Oxidized LDL during acute myocardial infarction

Thursday, 27 May 1999 Poster session: Mod!/ied lipoproteins, their receptors and potential roles in atherosclerosis reduced Cu2'-binding and 2-oxo-histidine formation are not sufficient to explain this behaviour of ascorbate. Such an anti-prooxidant" property of ascorbic acid would make an important contribution to the protection of lipids in ascorbate-containing biological fluids. References

[I] [2] [3] [4]

Retsky. K.L.. Freeman, M.W. & Frei, B. (1993) J. Biol. Chem. 268. 1304-1309 Otero. P., Viana, M.. Herrera, E. & Boner. B. (1997) Free Rad. Res. 27, 619-626 Albertini. R. & Abuja. P. M. (19983 Free Rad. Res. (in press) Retsky. KL., Chen. K.. Zeind. J. & Frei. B. {19983 Free Radical Biol. Med. 26, 90--98

DIABETES MELLITUS AND THE INTENSITY OF LIPID PEROXIDATION IN THE PATIENTS WITH ISCHAEMIC HEART DISEASE D. Dincic, P. Jovic t, D. Tavciovski, R. Raicevic2, P. Pavlovic, M. Prcovic.

I Clinic of Cardiology. Central Laborator3, of Clinical Chemisto,." 2Clinic of Neurology. Militar3, Medical Academy Belgrade, Yugoslavia One of the possible ways of atherogenic action of increased glycemia values in the blood is the damage of cells, release of free radicals and the intensification of the lipid peroxidation process that represents the initial moment in the generation of atherosclerotic processes. The aim of the study was to determine the intensity of~ipid peroxidation in the patients with ischemic heart disease (IHD), depending on the presence or absence of diabetes. Out of 105 examinees with performed selective coronary angiography, normal coronary angiography finding was found in 31 patients (control group), triple-vessel coronary disease was observed in 30 patients, double-vessel in 24 patients and single-vessel disease was found in 20 patients. The intensity of lipid peroxidation was determined by measuring malondialdehyde (MDH) concentration, that was significantly higher in all subgroups of the patients compared to the controls (p < 0.009, p < 0.03. p < 0.01 ). In the experimental group were 18 patients with diabetes, in triplevessel group were 8 patients with diabetes, and in single-vessel group were 4 patients with diabetes. In all of them, MDH concentration was significantly higher compared to those who had never had diabetes in the same groups (p < 0.04, p < 0.04, p < 0.013. MDH concentration was higher in the patients with double-vessel coronary disease, as in the control group, but without statistical significance. Obtained results revealed that the intensity of lipid peroxidation represented significant risk factor in the onset and development of ischemic heart disease, particularly in those patients with diabetes, as the risk factor.

OXIDIZED LIPOPROTEINS AND ANTIOXIDANTS HYPERTENSION AND HYPERCHOLESTEROLEMIA

IN

D.S.P. Abdalla I , P. Moriel 1, EL. Plavnik 2, M.T. Zanella 2, E.C. Pincinato I ,

M.C. Bertolami 3. IFCF - USP. 2UNIFESP. 3Inst. Dante Pazzanese de

CardioL, S.P.. Bra:il It is suggested that oxidized lipoproteins play a role in the pathogenesis of atherosclerosis and hypertension. In the present study, in vitro oxidative susceptibility o f LDL. the antioxidant status and the lipid peroxides content of blood plasma were determined in hypercholesterolemic (HC), hypertensive (H), hypercholesterolemic hypertensive (HH) and normolipidemic normotensive subjects (N). The kinetics of in vitro LDL oxidation was monitored and the lag time, lag rate, log rate and peak time were analysed. Cholesteryl ester (CE-OOHL triglyceride (TL-OOH), phospholipid (PLOOH) peroxides, as well as, ascorbate, urate, ct-tocopherol, lycopene and [~-carotene were determined by HPLC. Plasma ascorbate, lipid soluble antioxidants, the lag time and peak time were lower while CE-OOH and TL-OOH were higher in H, HC, and HH than in N. No difference among groups was observed for PL-OOH. The showed that: I) cholesterol of blood plasma is negatively correlated with the content of plasma antioxidants and positively correlated with the lipid peroxide content; 2) there was a positive correlation between the content of antioxidants and the resistance of LDL to oxidation; 3) a positive correlation between ct-tocopherol and PL-OOH suggests that ct-tocopherol may act as a pro-oxidant on lipoprotein surface; 4) the lipid peroxide content and the LDL susceptibility to oxidation are higher in hypercholesterolemic and hypertensive patients compared to normal subjects. Supported by FAPESP

