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Inhibition of low-density lipoprotein (LDL) lipid peroxidation by policosanol, a new cholesterol-lowering agent
Monday June 26, 2000: Poster Abstracts P: W29 Oxidation and Atherogenesis
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subjects with no CAD overlapped with 30% of N subjects characterized by the same low R1 and R2 values and low apoC-III (< 12.5 mg/dL) and representing an area of moderate risk. Conclusions: This study showed that OS levels critical for initiating CAD are expressed by low RI (<0.55) and R2 (<5.0) and that high OS in combination with high levels of apoC-III may be predictive of CAD risk in N and HTG subjects. MoP3:W29 J Inhibition of low-density iipoprotein (LDL) lipid peroxidation by policosanol, a new cholesterol-lowering agent R. Mentndez, R. Mds, A.M. Amor, R.M. Gonz~ilez, J.C. Fem~indez, I. Rodeiro, M. Zayas, S. Jimtnez. Center of Natural Products, National
Center for Scientific Research, P.O. Box 6880, Havana, Cuba Policosanol is a mixture of high molecular weight aliphatic alcohols isolated and purified from sugar cane wax with cholesterol-lowering effects demonstrated in healthy volunteers, patients with type II hypercholesterolemia and patients with dyslipidemia associated to non-insulin dependent diabetes mellitus. Policosanol acts by inhibiting cholesterol biosynthesis and increasing LDL-C processing. Because oxidation of LDL plays a crucial role in the pathogenesis of atherosclerosis and the resistance of LDL to in vitro oxidation has been possibly correlated with the extent of atherosclerosis, we investigated the effects of policosanol on LDL oxidation in a double-blinded, placebo-controlled study conducted in 69 healthy volunteers. We tested the susceptibility of LDL to lipid peroxidation in a cell-free system by the addition of copper ions and in a more physiological system, macrophage-mediated oxidation. Policosanol administered within its dosage for lowering cholesterol (5 and 10 rag/day), significantly increased lag phase (up to 1.5-fold), decreased the rate of conjugated diene generation and significantly inhibited macrophagemediated lipid peroxidation (up to 37%). We further studied the effects of policosanol in other double-blinded, randomized, placebo-controlled trial undertaken in 20 patients with type 11 hypercholesterolemia at high atherosclerotic risk. Even when these patients showed increased susceptibility of LDL to lipid peroxidation, policosanol administered at its starting dose (5 mg/day) significantly prolonged lag phase of diene generation curve (13.9%) when compared to placebo. Also, policosanol tended to decrease diene propagation rate, but such reduction (11.3%) did not reach statistical significance compared with placebo. In conclusion, this study demonstrated that policosanol in addition to its cholesterol-lowering effects has other properties that enables it to reduce the potential of LDL to undergo lipid peroxidation. Such effect can be considered of promissory value in the management of atherosclerosis since the reduction of both, plasma LDL concentration and its susceptibility to, oxidation may reduce the atherosclerotic risk. I
MoP4:W29 iI Mechanism of the antioxidant to prooxidant switch for dehydroascorbate in low density Upoprotein oxidation E.T.M. Horsley, L.K. Harris, N.J. Moss, D.S. Leake. The University of
Reading, Reading, UK There is a growing list of compounds that switch from being antioxidants for low density lipoprotein (LDL) to being pro-oxidant when the LDL becomes partially oxidised. The objective of this study was to elucidate the mechanism of this antioxidant to prooxidant switch using dehydroascorbate, the oxidation product of ascorbic acid, as an example. The oxidation of LDL by 5 /~M CuSO4 was followed daily after its isolation and it was found that dehydroascorbate (10/~M) switched from being a strong antioxidant to being a prooxidant 4-8 days after the LDL was isolated. HPLC analysis of LDL samples taken daily revealed that more than 80% of the original alpha-tocopherol was still present in LDL when the switch occurred. Lipid hydroperoxides, in the form of 13 (S)-hydroperoxyoctadeca-gZ, 11E-dienoic acid, were added to fresh LDL to confirm that it was the presence of lipid hydroperuxides in partially oxidised LDL and not the depletion of alpha-tocopherol that was responsible for the antioxidant to prooxidant switch in the activity of dehydroascorbate. In the presence of lipid hydroperoxides, dehydroascorbate (10/~M) had a prooxidative effect whereas in their absence it had a strong antioxidant effect. Dehydmascorbate (or more likely a degradation product of dehydroascorbate) caused a slow reduction of Cu2+ to Cu+, as determined using bathocuproine to detect Cu+. This reduction of Cu2+ occurred in either the presence or absence of bathocupmine. The production of Cu+ may explain the prooxidant effect of dehydroascorbate, as Cu+ decomposes lipid hydroperoxides into lipid alkoxyl and other radicals faster than does Cu2+. We thank MAFF and the BBSRC for their funding.
