Enhancement of D-chiro-inositol transport across intestinal cells by alpha-Lactalbumin peptides.

OBJECTIVE: This study aims to characterize in vitro D-chiro-inositol intestinal absorption and identify factors able to improve its bioavailability. D-chiro-inositol, one of the natural occurring stereoisomer of myo-inositol, acts as a second messenger in insulin-regulated glucose metabolism in complementary mode with myo-inositol. Because of their insulin-mimetic activities and safety, both myo-inositol and D-chiro-inositol are often employed as supplements in insulin-resistance treatment. MATERIALS AND METHODS: Trans-epithelial passage of D-chiro-inositol was evaluated in the human intestinal Caco-2 cell line differentiated on filter, a widely established in vitro model to study intestinal absorption. D-chiro-inositol transport was assayed in a concentration range corresponding to an estimated in vivo concentration following oral supplementation. α-Lactalbumin peptides, obtained by in vitro simulated gastrointestinal digestion, were tested as possible modulators of the intestinal permeability of D-chiro-inositol. RESULTS: The absorption of this stereoisomer was relatively low and presumably due to passive diffusion, while it was greatly enhanced by the presence of α-Lactalbumin digest. α-Lactalbumin peptides induced an increase in paracellular permeability that was completely reversible, indicating lack of cytotoxicity. This effect involved temporary rearrangement of F-actin apical cytoskeleton and of the tight junction protein ZO-1. CONCLUSIONS: Although further studies are required to identify and characterize the most effective peptides, the ability of α-Lactalbumin digest to act as absorption enhancers may have very interesting and promising applications in the fields of nutritional supplements and pharmacology.

Red wine prevents the postprandial increase in plasma cholesterol oxidation products: a pilot study.

Moderate wine consumption has been shown to lower cardiovascular risk. One of the mechanisms could involve the control of postprandial hyperlipaemia, a well-defined risk factor for atherosclerosis, reasonably by reducing the absorption of lipid oxidised species from the meal. The objective of the present study was to investigate whether wine consumption with the meal is able to reduce the postprandial increase in plasma lipid hydroperoxides and cholesterol oxidation products, in human subjects. In two different study sessions, twelve healthy volunteers consumed the same test meal rich in oxidised and oxidisable lipids (a double cheeseburger), with 300 ml of water (control) or with 300 ml of red wine (wine). The postprandial plasma concentration of cholesterol oxidation products was measured by GC-MS. The control meal induced a significant increase in the plasma concentration of lipid hydroperoxides and of two cholesterol oxidation products, 7-ß-hydroxycholesterol and 7-ketocholesterol. The postprandial increase in lipid hydroperoxides and cholesterol oxidation products was fully prevented by wine when consumed with the meal. In conclusion, the present study provides evidence that consumption of wine with the meal could prevent the postprandial increase in plasma cholesterol oxidation products.

Effect of coffee drinking on platelets: inhibition of aggregation and phenols incorporation.

Epidemiological studies indicate a J-shaped relationship linking coffee consumption and cardiovascular risk, suggesting that moderate coffee consumption can be beneficial. Platelet aggregation is of critical importance in thrombotic events, and platelets play a major role in the aetiology of several CVD. The aim of this study was to evaluate the effect of coffee drinking on platelet aggregation ex vivo, using caffeine as control. A crossover study was performed on ten healthy subjects. In two different sessions, subjects drank 200 ml coffee, containing 180 mg caffeine, or a capsule of caffeine (180 mg) with 200 ml water. Platelets were separated from plasma at baseline and 30 and 60 min after coffee drinking. Platelet aggregation was induced with three different agonists: collagen, arachidonic acid and ADP. Coffee drinking inhibited collagen (P < 0.05 from baseline at time 30 min) and arachidonic acid (P < 0.05 from baseline at time 60 min) induced platelet aggregation. Caffeine intake did not affect platelet aggregation induced by the three agonists. Coffee consumption induced a significant increase of platelet phenolic acids (likely present as glucuronate and sulphate derivatives), caffeic acid, the principal phenolic acid in coffee, raising from 0.3 (SEM 0.1) to 2.4 (SEM 0.6) ng/mg (P < 0.01). Caffeine was not detectable in platelets. Coffee drinking decreases platelet aggregation, and induces a significant increase in phenolic acid platelet concentration. The antiplatelet effect of coffee is independent from caffeine and could be a result of the interaction of coffee phenolic acids with the intracellular signalling network leading to platelet aggregation.

