Marie-Annette Carbonneau

Grape and grape seed extract capacities at protecting LDL against oxidation generated by Cu2+, AAPH or SIN-1 and at decreasing superoxide THP-1 cell production. A comparison to other extracts or compounds.

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 Cu 2+ , 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 Cu 2+ 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.

Red-wine beneficial long-term effect on lipids but not on antioxidant characteristics in plasma in a study comparing three types of wine--description of two O-methylated derivatives of gallic acid in humans.

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 sujects. 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 μmol/l) and that of gallic acid and caffeic acid was earlier (≤⃒1.5 and 0.3 μmol/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.

Dietary fatty acids modulate liver mitochondrial cardiolipin content and its fatty acid composition in rats with non alcoholic fatty liver disease

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.

A polyphenol extract modifies quantity but not quality of liver fatty acid content in high-fat–high-sucrose diet-fed rats: possible implication of the sirtuin pathway

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+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 PPARγ coactivator-1α (PGC-1α) 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.

Antioxidant properties of 3-deoxyanthocyanidins and polyphenolic extracts from Côte d’Ivoire’s red and white sorghums assessed by ORAC and in vitro LDL oxidisability tests

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.

Vitamin E Supplementation Increases LDL Resistance to ex vivo Oxidation in Hemodialysis Patients

BACKGROUND Oxidative stress and alterations in lipid metabolism observed in hemodialysis patients potentiate the low-density lipoprotein (LDL) oxidability, recognized as a key event during early atherogenesis. OBJECTIVE To explore the effects of an oral vitamin E supplementation on oxidative stress markers and LDL oxidability in hemodialysis patients. METHODS Fourteen hemodialysis patients and six healthy volunteers were given oral vitamin E (500 mg/day) for six months. Oxidative stress was assessed using: plasma and lipoprotein vitamin E levels [high-performance liquid chromatography (HPLC) procedure]; thiobarbituric acid reactive substances (TBARS, Yaggi method); and copper-induced LDL oxidation. All parameters were evaluated before initiation of vitamin E supplementation, and at three and six months thereafter. RESULTS At baseline, a significantly higher TBARS concentration and a higher LDL oxidability were observed in hemodialysis patients when compared to controls. After six months of vitamin E supplementation, TBARS and LDL oxidability were normalized in hemodialysis patients. CONCLUSION Our data confirm that hemodialysis patients are exposed to oxidative stress and increased susceptibility to ex vivo LDL oxidation. Since oral vitamin E supplementation prevents oxidative stress and significantly increases LDL resistance to ex vivo oxidation, supplementation by natural antioxidants such as vitamin E may be beneficial in hemodialysis patients.

A grape polyphenol extract modulates muscle membrane fatty acid composition and lipid metabolism in high-fat-high-sucrose diet-fed rats

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 of n-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.

Peroxynitrite mild nitration of albumin and LDL–albumin complex naturally present in plasma and tyrosine nitration rate–albumin impairs LDL nitration

In blood, peroxynitrite (ONOO− ) and CO2 react to form NO2√ and through the short-lived adduct , 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 . The human serum albumin (HSA)-bound tyrosine nitration rate was found to be 4.4 × 10− 3 mol/mol in the presence of . HSA decreased the LAC-tyrosine nitration rate from 2.5 × 10− 3 to 0.6 × 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.

New Insight on the Relationship between LDL Composition, Associated Proteins, Oxidative Resistance and Preparation Procedure

Oxidized low density lipoprotein (LDL) plays an important role in atherogenesis. It is generally thought that LDL is mainly oxidized in the intima of vessel walls, surrounded by hydrophilic antioxidants and proteins such as albumin. The aim of this study was to investigate the possible interrelationships between oxidation resistance of LDL and its protein and lipid moieties. Proteins and to a lesser extent lipids, appeared to be the major determinants in the LDL Cu 2+ -oxidation resistance, which in turn depend on the ultracentrifugation (UC) procedure used. Comparing high speed/short time (HS/ST, 4 u h), high speed/long time (HS/LT, 6-16 u h) and low speed/long time (LS/LT, 24 u h) conditions of UC, HS with the shortest time (4 u h) led to prepare LDL (named LDL·HS-4 u h) with higher total protein and triglyceride contents, unchanged total cholesterol, phospholipids and Vitamin E, and higher Cu 2+ -oxidation resistance. Among proteins, only albumin allows to explain changes. PAF acetyl hydrolase appeared to be unaffected, whereas its pro-oxidant role was established and found only in the absence of albumin. In contrast the pro-oxidant role of caeruloplasmin took place regardless of the albumin content of LDL. The antioxidant effect of albumin (the oxidation lag time was doubled for 20 u mol/mol albumin per LDL) is assumed to be due to its capacity at decreasing LDL affinity for Cu 2+ . Interestingly, the LDL·HS-4 u h albumin content mirrored the intrinsic characteristics of LDL in the plasma and was not affected by added free albumin. Moreover, it has been verified that in 121 healthy subjects albumin was the best resistance predictor of the Cu 2+ -oxidation of LDL·HS-4 u h, with a multiple regression equation: lag time (min)=62.1+0.67(HSA/apoB)+0.02 (TG/apoB) m 0.01(TC/apoB); r =0.54, P <0.0001. Accounted for by lag time, the oxidation resistance did not correlate with f -tocopherol and ubiquinol contents of LDL. The mean albumin content was about 10 u mol/mol, and highly variable (0-58 u mol/mol) with subjects. The LDL·HS-4 u h may account for the status of LDL in its natural environment more adequately than LDL resulting from other conditions of UC.

First Evidence for an LDL- and HDL-associated Nitratase Activity That Denitrates Albumin-Bound Nitrotyrosine—Physiological Consequences*

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‐NO2‐tyrosine (3NT). Nitrated albumin was incubated in the presence of lipoprotein fraction (LPF) to be tested, with or without Ca2+. 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 NO2−/NO3−‐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 Ca2+. γ‐Tocopherol loading of LPF inhibited 3NT loss. 3NT loss was found for the first time to be stoichiometrically equivalent to NO3−, proving that the 3NT loss must be ascribed to a 3NT‐denitrating nitratase activity. 3NT loss and NO3− 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 NO3−. But, in inflammatory conditions, xanthine oxidoreductase is expressed leading to detrimentally reduce O2 and NO3− into O2•− 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.