MoP5:W29 J Mechanisms by which cysteine can inhibit or promote the oxidation of low density lipoprotein by copper R.A. Patterson, D.J. Lamb, D.S. Leake. The University of Reading, Reading,
UK It is widely believed that oxidised low density lipoprotein (LDL) is pro-atherogenic. In vitro, cells often show a dependence upon micromolar levels of transition metal ions and thiols to oxidise LDL. In the absence of cells, however, LDL oxidation by copper ions in a simple phosphate buffer is inhibited by the thiol cysteine or its disulphide cystine. We have shown, using an equilibrium dialysis technique to measure copper binding to LDL, that one of the mechanisms by which cysteine (or more likely its oxidation products in the presence of copper) and cystine (each 100/zM) inhibit LDL oxidation is by decreasing the binding of copper to LDL (97% inhibition for each). It has been demonstrated previously that cysteine increased the oxidation of LDL that was already partially oxidised. We oxidised LDL with the azo initiator 2-2'-azobis (2-amidinopropane) dihydrochloride in the absence of copper, to enrich the LDL with lipid hydroperoxides, and studied the effects of both cysteine and cystine on the decomposition of lipid hydroperoxides by copper into thiobarbituric acid-reactive substances (TBARS). Only cysteine (100 #M) was able to increase the rate of lipid hydroperoxide decomposition, which we propose is a pro-oxidant activity as it would be expected to produce lipid alkoxyl radicals, as well TBARS and other products. Cysteine, but not cystine, rapidly reduced Cu2+ to Cu+, as shown by a bathocuproine assay. We therefore attribute cysteine's ability to decompose lipid hydroperoxides to its copper reducing activity as lipid hydroperoxide decomposition is faster with Cu+ than Cu2+. This work identifies some of the mechanisms involved in the antioxidant or pro-oxidant activities of cysteine and cystine. We thank the Wellcome Trust for financial support.
MoP6:W29 ]I In vivo complex formation of modified alphal-antitrypsin with LDL S. Mashiba I , Y, Wada2, M. Takeya 3, A. Sugiyama2, T. Hamakubo 2, T. Kodama2, K. Uchida 1. llkagaku Co. Ltd., Kyoto; 2Department of
Molecular Biology and Medicine, RCAST, University of Tokyo; 3Department of Pathology, University of Kumamoto, Japan Objective: Inactivated form of alphal-antitrypsin (AT) and LDL coelute in gel permeation chromatography, and to confirm the association of modified AT with LDL, monoclonal antibody against oxidized by chrolamine T and inactivated AT was established. Methods and Results: From several antibodies obtained, an antibody which did not react neither with native AT, trypsin or elastase-AT complex, but did with chloramine T treated AT and oxidized LDL-AT complex was selected and named as OxAT-4. Characterization of OxAT-4 was confirmed by binding assay with various types of aldehyde modified AT as competing ligands. Presence of modified AT within whole LDL fraction was shown by immunoprecipitation using OxAT-4. LDL with modified AT (AT-LDL) was purified using affinity chromatography with OxAT-4 coated carrier. Western blotting using anti apoB polyclonal antibody and OxAT-4 confirmed the collocalization of apo-B and modified AT in this purified fraction of LDL. By immunohistochemistry, localization of OxAT-4 antigen in atherosclerotic core was observed, but not completely identical that of macrophage. Degradation assay indicated the significant increase of uptake of AT-LDL by mouse peritoneal resident macrophage. Conclusion: Complex formation with modified AT and LDL in plasma was strongly suggested. Immunohistochemistry revealed the possibility that after the production by monocyte/macrophage, some degree of AT may be changed into AT-LDL in arterial wall, degraded there, and may contribute to atherogenesis. MoP7:W29 ] Induction of gene expression by phenolic antioxidants, BO-653 and probucol and BHQ in human endothelial cells W. Takabe 1, C. Mataki 1, Y. Wada 1, M. Ishii 1, Y. Okutani 2, O. Cynshi 3, N. Noguchi I , H. Aburatani 1, T. Hamakubo 1, T. Kodama I , E. Niki I . 1RCAST,
University of Tokyo; 2Daiichi pharmaceutical Co., Ltd Tokyo; 3Chugai pharmaceutical Co., Ltd, Shizuoka, Japan Objective: The effects of anti-atherogenic antioxidants on the gene expression in human endothelial cells were investigated using DNA chip. Methods: Human umbilical vein endothelial cells (HUVEC) were grown to confluent state to which 2,3-dihydru-5-hydroxy-2,2dipentyl-4,6-di-tert-butyl-
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000
54
!
