PubMed Health⌕ Search

Biomedical subjects

F Ursini

Publications and source records attributed to F Ursini.

At least 37 records · Page 2Linked to original sources
PubMed1999

Kinetic analysis of antioxidant capacity of wine.

A competition kinetics procedure for measuring total antioxidant capacity in wine is described. This procedure is based on the "crocin bleaching test" [28] as modified for analyzing the antioxidant capacity of complex mixtures [24]. The antioxidant capacity of white wines ranged from 0.08 to 1.2 mM equivalents of the reference antioxidant (Trolox C), while for red wines values ranging from 6.4 to 41.9 mM have been obtained. Although a correlation exists between antioxidant capacity and total phenol content of wines, due to the variable reactivity of different phenol groups, the analysis of the phenol content provides only a crude indication of the actual antioxidant capacity. The analysis of antioxidant capacity on different polyphenol classes, separated by solid phase extraction, indicated that anthocyanins are the major antioxidants of young red wines, and tannins of old red wines and white wines. Several vintages of the same grape have been analyzed and the expected decrease of antioxidant capacity upon ageing was not observed, although spectrophotometric analysis clearly demonstrated the shift from anthocyanin monomers to polymers. Artificial ageing by stirring under air produced a rapid decrease (30 min) of antioxidant capacity followed by an increase (up to two weeks), but not any significant modification of the spectrophotometric chemical age factor. Since neither natural nor artificial ageing present a clear-cut relationship with a decrease of antioxidant capacity, we could conclude that the a priori assumption that an old wine contains less antioxidant capacity, although popular, is not fully correct.

Air↗
PubMed1999

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.

Animals↗
PubMed1998

Postprandial plasma lipid hydroperoxides: a possible link between diet and atherosclerosis.

There is increasing evidence implicating a dietary source of plasma lipid peroxides that become elevated in the postprandial state. This phenomenon may be a contributing factor to the correlation found between postprandial hyperlipidemia and increased risk of cardiovascular disease. Using a newly developed method for measuring lipid hydroperoxides directly in plasma, a pilot study was performed which revealed that lipid hydroperoxides are indeed elevated following a fatty meal. Lipid hydroperoxides increased within 2-4 h after the meal and returned to basal levels, corresponding to the usual postprandial hyperlipidemia. A marked suppression of postprandial hydroperoxides was found when a meal was consumed with wine, suggesting that these hydroperoxides can be formed and then absorbed during the digestive process.

Adult↗
PubMed1998

Analysis of plasma antioxidant capacity by competition kinetics.

A competition kinetics procedure for measuring plasma antioxidant capacity is described. This procedure is based on the "crocin bleaching test" (Bors, W., et al. Biochim. Biophys. Acta 796:312-319; 1984) modified for analyzing the antioxidant capacity of complex mixtures (Tubaro, F., et al. J. Am. Oil Chem. Soc. 73:173-179; 1996). The information produced by this test is similar to that of the popular "total radical trapping antioxidant potential" (TRAP) analysis. However, the adopted kinetic approach is, in principle, more precise, taking into account both the concentration of antioxidants and their rate constant for the reaction with peroxy radical, which is overlooked in TRAP tests, as implied by the theory of the approach and confirmed by dynamic modeling. The kinetic analysis has also the advantage of accounting for the average between antioxidant effect (reduction of peroxy radicals) and possible prooxidant effect (oxidation by the radical of the antioxidant of the target supposed to be protected) if any. Thus, the result of this analysis provides a more precise evaluation of the efficiency of antioxidant defense. The intraassay variation resulted in less than 8% and, in young healthy subjects, the plasma antioxidant capacity, expressed as mM equivalents of a reference antioxidant (Trolox C), gave 1.59 +/- 0.28. The validated procedure has been used to show that plasma antioxidant capacity is deeply influenced by the consumption of wine.

Adult↗
PubMed1998

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.

Apolipoproteins B↗
PubMed1998

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.

Animals↗
PubMed1998

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.

Animals↗
PubMed1997

LDL- is a lipid hydroperoxide-enriched circulating lipoprotein.

