High plasma concentrations of triglycerides and low plasma concentrations of esterified cholester... more High plasma concentrations of triglycerides and low plasma concentrations of esterified cholesterol and lysolecithin, with an impaired rate of VLDL and LDL catabolism, have been reported in chronic uremic patients. An important contribution to these abnormalitites might be an impaired activity of the (LCAT). Serum LCAT activity and cholesteryl ester clearance were determined in 11 patients with chronic renal failure and in 10 controls. LCAT activity was determined by using the serum of each patient both as a source of enzyme and as a substrate ("intrinsic" activity) and was compared with the activity determined on a standard substrate ("extrinsic activity), so as to ascertain the presence of inhibitory factors in the patients' sera. Both activityes have been found to be significantly (P less than 0.01) lower in chronic uremic patients than in controls. The cholesteryl ester clearance apparently did not respond to the stimulatory effect of hypertriglyceridemia, as ...
Consumption of a meal containing oxidized and oxidizable lipids gives rise to an increased plasma... more Consumption of a meal containing oxidized and oxidizable lipids gives rise to an increased plasma concentration of lipid hydroperoxides, detectable by a sensitive chemiluminescence procedure. This is associated with increased susceptibility of LDL to oxidation, apparently due a structural perturbation at the particle surface brought about by lipid oxidation products. The postprandial modification of LDL is at least partially accounted for by an increase of LDL-, a subfraction containing lipid oxidation products where apoprotein-B-100 (apoB-100) is denatured. Consuming the meal with a suitable source of antioxidants, such as those found in red wine, minimizes this postprandial oxidative stress. The inhibition of peroxidation of lipids present in the meal during digestion is a possible mechanism for the observed protection of LDL. The in vivo oxidatively modified LDL- has numerous features that correspond to the atherogenic minimally modified LDL produced in vitro. These modified part...
... Plasma and L ipoproteinsby K inetic A nalysisof Photon Emission By ANTONIO M. PASTORINO, ADRI... more ... Plasma and L ipoproteinsby K inetic A nalysisof Photon Emission By ANTONIO M. PASTORINO, ADRIANA ZAMBURLINI, LucIo ZENNARO, MATILDE ... 13 and Rosen and Rauckman TM suggested a Fenton-like reaction, where hydroperoxides reduce ferric hematin to ferrous ...
Single and double site mutants affecting the presumed catalytic centre of the selenoenzyme PHGPx ... more 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 neut...
The lipid hydroperoxide content of isolated, native human plasma lipoproteins, was measured, by t... more 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...
... (1)]. The kinetic (1) 2RSH + ROOH ~ RSSR + H20 + ROH pattern is characterized as a Ping-Pong ... more ... (1)]. The kinetic (1) 2RSH + ROOH ~ RSSR + H20 + ROH pattern is characterized as a Ping-Pong mechanism with indefinite Michaelis constants, indefinite maximum velocities, 46'47'53-55 and negligible, if any, product inhibition. ...
The cell sap from pig liver contains a protein which protects phosphatidylcholine liposomes and b... more The cell sap from pig liver contains a protein which protects phosphatidylcholine liposomes and biomembranes from peroxidative degradation in the presence of glutathione. The activity of this protein has been assayed by measuring the inhibition of aged phosphatidylcholine liposome peroxidation induced by the Fe3+-triethylenetetramine complex. The peroxidation-inhibiting protein from pig liver has been purified 585-fold to homogeneity with overall recovery of activity of 12%. (NH4)2SO4 precipitation, ion-exchange chromatography on DEAE-Sepharose CL-6B and CM23-cellulose, affinity chromatography on glutathione-bromosulfophthalein-Sepharose and gel filtration on Sephadex G-50 were used. Gel filtration and SDS- polyacrylamide gel electrophoresis indicated a molecular weight of approximately 20 000. The protein inhibited peroxidation by Fe3+-triethylenetetramine following a 15 min preincubation of phosphatidylcholine liposomes in the presence of 5mM glutathione or 2-mercapthoethanol. The...
