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Haem proteins and oxidative modification of low-density lipoproteins
68 7.13
7.15
Oxidative Modification of Lipoproteins in Atherosclerosis NMR ANALYSIS OF LOW-DENSITY LIPOPROTEIN G:KIDATIVELY MODIFIED IN VITRO ~. Bradamante', L. Barenghi2, G. Giudici2 and C. Vergani 3 'CNR - Centro Speciali Sistemi Organici, 2Fondazione Rivetti, 31st. Medicina Interna, University of Milan, Italy.
THE NMR APPROACH TO RECOGNIZE HUMAN MODIFIED UPOPROTEINS IN VITRO AND IN VIVO S. Bradamante', I_ Barenghi2, G. Giudici2 and C. Vergani3
We studied oxidized LDL (ox-LDL) by the application of high resolution Nuclear Magnetic Resonance (NMR) spectroscopy ('H, '3C, and 3,p). Varian XL-200, Varian XL-300 and Bruker AM 500 instruments were used. Different models were considered for production of ox-LDL in vitro to test NMR specificity and sensitivity. Ox-LDL were produced by freeradical-mediated processes catalyzed by transition metals,ie. treatment of LDL (30-400 mg total cholesterol/dL) with different concentrations of copper (5, 20, and 100 ~.M) or iron salts (100-1000 ,~M), at 25-37 C for 24, 48, 69h. LDL modifications were monitored by classical biochemical methods for changes of lipid composition, decrease of the free lysine groups, increased agarose electrophoretic mobility etc. We focused on levels of lipid hydroperoxides (LPO), TBARS, oxo-dienes and conjugated dienes (CD). The NMR approach proved to be rapid, non disruptive and showed several lipid components at the molecular level. In particular information was obtained on: a) structure mobilities of lipids and on lipid-protein interactions in terms of variations of saturated fatty acyl chain and of transition temperature of cholesterol; b) production of diene-conjugated lipids (mainly cis-trans oriented) and oxygenated derivatives (in particular trans-epoxy system); c) polyunsaturated/monounsaturated fatty chains; d) lysophosphatidylcholine production. The results show that the NMR approach is sensitive and valuable for the assessment of structural changes in ox-LDL.
Previous studies showed that lipoprotein modifications play an important role in the mulUple pathogenic mechanisms of atherosclerosis` Oxidatively-modified low density lipoprotelns (ox-LDL) are potentially atherogenic because of altered interactions with the LDL receptor, massive deposition of cholesterol esters in macrophages, and cytotoxicity. We utilized high resolution NMR spectroscopy ('H, "C and "P) to investigate lipid peroxidation in high cardiovascular risk subjects, In particular we examined tissues (atherosclerotic lesions) and blood (whole plasma and lipoprotein fractions). Our in vivo data were compared with those obtained in vitro: a) by oxidizing plasma lipoproteins (HDL and LDL) at different stages by a free-radical-mediated process catalyzed by transition metals (Cu, Fe);(see accompanying abstracts) b) by modifying lipoproteins with aldehydes (MDA, 2nonenal...); c) by analyzing oxidized polyunsaturated fatty acids (linoleic and arachidonic acids) and some suitable commercially available standards (butadiene monoxide, cis9,1 1 -trans tetradecadienol ...); d) by oxidizing phosphatidylcholine and cholesterol using lipoxygenase. We gained evidence of altered lipoprotein composition (increased CD, LPO, and lyso-PC; decreased polyunsaturated chains,...) on examining the whole plasma and the lipoprotein fractions from our patients. Lesion lipids exhibit features similar to those shown by lipid extracts from LDL oxidized in vitro. The NMR approach appears highly applicable for investigating modified lipoproteins from humans. It is relatively rapid and not disruptive. It needs no or very little treatment of the sample, thus avoiding artifacts. Several lipid components are elaborated at the molecular levels, allowing for their quantification. Moreover NMR analysis provides information on the supramolecular ordering of complex systems.
'CNR - Centre Speciali Sistemi Organici, 2Fondazione Rivetti, 31st. Medicina Intema, University of Milan, Italy.
Richard Bruckdorfer,Catherine Rice-Evans D e p t of B i o c h e m i s t r y , Royal Free Hosp. School of Medicine, London NW3 2PF U.K.
INTERACTION OF LOW DENSITY LIPOPROTEINS(LDL) WITH ARTERIAL PROTEOGLYCANS MARKEDLY INCREASES ITS SUSCEPTIBILITY TO OXIDATION BY COPPER AND UPTAKE BY CELLS German CameJo, Eva Hurt, Sirgitta Rosengren and G6ran BondJers. Wallenberg Laboratory, University of Gothenburg, S-41345 and H~ssle Cardiovascular Research Laboratories, Sweden.
