Evidence of postprandial absorption of olive oil phenols in humans.

BACKGROUND AND AIM: Olive oil phenols are potent antioxidants in vitro. If this were to be also demonstrated in vivo, it would help to explain the beneficial effects of this typical ingredient of the Mediterranean diet. This study was designed to determine the presence in lipoprotein fractions of two phenolic compounds peculiar to extra virgin olive oil, namely tyrosol and OH-tyrosol, and whether their absorption induces an antioxidant effect in vivo. METHODS AND RESULTS: Two trials were performed. In the first (Long-term), 14 healthy volunteers followed two diets, each for one month. The only difference between the diets was that the first supplied 50 g of extra virgin olive oil per day, where-as the second one supplied 50 g of refined olive oil with no simple phenols, as demonstrated by GC-MS analysis. There were no changes in LDL oxidizability and tyrosol and OH-tyrosol were not recovered in lipoproteins and plasma from fasting samples drawn at the end of each diet period. In the second study (Postprandial), eight healthy volunteers received an oral fat load consisting of 100 g of extra virgin olive oil. Blood was drawn at times 0', 30', 60', 120', 240', 360', and major plasma lipoprotein classes were separated. The concentration of tyrosol, OH-tyrosol and vitamin E was determined in lipoprotein fractions. Plasma antioxidant capacity was measured by a crocin-bleaching test and expressed as mM Trolox C equivalents. Tyrosol and OH-tyrosol were recovered in all lipoprotein fractions, except VLDL, with concentrations peaking between 60' and 120'. However, a very high variability in tyrosol and OH-tyrosol absorption was observed among subjects. Vitamin E content of LDL and HDL did not vary significantly throughout the study. Plasma antioxidant capacity increased significantly at time 120' (baseline 0.96 mM Trolox; 120' 1.19 mM Trolox; p = 0.02), and then returned almost to baseline values after 360' (1.1 mM Trolox). CONCLUSIONS: These findings suggest that phenolic compounds in olive oil are absorbed from the intestine, though not through a pathway dependent on chylomicron formation, and may exert a significant antioxidant effect in vivo, probably in the postprandial phase.

Synergistic inhibition of LDL oxidation by phytoestrogens and ascorbic acid.

Increasing evidence indicates that oxidative modification of low-density lipoprotein (LDL) is an important determinant in atherogenesis, and following menopause, the incidence of coronary heart disease is as prevalent in women as it is in men. Estrogen has been demonstrated to inhibit the susceptibility of LDL to be oxidized, and more recently the use of phytoestrogens has been considered for estrogen replacement therapy. In this study the antioxidant activity of the three major phytoestrogens: genistein, daidzein, and equol were measured in terms of LDL oxidative susceptibility. Increasing levels of genistein, daidzein, and equol inhibited LDL oxidation, and this inhibitory effect was further enhanced in the presence of ascorbic acid. The synergism exhibited by these compounds is of clinical importance to phytoestrogen therapy since the efficacy of phytoestrogens as effective antioxidants is evident at concentration well within the range found in the plasma of subjects consuming soy products. However, this synergism, combined with the low reactivity of the phytoestrogens with peroxyl radicals, suggests that an antioxidant mechanism other then free radical scavenging reactions account for the phytoestrogen antioxidant effect. A structural basis for inhibition of LDL oxidation involving interaction of the phytoestrogens with apoB-100 is postulated.

Selenium, the element of the moon, in life on earth.

The present status of selenium biochemistry is reviewed with particular emphasis on biomedical problems related to the selenium status of humans and experimental animals. Historical milestones of selenium biochemistry starting from the identification of the first selenoenzymes up to the elucidation of prokaryotic and eukaryotic selenoprotein biosynthesis are compiled. Topical hypotheses on the biological role of selenium in general and of individual selenoproteins in respect to antioxidant defense, redox regulation of metabolic processes, thyroid function, spermatogenesis, oncogenesis, and atherogenesis are critically evaluated.

Reactive oxygen species and proinflammatory cytokine signaling in endothelial cells: effect of selenium supplementation.