A subclass of LDL described on the basis of its greater electronegativity and oxidative status is further characterized using a new, highly sensitive single photon counting technique to measure lipid hydroperoxides. We describe in this report that these particles, which we refer to as LDL-, are enriched in lipid peroxides and other peroxidation products as compared to the bulk of the unmodified, normal LDL (nLDL) recovered from human plasma. This chemiluminescence-based, single photon counting technique has unique advantages in that analyses are performed on whole LDL, thus avoiding artifactual lipid peroxidation during lipid extraction. Evidence for increased amounts of lipid hydroperoxides in LDL- versus nLDL are in agreement with other analytical methods such as measurement of conjugated dienes as well as cholesterol oxidation products. LDL- also has lower proportions of polyunsaturated fatty acids than nLDL. Analysis of the amino acid composition of apoB-100 and fatty acid composition of total LDL lipids also revealed major differences between nLDL and LDL- consistent with an oxidative modification of the latter. Thus, LDL- has significantly lower proportions of the oxidizable amino acids histidine and lysine, and marked differences in other neutral and acidic amino acids. The deficit in specific amino acids is in agreement with a reduced TNBS reactivity and increased relative electrophoretic mobility of LDL-. We postulate that LDL- is a major carrier of lipid hydroperoxides associated with plasma LDL and may arise from oxidative events in the vasculature and/ or by ingestion of peroxide-enriched meals.

Amino Acids↗
PubMed1997

Phospholipid hydroperoxide glutathione peroxidase (PHGPx): more than an antioxidant enzyme?

The family of glutathione peroxidases encompasses, as far, three tetrameric glutathione peroxidases (GPx) and the monomeric PHGPx. Although the overall homology between tetrameric enzymes and PHGPx is less than 30%, a pronounced similarity has been detected on clusters involved in the active site and a common catalytic triad (selenocysteine glutamine and tryptophan) has been defined by structural and kinetic data. A major peculiar feature of the reaction catalyzed by PHGPx is the possibility to accommodate large lipophilic substrates. This accounts for the observed dramatic antiperoxidant effect and the synergism with vitamin E. Moreover, the reduction of lipid hydroperoxides accounts also for the observed modulation of cycloxygenase and inhibition of 15-lipoxygenase. On the other hand, structural and kinetic data indicate that also the specificity of PHGPx for the donor substrate is not restricted to GSH and the recent observation the PHGPx binds to specific mitochondrial proteins, from which it is released by ionic strength and thiols, suggests a possible fole of this selenoenzyme in catalyzing the specific oxidation of protein thiols, thus modulating the activity of cellular regulatory elements. On this light, the selenium mojety of PHGPx, reacting much faster that thiols with a peroxide, and then oxidizing specific protein thiols, would channel the oxidation toward protein targets, thus providing, by protein-protein interaction, the specificity of the redox transition.

Animals↗
PubMed1996

The selenoenzyme phospholipid hydroperoxide glutathione peroxidase controls the activity of the 15-lipoxygenase with complex substrates and preserves the specificity of the oxygenation products.

Mammalian 15-lipoxygenases have been suggested to be involved in cell differentiation and atherogenesis because of their capability of oxygenating polyenoic fatty acids esterified to biomembranes and lipoproteins. We investigated the interaction of the lipid-peroxidizing 15-lipoxygenase and the hydroperoxy lipid-reducing phospholipid hydroperoxide glutathione peroxidase during their reaction with biomembranes and lipoproteins and obtained the following results. 1) Lipoxygenase treatment of submitochondrial membranes led to the formation of hydroperoxyphosphatidylethanolamine and hydroperoxyphosphatidylcholine as indicated by high performance liquid chromatography with chemiluminescence detection. In 15-lipoxygenase-treated low density lipoprotein cholesteryl hydroperoxylinoleate was the major oxygenation product. 2) Phospholipid hydroperoxide glutathione peroxidase was capable of reducing the hydroperoxy lipids formed by the 15-lipoxygenase to their corresponding alcohols. 3) Preincubation of low density lipoprotein and submitochondrial membranes with the phospholipid hydroperoxide glutathione peroxidase completely prevented the lipoxygenase reaction. However, addition of exogenous hydroperoxy lipids restored the oxygenase activity. 4) Short-term incubations of the complex substrates with the 15-lipoxygenase led to a specific pattern of oxidation products which was rendered more unspecific at long-term incubation or at high substrate concentrations. If the phosholipid hydroperoxide glutathione peroxidase was present during the reaction, the specific product pattern was preserved. These data indicate that the phospholipid hydroperoxide glutathione peroxidase is capable of reducing hydroperoxy ester lipids formed by a 15-lipoxygenase, and that it may down-regulate the 15-lipoxygenase pathways in mammalian cells. The specificity of 15-lipoxygenase-derived hydroperoxy lipids depends on their immediate reduction to the corresponding alcohols preventing postcatalytic isomerization.

Alcohols↗
PubMed1995

Direct measurement by single photon counting of lipid hydroperoxides in human plasma and lipoproteins.

