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In vivo oxidative stress in microvascular endothelium by endogenous nitric oxide suppression: Leukocyte-independent events
484
2:21
Session 2: Measurement of Oxidative Stress in vivo PROTEIN OXIDAT’IVE DAMAGE IS ASSOCIATED WITH LIFE EXPECTANCY OF ANIMALS Rajindar S. Sohal, Sanjiv AgarwaJ & William C. On, Department of Biological Sciences, Southern Methodist University, Dallas, Texas 75275 USA. This investigation was conducted to test the main prediction of the free radical hypothesis, namely that oxidative damage is a major causal factor in aging. Protein carbonyl content was found to be related to the life expectancy or physiological age of flies and mice rather than the chronological age. Distinction between physiological and chronological age was made using different strategies, e.g., flies exhibiting senescence-related loss of flying ability were comparcd with non-senescent cohorts of the same chronological age; or the maximum life spans of flies were experimentally altered by varying the metabolic rate. In mice, dietary restriction was used to extend the life span. Protein carbonyl content not only increased with age but was a predictor of the life expectancy of animals. Carbonyl content in tissues corresponded to the rate of mitochondrial Oz.- and Hz02 generation rather than the antioxidant defenses (SOD, catalase, and glutathionc peroxidase) in the tissues. Rates of mitochondriai pro-oxidant generation, tissue oxidative damage and rate of aging seem to be linked phenomena. (This research was supported by the grant ROlAG7657 from N.I.H.-N.I.A.)
IN VIVO OXIDATIVE STRESS IN MICROVASCULAR ENDOTHELIUM BY ENDOGENOUS NITRIC OXIDE SUPPRESSION: LEUKOCYTE-INDEPENDENT EVENTS M. Suematsu. H. Suzuki. M. Mivasaka*. Y Ishimura** and G.W Schmid-Schonbem Institute for Biomedical Engineering, Llniwrsity ot California. San Diego. Department of Immunolopy. Tokyo Metropolitan Institute for Medical Sctences* Department of Biochemtstry. School of Medicine. Keio IIniverslty**
2:22
This study was aimed to demonstrate mtcrovasculsr endothellal oxidattve changes Independent of leukocyte adhesion in viva The rat mesentetic microcirculation was superfused with NC-mtro-L-argmme methyl ester (L-NAME, 100 PM), and intracellular oxidant formatjon m several microcirculatory cellular components such as ilffertolar and venular endothelium and mast cells was visually monitored by digital microfluorography assisted by carboxydichlorofluorescein (CDCF). a hydroperoride-sensitive fluorogenic probe (I, 2). Time history of leukocyte adhesion was monitored simultaneously. L-NAME induced a significant increase in CDCF fluorescence in arteriolar and venular endothelium as well as m mast cells which wz followed by leukocyte adhesion. The level of hydroperoxide formation in arterioles and venules evoked by 60-min L-NAME superfusion was eqwalent to that induced bv the sunerfusion of about 800 UM ten-butvlhvdroperoxide for IO m;n. Pretrkatment with anti-ICAMor -Cdld monoclonal antibody attenuated L-NAME-elicited venular leukocyte adheston without. suppressing CDCF fluorescence m situ. Pretreatment with desferioxamine (50 mgikg. ii,, 1 hr prior to L-NAME superfusion) significantly dimimshed the hydroperoxide formation in arterioles and venules. but not in interstitial mast cells, as well as subsequent venular leukocyte adhesion. These findings Indicate that endogenous nitric oxide may modulate oxidative stress in mast cell% arteriolar and venular mtcrovascular endothelium and thereby can Induce cenular leukocyte recnntment as a consequence of vascular oxidative eventa. References: 1) Gastroenterolopy 103: 994, 1992. 2) Am J Physiol
264: H88l. 1993
2:23
SYNTHESIS AND QUANTITATION OF HYDROPEROXIDESOF DNA Chandrakala Menon and John E. Biaglow Department of Biochemistry & Radiation Oncology, University of Pennsylvania, Philadelphia, PA 19 104
LDL LIPID OXIDATION AND PHOSPHOLIPASE A2 ACTIVITY IN ATHEROGENESIS: NOVEL ANALYTICAL APPRGACHES J.J.M. van den Berg’, M.G. Lansberg*, N.E. Cook1 and D.L. TribbIe* 1Children’s Hospital Oakland Research Institute, Oakland, CA 94609 USA and *Lawrence Berkeley Laboratory, Berkeley, CA 94720 USA.
