Cesar G. Fraga

Lipid peroxidation measured as thiobarbituric acid-reactive substances in tissue slices: characterization and comparison with homogenates and microsomes

Liver slices were used to measure lipid peroxidation induced by bromotrichloromethane, tert-butyl hydroperoxide (t-BOOH), or ferrous iron. The responses of liver homogenates and microsomes to oxidative conditions were compared with the response of tissue slices. Lipid peroxidation was evaluated by the production of thiobarbituric acid-reactive substances (TBARS). As was observed in homogenates and microsomes, TBARS production by liver slices depended upon the amount of tissue, the incubation time, inducer, the amount of inducer, and the presence of antioxidant. Control liver slices incubated at 37 degrees C for 2 h produced 19 nmol of TBARS per g of liver. When slices were incubated in the presence of 1 mM BrCCl3, 1 mM t-BOOH, or 50 microM ferrous iron, TBARS production increased 4.6-, 8.2-, or 6.7-fold over the control value, respectively. Comparable induction of TBARS by liver homogenates and microsomes was observed when these preparations were incubated with the same inducers. Addition of 5 microM butylated hydroxytoluene (BHT) prevented the induction of TBARS by 50 microM ferrous iron by liver slices. The results indicate the usefulness of tissue slices to measure lipid peroxidation. The usefulness of tissue slices is emphasized when a number of compounds or tissues are studied and tissue integrity is desired as in toxicological, pharmacological, and nutritional studies where reduced numbers of experimental animals is a relevant issue.

Basic biochemical mechanisms behind the health benefits of polyphenols.

Polyphenols and consequently many flavonoids have several beneficial actions on human health. However, the actual molecular interactions of polyphenols with biological systems remain mostly speculative. This review addresses the potential mechanisms of action that have been so far identified, as well as the feasibility that they could occur in vivo. Those mechanisms include: i) non specific actions, based on chemical features common to most polyphenols, e.g. the presence of a phenol group to scavenge free radicals; and ii) specific mechanisms; based on particular structural and conformational characteristics of select polyphenols and the biological target, e.g. proteins, or defined membrane domains. A better knowledge about the nature and biological consequences of polyphenol interactions with cell components will certainly contribute to develop nutritional and pharmacological strategies oriented to prevent the onset and/or the consequences of human disease.

Epicatechin in Human Plasma: In Vivo Determination and Effect of Chocolate Consumption on Plasma Oxidation Status

Diets that are rich in plant foods have been associated with a decreased risk for specific disease processes and certain chronic diseases. In addition to essential macronutrients and micronutrients, the flavonoids in a variety of plant foods may have health-enhancing properties. Chocolate is a food that is known to be rich in the flavan-3-ol epicatechin and procyanidin oligomers. However, the bioavailability and the biological effects of the chocolate flavonoids are poorly understood. To begin to address these issues, we developed a method based on HPLC coupled with electrochemical (coulometric) detection to determine the physiological levels of epicatechin, catechin and epicatechin dimers. This method allows for the determination of 20 pg (69 fmol) of epicatechin, which translates to plasma concentrations as low as 1 nmol/L. We next evaluated the absorption of epicatechin, from an 80-g semisweet chocolate (procyanidin-rich chocolate) bolus. By 2 h after ingestion, there was a 12-fold increase in plasma epicatechin, from 22 to 257 nmol/L (P < 0.01). Consistent with the antioxidant properties of epicatechin, within the same 2-h period, there was a significant increase of 31% in plasma total antioxidant capacity (P < 0.04) and a decrease of 40% in plasma 2-thiobarbituric acid reactive substances (P < 0.01). Plasma epicatechin and plasma antioxidant capacity approached baseline values by 6 h after ingestion. These results show that it is possible to determine basal levels of epicatechin in plasma. The data support the concept that the consumption of chocolate can result in significant increases in plasma epicatechin concentrations and decreases in plasma baseline oxidation products.

A Dose-Response Effect from Chocolate Consumption on Plasma Epicatechin and Oxidative Damage

Evidence from epidemiological studies suggests that a diet high in plant foods and rich in polyphenols is inversely associated with a risk for cardiovascular and other chronic diseases. Chocolate, like red wine and green tea, is a polyphenol-rich food, primarily containing procyanidin polyphenols. These polyphenols are hypothesized to provide cardioprotective effects due to their ability to scavenge free radicals and inhibit lipid oxidation. Herein, we demonstrate that 2 h after the ingestion of a procyanidin-rich chocolate containing 5.3 mg total procyanidin/g, of which 1.3 mg/g was (-)-epicatechin (epicatechin), plasma levels of epicatechin increased 133 +/- 27, 258 +/- 29 and 355 +/- 49 nmol/L in individuals who consumed 27, 53 and 80 g of chocolate, respectively. That the rise in plasma epicatechin levels was functionally significant is suggested by observations of trends for dose-response increases in the plasma antioxidant capacity and decreases in plasma lipid oxidation products. The above data support the theories that in healthy adults, 1) a positive relationship exists between procyanidin consumption and plasma procyanidin concentration and 2) the rise in plasma epicatechin contributes to the ability of plasma to scavenge free radicals and to inhibit lipid peroxidation.

Flavonoid-membrane interactions: a protective role of flavonoids at the membrane surface?