Polyphenols synergistically inhibit oxidative stress in subjects given red and white wine.

Aim of this study was to analyse the relationship between the plasma levels of polyphenols and the antioxidant activity of red and white wine. Twenty healthy subjects (HS) were randomly allocated to drink 300 ml of red (n = 10) or white n = 10 wine for 15 days. Ten HS who refrained from any alcohol beverage for 15 days were used as control. Urinary PGF-2alpha-III, a marker of oxidative stress and plasma levels of polyphenols were measured. Urinary PGF-2alpha-III significantly fell in subjects taking wine with a higher percentage decrease in subjects given red wine (-38.5 +/- 6%, p < 0.001) than in those given white wine (-23.1 +/- 6%). Subjects taking red wine had higher plasma polyphenols than those taking white wine (1.9 +/- 0.6 microM versus 1.5 +/- 0.33 microM, p < 0.001). Plasma polyphenols were inversely correlated with urinary PGF2alpha (r = 0.77, p < 0.001). No changes of urinary isoprostanes were observed in subjects who refrained from wine intake. In vitro study demonstrated that only a mixture of polyphenols, all in a range corresponding to that found in human circulation, inhibited LDL oxidation and PKC-mediated NADPH oxidase activation. Such inhibitory effects were more marked using the concentrations of polyphenols detected in human circulation after red wine intake. This study shows that red wine is more antioxidant than white wine in virtue of its higher content of polyphenols, an effect that may be dependent upon a synergism among polyphenols.

Biphasic effect of iron on human intestinal Caco-2 cells: early effect on tight junction permeability with delayed onset of oxidative cytotoxic damage.

Treatment of differentiated human intestinal Caco-2 cells with Fe(II) ascorbate altered tight junction permeability in a dose and time-dependent way for up to 3 hr of treatment Upon iron removal and transfer to complete culture medium, the effect was reversible up to 10 microM Fe(II), while at higher concentrations a late phase toxic effect was observed. Reduction of intracellular energy abolished the short term effect of iron on tight junction permeability without affecting its cellular uptake, suggesting that active processes, other than transport, were involved. The short term effect of iron the permeability of tight junctions did not appear to result from the generation of reactive oxygen species, as it was not prevented by antioxidant treatment under normal energy conditions. Conversely, the late phase effect leading to both apoptosis and necrosis during the 24 hr following iron removal could be reduced by antioxidant treatment and was exacebated by GSH depletion. Iron induced oxidative stress may therefore be responsible for membrane damage and cellular death occurring in the late phase. The reported effects of iron on intestinal tight junction permeability followed by more widespread cytotoxicity from oxidative events should be considered in light of the extensive use of iron supplementation in different phases of human life.

Selective changes in DNA binding activity of transcription factors in UM-X7.1 cardiomyopathic hamsters.

UM-X7.1 hamsters (CH) are considered a representative model for human cardiomyopathy. CH display the loss of the cytoskeletal delta-sarcoglycan protein, associated with myocardium remodeling and fatal reduction of heart functional efficiency. Even though altered redox balance and calcium homeostasis have already been reported to affect cardiomyocyte function, the molecular mechanisms underlying this pathology are largely unknown. We found no significant differences in DNA binding activity of redox-related (NF-kappaB, Sp1, AP-1 and AP-2) transcription factors in heart ventricles of 90 day-old CH, compared to normal animals. On the other hand, DNA binding activity of calcium-dependent transcription factors NF-AT3 and CREB were increased and decreased respectively in CH vs. normal ventricles. Western blot experiments confirmed the down regulation of CREB levels and suggest a novel regulation mechanism for this transcription factor in the heart. Our results are consistent with recent studies on NF-AT3, GATA4 and CREB transgenic mice, and provide clues for the comprehension of pathogenetic mechanisms of hamster hereditary cardiomyopathy.