The release of superoxide (O(2)(*-)) and hydrogen peroxide (H(2)O(2)), induced by tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta (IL-1beta), has been studied in the endothelial cell line ECV 304 in the presence and absence of selenium (Se) supplementation. Both cytokines elicit the production of both species. Selenium supplementation, which increases Se-enzyme activity, decreases the amount of H(2)O(2) but not O(2)(*-) detectable in the extracellular medium. Inhibition of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase by diphenyliodonium (DPI) or phenylarsine oxide (PAO), largely prevents O(2)(*-) production, whereas H(2)O(2) remains above the amount accounted for by disproportion of residual O(2)(*-). Thus, a fraction of H(2)O(2) found in the medium, derives from an intracellular pool, which is under control of selenium-dependent peroxidases. This is further supported by the observation that in Se-supplemented cells, the rate of intracellular glutathione (GSH) depletion induced by cytokine treatment is faster and more extensive. Because Se supplementation decreases cytokine-induced NF-kappaB activity, whereas added H(2)O(2) is inactive and catalase does not affect the activation induced by TNF-alpha, it is concluded that only intracellularly generated H(2)O(2) has a role in transcription factor activation by both TNF-alpha and IL-1beta.

Optimization of nutrition: polyphenols and vascular protection.

The role of polyphenols in human nutrition is discussed on the basis of their redox chemistry, which accounts for the observed antioxidant effect and in turn for their protective effect against atherosclerosis. Epidemiologic data, together with experimental pathology and cell biology, support the recommendation that optimal nutrition should contain polyphenols in amounts that may be better described as a "Recommended Optimal Intake" (ROI) than as a "Recommended Dietary Allowance" (RDA). Because a valid procedure to identify polyphenols in plasma is not available, analysis of plasma antioxidant capacity is instead suggested as a suitable index to define the optimal nutritional intake.

Dual function of the selenoprotein PHGPx during sperm maturation.

The selenoprotein phospholipid hydroperoxide glutathione peroxidase (PHGPx) changes its physical characteristics and biological functions during sperm maturation. PHGPx exists as a soluble peroxidase in spermatids but persists in mature spermatozoa as an enzymatically inactive, oxidatively cross-linked, insoluble protein. In the midpiece of mature spermatozoa, PHGPx protein represents at least 50 percent of the capsule material that embeds the helix of mitochondria. The role of PHGPx as a structural protein may explain the mechanical instability of the mitochondrial midpiece that is observed in selenium deficiency.

Testosterone mediates expression of the selenoprotein PHGPx by induction of spermatogenesis and not by direct transcriptional gene activation.

Selenium deficiency is known to be associated with male infertility, and the selenoprotein PHGPx has been shown to increase in rat testis after puberty and to depend on gonadotropin stimulation in hypophysectomized rats [Roveri et al. (1992) J. Biol. Chem. 267, 6142 6146]. Exposure of decapsulated whole testis, however, failed to reveal any transcriptional activation or inhibition of the PHGPx gene by testosterone, human chorionic gonadotropin, or forskolin. Nevertheless, it was verified that the specific activity of PHGPx in testis, but not of cGPx, correlated with sexual maturation. Leydig cell destruction in vivo by ethane dimethane sulfonate (EDS) resulted in a delayed decrease in PHGPx activity and mRNA that could be completely prevented by testosterone substitution. cGPx transiently increased upon EDS treatment, probably as a result of reactive macrophage augmentation. In situ mRNA hybridization studies demonstrated an uncharacteristic low level of cGPx transcription in testis, whereas PHGPx mRNA was abundantly and preferentially expressed in round spermatids. The data show that the age or gonadotropin-dependent expression of PHGPx in testis does not result from direct transcriptional gene activation by testosterone, but is due to differentiation stage-specific expression in late spermatids, which are under the control of Leydig cell-derived testosterone. The striking burst of PHGPx expression at the transition of round to elongated spermatids suggests an involvement of this selenoprotein in sperm maturation.

Probing the presumed catalytic triad of a selenium-containing peroxidase by mutational analysis.

Glutathione peroxidases (GPx) are characterized by a catalytically active selenium which forms the center of a strictly conserved triad composed of selenocysteine, glutamine, and tryptophan. In order to check the functional relevance of this structural peculiarity, six molecular mutants of phospholipid hydroperoxide glutathione peroxidase (PHGPx) were designed, isolated, and investigated kinetically. Replacement of the selenocysteine in position 46 by cysteine decreased k + 1, i.e., the reaction rate of reduced enzyme with hydroperoxide, by three orders of magnitude. The rate of regeneration of the reduced enzyme by glutathione (k' + 2) was similarly affected. Additional substitution of Gln81 or Trp136 by acid residues resulted in a further decrease of k + 1 by three orders of magnitude, whereas histidine or neutral residues in these positions proved to be less deleterious. The data support the hypothesis that the typical triad of selenocysteine, glutamine, and tryptophan is indeed a novel catalytic center in which the reactivity of selenium is optimized by hydrogen bonding provided by the adjacent glutamine and tryptophan residues.

