to luteolinidin and apigeninidin, respectively. Red sorghum PCs and the two 3-deoxyanthocyanidins were also effective at preventing LDL vitamin E depletion and conjugated diene production. Red sorghum flour exhibits antioxidant capacity suggesting that it may be a valuable health-promoting food.

Accumulation of muscle TAG content and modification of muscle phospholipid fatty acid pattern may have an impact on lipid metabolism, increasing the risk of developing diabetes. Some polyphenols have been reported to modulate lipid metabolism, in particular those issued from red grapes. The present study was designed to determine whether a grape polyphenol extract (PPE) modulates skeletal muscle TAG content and phospholipid fatty acid composition in high-fat–high-sucrose (HFHS) diet-fed rats. Muscle plasmalemmal and mitochondrial fatty acid transporters, GLUT4 and lipid metabolism pathways were also explored. The PPE decreased muscle TAG content in HFHS/PPE diet-fed rats compared with HFHS diet-fed rats and induced higher proportions ofn-3 PUFA in phospholipids. The PPE significantly up-regulated GLUT4 mRNA expression. Gene and protein expression of muscle fatty acid transporter cluster of differentiation 36 (CD36) was increased in HFHS diet-fed rats but returned to control values in HFHS/PPE diet-fed rats. Carnitine palmitoyltransferase 1 protein expression was decreased with the PPE. Mitochondrial β-hydroxyacyl CoA dehydrogenase was increased in HFHS diet-fed rats and returned to control values with PPE supplementation. Lipogenesis, mitochondrial biogenesis and mitochondrial activity were not affected by the PPE. In conclusion, the PPE modulated membrane phospholipid fatty acid composition and decreased muscle TAG content in HFHS diet-fed rats. The PPE lowered CD36 gene and protein expression, probably decreasing fatty acid transport and lipid accumulation within skeletal muscle, and increased muscle GLUT4 expression. These effects of the PPE are in favour of a better insulin sensibility.

Corresponding author: Dr Christine Feillet-Coudray, fax : + 33 4 67 54 56 94, email : cfeillet@supagro.inra.fr; International audience; High-fat or high-fat–high-sucrose diets are known to induce non-alcoholic fatty liver disease and this is emerging as one of the most common liver diseases worldwide. Some polyphenols have been reported to decrease rat hepatic lipid accumulation, in particular those extracted from red grapes such as resveratrol. The present study was designed to determine whether a polyphenol extract (PPE), from red grapes, modulates liver fatty acid composition and desaturase activity indexes in rats fed a high-fat–high-sucrose (HFHS) diet, and to explore whether sirtuin-1 deacetylase activation was implicated in the effect of the PPE against liver steatosis. The effect of this PPE on mitochondriogenesis and mitochondrial activity was also explored. The PPE decreased liver TAG content in HFHS þ PPE diet-fed rats in comparison with HFHS diet-fed rats. The PPE had no effect on liver fatty acid composition, desaturase activity indexes and stearoyl-CoA desaturase 1 (SCD1) gene expression. Sirtuin-1 deacetylase protein expression was significantly increased with the PPE; AMP kinase protein expression was higher with the PPE in comparison with the HFHS rats, but no modification of phosphorylated AMP kinase was observed. Protein expression of phospho-acetyl-CoA carboxylase was decreased in HFHS rats and returned to basal values with the PPE. Finally, the PPE modulated PPARg coactivator-1a (PGC-1a) but did not modify mitochondriogenesis and mitochondrial activity. In conclusion, the PPE partially prevented the accumulation of TAG in the liver by regulating acetyl-CoA carboxylase phosphorylation, a key enzyme in lipid metabolism, probably via sirtuin-1 deacetylase activation. However, the PPE had no effect on the qualitative composition of liver fatty acids.

In the systemic circulation, LDL occurs in the form of a weakly nitrated LDL-albumin complex (LAC). The question here is whether LAC (or HDL) is able to denitrate the albumin-bound 3-NO(2)-tyrosine (3NT). Nitrated albumin was incubated in the presence of lipoprotein fraction (LPF) to be tested, with or without Ca(2+). After precipitation and centrifugation, supernatants (SNs) and protein pellets (PP) were collected. HCl proteolysis was carried out with deuterated 3NT as an internal standard, and amino acids were derivatized for GC-MS analysis, whereas SNs were used for NO(2) (-)/NO(3) (-)-fluorimetric assays. A loss of 3NT, higher with albumin-low LDL than with albumin-rich LDL or HDL, was found in PP only in the presence of Ca(2+). gamma-Tocopherol loading of LPF inhibited 3NT loss. 3NT loss was found for the first time to be stoichiometrically equivalent to NO(3) (-), proving that the 3NT loss must be ascribed to a 3NT-denitrating nitratase activity. 3NT loss and NO(3) (-) production that clearly cannot be attributed to PON-1 were impaired by D-penicillamine and phenylacetate, inhibitor, and substrate of PON-1, respectively, leading to speculate on the active site. Finally, nitratase activity and albumin contribute to beneficially convert peroxynitrite (ONOO(-)) into nonbioactive NO(3) (-). But, in inflammatory conditions, xanthine oxidoreductase is expressed leading to detrimentally reduce O(2) and NO(3) (-) into O(2) (*) (-) and NO(*) that may interact, reconstituting the ONOO(-) pool. The real consequence of nitratase activity and the physiological significance of nitration/denitration processes remain to be explored.