T h e n a t u r e of t h e r a d i c a l species which induces the oxidation of L D L i n v i v o is u n c e r t a i n , m a n y t y p e s of r a d i c a l having the potential to i n i t i a t e this process. We propose that haem proteins have a potent action on LDL. Ferryl myoglobin radicals generated in the presence of h y d r o g e n peroxide, are potent initiators of LDL oxidation. Similar radicals are formed from other haem proteins. Nitric o x i d e , w h i c h is g e n e r a t e d by endothelial cells or activated macrophages, can modulate the formation of t h e s e r a d i c a l s and the oxidation of LDL. This depends o n t h e l e v e l s of H=O=, suppression at l o w e r l e v e l s a n d amplification in excess. Metmyoglobin alone slowly oxidises LDL. This may due to the presence of s m a l l a m o u n t s of e n d o g e n o u s hydroperoxides in LDL which leads to the formation of e i t h e r lipid peroxyor alkoxyradicals which propagate the reaction. We propose that haem proteins,leaking from damaged tissue, are potential stimulators of LDL peroxidation and may play a significant role in atherosclerosis.
Proteoglycans (PG) appear to contribute to extracellular retention of low density lipoproteins (LDL) in atherosclerotic lesions. In vitro, PGinduced modification of LDL increase its uptake by human macrophages transforming them to foam cells resembling those in lesions. We found that LDL once has been complex to human arterial chondroitin sulfate-rich PG (CSPG) and dissociated from them becomes more susceptible to oxidative modifications catalyzed by Cu(II). The rate and final level of thiobarbituric acid-reactive substances (TBARS) produced after incubation of 200 ug/ml of LDL protein with 5 uM CuSO 4 was 2 to 4 times higher for CSPG-treated LDL (LDL-PG) than for native LDL (LDLn) (64 vs 22 nMols/mg prot after 24 h). Also in LDL-PG the lag period for conjugated diene formation was eliminated which indicates a 20 fold increase in its formation as compared to LDLn. The rate and final extent of LDL lipide modification and formation of apoB aggregates was m u c h ~ i g h e r in LDL-PG than in LDLn. The degradation of ~L~Ilabeled LDL-PG and LDLn that have been exposed to Cu(II) for 4 h was 1.9 ug/mg prot and 0.42 ug/mg prot respectively. Thus reflecting the higher extent of modification taken place in LDL-PG as compared to native LDL when exposed to similar oxidative conditions. Competition experiments suggest that the modified LDL is taken up via scavenger receptors. The increased susceptibility to oxidation induced by proteoglycans may be related to alterations that the sulfated polysaccharidee cause in the surface of LDL particles. The effects described may increase the potential atherogenicity of LDL that becoams focally associated to proteoglycans of the intima.
HAEM PROTEINS OF LOW-DENSITY
AND OXIDATIVE LIPOROTEINS.
MODIFICATION
7.14
7.16
7.15
Oxidative Modification of Lipoproteins in Atherosclerosis NMR ANALYSIS OF LOW-DENSITY LIPOPROTEIN G:KIDATIVELY MODIFIED IN VITRO ~. Bradamante', L. Barenghi2, G. Giudici2 and C. Vergani 3 'CNR - Centro Speciali Sistemi Organici, 2Fondazione Rivetti, 31st. Medicina Interna, University of Milan, Italy.
THE NMR APPROACH TO RECOGNIZE HUMAN MODIFIED UPOPROTEINS IN VITRO AND IN VIVO S. Bradamante', I_ Barenghi2, G. Giudici2 and C. Vergani3
We studied oxidized LDL (ox-LDL) by the application of high resolution Nuclear Magnetic Resonance (NMR) spectroscopy ('H, '3C, and 3,p). Varian XL-200, Varian XL-300 and Bruker AM 500 instruments were used. Different models were considered for production of ox-LDL in vitro to test NMR specificity and sensitivity. Ox-LDL were produced by freeradical-mediated processes catalyzed by transition metals,ie. treatment of LDL (30-400 mg total cholesterol/dL) with different concentrations of copper (5, 20, and 100 ~.M) or iron salts (100-1000 ,~M), at 25-37 C for 24, 48, 69h. LDL modifications were monitored by classical biochemical methods for changes of lipid composition, decrease of the free lysine groups, increased agarose electrophoretic mobility etc. We focused on levels of lipid hydroperoxides (LPO), TBARS, oxo-dienes and conjugated dienes (CD). The NMR approach proved to be rapid, non disruptive and showed several lipid components at the molecular level. In particular information was obtained on: a) structure mobilities of lipids and on lipid-protein interactions in terms of variations of saturated fatty acyl chain and of transition temperature of cholesterol; b) production of diene-conjugated lipids (mainly cis-trans oriented) and oxygenated derivatives (in particular trans-epoxy system); c) polyunsaturated/monounsaturated fatty chains; d) lysophosphatidylcholine production. The results show that the NMR approach is sensitive and valuable for the assessment of structural changes in ox-LDL.
