3.P.69 Gemfibrozil metabolite inhibits in vitro LDL oxidation and diminishes cytotoxicity induced by oxidized LDL

Wednesday 8 October 1997 : Posters Lipid peroxidation antibody was developed . The reactivity in 5 ttg of LDL from healthy people was equal to 0 .52 ng of OxLDL. OxLDL levels in patients for cardiovascular diseases, LCAT deficiency and hemodialysis treatment were about 2-, 3- and 8-fold higher than that of normal, respectively . The OxLDL levels, however, were not correlated with plasma total cholesterol levels . These results indicate that 1) OxPC is present in plasma LDL and in foam cells, 2) the OxLDL levels can be increased in certain pathological conditions, and 3) OxLDL may be a risk factor for cardiovascular diseases independent of cholesterol .

3 P.68

Covalent binding of lipids to apolipoprotein B as a marker of LDL oxidation

VV Kaplun, V.V. Tertov, A .N . Orekhov . Institute of Experimental Cardiology, Cardiology Research Center, Institute for Atherosclerosis Research, Ltd., Moscow, Russia At present, it is generally recognized that oxidatively modified LDL plays an important role in atherogenesis . However, determination of the degree of LDL oxidation in vivo is complicated due to a number of various lipoperoxidation products with the short life time as well as their loss during LDL isolation . In this investigation we studied the possibility of measurement the degree of LDL oxidation by determination of lipids covalently bound to apolipoprotein B . To demonstrate the existence of adducts of lipids with apo-B, LDL was oxidized by copper ions, 2.2'-azobis-(2-amidinopropane hydrochloride) as well as by macrophages . After the oxidation, LDL was delipidated by repeated extraction with isopropanol, chloroform-methanol mixture and diethyl ether . Accumulation of protein-bound sterols in oxidized LDL was demonstrated by Chugaev's reaction and high performance liquid chromatography after 30 min alkaline hydrolysis at 100°C . Presence of protein-bound phospholipids in oxidized LDL was registered by nuclear magnetic resonance technique as well as by calorimetric phosphate determination . Kinetic study has shown that in contrast to conjugated dienes, TBA-reactive substances and hydroperoxides, the content of protein-bound lipids was increasing monotonously up to 36 h of oxidation . Obtained data suggest that the content of protein-bound lipids is a parameter reflecting the degree of LDL oxidation and can be used to evaluate the role of lipoprotein oxidation in atherogenesis .

3 .P.69

Gemfibrozil metabolite inhibits in vitro LDL oxidation and diminishes cytotoxicity induced by oxidized LDL

Mitsunobu Kawamura, Shigeru Miyazaki, Tamio Teramoto l , Makoto Kinoshita', Kazumasa Hase . Dept. of Intern. Med., Tokyo Teishin Hosp . ; 'Dept. of Med., Teikyo Univ. School of Med., Japan Purpose : M1, a metabolite of gemfibrozil (Gem) may have antioxidant properties, because it is hydroxylated on a phenol ring, i .e ., 5-(4-hydroxy-2,5-dimethyl-phenoxy)-2,2-dimethyl pentanoic acid . The antioxidant properties may provide antiatherogenic effect with Gem. Methods: Susceptibility of LDL to oxidative modification was evaluated by the system using Cu2+ or V-70 (new azo compound) as previously reported . Conjugated dienes, lipid hydroperoxide and thiobarbituric acid reactive substances were measured to evaluate the degree of LDL oxidation . The oxidized LDL (Ox-LDL), which is used for cytotoxicity studies, was prepared by the dialyzing method using Cu2+ as the inducer. Cytotoxicity of Ox-LDL was studied on J 774 macrophages by MTT assay using 3-[4,5-dimethyl-thiazol-2-ylj-2,5-diphenyl tetrazolium bromide . Results : (1) The oxidative modification of LDL was inhibited by Ml in + dose-dependent manner . (2) Ml inhibited LDL oxidation mediated by Cu 2 for three hours. (3) When the LDL incubated with Ml was dialyzed against PBS for 20 hours, antioxidant effect of Ml on LDL oxidation was diminished . (4) Ml diminished cytotoxicity of oxidized LDL, although it was milder than probucol. Conclusion : The present study suggested that Gem has antioxidant effect on LDL and protective effect on cytotoxicity of Ox-LDL through M1, Gem metabolite, leading to antiatherogenic effect .