We have succeeded in making hydroperoxides of These DNA and quantitating them accurately. products are associated with the pyrimidine bases in Thymidine particularly thymidine. DNA, hydroperoxides were synthesized by exposing 2mM solutions of thymidine to 200 gray gamma radiation from a cesium source, dose rate = lSgy/min. H202 in the solution was removed with catalase. Under these conditions, a maximum yield of 23 mM thymidine hydroperoxide was obtained. This accounted for about 1.15% of the nucleotide present in solution. thymine a hydroperoxide was Alternatively, chemically synthesized from Shydroxymethyluracil. We are exploring the possibility of making DNA hydroperosides by, reacting DNA with NaOCl and H202. We have also s: cceeded in quantitating the hydroperoxides accurately. The hydroperoxides were quantitated by the potassium iodide method. H202 was measured by the Fricke’s method or by the peroxide probe. Alternatively, DNA hydroperoxide was quantitated by a coupled assay consisting of reaction of the hydroperoxide with a known concentration of GSH in the presence of Se dependent glutathione peroxidase. The remaining GSH was reacted with stoichiometric quantities of DTNB and the absorbance was read at 412 nm. 1~202, cumene hydroperoxide and t-butyl hydroperoxide were used as standards in both methods.
Research in atberogenesis currently focuses on the hypothesis that the conversion of low density lipoproteins (LDL) into atherogenic panicles is mediated by oxidative processes. ModiIicaiion of the apoB protein appears to he crucial, but lipid oxidation products may initiate apoB modification. Pbospholipase A2 (PLA2) activity has been suggested to release reactive oxidized fatty acids that subsequently modify apoB. Studies thus far have provided mostly nonspecific and incomplete information regarding the mechanisms by which oxidation of LDL lipids may be involved in atherogenesis. This poster highlights the analytical approaches we use to study three areas of interest: (1) the identity of LDL lipid oxidation products; (2) the role of PLA2 in LDL atherogenicity; (3) the kinetics of LDL oxidation processes. (1) Gas chromatography-mass spectrometry is used to separate and identify oxidized lipid species. (2) A lipid monolayer technique is used to study the changes in physicochemical behavior of LDL surface phospholipids upon oxidation. We show that oxidized phospholipids adopt a drastically different conformation in the monolayer, which is linked to recognition and preferential cleavage of oxidized fatty acyl chains by PLA2. Monolayer studies provide mechanistic information on phospholipase activity and can be used to establish if PLA2 has a specificity for certain oxidized substrates. Purified oxidized linids will be inuoduced into LDL in vifro with or without PLA2 inhibition to study their potential for initiating atherogenic changes in LDL. (3) Oxidative susceptibility of LDL is studied using the fiuorescent polyunsaturated fatty -&id, psinarlc acid @A), and its methylated derivative, PnA methyl ester @ME), as oxidation probes specific for LDL surface and core regions, respectively. Differences in lipid composition, antioxidant content, and accessibility to oxidants of these two pools could determine the overall response of the LDL particle to oxidative stress, and the PnA/pnME probe combination provides a unique 1001 to investigate this. These studies will generate new information with regard to the oxidative metabolism of LDL lipids and their involvement in atherogenesis.
2:24
2:21
Session 2: Measurement of Oxidative Stress in vivo PROTEIN OXIDAT’IVE DAMAGE IS ASSOCIATED WITH LIFE EXPECTANCY OF ANIMALS Rajindar S. Sohal, Sanjiv AgarwaJ & William C. On, Department of Biological Sciences, Southern Methodist University, Dallas, Texas 75275 USA. This investigation was conducted to test the main prediction of the free radical hypothesis, namely that oxidative damage is a major causal factor in aging. Protein carbonyl content was found to be related to the life expectancy or physiological age of flies and mice rather than the chronological age. Distinction between physiological and chronological age was made using different strategies, e.g., flies exhibiting senescence-related loss of flying ability were comparcd with non-senescent cohorts of the same chronological age; or the maximum life spans of flies were experimentally altered by varying the metabolic rate. In mice, dietary restriction was used to extend the life span. Protein carbonyl content not only increased with age but was a predictor of the life expectancy of animals. Carbonyl content in tissues corresponded to the rate of mitochondrial Oz.- and Hz02 generation rather than the antioxidant defenses (SOD, catalase, and glutathionc peroxidase) in the tissues. Rates of mitochondriai pro-oxidant generation, tissue oxidative damage and rate of aging seem to be linked phenomena. (This research was supported by the grant ROlAG7657 from N.I.H.-N.I.A.)