Journal of free radicals in biology & medicine, 1985
The recently purified "phospholipid hydroperoxide glutathione peroxidase" has been used... more The recently purified "phospholipid hydroperoxide glutathione peroxidase" has been used to measure the membrane hydroperoxides formed during lipid peroxidation that are not substrates for the "classical" glutathione peroxidase. A spectrophotometric test in the presence of glutathione, glutathione reductase and NADPH has been used. The peroxidized membranes were added directly to the reaction mixture and the reaction was started by the addition of the enzyme. Triton X-100 exerted a stimulatory effect. Phospholipid hydroperoxide glutathione peroxidase allows a rapid, sensitive, accurate and specific determination of membrane hydroperoxides, the most quantitative index of lipid peroxidation. Glutathione peroxidase can be used in the same test to measure other hydroperoxides such as the cumene hydroperoxide used to induce the peroxidation.
The effect of diets supplemented with three different fats (olive oil, sunflower oil, pork fat) o... more The effect of diets supplemented with three different fats (olive oil, sunflower oil, pork fat) on the susceptibility of the rat heart to oxidative stress and on the rate of eicosanoid release were studied. Our results show that when fatty-acid unsaturation of heart lipids is increased or vitamin E is decreased, even to a low degree, a marked enhancement of the susceptibility to hydroperoxide-induced oxidative stress (measured by chemiluminescence emission) occurs, which is associated with an increase of eicosanoid release from the heart.
Lipid hydroperoxides (LOOHs) in various lipid assemblies are shown to be efficiently reduced and ... more Lipid hydroperoxides (LOOHs) in various lipid assemblies are shown to be efficiently reduced and deactivated by phospholipid hydroperoxide glutathione peroxidase (PHGPX), the second selenoperoxidase to be identified and characterized. Coupled spectrophotometric analyses in the presence of NADPH, glutathione (GSH), glutathione reductase and Triton X-100 indicated that photochemically generated LOOHs in small unilamellar liposomes are substrates for PHGPX, but not for the classical glutathione peroxidase (GPX). PHGPX was found to be reactive with cholesterol hydroperoxides as well as phospholipid hydroperoxides. Kinetic iodometric analyses during GSH/PHGPX treatment of photoperoxidized liposomes indicated a rapid decay of total LOOH to a residual level of 35-40%; addition of Triton X-100 allowed the reaction to go to completion. The non-reactive LOOHs in intact liposomes were shown to be inaccessible groups on the inner membrane face. In the presence of iron and ascorbate, photoperoxi...
High plasma concentrations of triglycerides and low plasma concentrations of esterified cholester... more High plasma concentrations of triglycerides and low plasma concentrations of esterified cholesterol and lysolecithin, with an impaired rate of VLDL and LDL catabolism, have been reported in chronic uremic patients. An important contribution to these abnormalitites might be an impaired activity of the (LCAT). Serum LCAT activity and cholesteryl ester clearance were determined in 11 patients with chronic renal failure and in 10 controls. LCAT activity was determined by using the serum of each patient both as a source of enzyme and as a substrate ("intrinsic" activity) and was compared with the activity determined on a standard substrate ("extrinsic activity), so as to ascertain the presence of inhibitory factors in the patients' sera. Both activityes have been found to be significantly (P less than 0.01) lower in chronic uremic patients than in controls. The cholesteryl ester clearance apparently did not respond to the stimulatory effect of hypertriglyceridemia, as ...
Consumption of a meal containing oxidized and oxidizable lipids gives rise to an increased plasma... more Consumption of a meal containing oxidized and oxidizable lipids gives rise to an increased plasma concentration of lipid hydroperoxides, detectable by a sensitive chemiluminescence procedure. This is associated with increased susceptibility of LDL to oxidation, apparently due a structural perturbation at the particle surface brought about by lipid oxidation products. The postprandial modification of LDL is at least partially accounted for by an increase of LDL-, a subfraction containing lipid oxidation products where apoprotein-B-100 (apoB-100) is denatured. Consuming the meal with a suitable source of antioxidants, such as those found in red wine, minimizes this postprandial oxidative stress. The inhibition of peroxidation of lipids present in the meal during digestion is a possible mechanism for the observed protection of LDL. The in vivo oxidatively modified LDL- has numerous features that correspond to the atherogenic minimally modified LDL produced in vitro. These modified part...
... Plasma and L ipoproteinsby K inetic A nalysisof Photon Emission By ANTONIO M. PASTORINO, ADRI... more ... Plasma and L ipoproteinsby K inetic A nalysisof Photon Emission By ANTONIO M. PASTORINO, ADRIANA ZAMBURLINI, LucIo ZENNARO, MATILDE ... 13 and Rosen and Rauckman TM suggested a Fenton-like reaction, where hydroperoxides reduce ferric hematin to ferrous ...