Absorption of phenolic acids in humans after coffee consumption.

Despite extensive literature describing the biological effects of polyphenols, little is known about their absorption from diet, one major unresolved point consisting of the absorption of the bound forms of polyphenols. In this view, in the present work we studied the absorption in humans of phenolic acids from coffee, a common beverage particularly rich in bound phenolic acids, such as caffeic acid, ferulic acid, and p-coumaric acid. Coffee brew was analyzed for free and total (free + bound) phenolic acids. Chlorogenic acid (5'-caffeoylquinic acid), a bound form of caffeic acid, was present in coffee at high levels, while free phenolic acids were undetectable. After alkaline hydrolysis, which released bound phenolic acids, ferulic acid, p-coumaric acid, and high levels of caffeic acid were detected. Plasma samples were collected before and 1 and 2 h after coffee administration and analyzed for free and total phenolic acid content. Two different procedures were applied to release bound phenolic acids in plasma: beta-glucuronidase treatment and alkaline hydrolysis. Coffee administration resulted in increased total plasma caffeic acid concentration, with an absorption peak at 1 h. Caffeic acid was the only phenolic acid found in plasma samples after coffee administration, while chlorogenic acid was undetectable. Most of caffeic acid was present in plasma in bound form, mainly in the glucuronate/sulfate forms. Due to the absence of free caffeic acid in coffee, plasma caffeic acid is likely to be derived from hydrolysis of chlorogenic acid in the gastrointestinal tract.

Red wine mitigates the postprandial increase of LDL susceptibility to oxidation.

The aim of the present study was to verify the extent of oxidative stress induced by a meal at plasma and LDL level, and to investigate the capacity of red wine to counteract this action. In two different sessions, six healthy men ate the same test meal consisting of "Milanese" meat and fried potatoes. The meal was taken either with 400 ml red wine or with an isocaloric hydroalcoholic solution. Oxidative stress at plasma level was estimated through the measure of ascorbic acid, alpha-tocopherol, protein SH groups, uric acid, and antioxidant capacity, measured before and 1 and 3 h after the meal. The change in the resistance of LDL to oxidative modification was taken as an index of exposure to pro-oxidants. The susceptibility to Cu(II)-catalyzed oxidation of baseline and postprandial LDL was measured as conjugated dienes formation, tryptophan residues, and relative electrophoretic mobility. The experimental meal taken with wine provoked a significant increase in the total plasma antioxidant capacity and in the plasma concentration of alpha-tocopherol and SH groups. Postprandial LDL was more susceptible to metal-catalyzed oxidation than the homologous baseline LDL after the ethanol meal. On the contrary, postprandial LDL obtained after the wine meal was as resistant or more resistant to lipid peroxidation than fasting LDL.

Total antioxidant capacity as a tool to assess redox status: critical view and experimental data.

The measure of antioxidant capacity (AC) considers the cumulative action of all the antioxidants present in plasma and body fluids, thus providing an integrated parameter rather than the simple sum of measurable antioxidants. The capacity of known and unknown antioxidants and their synergistic interaction is therefore assessed, thus giving an insight into the delicate balance in vivo between oxidants and antioxidants. Measuring plasma AC may help in the evaluation of physiological, environmental, and nutritional factors of the redox status in humans. Determining plasma AC may help to identify conditions affecting oxidative status in vivo (e.g., exposure to reactive oxygen species and antioxidant supplementation). Moreover, changes in the plasma AC after supplementation with galenic antioxidants or with antioxidant-rich foods may provide information on the absorption and bioavailability of nutritional compounds. Consequently, this review discusses the rationale, interpretation, confounding factors, measurement limits, and human applications of the measure of plasma AC.