Flavonoids can exert beneficial health effects through multiple mechanisms. In this paper, we address the important, although not fully understood, capacity of flavonoids to interact with cell membranes. The interactions of polyphenols with bilayers include: (a) the partition of the more non-polar compounds in the hydrophobic interior of the membrane, and (b) the formation of hydrogen bonds between the polar head groups of lipids and the more hydrophilic flavonoids at the membrane interface. The consequences of these interactions are discussed. The induction of changes in membrane physical properties can affect the rates of membrane lipid and protein oxidation. The partition of certain flavonoids in the hydrophobic core can result in a chain breaking antioxidant activity. We suggest that interactions of polyphenols at the surface of bilayers through hydrogen bonding, can act to reduce the access of deleterious molecules (i.e. oxidants), thus protecting the structure and function of membranes.

Iron toxicity and antioxidant nutrients

Iron is an essential nutrient for the growth, development, and long-term survival of most organisms. High tissue iron concentrations have been associated with the development and progression of several pathological conditions, including certain cancers, liver and heart disease, diabetes, hormonal abnormalities, and immune system dysfunctions. In this review we discuss the relevance of iron toxicity on free radical-mediated tissue damage, and how iron interactions with nutrient antioxidants and other metals can affect the extent of oxidative damage to different biomolecules. It can be concluded that the ingestion of antioxidant rich foods may prevent or delay primary and secondary effects associated with iron overload-related diseases.

Increased chemiluminescence and superoxide production in the liver of chronically ethanol-treated rats

Rats fed ethanol (1.74 +/- 0.12 g/day/100 g body wt for 12 weeks) showed a 45% increased microsomal production of O-2 (2.23 +/- 0.14 nmol/min/mg protein) and a 28% increased content of endoplasmic reticulum protein (26.8 +/- 1.4 mg/g liver). This could lead, at substrate saturation, to a 86% increased cytosolic production of O-2 which is not compensated by cytosolic superoxide dismutase levels that remain normal. It is claimed that this unbalance between O-2 production and superoxide dismutase leads to a peroxidative stress in agreement with the 54% increased spontaneous liver chemiluminescence (37 +/- 2 cps/cm2) measured in the ethanol-treated rats. Hydroperoxide-induced chemiluminescence was 57, 43, and 28% higher, respectively, in homogenates, mitochondria, and microsomes isolated from ethanol-treated rats as compared with controls. Vitamins E and A were more effective inhibitors of the hydroperoxide-stimulated chemiluminescence in the liver homogenates from ethanol-treated rats as compared with the effect on the homogenates from control animals. The results are consistent with a peroxidative stress in chronic alcoholism leading to increased lipoperoxidation and decreased levels of antioxidants.

Cocoa antioxidants and cardiovascular health

An increasing body of epidemiologic evidence supports the concept that diets rich in fruits and vegetables can promote health and attenuate, or delay, the onset of various diseases. Epidemiologic data support the idea that these health benefits are causally linked to the consumption of certain flavonoids present in fruit and vegetables. In the context of cardiovascular health, a particular group of flavonoids, namely, the flavan-3-ols (flavanols), has received attention. Flavanol-rich, plant-derived foods and beverages include wine, tea, and various fruits and berries, as well as cocoa and cocoa products. Numerous dietary intervention studies in humans and animals indicate that flavanol-rich foods and beverages might exert cardioprotective effects with respect to vascular function and platelet reactivity. This review discusses the bioactivity of flavanols in the context of cardiovascular health, with respect to their bioavailability, their antioxidant properties, and their vascular effects.

Inhibition of angiotensin converting enzyme (ACE) activity by flavan-3-ols and procyanidins.

It was determined that flavan‐3‐ols and procyanidins have an inhibitory effect on angiotensin I converting enzyme (ACE) activity, and the effect was dependent on the number of epicatechin units forming the procyanidin. The inhibition by flavan‐3‐ols and procyanidins was competitive with the two substrates assayed: N‐hippuryl‐L‐histidyl‐L‐leucine (HHL) and N‐[3‐(2‐furyl)acryloyl]‐L‐phenylalanylglycylglycine (FAPGG). Tetramer and hexamer fractions were the more potent inhibitors, showing Ki of 5.6 and 4.7 μM, respectively. As ACE is a membrane protein, the interaction of flavanols and procyanidins with the enzyme could be related to the number of hydroxyl groups on the procyanidins, which determine their capacity to be adsorbed on the membrane surface.

Epicatechin, catechin, and dimeric procyanidins inhibit PMA-induced NF-κB activation at multiple steps in Jurkat T cells

The capacity of the flavan‐3‐ols [(–)‐epicatechin (EC) and (+)‐catechin (CT)] and a B dimeric procyanidin (DP‐B) to modulate phorbol 12‐myristate 13‐acetate (PMA)‐induced NF‐κB activation in Jurkat T cells was investigated. The classic PMA‐triggered increase in cell oxidants was prevented when cells were preincubated for 24 h with EC, CT, or DP‐B (1.7–17.2 μM). PMA induced the phosphorylation of IKKβ and the subsequent degradation of IκBα: These events were inhibited in cells pretreated with the flavonoids. PMA induced a 4.6‐fold increase in NF‐κB nuclear binding activity in control cells. Pretreatment with EC, CT, or DP‐B decreased PMA‐induced NF‐κB binding activity and the transactivation of the NF‐κB‐driven gene IL‐2. EC, CT, and DP‐B inhibited, in vitro, NF‐κB binding to its DNA consensus sequence, but they had no effect on the binding activity of CREB or OCT‐1. Thus, EC, CT, or DP‐B can influence the immune response by modulating NF‐κB activation. This modulation can occur at early (regulation of oxidant levels, IKK activation) as well as late (binding of NF‐κB to DNA) stages of the NF‐κB activation cascade. A model is presented for possible interactions between DP‐B and NF‐κB proteins, which could lead to the inhibition of NF‐κB binding to κB sites.