Single and double site mutants affecting the presumed catalytic centre of the selenoenzyme PHGPx ... more 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 neut...
The lipid hydroperoxide content of isolated, native human plasma lipoproteins, was measured, by t... more 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...
... (1)]. The kinetic (1) 2RSH + ROOH ~ RSSR + H20 + ROH pattern is characterized as a Ping-Pong ... more ... (1)]. The kinetic (1) 2RSH + ROOH ~ RSSR + H20 + ROH pattern is characterized as a Ping-Pong mechanism with indefinite Michaelis constants, indefinite maximum velocities, 46'47'53-55 and negligible, if any, product inhibition. ...
The cell sap from pig liver contains a protein which protects phosphatidylcholine liposomes and b... more The cell sap from pig liver contains a protein which protects phosphatidylcholine liposomes and biomembranes from peroxidative degradation in the presence of glutathione. The activity of this protein has been assayed by measuring the inhibition of aged phosphatidylcholine liposome peroxidation induced by the Fe3+-triethylenetetramine complex. The peroxidation-inhibiting protein from pig liver has been purified 585-fold to homogeneity with overall recovery of activity of 12%. (NH4)2SO4 precipitation, ion-exchange chromatography on DEAE-Sepharose CL-6B and CM23-cellulose, affinity chromatography on glutathione-bromosulfophthalein-Sepharose and gel filtration on Sephadex G-50 were used. Gel filtration and SDS- polyacrylamide gel electrophoresis indicated a molecular weight of approximately 20 000. The protein inhibited peroxidation by Fe3+-triethylenetetramine following a 15 min preincubation of phosphatidylcholine liposomes in the presence of 5mM glutathione or 2-mercapthoethanol. The...
Journal of free radicals in biology & medicine, 1985
The recently purified "phospholipid hydroperoxide glutathione peroxidase" has been used... more The recently purified "phospholipid hydroperoxide glutathione peroxidase" has been used to measure the membrane hydroperoxides formed during lipid peroxidation that are not substrates for the "classical" glutathione peroxidase. A spectrophotometric test in the presence of glutathione, glutathione reductase and NADPH has been used. The peroxidized membranes were added directly to the reaction mixture and the reaction was started by the addition of the enzyme. Triton X-100 exerted a stimulatory effect. Phospholipid hydroperoxide glutathione peroxidase allows a rapid, sensitive, accurate and specific determination of membrane hydroperoxides, the most quantitative index of lipid peroxidation. Glutathione peroxidase can be used in the same test to measure other hydroperoxides such as the cumene hydroperoxide used to induce the peroxidation.
The effect of diets supplemented with three different fats (olive oil, sunflower oil, pork fat) o... more The effect of diets supplemented with three different fats (olive oil, sunflower oil, pork fat) on the susceptibility of the rat heart to oxidative stress and on the rate of eicosanoid release were studied. Our results show that when fatty-acid unsaturation of heart lipids is increased or vitamin E is decreased, even to a low degree, a marked enhancement of the susceptibility to hydroperoxide-induced oxidative stress (measured by chemiluminescence emission) occurs, which is associated with an increase of eicosanoid release from the heart.
Lipid hydroperoxides (LOOHs) in various lipid assemblies are shown to be efficiently reduced and ... more Lipid hydroperoxides (LOOHs) in various lipid assemblies are shown to be efficiently reduced and deactivated by phospholipid hydroperoxide glutathione peroxidase (PHGPX), the second selenoperoxidase to be identified and characterized. Coupled spectrophotometric analyses in the presence of NADPH, glutathione (GSH), glutathione reductase and Triton X-100 indicated that photochemically generated LOOHs in small unilamellar liposomes are substrates for PHGPX, but not for the classical glutathione peroxidase (GPX). PHGPX was found to be reactive with cholesterol hydroperoxides as well as phospholipid hydroperoxides. Kinetic iodometric analyses during GSH/PHGPX treatment of photoperoxidized liposomes indicated a rapid decay of total LOOH to a residual level of 35-40%; addition of Triton X-100 allowed the reaction to go to completion. The non-reactive LOOHs in intact liposomes were shown to be inaccessible groups on the inner membrane face. In the presence of iron and ascorbate, photoperoxi...
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Papers by Fulvio Ursini