47

LYSOSOMAL DESTABILIZATION DURING MACROPHAGE DAMAGE INDUCED BY CHOLESTEROL OXIDATION PRODUCTS X.M. Yuan I , W Li 1 , U.T. Brunk 1 , H. Dalen 2, Y.H. Chang 3, A. Sevanian3

/Diuision of Pathology 1I. Link~ping University, S-581 85 Linlc~ping, Sweden; 2Department of Pathology, The Gade Institute, University of Bergen, Bergen, Nomvay; 3Department of Molecular Pharmacology and Toxicology, School of Pharmacy. University of Southern California, Los Angeles, USA We have previously shown that oxidized LDL induces damage to lysosomal membranes of macrophages, with leakage of lysosomal contents resulting in apoptotic cell death. In the present study we characterized the initial events during macrophage apoptosis induced by cholesterol oxidation products (ChOx). These compounds have been reported to be the major cytotoxic components of oxidized LDL and also stimulate cholesterol accumulation in vascular cells. Within 24 hours of exposure, ChOx caused lysosomal destabilisation, cellular retraction and shrinkage, mitochondrial enlargement, and multivesicular cytosolic inclusions resulting in an enlarged lysosomal compartment indicative of reparative autophagocytosis. The latter is suggested to be a result of leakage of hydrolytic enzymes into the cytosol. Such leakage may lead to activation of the caspase cascade. MnSOD mRNA levels were markedly increased after 24 hours of exposure to ChOx, suggesting associated induction of mitochondrial protection, repair or turnover. Since ChOx have been shown previously to disrupt membrane structure, ion homeostasis and cytoskeletal assembly, it is plausible that damage to lysosomes and mitochondria are sequelae to the cascade of cytotoxic events arising from compromised membrane-cytoskeletal integrity. Taken together, the findings indicate that ChOx induce profound cellular alterations in macrophages, including early disruption of lysosomes with resultant reparative autophagocytosis which may be followed by apoptotic or necrotic cell death. OXIDIZED LDL IN ACUTE CEREBROVASCULAR DISEASE U Ferlito, M. Motta, G. Pistone, P. Ruello, M. Rizzo, M.P. Panebianco,

I. Giugno, M. Malaguarnera. h;stitute of Internal Medicine and Geriatrics, University q/" Catania. Italy Acute cerebrovascular disease is the main cause of death in industrialized countries. The pathogenesis of arteriosclerosis is still partially unknown. Epidemiological and clinical studies ascertained the strong relationship between serum lipidic pattern and incidence o f the disease. Recently, one observed that low density lipoprotein oxidation may have a fundamental role in the pathogenesis of the atherosclerosis. In ord& to assess this mechanism, we carried out a study involving 46 patients, 17 males and 29 females (mean age 70.8+8.4 years) with acute cerebrovascular accident (30 ischemic and 5 haemorragic strokes and 11 TIA) and 30 control subjects (15 males and 15 females, mean age 69.33+6.85 years). Using the common laboratory methods, we evaluated the following parameters: glycaemia, azotemia, ereatininemia, uricaemia, AST, ALT, Alkaline phosphatase, gamma-glutamyltranspeptidase, Lactic-dehydrogenase, CPK, total cholesterol, HDL-c and triglycerides serum levels. We also take into account PT, PTT, AP and serum fibrinogen levels. Serum LDL-c was quantified using Friedwald's formula; antibodies against oxidized LDL (oLDL Ab) were detected by ELISA method (oLAB reactive, Biomedica, Austria). Statistical analysis was carried out using Student's t test. The patients with acute cerebrovascular accident showed a mean serum oLDL Ab value of 151.15+65.62 mU/ml compared to 438.93+132.03 mU/ml in control subjects (p = 0.001). All other assessed parameters were within the normal range. Acute cerebrovascular disease provokes an increase of the free radical production. This phenomenon seems to influence the LDL structure, lowering the serum levels of these lipoproteins. OXIDIZED LDL DURING ACUTE MYOCARDIAL INFARCTION G. Pistone, I. Giugno, M. Rizzo, M.P. Panebianco, E Ruello, S. Bosco,

