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C33 Intestinal cholesterol absorption and oxidative status of enterocytes
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INTESTINAL CBOLESTEROL ABSORFI’ION AND OXIDATlVE STATUS OF ENTEROCYTES Elena Bravo, Medo Cantafora, and Roberto Rivabene Laboratmy of Metabolism and Pathological Biochemistry, lstituto Superiore di Sat&a. Viale Regina Elena, 299 00161 Rome,Italy. Dietary fatty acld composition and enterczyte (dys)fun&on influence both intestinal chnlesterol absorption and and its level in the plasma. The redox balance within the cells has profound consequences forseveral cellular functions, includmg metabolic processes However, little has been reported on the cellular redox regulation of cholesterol metabolism at the intestinal level ‘l?as in v&o study was performed to evaluate w&her the fate of cholesterol esterification within the mtemcytes, a key step m the absorption of diet any cholesterol, is intluenced by changes of the redox state of the enterocytes. Caco-2 human intestinal ceils, a suitable model for the study of enterocyte lipid metabolism, were used To induce a “mild“ pro-oxidant or anti-oxidant condition, cells were pretreated for 24 hrs with 2.5 FM CuSQ or 5 mM N-acetylcysteme (NAC), respectively Cholesterol esterificatton was then e&mat& by the incorporation of labeled 01&c acid-albumin complex (4 hrs at 37’C) into cholcsteryl esters. The effect of two different concentrations ofoleic acid (50 and 500 &I) and the addition of cholesterol (0.129 pmoliml with 50 PM oleic acid) to the incubation m&a, was also studied. The rate of cholesteryl ester formation was not significantly affected by CuSO., (increases cellular levels of lipld peroxldes and the oxidized/reduced glutathione ratio) and NAC ( increases the internal reductive potential in cell) treatments, with respect to untreated cells. However, the change in oleate concentration from 50 to 500 uM strongly increased cholesteryl esters synthesis. These results suggest that cholesterol esterification does not critically depend on variations of redox state of the enterocytes and that olnc aad. but not cholesterol, enhances enterocyte cholesterol estenficatlon This work was kindly supponedby 8 grantfrom Ante BancaNaz~~naledelle Comunicazlm (An I.% 194s)
DIFFERENT OXIDATION PRODUCTS FROM INDIVIDUAL FAlTY ACIDS Francesco Visioli and Claudio Galli Institute of Pharmacological Sciences, Via Balzaretti 9, 20133 Milan, Italy. email: francesco.visioliQunimi.it Free radical-induced lipid peroxidation is related to the onset of several diseases, including atherosclerosis. It is generally assumed that the oxidizability of fatty acids is correlated to their degree of unsaturation. Also, most studies of lipid peroxidation employ only few markers of oxidative eg the generation of malondialdehyde. We stress, investigated the oxidizability of individual fatty acids in a model of AAPH-mediated peroxidation of micelles by assessing several indices of oxidative stress. The results show that the generation of oxidation products by individual fatty acids is not directly related to their degree of unsaturation. For instance, while linoleic acid levels rapidly
decrease during AAPH-Fediated oxidation, yielding high amounts of conjugated dznndes,TBARS are mo,stly derived acids. docosahexanotc arachidonic from Eicosapentanoic acid yields lower lipid peroxide and malondialdehyde levels than arachidonic acid, in spite of the fact that the latter has equal chain length and just one less double bond. Finally, most in vitro studies employ cells that are almost devoid of antioxidant vitamins, eg tocopherols and ascorbate, in addition to being very low in polyunsaturated fatty acids. In turn, the differential oxidizability of individual fatty acids and the composition of cellular models should be taken into account during studies of lipid peroxidation.