Probing the presumed catalytic triad of selenium-containing peroxidases by mutational analysis of phospholipid hydroperoxide glutathione peroxidase (PHGPx).

Single and double site mutants affecting the presumed catalytic centre of the selenoenzyme PHGPx were subjected to functional analysis. The rate constants k+1 and k'+2, for the oxidation and the regeneration of the ground state enzyme were estimated, respectively. Moreover, the alkylation rate of the reactive centre by iodoacetate (kinact.) was also analysed. The substitution of the catalytically competent selenocysteine 46 by cysteine (PHGPxcys46) decreased k+1 and k'+2 by about three orders of magnitude, although leaving unaffected kinact.. Furthermore, mutations of PHGPxcys46 involving the other residues of the triad decreased both kinact. and k+1, thus highlighting the involvement of Gln 81 and Trp 136 in the dissociation/activation of the nucleophilic cysteine thiol. In general, substitutions of Gln 81 or Trp 136 by acidic residues in PHGPxcys46 most dramatically depressed the k+1 values, because they practically prevented the dissociation of the thiol group, while neutral or positively charged residues in these positions allowed an intermediate dissociation and induced a corresponding reactivity of the thiol. Our data, for the first time, reveal that the presumed triad of selenocysteine, glutamine and tryptophan residues represents a novel type of catalytic centre, whose integrity is essential for the full catalytic function of glutathione peroxidases.

Protective effect of dietary selenium supplementation on delayed cardiotoxicity of adriamycin in rat: is PHGPX but not GPX involved?

The involvement of Se enzymes in the protection against the oxidative stress induced by adriamycin (ADR) in rat heart has been studied in animals fed for 10 weeks at three different levels of Se content (low = 0.02 ppm; normal = 0.5 ppm; high = 1.0 ppm) and receiving a weekly injection of 3 mg/kg ADR for 4 weeks. ECG (QaT duration) and contractility of isolated atria were measured. The high-Se diet showed a significant protection on both parameters. To assess the hypothesis that an increase of specific activity of antioxidant Se enzymes may account for the cardioprotective effect of selenium, glutathione peroxidase (GPX), and phospholipid hydroperoxide glutathione peroxidase (PHGPX) were tested. The assays were performed on ventricles isolated from treated rats. At the end of the experimental period, GPX (cytosolic enzyme) did not show any significant difference between controls and ADR-treated at any level of Se content, thus excluding its involvement in the cardioprotection observed in high-Se ADR-treated animals. PHGPX, which is present both in cytosol and in the cell membrane, showed a trend to increase its activity in the presence of ADR treatment only in the membrane fraction; however, the statistical significance was reached only in the low-Se group (+100%). This observation suggests that membrane PHGPX might be involved in the cellular mechanism of adaptation of the heart to the toxic effects of ADR; however, the behavior of these enzymes does not seem to account for the significant protection of selenium supplementation both on ECG and on contractile indices of ADR cardiotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of dietary monounsaturated and polyunsaturated fatty acids on the susceptibility of plasma low density lipoproteins to oxidative modification.

Oxidized low density lipoproteins (LDLs) are thought to play an important role in atherogenesis. Nutritional and biochemical studies suggest that diet can modulate the susceptibility of plasma LDL to undergo oxidative degradation by affecting the concentration of polyunsaturated fatty acids and antioxidants in the lipoprotein particle. In the present study 11 healthy male volunteers underwent two diet phases. In one phase the diet was enriched in oleic acid (mono), while in the other it was high in linoleic acid (poly). Both diets lowered plasma total and LDL cholesterol contents. The sensitivity of plasma LDL to oxidation was estimated by challenging these lipoproteins with 2,2'-azobis(2-amidinopropane)dihydrochloride, a free-radical initiator. Although neither diet affected the antioxidant content of plasma LDL, the resistance to lipid peroxidation, measured after the consumption of antioxidants present in the lipoprotein, was higher during the mono phase. Indeed, the peroxidation rate of plasma LDL was inversely correlated with the oleic acid to linoleic acid ratio in the LDL particle. These results support the thesis that diets rich in monounsaturated fatty acids increase the resistance of plasma LDL to oxidative modification, independent of their content of antioxidants. This effect could lower the atherogenicity of these lipoproteins.

