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LDL oxidation in patients with severe carotid atherosclerosis
Monday 10 October 1994: Poster Abstracts Modified lipoproteins 400 CNRS, Univ. RenC Descartes, 45, rue des Saints-Peres, 75006 Paris, France
Lp(a) can be distinguished from LDL by the presence of an additional apolipoprotein, ape(a). As with LDL, oxidative modification of this particular lipoprotein appears to be involved in the development of atherosclerotic lesions. We compared the in vitro resistance to gamma radiolysis-induced oxidation of Lp(a) to that of LDL isolated from the same blood samples. Purified preparations of Lp(a) and LDL were obtained by sequential ultracentrifugation (d = 1.050-I. 12) and exclusion gel chromatography. Lipoprotein preparations (0.40 mm01 phospholipidsfl) were dialyzed against lo- M sodium phosphate/lO- M sodium formate buffer and the action of 02’YHO~ free radicals was studied at two pHs: pH 7.0, where the ratio [02’7/[HO2’] is close to 100, and pH 5.7, where this ratio is close to 10. Four markers of the lipid peroxidation have been followed as a function of the irradiation doses (up to 600 Gy): vitamin E decrease, thiobarbituric acid-reactive substances (TBARS) formation, conjugated diene production and modification of electrophoretic mobility of oxidized lipoproteins. In Lp(a) as in LDL, we observed an important pH effect: vitamin E depletion was slower and lag dose before appearance of lipid peroxidation products was higher at pH 7.0 than at pH 5.7, demonstrating a higher oxidizing ability of HO*’ than Oz’- radicals. Moreover, at both pHs, Lp(a) preparations exhibited a greater resistance to oxidation than LDL, as indicated by a slower vitamin E decrease and a diminished production of both conjugated dienes and TBARS. LDL oxidation in patients with severe carotid atherosclerosis &~gg,& Chiesa R, Melissano G, Caste&no R, Astore D, Grossi A, Finardi G, Bellomo G, Dem. of Internal Medicine, First Medi-
/1611
cal Clinic, Policlinico S. Matteo,” Univ. of Pavia, I-27100 Pavia, Italy
Among the various risk factors involved in the development and progression of carotid atherosclerosis, the oxidation of LDL is considered to play a relevant role. LDL oxidation has been investigated in 94 patients with severe carotid atherosclerosis undergoing elective carotid artery endarterectomy and in 42 matched control subjects. LDL oxidation was evaluated in all patients as: (i) the susceptibility to in vitro oxidation, (ii) vitamin E concentration and its antioxidant efficiency and (iii) the presence of autoantibodies against oxidatively modified lipoprotein, to monitor the occurrence of the oxidative processes taking place in vivo. No difference was detected between control subjects and patients as concerning vitamin E concentration and the kinetics of conjugated diene formation in isolated LDL exposed to CuSO4. However, vitamin E efficiency was lower (9.6 + 4.2 vs 30.2 * 7.6 min/nmol vitamin E) and the duration of the vitamin Eindependent lag-phase was longer (105.5 f 16.5 vs 58 f 11.8 min) in the patient group. Autoantibodies against oxidatively modified lipoproteins were measured by ELISA using native LDL, Cu2+oxidized LDL (oxLDL), or malondialdehyde-derivatized LDL (MDA-LDL) as antigens. The antibody titer was calculated as the ratio of antibodies against modified vs native proteins. Patients with carotid atherosclerosis had an antibody ratio significantly higher than control subjects both for anti-oxLDL IgG (1.78 f 0.39 vs 1.05zt 0.3) and IgM (1.98 f 0.83 vs 1.40 f 0.09) and for antiMDA-LDL IgG (2.39 zb0.5 1 vs 2.04 f 0.11) and IgM (4.18 zb 1.89 vs 2.9 f 0.15). The highest titers were found in patients with associated hyperlipidemia and hypertension, alone or in combination. These data indicate that patients with severe carotid atherosclerosis develop autoantibodies against oxidatively modified
43
LDL and experience, despite an apparently ‘normal’ oxidation profile in vitro, an enhanced LDL oxidation in vivo. Ef&ct uf the antioxidant probuad on experimental transplantation atherosclerosis Andersen H0, Helm P, &I&& Hansen BF, Kjeldsen K, Nordestgaard BG, Dept. of Clinical Chemistry, RASK, K&e, Denmark
1
Attenuation of atherogenesis by oral probucol treatment has been demonstrated in several animal studies. This effect is not mediated solely by lowering plasma cholesterol, but has been attributed primarily to the antioxidative properties of probucol that is carried within the lipoprotein particle. By inhibiting oxidation of LDL, probucol decreases the development of foam cells and thereby the formation of fatty streaks, the early step in atherogenesis. In the present study we have investigated whether pmbuco1 has the same effect on experimental transplantation atherosclerosis. The thoracic aorta from one rabbit was transplanted as a bypass graft onto the abdominal aorta of another rabbit. Eight rabbits were treated with 1% dietary probucol and nine animals were