Ellagic acid and its methyl-derivatives inhibit a newly found nitratase activity.

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.

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-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.

Regional Brazilian diet-induced pre-natal malnutrition in rats is correlated with the proliferation of cultured vascular smooth muscle cells.

OBJECTIVES: Pre-natal malnutrition induces hypertension and insulin resistance, pathologies commonly linked to atherosclerotic disease. The proliferation of vascular smooth muscle cells (SMCs) is important during development of the atherosclerotic plaque. In this work, we investigated whether the serum of pre-natal malnourished Wistar rats could alter the proliferation of aortic and renal artery SMCs in culture. Malnutrition was induced by feeding a basic regional diet available in a rural area of Pernambuco State, Brazil. This diet was rich in carbohydrates and deficient in proteins, lipids, vitamins and minerals, including sodium chloride. METHODS AND RESULTS: Serum was obtained from the blood of 90-day-old control and pre-natal undernourished rats. SMCs from control Wistar rats at the 6th passage were allowed to adhere to plates in Dulbecco's modified Eagle's medium (DMEM) supplemented with fetal calf serum (10%). Subsequently, the SMCs were maintained in DMEM supplemented with rat serum (10%). The number of cells was counted on the 3rd, 6th and 8th days of culture into rat serum. [3H]-thymidine incorporation into SMCs was evaluated after 20 h or 6 days of incubation. The birth weight of male and female undernourished offspring was 25% (p<0.05) and 46% (p<0.05) lower, respectively, than their corresponding control groups. On the 8th day of culture, the number of aortic SMCs in the serum of undernourished male and female rats, as well as renal artery SMCs in the serum of undernourished female rats, was higher than in the serum of control rats. The [3H]-thymidine incorporation was higher in aortic SMCs incubated for 6 days in the serum of undernourished male and female rats. At confluence, the density of aortic SMCs was higher than that of renal artery SMCs. CONCLUSIONS: Pre-natal malnutrition produces serum with altered properties that can affect the proliferation of SMCs and may contribute to atherosclerotic disease.

Fluorescence spectroscopy for monitoring deterioration of extra virgin olive oil during heating.

The potential of fluorescence spectroscopy for characterizing the deterioration of extra virgin olive oil (EVOO) during heating was investigated. Two commercial EVOO were analysed by HPLC to determine changes in EVOO vitamin E and polyphenols as a result of heating at 170 degrees C for 3 h. This thermal oxidation of EVOO caused an exponential decrease in hydroxytyrosol and vitamin E (R(2)=0.90 and 0.93, respectively) whereas the tyrosol content was relatively stable. At the same time, amounts of preformed hydroperoxides (ROOH), analysed by an indirect colorimetric method, decreased exponentially during the heating process (R(2)=0.94), as a result of their degradation into secondary peroxidation products. Fluorescence excitation spectra with emission at 330 and 450 nm were recorded to monitor polyphenols and vitamin E evolution and ROOH degradation, respectively. Partial least-squares calibration models were built to predict these indicators of EVOO quality from oil fluorescence spectra. A global approach was then proposed to monitor the heat charge from the overall fluorescence fingerprint. Different data pretreatment methods were tested. This study indicates that fluorescence spectroscopy is a promising, rapid, and cost-effective approach for evaluating the quality of heat-treated EVOO, and is an alternative to time-consuming conventional analyses. In future work, calibration models will be developed using a wide range of EVOO samples.

Potential anti-atherogenic cell action of the naturally occurring 4-O-methyl derivative of gallic acid on Ang II-treated macrophages.

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 < or = 1 microM) 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.

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 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.

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 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.

Synergistic antioxidative properties of phenolics from natural origin toward low-density lipoproteins depend on the oxidation system.

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.

Cord blood essential fatty acid and alpha-tocopherol in full-term newborns in a Northeast Brazil area.

Malnutrition of children during the first two years of life constitutes a public health concern in Brazil, particularly in the Northeast. Most of the nutrition data are concerned with protein-energy malnutrition and hypovitaminosis A. The purpose of this cross-sectional study was to assess the essential fatty acid (EFA) status, which is crucial in physical and mental development, and that of vitamin E which prevents against the oxidative loss of EFA physiological properties, in 81 full-term newborns. Blood samples were obtained from the residual blood of the umbilical cord (UC) at delivery. Fatty acid composition of UC plasma did not show any sign of EFA deficiency. The levels of docosahexanoic (DHA) and arachidonic acid (AA) appeared to be quite similar to those obtained in European populations. UC plasma vitamin E content was 6.31 +/- 1.99 mumol/L whereas the lipid-normalized vitamin E was 2.36 mumol/mmol of lipids. An interesting point was that newborns with vitamin E inferior to the median value (5.80 mumol/L) revealed significantly lower contents of linoleic acid and DHA in UC than newborns superior to the median value. Together with the absolute or normalized plasma level of vitamin E, this supports the observation that one quarter of the community's newborns is deficient in vitamin E.

A diet high in cholesterol and deficient in vitamin E induces lipid peroxidation but does not enhance antioxidant enzyme expression in rat liver.

Expression of antioxidant enzymes (AOE), an important mechanism in the protection against oxidative stress, could be modified by the redox status of the cells. The aim of this project was to evaluate the role of vitamin E deficiency in association with a high-cholesterol diet in the hepatic lipid peroxidation and the expression of AOE. Two groups of 6 male rats were fed with a high-cholesterol or a high-cholesterol vitamin E-deficient diet. All animals were sacrificed at 72 days of treatment. Liver lipid peroxidation index (Malondialdehyde; MDA) and hepatic AOE were evaluated. Total liver RNA was extracted, and the steady state messenger RNA (mRNA) levels of glutathion peroxydase, manganese superoxide dismutase, Cu/Zn superoxide dismutase and catalase were examined by northern blot. After 72 days on the diet, a significant increase in the lipid peroxidation index was observed in the vitamin E deficient group (MDA : 4.45 +/- 0.29 nmol/mg protein versus 3.65 +/- 0.1 nmol/mg protein in vitamin E normal group). Despite this oxidative stress, the activities and mRNA levels of liver AOE were not significantly different in the 2 groups. These preliminary results show that chronic vitamin E deficiency associated with high cholesterol diet is able to increase lipid peroxidation without modulation of AOE expression and activity in the liver. This suggests that beneficial effects of dietary vitamin E are due to a plasma antioxidant effect or a cell mediated action, rather than to a specific modulation of cellular enzymes.