Previous studies showed that lipoprotein modifications play an important role in the mulUple pathogenic mechanisms of atherosclerosis` Oxidatively-modified low density lipoprotelns (ox-LDL) are potentially atherogenic because of altered interactions with the LDL receptor, massive deposition of cholesterol esters in macrophages, and cytotoxicity. We utilized high resolution NMR spectroscopy ('H, "C and "P) to investigate lipid peroxidation in high cardiovascular risk subjects, In particular we examined tissues (atherosclerotic lesions) and blood (whole plasma and lipoprotein fractions). Our in vivo data were compared with those obtained in vitro: a) by oxidizing plasma lipoproteins (HDL and LDL) at different stages by a free-radical-mediated process catalyzed by transition metals (Cu, Fe);(see accompanying abstracts) b) by modifying lipoproteins with aldehydes (MDA, 2nonenal...); c) by analyzing oxidized polyunsaturated fatty acids (linoleic and arachidonic acids) and some suitable commercially available standards (butadiene monoxide, cis9,1 1 -trans tetradecadienol ...); d) by oxidizing phosphatidylcholine and cholesterol using lipoxygenase. We gained evidence of altered lipoprotein composition (increased CD, LPO, and lyso-PC; decreased polyunsaturated chains,...) on examining the whole plasma and the lipoprotein fractions from our patients. Lesion lipids exhibit features similar to those shown by lipid extracts from LDL oxidized in vitro. The NMR approach appears highly applicable for investigating modified lipoproteins from humans. It is relatively rapid and not disruptive. It needs no or very little treatment of the sample, thus avoiding artifacts. Several lipid components are elaborated at the molecular levels, allowing for their quantification. Moreover NMR analysis provides information on the supramolecular ordering of complex systems.
'CNR - Centre Speciali Sistemi Organici, 2Fondazione Rivetti, 31st. Medicina Intema, University of Milan, Italy.
Richard Bruckdorfer,Catherine Rice-Evans D e p t of B i o c h e m i s t r y , Royal Free Hosp. School of Medicine, London NW3 2PF U.K.
INTERACTION OF LOW DENSITY LIPOPROTEINS(LDL) WITH ARTERIAL PROTEOGLYCANS MARKEDLY INCREASES ITS SUSCEPTIBILITY TO OXIDATION BY COPPER AND UPTAKE BY CELLS German CameJo, Eva Hurt, Sirgitta Rosengren and G6ran BondJers. Wallenberg Laboratory, University of Gothenburg, S-41345 and H~ssle Cardiovascular Research Laboratories, Sweden.
T h e n a t u r e of t h e r a d i c a l species which induces the oxidation of L D L i n v i v o is u n c e r t a i n , m a n y t y p e s of r a d i c a l having the potential to i n i t i a t e this process. We propose that haem proteins have a potent action on LDL. Ferryl myoglobin radicals generated in the presence of h y d r o g e n peroxide, are potent initiators of LDL oxidation. Similar radicals are formed from other haem proteins. Nitric o x i d e , w h i c h is g e n e r a t e d by endothelial cells or activated macrophages, can modulate the formation of t h e s e r a d i c a l s and the oxidation of LDL. This depends o n t h e l e v e l s of H=O=, suppression at l o w e r l e v e l s a n d amplification in excess. Metmyoglobin alone slowly oxidises LDL. This may due to the presence of s m a l l a m o u n t s of e n d o g e n o u s hydroperoxides in LDL which leads to the formation of e i t h e r lipid peroxyor alkoxyradicals which propagate the reaction. We propose that haem proteins,leaking from damaged tissue, are potential stimulators of LDL peroxidation and may play a significant role in atherosclerosis.
Proteoglycans (PG) appear to contribute to extracellular retention of low density lipoproteins (LDL) in atherosclerotic lesions. In vitro, PGinduced modification of LDL increase its uptake by human macrophages transforming them to foam cells resembling those in lesions. We found that LDL once has been complex to human arterial chondroitin sulfate-rich PG (CSPG) and dissociated from them becomes more susceptible to oxidative modifications catalyzed by Cu(II). The rate and final level of thiobarbituric acid-reactive substances (TBARS) produced after incubation of 200 ug/ml of LDL protein with 5 uM CuSO 4 was 2 to 4 times higher for CSPG-treated LDL (LDL-PG) than for native LDL (LDLn) (64 vs 22 nMols/mg prot after 24 h). Also in LDL-PG the lag period for conjugated diene formation was eliminated which indicates a 20 fold increase in its formation as compared to LDLn. The rate and final extent of LDL lipide modification and formation of apoB aggregates was m u c h ~ i g h e r in LDL-PG than in LDLn. The degradation of ~L~Ilabeled LDL-PG and LDLn that have been exposed to Cu(II) for 4 h was 1.9 ug/mg prot and 0.42 ug/mg prot respectively. Thus reflecting the higher extent of modification taken place in LDL-PG as compared to native LDL when exposed to similar oxidative conditions. Competition experiments suggest that the modified LDL is taken up via scavenger receptors. The increased susceptibility to oxidation induced by proteoglycans may be related to alterations that the sulfated polysaccharidee cause in the surface of LDL particles. The effects described may increase the potential atherogenicity of LDL that becoams focally associated to proteoglycans of the intima.
HAEM PROTEINS OF LOW-DENSITY
AND OXIDATIVE LIPOROTEINS.
MODIFICATION
7.14
7.16