A single photon counting procedure for measuring lipid hydroperoxides in human plasma or LDL-VLDL, escaping from extraction and chromatography, is described. This appears to be a relevant procedure because the recovery of phospholipid hydroperoxides from plasma is a critical point which, in our hands, was limited and poorly reproducible. The sample is added to a reaction mixture containing luminol, hemin, and Triton X-100 in an alkaline buffer, the photon emission is recorded, and the data are processed using the monoexponential decay of the photon emission rate. The measurement is applied to (a) plasma passed through a "desalting" cartridge to eliminate the small water-soluble antioxidants which inhibit the chemiluminescent process or (b) apo-B-containing lipoproteins (LDL-VLDL) isolated by heparin-Sepharose affinity chromatography. The content of lipid hydroperoxides is calculated using an internal calibration with palmitoyllinoleoylphosphatidylcholine hydroperoxide. This procedure, based on a single photon counting technology, was adopted to produce reliable results using samples from which inhibitors of the photon emission process have not been completely eliminated. The specificity of the signal for lipid hydroperoxides was validated by its complete disappearance following incubation of the sample with glutathione and phospholipid-hydroperoxide glutathione peroxidase (EC 1.11.1.12), the sole enzyme specific for all classes of lipid hydroperoxides in lipoproteins. The interassay variability was < 10%. The results indicated that the concentration of lipid hydroperoxides in the plasma of 20 healthy subjects was 353 +/- 78 nM. In different subjects, LDL-VLDL accounted for 40-80% of the lipid hydroperoxides in plasma.

Apolipoproteins B↗
PubMed1995

Measurement of lipid hydroperoxides in plasma lipoproteins by a new highly-sensitive 'single photon counting' luminometer.

The lipid hydroperoxide content of isolated, native human plasma lipoproteins, was measured, by the luminol-based chemiluminescent reaction, using a highly sensitive single photon counting instrument. The reaction was specific for lipid hydroperoxides since the signal completely disappeared after treatment with the selenoperoxidase specific for lipidic substrates. In this analytical procedure the whole kinetic of photon emission induced by lipid hydroperoxides and hemin in the presence of luminol is integrated, taking advantage of the mono-exponential fitting of the decay of photon emission. The addition of a detergent to the reaction mixture improved the precision of the measurements apparently by preventing oxidative chain reactions affecting the shape of the decay of photon emission. The sensitivity of the instrument allowed measurements on samples containing just a few picomoles of hydroperoxides, small enough to minimize the effect of antioxidants and quenchers possibly present in the sample (as in the case of lipoproteins). Thus, by using an internal calibration with a phospholipid hydroperoxide, the evaluation of the lipid hydroperoxide content in whole, native lipoproteins was possible without previous extraction and chromatographic separation. Data obtained from plasma lipoproteins isolated by different procedures suggest that lipid hydroperoxide content increases during ultracentrifugation.

Centrifugation↗
PubMed1995

Copper-induced lipid peroxidation in liposomes, micelles, and LDL: which is the role of vitamin E?

Liposomes, containing phospholipid hydroperoxides, are peroxidised in the presence of Cu++. Peroxidation starts after a period of resistance to oxidation, which is abolished by the shift of lipid organisation from bilayer to micellar dispersion. Independently from ongoing peroxidation, vitamin E in liposomes also reacts with Cu++, and it is consumed. The evidence that phospholipid hydroperoxides induce an acceleration of vitamin E consumption rate and that the consumption of vitamin E and phospholipid hydroperoxides are stoichiometric indicates that, in liposomes, the rate-limiting reaction is the interaction between radicals generated by copper from vitamin E and phospholipid hydroperoxides. In micelles, on the other hand, vitamin E is directly oxidised by copper at a much faster rate; thus, the concerted consumption of phospholipid hydroperoxides does not take place. Moreover, in micelles challenged with Cu++, vitamin E plays a pro-oxidant effect (M. Maiorino et al. FEBS Letts., 330(2):174-176; 1993). In LDL, incubation with Cu++ promotes vitamin E consumption at a fast rate, as in micelles, but not the concerted disappearance of lipid hydroperoxides, as in liposomes. However, the direct vitamin E oxidation by Cu++, observed in micelles and liposomes, does not lead to a pro-oxidant effect in LDL. The kinetics of peroxidation, indeed, is identical in native and vitamin E-depleted LDL. These results argue against an involvement of vitamin E, both as antioxidant or pro-oxidant in LDL challenged with Cu++, and suggest that other factors, besides antioxidant content, must be relevant in determining LDL oxidative resistance.

Copper↗
PubMed1995

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.

Animals↗