C36 c34 Endothelial dysfunction after an oral fat tolerance test (OFTT) Marchesi S, Lupattelli G, Palumbo B, Pirro M, Siepi D, Casciti Manuarino E. Dipartimento di Medicina C!ica e Sperimentale.University of Pen@
C,
The early stages of atherosclerosis are characterized by endothelial dysfunction evidence of which is a reduction in flow-mediated vaoactivity, as shorn by a non-invasive ultrasound of the brachial artery. Post-prandial biglyceride concentration is closely related to athcrogenic risk. It is not completely understood whether post-prandial hyperlipemia is correlated with endothehal dysfunction In 10 young (23i2 years), healthy, normoiipcmic mm, flow-mediated vasoactivity (expressedas a percentagechangein arterial diameter 60 secondsa& a 3 minute upper arm arterial occlmion), along with total cholesterol, LDL cholesterol, HDL cholesterol, triglyceride levels and LDL size were evaluated before and 2, 4 and 6 hours after an oral fat tolerance test (700 kcal/mq: 3% protein, 14% carbohydrate, 83% fat). The Hind III polymorphism of LPL gene was also evaluated. Triglyceride levels rose by 44f25 mgidl at the 2d hour, 7If47 m&U at the 4* hour and 52+23 mg/dl at the 6* hour (pcO.05). The mean value of rest flow-mediated vasoactivity was 18.5&6.5%, which decreased significantIy (~~0.05) to 3.4i2.2% and 4.9~?1.8% at the 2”dand the 4* post-prandial hours. At the 6’ hour the meanvalue overlapped the basal: 17.3i6.5%. LDL size did not viuy significantly at any time. Linear regression analysis show a significant correlation between triglyceride increasesand flow mediatedvasoactivity (I-=. 0.5, p
ANTIOXIDANT CONTENT AND LIPID COMPOSITION AFFECT THE KINETICS OF COPPER REDUCTION AND COPPERDEPENDENT OXIDATION OF HUMAN LDL ‘F.Visioli, R. Bordone, C. Perugini, M. Bagnati, and G. Beliomo Institute of Pharmacological Sciences, ‘Institute of Pharmacological Sciences, University of Milan0 and Department of Medical Sciences, University of Piemonte Orientale, Novara, Italy The precise molecular mechanisms involved in copperinduced peroxidation of LDL are still under investigation, although the dynamic reduction of Cu(ll) to Cu(l) seem to be an absolute prerequisite. Here we used intact human LDL, phospholipidcontaining delipidated LDL ghosts and trilinoleil-reconstituted LDL to investigate both the process of copper reduction and LDL. Both LDL ghosts and trilinoleil-reconstituted LDL were deprived of antioxidants and were extremely susceptible to free-radical mediated oxidation but, paradoxically, rather resistant to copperpromoted oxidation. The dynamic reduction of Cu(ll) to Cu(l) was quantitatively decreased in LDL ghosts and trilinoleil-LDL and the kinetics of the reductive process was also changed, lacking the initial rapid reduction and the subsequent inhibition phases, due to the absence of the endogenous antioxidants. Conversely, the rate of reduction was rather linear and mainly due to lipid peroxides already present or newly-formed during copper-catalyzed lipid peroxidation. In a series of dosedependency experiments it also emerged that the amount of Cu(ll)-reducing equivalents associated with trilinoleil-LDL was linear and dependent on the concentration of triglycerides in the reconstituted particles. These data indicate that copper undergoes redox transitions in LDL by utilizing reducing equivalents originating either from endogenous antioxidants or/and from the lipid peroxides formed in the hydrophobic core of the lipoprotein particle.
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c35
c33
INTESTINAL CBOLESTEROL ABSORFI’ION AND OXIDATlVE STATUS OF ENTEROCYTES Elena Bravo, Medo Cantafora, and Roberto Rivabene Laboratmy of Metabolism and Pathological Biochemistry, lstituto Superiore di Sat&a. Viale Regina Elena, 299 00161 Rome,Italy. Dietary fatty acld composition and enterczyte (dys)fun&on influence both intestinal chnlesterol absorption and and its level in the plasma. The redox balance within the cells has profound consequences forseveral cellular functions, includmg metabolic processes However, little has been reported on the cellular redox regulation of cholesterol metabolism at the intestinal level ‘l?as in v&o study was performed to evaluate w&her the fate of cholesterol esterification within the mtemcytes, a key step m the absorption of diet any cholesterol, is intluenced by changes of the redox state of the enterocytes. Caco-2 human intestinal ceils, a suitable model for the study of enterocyte lipid metabolism, were used To induce a “mild“ pro-oxidant or anti-oxidant condition, cells were pretreated for 24 hrs with 2.5 FM CuSQ or 5 mM N-acetylcysteme (NAC), respectively Cholesterol esterificatton was then e&mat& by the incorporation of labeled 01&c acid-albumin complex (4 hrs at 37’C) into cholcsteryl esters. The effect of two different concentrations ofoleic acid (50 and 500 &I) and the addition of cholesterol (0.129 pmoliml with 50 PM oleic acid) to the incubation m&a, was also studied. The rate of cholesteryl ester formation was not significantly affected by CuSO., (increases cellular levels of lipld peroxldes and the oxidized/reduced glutathione ratio) and NAC ( increases the internal reductive potential in cell) treatments, with respect to untreated cells. However, the change in oleate concentration from 50 to 500 uM strongly increased cholesteryl esters synthesis. These results suggest that cholesterol esterification does not critically depend on variations of redox state of the enterocytes and that olnc aad. but not cholesterol, enhances enterocyte cholesterol estenficatlon This work was kindly supponedby 8 grantfrom Ante BancaNaz~~naledelle Comunicazlm (An I.% 194s)
DIFFERENT OXIDATION PRODUCTS FROM INDIVIDUAL FAlTY ACIDS Francesco Visioli and Claudio Galli Institute of Pharmacological Sciences, Via Balzaretti 9, 20133 Milan, Italy. email: francesco.visioliQunimi.it Free radical-induced lipid peroxidation is related to the onset of several diseases, including atherosclerosis. It is generally assumed that the oxidizability of fatty acids is correlated to their degree of unsaturation. Also, most studies of lipid peroxidation employ only few markers of oxidative eg the generation of malondialdehyde. We stress, investigated the oxidizability of individual fatty acids in a model of AAPH-mediated peroxidation of micelles by assessing several indices of oxidative stress. The results show that the generation of oxidation products by individual fatty acids is not directly related to their degree of unsaturation. For instance, while linoleic acid levels rapidly
decrease during AAPH-Fediated oxidation, yielding high amounts of conjugated dznndes,TBARS are mo,stly derived acids. docosahexanotc arachidonic from Eicosapentanoic acid yields lower lipid peroxide and malondialdehyde levels than arachidonic acid, in spite of the fact that the latter has equal chain length and just one less double bond. Finally, most in vitro studies employ cells that are almost devoid of antioxidant vitamins, eg tocopherols and ascorbate, in addition to being very low in polyunsaturated fatty acids. In turn, the differential oxidizability of individual fatty acids and the composition of cellular models should be taken into account during studies of lipid peroxidation.