Phospholipid hydroperoxide glutathione peroxidase of rat testis. Gonadotropin dependence and immunocytochemical identification.

A high glutathione peroxidase activity toward phospholipid hydroperoxides is present in rat testis. The attribution of this activity to the selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPX) was supported by cross-reactivity with antibodies raised against pig heart PHGPX which had been purified and characterized. Rat testis PHGPX is partially cytosolic and partially linked to nuclei and mitochondria. The soluble and organelle-bound enzymes appear identical by Western blot analysis. PHGPX, but neither selenium-dependent nor non-selenium-dependent glutathione peroxidase activity, is expressed in testes only after puberty, disappears after hypophysectomy, and is partially restored by gonadotropin treatment. Specific immunostaining of testes by antiserum against PHGPX appears as a fine granular brown pattern localized throughout the cytoplasm in more immature cells but is confined to the peripheral part of the cytoplasm, the nuclear membrane, and mitochondria in maturating spermatogenic cells. As expected, immunostaining of spermatogenic cells in hypophysectomized animals was negative, but gonadotropin treatment only marginally increased the immunoreactivity. The expression of PHGPX in testes is consistent with the previously described specific requirement for selenium for synthesis of a 15-20-kDa selenoprotein which is related to the production of functional spermatozoa.

Phospholipid hydroperoxide glutathione peroxidase is the 18-kDa selenoprotein expressed in human tumor cell lines.

Human tumor cell lines cultured in 75Se-containing media demonstrate four major 75Se-labeled cellular proteins (57, 22, 18, and 12 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Among these selenoproteins, an enzymatic activity is known only for the 22-kDa protein, since this protein has been identified as the monomer of glutathione peroxidase. However, all tested cell lines also contained a peroxidase activity with phospholipid hydroperoxides that is completely accounted for by the other selenoenzyme, phospholipid hydroperoxide glutathione peroxidase (PHGPX) (Ursini, F., Maiorino, M., and Gregolin, C. (1985) Biochim. Biophys. Acta 839, 62-70). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of 75Se-labeled proteins separated by gel permeation chromatography supported the identification of PHGPX as the monomeric protein matching the 18 kDa band. This paper is the first report on the identification of PHGPX in human cells.

Effect of fish oil and coconut oil on antioxidant defence system and lipid peroxidation in rat liver.

Diets high in fish oil containing polyunsaturated fatty acids of the n-3 family, have been suggested to decrease the risk of cardiovascular disease. However these lipids are highly susceptible to oxidative deterioration. In order to investigate the influence of n-3 fatty acids on oxidative status, the effect of feeding rats with fish oil or coconut oil diets was studied by measuring different parameters related to an oxidative free radical challenge. Synthetic diets containing 15% (w/v) fish oil or coconut oil were used to feed growing rats for 4 weeks. As compared to control diet, the fish oil containing diet produced a significant decrease of cholesterol and triglyceride concentration in serum, however there was a significant increase in lipid peroxidation products. In addition, in fish oil fed animals, there was also a decrease in vitamin E and A concentration. Furthermore, the rate of lipid peroxidation in isolated microsomes was three fold higher in rats fed fish oil as compared to rats with coconut oil diet. No significant differences between the two experimental groups were observed in superoxide dismutase (SOD) and phospholipid hydroperoxide glutathione peroxidase (PHGPX) activities. However, there was a decrease in glutathione peroxidase (GPX) activity. These results suggest that fish oil feeding at an amount compatible with human diet, although decreasing plasma lipids, actually challenge the antioxidant defence system, thus increasing the susceptibility of tissues to free radical oxidative damage.

Phospholipid hydroperoxide glutathione peroxidase in various mouse organs during selenium deficiency and repletion.