Ferulic acid excretion as a marker of consumption of a French maritime pine (Pinus maritima) bark extract.

French maritime pine (Pinus maritima) bark extract (PBE) is a polyphenol-rich food supplement patented under the name of Pycnogenol and known to have strong antioxidant activity and different beneficial effects on human health. Although its biological properties have begun to be extensively studied both in vitro, in laboratory animals and more recently in humans, little is known about its bioavailability. The present study investigated the urinary excretion of free and conjugated ferulic acid, present in quantitatively detectable amounts in PBE, after oral PBE administration to human subjects. Eleven healthy adult subjects (4 women and 7men) consumed either a single dose (200 mg PBE) or two doses of PBE (100 and 200 mg, respectively) within a 48-h interval. Two days before the oral administration of PBE and during the urine sample collection period volunteers adhered to a diet low in polyphenols. Aliquots of all urine production were collected over 24 h. Free and conjugated ferulic acid was assessed in urine by HPLC using diode array detection. A close association between the dietary intake of PBE and the urinary excretion of ferulic acid was detected. Moreover, the results indicate that a considerable proportion of ferulic acid is excreted as glucuronide or sulfate after PBE consumption, varying over the range 2 to 20% between individuals. The kinetics of excretion associated with the administration of 100 mg PBE was quite similar to that obtained after 200 mg PBE. A a biphasic trend was evident in a number of subjects. All subjects studied here displayed a significant, although variable level of excretion of ferulic acid after supplementation with PBE, Thus, the data provide evidence that at least a part of the phenolic components of PBE are absorbed, metabolized, and eliminated by humans.

Beer increases plasma antioxidant capacity in humans.

The positive association of a moderate intake of alcoholic beverages with a low risk for cardiovascular disease, in addition to ethanol itself, may be linked to their polyphenol content. This article describes the effect of acute ingestion of beer, dealcoholized beer, and ethanol (4.5% v/v) on the total plasma antioxidant status of subjects, and the change in the high performance liquid chromatography profile of some selected phenolic acids (caffeic, sinapic, syringic, and vanillic acids) in 14 healthy humans. Plasma was collected at various times: before (T0), 1 hour after (T1), and 2 hours after (T2) drinking. The study is part of a larger research planned to identify both the impact of brewing on minor components potentially present in beer and their metabolic fate in humans. Beer was able to induce a significant (P < 0.05) increase in plasma antioxidant capacity at T1 (mean +/- SD: T0 1,353 +/- 320 microM; T1 1,578 +/- 282 microM), returning close to basal values at T2. All phenolic acids measured in plasma tended to increase after beer intake (20% at T1, 40% at T2). Syringic and sinapic acid reached statistical significance (P < 0.05 by one-way analysis of variance-Fisher's test) at T1 and T2, respectively. Plasma metabolic parameters (glucose, total cholesterol, triglycerides, and uric acid) and plasma antioxidants (alpha-tocopherol and glutathione) remained unchanged. Ethanol removal impaired the absorption of phenolic acids, which did not change over the time of the experiment, accounting for the low (and not statistically significant) increase in plasma antioxidant capacity after dealcoholized beer drinking. Ethanol alone did not affect plasma antioxidant capacity or any of the antioxidant and metabolic parameters measured.

Benzoic and cinnamic acid derivatives as antioxidants: structure-activity relation.