treated with placebo. After a recovery period of 2 weeks the rabbits were fed individual amounts of cholesterol in order to raise plasma cholesterol levels to 6-7 nunovl in both groups. However, to obtain similar plasma cholesterol levels the amount of dietary cholesterol tended to be higher in the probucol-treated animals than in the placebo group. The distribution of plasma cholesterol between the lipoprotein classes was similar except for the concentration of HDL that was significantly lowered by probucol treatment. Probucol decreased markedly the susceptibility of LDL particles to oxidation, as measured by the production of conjugated dienes by added copper. However, the cholesterol content in the aortic grafts was without significant difference between the probucol and the placebo group (1879 f 308 versus 2086 + 440 nmol/cm2, Xlf SEM). These amounts were about 20 times higher than the corresponding values for the host aorta. Thus, our results confirm the autioxidative effect of probucol on LDL particles, but this was not accompanied by a retardation of transplantation atherosclerosis in rabbits. 11631 w,
influence of the fatty acid profile of LDL subfractions on their resistance to in vitro oxidation Aristequi R, K&rig D, Keul J, Berg A, Center of
Internal Medicine, Hugstetter Str. 55, D-79106
Freiburg,
Ger-
many
It is widely assumed that high concentrations of small dense LDL are an important risk factor for the development of atherosclerosis. One possible reason is a lower resistance to oxidation of small dense LDL. To test this hypothesis, LDL particles of 16 healthy persons were separated into 6 subfractions by equilibrium density gradient uhracentrifugation. In these subfractions we determined the fatty acid profile by gas liquid chromatography; additionally vitamin E and /I-carotene concentrations were measured by HPLC. The lag phase during Cu2+-induced oxidation in vitro served as a measure of the resistance to oxidation. Multivariate analysis demonstrated that the PUFA content of LDL (‘%IPUFA) is the most important factor in determining the lag time (r = -O.47, r2 = 0.22, P < 0.0001). The next important factor is the amount of vitamin E oer LDL oarticle (? = 0.26. for both factors, univariate: r=+O.3b) No other variables such as LDL-density, /J-carotene concentration, or LDL composition contributed significantly to LDL oxidizability in this study. We conclude that the resistance to oxidation of LDL is mainly determined by LDL composition in terms of fatty acids and antioxidants and only to a minor extent by their density.
Atherosclerosis X, Montreal, October I994
Lp(a) can be distinguished from LDL by the presence of an additional apolipoprotein, ape(a). As with LDL, oxidative modification of this particular lipoprotein appears to be involved in the development of atherosclerotic lesions. We compared the in vitro resistance to gamma radiolysis-induced oxidation of Lp(a) to that of LDL isolated from the same blood samples. Purified preparations of Lp(a) and LDL were obtained by sequential ultracentrifugation (d = 1.050-I. 12) and exclusion gel chromatography. Lipoprotein preparations (0.40 mm01 phospholipidsfl) were dialyzed against lo- M sodium phosphate/lO- M sodium formate buffer and the action of 02’YHO~ free radicals was studied at two pHs: pH 7.0, where the ratio [02’7/[HO2’] is close to 100, and pH 5.7, where this ratio is close to 10. Four markers of the lipid peroxidation have been followed as a function of the irradiation doses (up to 600 Gy): vitamin E decrease, thiobarbituric acid-reactive substances (TBARS) formation, conjugated diene production and modification of electrophoretic mobility of oxidized lipoproteins. In Lp(a) as in LDL, we observed an important pH effect: vitamin E depletion was slower and lag dose before appearance of lipid peroxidation products was higher at pH 7.0 than at pH 5.7, demonstrating a higher oxidizing ability of HO*’ than Oz’- radicals. Moreover, at both pHs, Lp(a) preparations exhibited a greater resistance to oxidation than LDL, as indicated by a slower vitamin E decrease and a diminished production of both conjugated dienes and TBARS. LDL oxidation in patients with severe carotid atherosclerosis &~gg,& Chiesa R, Melissano G, Caste&no R, Astore D, Grossi A, Finardi G, Bellomo G, Dem. of Internal Medicine, First Medi-
/1611
cal Clinic, Policlinico S. Matteo,” Univ. of Pavia, I-27100 Pavia, Italy