C. Scuderi, M. Malaguarnera. h~stitute of lnternal Medicine and Geriatrics.

University of Catania. Italy Some author suggested that oxidized LDL (oLDL) play an important role in the occurrence of ischemic cardiovascular disease. Our study was aimed to evaluate the behaviour o f serum oLDL levels in acute myocardial infarction (AMI) patients to assess the changes o f lipoprotein oxidation during condition of acute stress. We enrolled 28 patients (22 males, 6 females; mean age 60.64+14.28 years) with AMI. We performed a blood withdrawal on days I, 3 and 7 from acute event. We administered to all

71st EAS Conglwss and Satellite Symposia

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Thursday. 27 May 1999 Poster session: Modified lipoproteins, their receptors and potential tr~les in atherosclerosis

patients rt-PA at dose of 100 mg within the first 3 hours from diagnosis. The baseline blood withdrawal was performed before rt-PA administration. In all patients, we evaluated the following hematochemical parameters: glycaemia, azotemia and creatininemia when the diagnosis was performed. We also evaluated CPK, AST, ALT, LDH, total cholesterol, c-HDL, oLDL, triglycerides, fibrinogen serum levels and prothrombinic activity with partial thromboplastine time on days 1,3 and 7 from diagnosis. Serum LDL-c level was quantified by Friedwald's method, oLDL were evaluated by ELISA method and OLAB reactive (Biomedica, Austria). All remaining parameters were determined using the common laboratory methods. We statistically analysed the data obtained using Wilcoxon's test for oLDL and bivariate regression test. Total cholesterol and c-LDL were significantly reduced on day 7 when compared to basal values (p = 0.05). Serum triglycerides levels slightly lowered on day 7. c-HDL levels did not change, oLDL lowered temporarily on day 3 and increased to the basal levels on day 7 (p = 0.05). We found a significant relationship between CPK serum values on days 1, 3 and oLDL values on the same days (p = 0.09; r = 0.374 and 0.371 respectively). Oxidative stress in acute phase of AMI provokes a transient reduction of serum oLDL levels, due to the physiological enhancement of the free radicals production. This datum is also confirmed by the correlation between CPK and oLDL serum levels observed by us. LIPID PEROXIDATION DURING AGING IN WATANABE RABBITS AS DETERMINED BY LIPOPROTEIN AND TISSUE OXYSTEROLS G. Galli, D. Caruso, E. De Fabiani, A. Toia, M. Galli Kienle 1. Inst. of

Pharmacological Sciences; t Dept. of Medical Chemistr3" and Biochemisto; University of Milan. Italy The Watanabe heritable hyperlipidemic (WHHL) rabbit is characterized by a deficiency of LDL receptors resulting in high plasma cholesterol. It spontaneously develops atherosclerotic lesions and is therefore widely used as a model of genetically based atherosclerosis. Objective of this study was to establish whether a correlation between circulating oxysterols and those found in atheromas of WHHL rabbits exists at different stages o f atherosclerosis. For this purpose we evaluated oxysterol (OXYs) distribution and levels in plasma, lipoprotein fractions (VLDL and LDL) and atheromas from WHHL rabbits in aging. We focused on OXYs related to lipid peroxidation, such as 7a-hydroxycholesterol (7ct), 7 [~-hydroxycholesterol (71~), 7-ketocholesterol (7CO), 5, 6 ct- and 5, 6 I~epoxycholesterol, and on 27-hydroxycholesterol (27OH), which represents an index of the catabolic pathway allowing cholesterol elimination from peripheral tissues. The sterolic fraction was extracted from biological matrices either without or with saponification under nitrogen (free OXYs and total OXYs, respectively). Quantitative analyses were performed using gaschromatographymass spectrometry. The main total OXYs found in plasma were 7a, 71~, 7CO, 5, 6 ~t- and 5, 6 ~-epoxycholesterol in both WHHL and New Zealand White (NZW) rabbits used as controls but levels in WHHL rabbits were higher than in NZW. Moreover during aging VLDL- and LDL-like panicles from WHHL, showed decreasing levels of cholesterol and became enriched in OXYs, mainly, 7ct, 7[~ and 7CO. In all tested segments of aortas from WHHL rabbits, levels of 7¢t and 7[~ were higher than in NZW, but no significant differences in OXYs levels were observed during aging. 27-hydroxycholesterol which is usually directly correlated with plasma cholesterol, was also unmodified with age in aorta despite the reduction of hypercholesterolemia, thus suggesting a constant ability to eliminate cholesterol through the 2 7 0 H-pathway in the Watanabe model. Since it is known that atherosclerosis is worsen in WHHL rabbits with age the present results suggest that lipoprotein associated-OXYs may represent an index of the progression. Acknowledgement: This work was financially supported by the Italian Ministry of University and Scientific Research for the National Program of Drug Research (PNF2).