subjects with no CAD overlapped with 30% of N subjects characterized by the same low R1 and R2 values and low apoC-III (< 12.5 mg/dL) and representing an area of moderate risk. Conclusions: This study showed that OS levels critical for initiating CAD are expressed by low RI (<0.55) and R2 (<5.0) and that high OS in combination with high levels of apoC-III may be predictive of CAD risk in N and HTG subjects. MoP3:W29 J Inhibition of low-density iipoprotein (LDL) lipid peroxidation by policosanol, a new cholesterol-lowering agent R. Mentndez, R. Mds, A.M. Amor, R.M. Gonz~ilez, J.C. Fem~indez, I. Rodeiro, M. Zayas, S. Jimtnez. Center of Natural Products, National
Center for Scientific Research, P.O. Box 6880, Havana, Cuba Policosanol is a mixture of high molecular weight aliphatic alcohols isolated and purified from sugar cane wax with cholesterol-lowering effects demonstrated in healthy volunteers, patients with type II hypercholesterolemia and patients with dyslipidemia associated to non-insulin dependent diabetes mellitus. Policosanol acts by inhibiting cholesterol biosynthesis and increasing LDL-C processing. Because oxidation of LDL plays a crucial role in the pathogenesis of atherosclerosis and the resistance of LDL to in vitro oxidation has been possibly correlated with the extent of atherosclerosis, we investigated the effects of policosanol on LDL oxidation in a double-blinded, placebo-controlled study conducted in 69 healthy volunteers. We tested the susceptibility of LDL to lipid peroxidation in a cell-free system by the addition of copper ions and in a more physiological system, macrophage-mediated oxidation. Policosanol administered within its dosage for lowering cholesterol (5 and 10 rag/day), significantly increased lag phase (up to 1.5-fold), decreased the rate of conjugated diene generation and significantly inhibited macrophagemediated lipid peroxidation (up to 37%). We further studied the effects of policosanol in other double-blinded, randomized, placebo-controlled trial undertaken in 20 patients with type 11 hypercholesterolemia at high atherosclerotic risk. Even when these patients showed increased susceptibility of LDL to lipid peroxidation, policosanol administered at its starting dose (5 mg/day) significantly prolonged lag phase of diene generation curve (13.9%) when compared to placebo. Also, policosanol tended to decrease diene propagation rate, but such reduction (11.3%) did not reach statistical significance compared with placebo. In conclusion, this study demonstrated that policosanol in addition to its cholesterol-lowering effects has other properties that enables it to reduce the potential of LDL to undergo lipid peroxidation. Such effect can be considered of promissory value in the management of atherosclerosis since the reduction of both, plasma LDL concentration and its susceptibility to, oxidation may reduce the atherosclerotic risk. I
MoP4:W29 iI Mechanism of the antioxidant to prooxidant switch for dehydroascorbate in low density Upoprotein oxidation E.T.M. Horsley, L.K. Harris, N.J. Moss, D.S. Leake. The University of
Reading, Reading, UK There is a growing list of compounds that switch from being antioxidants for low density lipoprotein (LDL) to being pro-oxidant when the LDL becomes partially oxidised. The objective of this study was to elucidate the mechanism of this antioxidant to prooxidant switch using dehydroascorbate, the oxidation product of ascorbic acid, as an example. The oxidation of LDL by 5 /~M CuSO4 was followed daily after its isolation and it was found that dehydroascorbate (10/~M) switched from being a strong antioxidant to being a prooxidant 4-8 days after the LDL was isolated. HPLC analysis of LDL samples taken daily revealed that more than 80% of the original alpha-tocopherol was still present in LDL when the switch occurred. Lipid hydroperoxides, in the form of 13 (S)-hydroperoxyoctadeca-gZ, 11E-dienoic acid, were added to fresh LDL to confirm that it was the presence of lipid hydroperuxides in partially oxidised LDL and not the depletion of alpha-tocopherol that was responsible for the antioxidant to prooxidant switch in the activity of dehydroascorbate. In the presence of lipid hydroperoxides, dehydroascorbate (10/~M) had a prooxidative effect whereas in their absence it had a strong antioxidant effect. Dehydmascorbate (or more likely a degradation product of dehydroascorbate) caused a slow reduction of Cu2+ to Cu+, as determined using bathocuproine to detect Cu+. This reduction of Cu2+ occurred in either the presence or absence of bathocupmine. The production of Cu+ may explain the prooxidant effect of dehydroascorbate, as Cu+ decomposes lipid hydroperoxides into lipid alkoxyl and other radicals faster than does Cu2+. We thank MAFF and the BBSRC for their funding.