An assay for the determination of the newly discovered selenoenzyme, phospholipid hydroperoxide glutathione peroxidase (PH-GPx) in biological material is described. Dietary selenium deficiency and repletion was used as a tool in order to modify this enzyme activity in various mouse organs and to compare it to the activity of the 'classical' selenium-dependent glutathione peroxidase (GPx) (EC 1.11.1.9). A semipurified diet containing less than 12 ppb Se was used for depletion. Controls received this diet supplemented with 500 ppb Se in the form of Na2SeO3. The results showed that a rapid loss of GPx activity occurred in liver, kidney and lungs of selenium-deficient mice which reached undetectable levels within 130 days. In the heart, about 24% of control GPx activity was still present. In contrast, PH-GPx activity was more slowly depleted by Se deficiency and resulted in residual activities ranging from 30 to 70% in the different organs even after 250 days of depletion. In repletion experiments with a single application of 10 or 500 micrograms/kg Se, only the high dose restored either enzyme activity. The data demonstrate that the need for selenium of the two glutathione peroxidases is different. A markedly distinct organ distribution of both enzymes suggests that the heart may be the organ more sensitive to oxidative stress.

Therapeutic, hemodynamic, and metabolic effects of hyperbaric oxygenation in peripheral vascular disease.

Hyperbaric oxygen therapy (HBT) consists of inhaling 100% oxygen under pressure exceeding atmospheric pressure. Patients with various degrees of peripheral vascular disease (stages II, III, and IV according to Fontaine's classification) were treated with HBT. Clinical symptoms improved in 70%. Ankle/brachial index increased by more than 0.10 in 47%. Photoplethysmographic pulse appearance has been detected in roughly one third of patients. Transcutaneous oxygen tension did not, however, improve significantly. Plasma malondialdehyde (MDA) was evaluated as a marker of lipid peroxidation, possibly influenced by hyperbaric oxygen. Surprisingly, the authors found that MDA levels decreased after HBT, probably as a result of the activation of antioxidant systems. These results show that HBT represents a useful approach in the treatment of peripheral vascular disease, since it improves clinical and hemodynamic parameters, with no potentially harmful side effects, such as enhanced lipid peroxidation, at least in the short term.

Kinetic mechanism and substrate specificity of glutathione peroxidase activity of ebselen (PZ51).

The glutathione peroxidase activity of ebselen (PZ51) was studied using different hydroperoxidic substrates. The single progression curves obtained in the spectrophotometric test were processed by a computer to fit the integrated rate equation that describes the ping pong reaction of the Se glutathione peroxidase. Ebselen catalyzes the GSH peroxidase reaction with a mechanism that appears kinetically identical to the mechanism of the enzymes. The inactivation of the catalytic properties of ebselen by iodoacetate suggests that a selenol moiety is involved. Among the substrates tested, the best hydroperoxidic substrates are the hydroperoxy derivatives of phosphatidyl choline. Ebselen is active also on membrane hydroperoxides as does phospholipid hydroperoxide glutathione peroxidase but not glutathione peroxidase.

The role of selenium peroxidases in the protection against oxidative damage of membranes.

The present review deals with the chemical properties of selenium in relation to its antioxidant properties and its reactivity in biological systems. The interaction of selenite with thiols and glutathione and the reactivity of selenocompounds with hydroperoxides are described. After a short survey on distribution, metabolism and organification of selenium, the role of this element as a component of the two seleno-dependent glutathione peroxidases is described. The main features of glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase are also reviewed. Both enzymes reduce different hydroperoxides to the corresponding alcohols and the major difference is the reduction of lipid hydroperoxides in membrane matrix catalyzed only by the phospholipid hydroperoxide glutathione peroxidase. However, in spite of the different specificity for the peroxidic substrates, the kinetic mechanism of both glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase seems identical and proceeds through a tert-uni ping pong mechanism. In the reaction cycle, indeed, as supported by the kinetic data, the oxidation of the ionized selenol by the hydroperoxide yields a selenenic acid that in turn is reduced back by two reactions with reduced glutathione. Special emphasis has been given to the role of selenium-dependent glutathione peroxidases in the prevention of membrane lipid peroxidation. While glutathione peroxidase is able to reduce hydrogen peroxide and other hydroperoxides possibly present in the soluble compartment of the cell, this enzyme fails to inhibit microsomal lipid peroxidation induced by NADPH or ascorbate and iron complexes. On the other hand, phospholipid hydroperoxide glutathione peroxidase, by reducing the phospholipid hydroperoxides in the membranes, actively prevents lipid peroxidation, provided a normal content of vitamin E is present in the membranes. In fact, by preventing the free radical generation from lipid hydroperoxides, phospholipid hydroperoxide glutathione peroxidase decreases the vitamin E requirement necessary to inhibit lipid peroxidation. Finally, the possible regulatory role of the selenoperoxidases on the arachidonic acid cascade enzymes (cyclooxygenase and lipoxygenase) is discussed.