C36 c34 Endothelial dysfunction after an oral fat tolerance test (OFTT) Marchesi S, Lupattelli G, Palumbo B, Pirro M, Siepi D, Casciti Manuarino E. Dipartimento di Medicina C!ica e Sperimentale.University of Pen@
C,
The early stages of atherosclerosis are characterized by endothelial dysfunction evidence of which is a reduction in flow-mediated vaoactivity, as shorn by a non-invasive ultrasound of the brachial artery. Post-prandial biglyceride concentration is closely related to athcrogenic risk. It is not completely understood whether post-prandial hyperlipemia is correlated with endothehal dysfunction In 10 young (23i2 years), healthy, normoiipcmic mm, flow-mediated vasoactivity (expressedas a percentagechangein arterial diameter 60 secondsa& a 3 minute upper arm arterial occlmion), along with total cholesterol, LDL cholesterol, HDL cholesterol, triglyceride levels and LDL size were evaluated before and 2, 4 and 6 hours after an oral fat tolerance test (700 kcal/mq: 3% protein, 14% carbohydrate, 83% fat). The Hind III polymorphism of LPL gene was also evaluated. Triglyceride levels rose by 44f25 mgidl at the 2d hour, 7If47 m&U at the 4* hour and 52+23 mg/dl at the 6* hour (pcO.05). The mean value of rest flow-mediated vasoactivity was 18.5&6.5%, which decreased significantIy (~~0.05) to 3.4i2.2% and 4.9~?1.8% at the 2”dand the 4* post-prandial hours. At the 6’ hour the meanvalue overlapped the basal: 17.3i6.5%. LDL size did not viuy significantly at any time. Linear regression analysis show a significant correlation between triglyceride increasesand flow mediatedvasoactivity (I-=. 0.5, p
ANTIOXIDANT CONTENT AND LIPID COMPOSITION AFFECT THE KINETICS OF COPPER REDUCTION AND COPPERDEPENDENT OXIDATION OF HUMAN LDL ‘F.Visioli, R. Bordone, C. Perugini, M. Bagnati, and G. Beliomo Institute of Pharmacological Sciences, ‘Institute of Pharmacological Sciences, University of Milan0 and Department of Medical Sciences, University of Piemonte Orientale, Novara, Italy The precise molecular mechanisms involved in copperinduced peroxidation of LDL are still under investigation, although the dynamic reduction of Cu(ll) to Cu(l) seem to be an absolute prerequisite. Here we used intact human LDL, phospholipidcontaining delipidated LDL ghosts and trilinoleil-reconstituted LDL to investigate both the process of copper reduction and LDL. Both LDL ghosts and trilinoleil-reconstituted LDL were deprived of antioxidants and were extremely susceptible to free-radical mediated oxidation but, paradoxically, rather resistant to copperpromoted oxidation. The dynamic reduction of Cu(ll) to Cu(l) was quantitatively decreased in LDL ghosts and trilinoleil-LDL and the kinetics of the reductive process was also changed, lacking the initial rapid reduction and the subsequent inhibition phases, due to the absence of the endogenous antioxidants. Conversely, the rate of reduction was rather linear and mainly due to lipid peroxides already present or newly-formed during copper-catalyzed lipid peroxidation. In a series of dosedependency experiments it also emerged that the amount of Cu(ll)-reducing equivalents associated with trilinoleil-LDL was linear and dependent on the concentration of triglycerides in the reconstituted particles. These data indicate that copper undergoes redox transitions in LDL by utilizing reducing equivalents originating either from endogenous antioxidants or/and from the lipid peroxides formed in the hydrophobic core of the lipoprotein particle.