3 .P.70

Protection of LDL by lipophilic antioxidants against oxidation induced by y-radiolysis of water

A. Khalil, J .-G . Lehoux, J .-P. Jay-Gerin, A . Fortun, A. El Abbouyi, J. Wallach, T. Fulop. Institut Universitaire de Geriatrie, Sherbrooke, (Qc), Canada; Universite Claude Bernard, Lyon, France ; Universite Chouaib Doukkali, El Jadida, Maroc The aim of this work was to study the role of novel lipophilic antioxidant

213

such as Dehydroepiandrosterone (DHEA) and U-74389G (trilazad mesylate analog) in the modulation of LDL peroxidation . Oxygenated aqueous solution of LDL with DHEA (10 sM) and U-74389G (20 µM), were submitted to the action of OH and 02 - produced by y-radiolysis. The antioxidant activity of DHEA and U-74389G was studied by determining then effect on LDL peroxidation as measured by the production of conjugated dienes, thiobarbituric acid-reactive substances (TBARS), electrophoretic migration as well as endogenous vitamin E concentration and fluorescence appearance at 440 nm. DHEA and U-74389G inhibited the oxidative modification of LDL by a decrease in the formation of lipid oxidation products, the electrophoretic migration and the fluorescence appearance, as well as the chemotactic activity of oxidized LDL towards monocytes . U-74389G was also able to reduce the macrophage uptake of oxidized LDL . Our results suggest that these lipophilic antioxidants could be important in the prevention of atherosclerosis and should be evaluated in animal models as a potential therapy to retard atherogenesis .

3 .R71

Copper induced oxidation to serum constituents in hyperlidemia and diabetes mellitus

Masayoshi Kibata, Ayako Kuyama, Mituru Ando, Masami Takeyama, Yasuo Mishima . Department of Internal Medicine and Clinical Laboratory National Sanatorium Minamiokayama Hospital, 4066, Hayashima, Okayama, 701-03, Japan Instead of isolated LDL, whole serum was exposed to copper induced oxistress . Serum constituents were measured at the two states, one of which serum kept at 4°C for 24 hours (stage 1) and the other kept at 37°C with addition of :500 uM CuSO4 (stage 3) . At stage 3 albumin, total bilirubin, uric acid and most of members of lipoproteins reduced, but TBARS increased intensively and apoprotein B and A-2 elevated slightly . In hyperlipidemia, because originally lipoprotein constituents increased the effect of oxidation was stronger than control, especially it was observed in triglyceride-rich hyperlipidemias which demonstrated abnormal shape (nodule formation or disrupted) LDL which were depicted by densitography of PAGE. In diabetes mellitus, vitamin E showed significant reduction rate from stage 1 to stage 3 . It depends on partly lower level of vitamin E but reduction rate was not correlated with it. Further, specially invented antibody to AGE showed significant increase in DM serum at stage 3 which meant that AGE is formed easily by oxidation especially in DM state . These observation would give us a lot of inforrnations to avoid atherosclerotic development and the significance of extrinsic antioxidants in practice .

3 .R72

Elevation of plasma lipid peroxidation products in patients with unstable coronary artery disease

K . Kostner, T. Neunteufl, P. Yang, D . Glogar, K . Huber, G . Maurer. Department of Cardiology, University of Vienna, Austria There is increasing evidence that oxidatively modified lipoproteins play a key role in the pathogenesis of atherosclerosis and that antioxidants may prevent atherosclerosis by inhibiting lipid peroxidation . Since there exist few in vivo studies on lipid peroxidation in CAD, we measured different parameters of lipid perox:idation (peroxides, conjugated dienes and TBARS) in 100 CAD patients and compared them to 115 control subjects . As expected, CAD patients had higher triglyceride (192 vs 107 mg/dl), total cholesterol (220 vs 189 mg/dl), LDL-C (148 vs 125 mg/dl) and lower HDL-C (35 vs 47 mg/dl) values as compared to the control group, respectively . All these differences were statistically highly significant (p < 0 .001) . The mean concentration of lipid peroxides in patients with CAD was 25 .2 sg/dl, the mean concentration of conjugated dienes was 14 .7 zg/dl and the mean concentration of TBARS was 2.0 jsg/dl . In controls these values were 21 .8, 11 .4 and 1 .6 .cg/dl respectively. Significant differences were observed in both conjugated dienes and TBARS (p = 0 .01), but not in peroxides . When we divided the CAD group into patients with stable (n = 54) and patients with unstable (n = 46) angina, we found significantly higher levels in the latter group. Patients with unstable angina were also found to have significantly lower alpha tocopherol/LDL levels when compared to both patients with stable angina and controls . Unstable CAD Stable CAD

Peroxides

Conj . Dienes

TBARS

Tocoph ./LDL

31 .2 µg/dl 18 .1 4g/dl p=0.0001

13 .4 sg/dl 8 .9 µg/dl p=0 .0001

2.54 µgldl 1 .5 µg/dl p=0.003

6 .1 µgldl 6 .7 µgldl

p=0 .01

In conclusion we report here for the first time that plasma lipid peroxidation

11th International Symposium on Atherosclerosis, Paris, October 1997