We have recently established that the blood concentrations of gallic acid (GA), a polyphenolic component naturally found in food, and its O-methyl derivatives are very low (practically ⩽1 μM) in physiological (postprandial) condition. Using acellular oxidant systems and macrophage-differentiated promonocytes (MDPs) THP-1, we show here that the direct and indirect (through depressing effect on the superoxide cell production) antioxidant properties of these components were not effective at these concentrations. In contrast, 4-O-methyl GA was the most efficient component to depress AT1R and CD36 mRNA expression in Ang II-treated MDPs, suggesting a strong inhibition of Ang II-triggered pro-atherogenic mechanisms of foam cell formation.

A large body of evidence supports the key role of oxidized low-density lipoprotein in atherosclerosis. The aim of this study was to compare the capacity of natural polyphenols (PP) from Vitis vinifera and Olea europea at protecting LDL against oxidation brought about by Cu2+, oxygen-centered radical-generating AAPH, or peroxynitrite-generating SIN-1 in vitro systems, or at impairing superoxide production in promonocyte cells (THP-1) conveniently differentiated into adherent macrophages. PP were either from the whole grape (fraction A) containing mainly procyanidins, (epi)-catechin and anthocyanins, or from grape seed extracts (fractions B and C) consisting of tannins and procyanidin oligomers with a higher content in B than in C, or from a grape skin extract (fraction D) consisting mainly of anthocyanins, or from a hydrosoluble olive mill wastewater PP extract (fraction E) containing hydroxytyrosol and oleuropein. Chlorogenic acid (F) and catechin (G) were taken as archetypes of PP preventing oxidation partly as copper scavenger and as radical scavenger only, respectively. All grape fractions were efficient towards Cu2+ system (equally or more efficient than F), whereas they were rather poorly efficient towards AAPH and SIN-1 (less efficient than G but as efficient as F). Among the PP fractions, B was the most effective at protecting LDL in the SIN-1 system and at impairing THP-1 superoxide production. Taken together, these data suggest that the PP fraction from grape seed rich in procyanidins achieves the best compromise between the direct and indirect (i.e. cell-mediated) types of action in protecting LDL against oxidation, strengthening the need for improving the knowledge of its bioavailability in humans.

Specific antioxidant activity (SAA) (i.e., activity related to the molar or gallic acid equivalent amount of antioxidant) of natural polyphenolic mixtures or pure phenolic compounds was studied using their capacity to delay the conjugated diene production brought about by in vitro LDL copper-mediated or AAPH-mediated oxidation. The cinnamic acid series (caffeic, sinapic, ferulic acids) displayed a constant SAA over a large range of concentrations, whereas the benzoic acid series (gallic and protocatechuic acids) showed much higher SAA at low concentrations. The natural phenolic mixtures had a constant SAA. The highest SAA was obtained with caffeoyl esters (caffeoylquinic, rosmarinic, and caffeoyltartaric acids) and catechin for the copper-oxidation and the AAPH-oxidation system, respectively. Phenolic mixtures and acids delayed vitamin E depletion and decreased proinflammatory lysophosphatidylcholine production. As with polyphenols, probucol delayed lysophosphatidylcholine and conjugated dienes production, at higher concentrations, but was not effective at preventing vitamin E depletion. Polyphenols prevent the oxidation of LDL and its constituents (vitamin E, phosphatidylcholine), which is compatible with an antiinflammatory and antiatherosclerotic role in pathophysiological conditions.

Objectives: To evaluate the effect of the red wine phenolic compound (RWPC) dietary supplementation without alcohol interference on: (1) some of the biochemical characteristics of LDL, (2) the oxidative susceptibility of LDL and (3) the antioxidant capacity of total plasma (Pl-AOC). In order to account for discrepancies between the three series of data, the in vitro stability of the association of phenolic compounds and LDL was tested. Design: An intervention study with 20 volunteers. Each served as his own control. Cu2+-oxidizability of LDL and Pl-AOC were tested on blood samples before and after dietary supplementation. Cu2+-oxidizability of LDL was also tested by co-incubation in the presence of RWPC or phenolic acids with or without extensive dialysis. Setting: The Laboratory of Lipid Biochemistry and Biology, School of Medicine, and the Laboratory of Metabolic Diseases, Lapeyronie Hospital, University of Montpellier, France. Subjects: Healthy males, nonsmokers and moderate drinkers, submitted to a dietary regimen deprived of vitamin E and C for a period of 10 d before supplementation. They also abstained from alcohol, wine, fruit juices, coffee, tea and cola beverages during this period. Intervention: Six 0.33 g capsules/d (namely two capsules at each meal) of a preparation of red wine phenolic compounds in a dry powder form were given to the volunteers over a period of two weeks. Blood samples were drawn in fasting conditions at day 0 and day 14 of the supplementation period. Results: Supplementation led to: (1) in LDL, a significant increase in vitamin E content (n=20, P=0.01) or vitamin E/total fatty acid bis-allylic carbon number ratio (n=20, P=0.006) without modification in the other biochemical characteristics or Cu2+-oxidizability; (2) in plasma, a significant increase in the antioxidant capacity (n=11, P=0.01). In vitro studies showed that RWPC or sinapic, caffeic or ferulic acids incubated in the presence of LDL increased the protection of the lipoparticle against oxidation (caffeic>sinapic>ferulic). This effect, however, was totally lost after extensive dialysis. Conclusions: The enhancing effect of the RWPC supplementation on Pl-AOC may be due to a phenolic-compound action both in the aqueous phase of plasma and at the surface of lipoprotein particles. Surface location possibly explains the enhancing-sparing effect of supplementation on LDL vitamin E and the absence of effect on dialysed-LDL oxidizability. Sponsorship: Supported by a grant from the Scientific Committee ‘Vin et Sante: Pathologies Vasculaires’.