IN VIVO OXIDATIVE STRESS IN MICROVASCULAR ENDOTHELIUM BY ENDOGENOUS NITRIC OXIDE SUPPRESSION: LEUKOCYTE-INDEPENDENT EVENTS M. Suematsu. H. Suzuki. M. Mivasaka*. Y Ishimura** and G.W Schmid-Schonbem Institute for Biomedical Engineering, Llniwrsity ot California. San Diego. Department of Immunolopy. Tokyo Metropolitan Institute for Medical Sctences* Department of Biochemtstry. School of Medicine. Keio IIniverslty**
2:22
This study was aimed to demonstrate mtcrovasculsr endothellal oxidattve changes Independent of leukocyte adhesion in viva The rat mesentetic microcirculation was superfused with NC-mtro-L-argmme methyl ester (L-NAME, 100 PM), and intracellular oxidant formatjon m several microcirculatory cellular components such as ilffertolar and venular endothelium and mast cells was visually monitored by digital microfluorography assisted by carboxydichlorofluorescein (CDCF). a hydroperoride-sensitive fluorogenic probe (I, 2). Time history of leukocyte adhesion was monitored simultaneously. L-NAME induced a significant increase in CDCF fluorescence in arteriolar and venular endothelium as well as m mast cells which wz followed by leukocyte adhesion. The level of hydroperoxide formation in arterioles and venules evoked by 60-min L-NAME superfusion was eqwalent to that induced bv the sunerfusion of about 800 UM ten-butvlhvdroperoxide for IO m;n. Pretrkatment with anti-ICAMor -Cdld monoclonal antibody attenuated L-NAME-elicited venular leukocyte adheston without. suppressing CDCF fluorescence m situ. Pretreatment with desferioxamine (50 mgikg. ii,, 1 hr prior to L-NAME superfusion) significantly dimimshed the hydroperoxide formation in arterioles and venules. but not in interstitial mast cells, as well as subsequent venular leukocyte adhesion. These findings Indicate that endogenous nitric oxide may modulate oxidative stress in mast cell% arteriolar and venular mtcrovascular endothelium and thereby can Induce cenular leukocyte recnntment as a consequence of vascular oxidative eventa. References: 1) Gastroenterolopy 103: 994, 1992. 2) Am J Physiol
264: H88l. 1993
2:23
SYNTHESIS AND QUANTITATION OF HYDROPEROXIDESOF DNA Chandrakala Menon and John E. Biaglow Department of Biochemistry & Radiation Oncology, University of Pennsylvania, Philadelphia, PA 19 104
LDL LIPID OXIDATION AND PHOSPHOLIPASE A2 ACTIVITY IN ATHEROGENESIS: NOVEL ANALYTICAL APPRGACHES J.J.M. van den Berg’, M.G. Lansberg*, N.E. Cook1 and D.L. TribbIe* 1Children’s Hospital Oakland Research Institute, Oakland, CA 94609 USA and *Lawrence Berkeley Laboratory, Berkeley, CA 94720 USA.
We have succeeded in making hydroperoxides of These DNA and quantitating them accurately. products are associated with the pyrimidine bases in Thymidine particularly thymidine. DNA, hydroperoxides were synthesized by exposing 2mM solutions of thymidine to 200 gray gamma radiation from a cesium source, dose rate = lSgy/min. H202 in the solution was removed with catalase. Under these conditions, a maximum yield of 23 mM thymidine hydroperoxide was obtained. This accounted for about 1.15% of the nucleotide present in solution. thymine a hydroperoxide was Alternatively, chemically synthesized from Shydroxymethyluracil. We are exploring the possibility of making DNA hydroperosides by, reacting DNA with NaOCl and H202. We have also s: cceeded in quantitating the hydroperoxides accurately. The hydroperoxides were quantitated by the potassium iodide method. H202 was measured by the Fricke’s method or by the peroxide probe. Alternatively, DNA hydroperoxide was quantitated by a coupled assay consisting of reaction of the hydroperoxide with a known concentration of GSH in the presence of Se dependent glutathione peroxidase. The remaining GSH was reacted with stoichiometric quantities of DTNB and the absorbance was read at 412 nm. 1~202, cumene hydroperoxide and t-butyl hydroperoxide were used as standards in both methods.
Research in atberogenesis currently focuses on the hypothesis that the conversion of low density lipoproteins (LDL) into atherogenic panicles is mediated by oxidative processes. ModiIicaiion of the apoB protein appears to he crucial, but lipid oxidation products may initiate apoB modification. Pbospholipase A2 (PLA2) activity has been suggested to release reactive oxidized fatty acids that subsequently modify apoB. Studies thus far have provided mostly nonspecific and incomplete information regarding the mechanisms by which oxidation of LDL lipids may be involved in atherogenesis. This poster highlights the analytical approaches we use to study three areas of interest: (1) the identity of LDL lipid oxidation products; (2) the role of PLA2 in LDL atherogenicity; (3) the kinetics of LDL oxidation processes. (1) Gas chromatography-mass spectrometry is used to separate and identify oxidized lipid species. (2) A lipid monolayer technique is used to study the changes in physicochemical behavior of LDL surface phospholipids upon oxidation. We show that oxidized phospholipids adopt a drastically different conformation in the monolayer, which is linked to recognition and preferential cleavage of oxidized fatty acyl chains by PLA2. Monolayer studies provide mechanistic information on phospholipase activity and can be used to establish if PLA2 has a specificity for certain oxidized substrates. Purified oxidized linids will be inuoduced into LDL in vifro with or without PLA2 inhibition to study their potential for initiating atherogenic changes in LDL. (3) Oxidative susceptibility of LDL is studied using the fiuorescent polyunsaturated fatty -&id, psinarlc acid @A), and its methylated derivative, PnA methyl ester @ME), as oxidation probes specific for LDL surface and core regions, respectively. Differences in lipid composition, antioxidant content, and accessibility to oxidants of these two pools could determine the overall response of the LDL particle to oxidative stress, and the PnA/pnME probe combination provides a unique 1001 to investigate this. These studies will generate new information with regard to the oxidative metabolism of LDL lipids and their involvement in atherogenesis.
2:24