MoP5:W29 J Mechanisms by which cysteine can inhibit or promote the oxidation of low density lipoprotein by copper R.A. Patterson, D.J. Lamb, D.S. Leake. The University of Reading, Reading,
UK It is widely believed that oxidised low density lipoprotein (LDL) is pro-atherogenic. In vitro, cells often show a dependence upon micromolar levels of transition metal ions and thiols to oxidise LDL. In the absence of cells, however, LDL oxidation by copper ions in a simple phosphate buffer is inhibited by the thiol cysteine or its disulphide cystine. We have shown, using an equilibrium dialysis technique to measure copper binding to LDL, that one of the mechanisms by which cysteine (or more likely its oxidation products in the presence of copper) and cystine (each 100/zM) inhibit LDL oxidation is by decreasing the binding of copper to LDL (97% inhibition for each). It has been demonstrated previously that cysteine increased the oxidation of LDL that was already partially oxidised. We oxidised LDL with the azo initiator 2-2'-azobis (2-amidinopropane) dihydrochloride in the absence of copper, to enrich the LDL with lipid hydroperoxides, and studied the effects of both cysteine and cystine on the decomposition of lipid hydroperoxides by copper into thiobarbituric acid-reactive substances (TBARS). Only cysteine (100 #M) was able to increase the rate of lipid hydroperoxide decomposition, which we propose is a pro-oxidant activity as it would be expected to produce lipid alkoxyl radicals, as well TBARS and other products. Cysteine, but not cystine, rapidly reduced Cu2+ to Cu+, as shown by a bathocuproine assay. We therefore attribute cysteine's ability to decompose lipid hydroperoxides to its copper reducing activity as lipid hydroperoxide decomposition is faster with Cu+ than Cu2+. This work identifies some of the mechanisms involved in the antioxidant or pro-oxidant activities of cysteine and cystine. We thank the Wellcome Trust for financial support.
MoP6:W29 ]I In vivo complex formation of modified alphal-antitrypsin with LDL S. Mashiba I , Y, Wada2, M. Takeya 3, A. Sugiyama2, T. Hamakubo 2, T. Kodama2, K. Uchida 1. llkagaku Co. Ltd., Kyoto; 2Department of
Molecular Biology and Medicine, RCAST, University of Tokyo; 3Department of Pathology, University of Kumamoto, Japan Objective: Inactivated form of alphal-antitrypsin (AT) and LDL coelute in gel permeation chromatography, and to confirm the association of modified AT with LDL, monoclonal antibody against oxidized by chrolamine T and inactivated AT was established. Methods and Results: From several antibodies obtained, an antibody which did not react neither with native AT, trypsin or elastase-AT complex, but did with chloramine T treated AT and oxidized LDL-AT complex was selected and named as OxAT-4. Characterization of OxAT-4 was confirmed by binding assay with various types of aldehyde modified AT as competing ligands. Presence of modified AT within whole LDL fraction was shown by immunoprecipitation using OxAT-4. LDL with modified AT (AT-LDL) was purified using affinity chromatography with OxAT-4 coated carrier. Western blotting using anti apoB polyclonal antibody and OxAT-4 confirmed the collocalization of apo-B and modified AT in this purified fraction of LDL. By immunohistochemistry, localization of OxAT-4 antigen in atherosclerotic core was observed, but not completely identical that of macrophage. Degradation assay indicated the significant increase of uptake of AT-LDL by mouse peritoneal resident macrophage. Conclusion: Complex formation with modified AT and LDL in plasma was strongly suggested. Immunohistochemistry revealed the possibility that after the production by monocyte/macrophage, some degree of AT may be changed into AT-LDL in arterial wall, degraded there, and may contribute to atherogenesis. MoP7:W29 ] Induction of gene expression by phenolic antioxidants, BO-653 and probucol and BHQ in human endothelial cells W. Takabe 1, C. Mataki 1, Y. Wada 1, M. Ishii 1, Y. Okutani 2, O. Cynshi 3, N. Noguchi I , H. Aburatani 1, T. Hamakubo 1, T. Kodama I , E. Niki I . 1RCAST,
University of Tokyo; 2Daiichi pharmaceutical Co., Ltd Tokyo; 3Chugai pharmaceutical Co., Ltd, Shizuoka, Japan Objective: The effects of anti-atherogenic antioxidants on the gene expression in human endothelial cells were investigated using DNA chip. Methods: Human umbilical vein endothelial cells (HUVEC) were grown to confluent state to which 2,3-dihydru-5-hydroxy-2,2dipentyl-4,6-di-tert-butyl-
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000