Among the various risk factors involved in the development and progression of carotid atherosclerosis, the oxidation of LDL is considered to play a relevant role. LDL oxidation has been investigated in 94 patients with severe carotid atherosclerosis undergoing elective carotid artery endarterectomy and in 42 matched control subjects. LDL oxidation was evaluated in all patients as: (i) the susceptibility to in vitro oxidation, (ii) vitamin E concentration and its antioxidant efficiency and (iii) the presence of autoantibodies against oxidatively modified lipoprotein, to monitor the occurrence of the oxidative processes taking place in vivo. No difference was detected between control subjects and patients as concerning vitamin E concentration and the kinetics of conjugated diene formation in isolated LDL exposed to CuSO4. However, vitamin E efficiency was lower (9.6 + 4.2 vs 30.2 * 7.6 min/nmol vitamin E) and the duration of the vitamin Eindependent lag-phase was longer (105.5 f 16.5 vs 58 f 11.8 min) in the patient group. Autoantibodies against oxidatively modified lipoproteins were measured by ELISA using native LDL, Cu2+oxidized LDL (oxLDL), or malondialdehyde-derivatized LDL (MDA-LDL) as antigens. The antibody titer was calculated as the ratio of antibodies against modified vs native proteins. Patients with carotid atherosclerosis had an antibody ratio significantly higher than control subjects both for anti-oxLDL IgG (1.78 f 0.39 vs 1.05zt 0.3) and IgM (1.98 f 0.83 vs 1.40 f 0.09) and for antiMDA-LDL IgG (2.39 zb0.5 1 vs 2.04 f 0.11) and IgM (4.18 zb 1.89 vs 2.9 f 0.15). The highest titers were found in patients with associated hyperlipidemia and hypertension, alone or in combination. These data indicate that patients with severe carotid atherosclerosis develop autoantibodies against oxidatively modified
43
LDL and experience, despite an apparently ‘normal’ oxidation profile in vitro, an enhanced LDL oxidation in vivo. Ef&ct uf the antioxidant probuad on experimental transplantation atherosclerosis Andersen H0, Helm P, &I&& Hansen BF, Kjeldsen K, Nordestgaard BG, Dept. of Clinical Chemistry, RASK, K&e, Denmark
1
Attenuation of atherogenesis by oral probucol treatment has been demonstrated in several animal studies. This effect is not mediated solely by lowering plasma cholesterol, but has been attributed primarily to the antioxidative properties of probucol that is carried within the lipoprotein particle. By inhibiting oxidation of LDL, probucol decreases the development of foam cells and thereby the formation of fatty streaks, the early step in atherogenesis. In the present study we have investigated whether pmbuco1 has the same effect on experimental transplantation atherosclerosis. The thoracic aorta from one rabbit was transplanted as a bypass graft onto the abdominal aorta of another rabbit. Eight rabbits were treated with 1% dietary probucol and nine animals were treated with placebo. After a recovery period of 2 weeks the rabbits were fed individual amounts of cholesterol in order to raise plasma cholesterol levels to 6-7 nunovl in both groups. However, to obtain similar plasma cholesterol levels the amount of dietary cholesterol tended to be higher in the probucol-treated animals than in the placebo group. The distribution of plasma cholesterol between the lipoprotein classes was similar except for the concentration of HDL that was significantly lowered by probucol treatment. Probucol decreased markedly the susceptibility of LDL particles to oxidation, as measured by the production of conjugated dienes by added copper. However, the cholesterol content in the aortic grafts was without significant difference between the probucol and the placebo group (1879 f 308 versus 2086 + 440 nmol/cm2, Xlf SEM). These amounts were about 20 times higher than the corresponding values for the host aorta. Thus, our results confirm the autioxidative effect of probucol on LDL particles, but this was not accompanied by a retardation of transplantation atherosclerosis in rabbits. 11631 w,
influence of the fatty acid profile of LDL subfractions on their resistance to in vitro oxidation Aristequi R, K&rig D, Keul J, Berg A, Center of
Internal Medicine, Hugstetter Str. 55, D-79106
Freiburg,
Ger-
many
It is widely assumed that high concentrations of small dense LDL are an important risk factor for the development of atherosclerosis. One possible reason is a lower resistance to oxidation of small dense LDL. To test this hypothesis, LDL particles of 16 healthy persons were separated into 6 subfractions by equilibrium density gradient uhracentrifugation. In these subfractions we determined the fatty acid profile by gas liquid chromatography; additionally vitamin E and /I-carotene concentrations were measured by HPLC. The lag phase during Cu2+-induced oxidation in vitro served as a measure of the resistance to oxidation. Multivariate analysis demonstrated that the PUFA content of LDL (‘%IPUFA) is the most important factor in determining the lag time (r = -O.47, r2 = 0.22, P < 0.0001). The next important factor is the amount of vitamin E oer LDL oarticle (? = 0.26. for both factors, univariate: r=+O.3b) No other variables such as LDL-density, /J-carotene concentration, or LDL composition contributed significantly to LDL oxidizability in this study. We conclude that the resistance to oxidation of LDL is mainly determined by LDL composition in terms of fatty acids and antioxidants and only to a minor extent by their density.
Atherosclerosis X, Montreal, October I994