The antioxidant activity of four derivatives of benzoic acid was systematically compared with the activity of the four homologous derivatives of cinnamic acid. The couples of compounds differed for the kind of aromatic substitution (p-hydroxy, p-hydroxymethoxy, p-hydroxydimethoxy, dihydroxy). The antioxidant activity was measured using (i) a competition kinetic test, to measure the relative capacity to quench peroxyl radical and (ii) the in vitro oxidative modification of human low-density lipoprotein (LDL), initiated by 2,2'-azobis(amidinopropane) dihydrochloride or catalyzed by Cu(II). In both models, cinnamic acids were more efficient than their benzoic counterparts. As for the influence of the aromatic substitution, in the kinetic test the antioxidant activity increased in the sequence p-hydroxy < p-hydroxymethoxy < dihydroxy < p-hydroxydimethoxy. In contrast, in the LDL system, the dihydroxy acids had an antioxidant capacity equal to or higher than that of the p-hydroxydimethoxy acids.

Effect of caffeic acid on tert-butyl hydroperoxide-induced oxidative stress in U937.

Nonvitamin phenolic compounds are ubiquitous in food plants and therefore potentially present in human plasma in a diet-dependent concentration. The aim of this study was to evaluate the ability of caffeic acid, a phenolic acid with antioxidant activity, to affect cellular response in U937 human monocytic cells to t-butyl hydroperoxide-induced oxidative stress. In our experimental conditions caffeic acid was incorporated into cells without any cytotoxic effect. Caffeic acid-treated cells showed an increased resistance to oxidative challenge, as revealed by an higher percent of survival and the maintenance of an higher proliferative capacity in respect to control cells. This effect seems to be due to the ability of caffeic acid to reduce glutathione depletion and to inhibit lipid peroxidation during tBOOH treatment. It can be concluded that caffeic acid exerts an antioxidant action inside the cell, responsible for the observed modulation of the cellular response to oxidative challenge. Due to its presence in the diet, therefore, caffeic acid may play a role in the modulation of oxidative processes in vivo.

Oxidative modification of human low-density lipoprotein by horseradish peroxidase in the absence of hydrogen peroxide.

Heme-peroxidases, such as horseradish peroxidase (HRP), are among the most popular catalysts of low density lipoprotein (LDL) peroxidation. In this model system, a suitable oxidant such as H2O2 is required to generate the hypervalent iron species able to initiate the peroxidative chain. However, we observed that traces of hydroperoxides present in a fresh solution of linoleic acid can promote lipid peroxidation and apo B oxidation, substituting H2O2. Spectral analysis of HRP showed that an hypervalent iron is generated in the presence of H2O2 and peroxidizing linoleic acid. Accordingly, careful reduction of the traces of linoleic acid lipid hydroperoxide prevented formation of the ferryl species in HRP and lipid peroxidation. However, when LDL was oxidized in the presence of HRP, the ferryl form of HRP was not detectable, suggesting a Fenton-like reaction as an alternative mechanism. This was supported by the observation that carbon monoxide, a ligand for the ferrous HRP, completely inhibited peroxidation of LDL. These results are in agreement with previous studies showing that myoglobin ferryl species is not produced in the presence of phospholipid hydroperoxides, and emphasize the relevance of a Fenton-like chemistry in peroxidation of LDL and indirectly, the role of pre-existing lipid hydroperoxides.

Effect of caffeic acid dietary supplementation on the antioxidant defense system in rat: an in vivo study.

Dietary supplementation of caffeic acid (0.2 and 0.8% w/w) in rats resulted in a statistically significant increase of alpha-tocopherol both in plasma and lipoprotein. While caffeic acid was not detectable in plasma under fasting conditions, in postprandial plasma it was present at micromole concentrations, doubling plasma total antioxidant capacity. Lipoproteins from caffeic acid-fed rats were more resistant than control to Cu2+-catalyzed oxidation, despite the lack of incorporation of caffeic acid in the particles. No significant effects on plasma and liver copper concentration, nor the increase in liver of Mn-superoxide dismutase reported in copper deficiency, were detected. These results demonstrate the physiological relevance of caffeic acid and its antioxidant action in vivo, through both a direct contribution to the antioxidant defense system and a sparing effect on alpha-tocopherol.