C O R R E L A T I O N B E T W E E N SERUM OXIDIZED LDL AND E C H O A N G I O G R A P H I C A L L Y DEFINED C A R O T I D ATHEROSCLEROSIS P. Jovit, Jovi~i6, S. Spasit, T. Lepi¢3, R. Rai~evi¢3. Central Laborator3' of Clinical Chemistr3, Clinic ~["NeuroloKv. Militar3, Medical Acadeno: FaculO, t f Pharmacl; Belgrade. Yugoslavia The purpose of this study was to estimate the level of oxidized LDL in the patients with ischemic brain disease and its association with degree of carotid atherosclerosis. Fifty two patients (33 men and 19 women, mean age 665:7 yrs) were studied. The degree of carotid atherosclerosis was determined by B-mode real-time ultrasonography. According to the echoangiographic finding (EF) the patients were divide into four groups: O-normal (N = 12), I-without atherosclerotic plaque (N = 15), 2-with atherosclerotic plaque (N = 15 and 3-stenosis. The level of oxidized LDL was expressed as a content of malondialdehyde (MDA) in LDL fraction. The content of MDA was determined spectro photometrically by thiobarbituric acid. Data were compared by an analysis of variance and statistical significance of di fference between the mean values were tested by Bonferroni t-test. The mean values of MDA in LDL fraction of patients with normal (O) and pathological EF (1, 2, 3) were: 0.30:L0.11: 033+0.11; 0.55_+0.22 and 0.6±0.16/umol/L respectively. The difference in means of oxidized LDL between group with normal and pathological EF was significant, except the I group. There was no significant difference in the means between 2 and 3 groups. It was concluded that the level of oxidized LDL in patients with ischemic brain disease rises with the degree of carotid atherosclerosis. The level of oxidized LDL may be useful in discrimining between normal and advanced lesions in carotid arteries. INVOLVEMENT OF P H O S P H O L I P I D S ON A P O L I P O P R O T E I N B MODIFICATION DURING LOW DENSITY LIPOPROTEIN OXIDATION D.C. Tsoukatos t, A.I. Karakatsani t, T.A. Liapikos 1, A.N. Troganis 2.

I Laborator3' ~fBiochemistr3"." 2NMR Centre, Department ~l" Chemist~% Universi O, of loannina, 45110 Ioannina. Greece The oxidation of the LDL leads to oxidised phospholipids, containing shortchain acyl groups at sn-2 position. The aim of the study was to investigate the possible involvement of oxidised Phospholipids. generated during the oxidation of LDL, in the modification of apolipiprotein B (apoB). LDL was isolated from human plasma by ultracentifugation and was oxidised by Cu 2" , in the presence or absence of platelet activating factor (PAF) acetylhydrolase inhibitors. Phospholipids of native and oxidised under different conditions LDL were extracted and analysed by TLC and phosphorus assay. Delipidated apoB derived from native and oxidised LDL was submitted to phosphorus assay and to 31P-NMR analysis. Our Results have shown a marked loss of the PC content and an increase in the amount of lyso - PC of LDL after oxidation. There was no significant change in the amount of the other phospholipids. The lyso - PC formation was inhibited when LDL was oxidised in the presence of PAF acetylhydrolase inhibitors. The sum of the amount PC + lyso - PC was significantly higher on native LDL than on oxidised LDL. An increased amount of phospholipids remained attached on delipidated originated from oxidised LDL. 3Jp NMR analysis of such apolipoprotein showed an organic phosphorus peak at -0.55 ppm, which was not affected by PLA2 treatment and suggested the formation of Schiff base adducts ofoxidised phospholipids with apoB. The above reaction occurs in parallel with the hydrolysis of oxidised phospholipids, catalyzed by the LDL attached PAF acetylhydrolase and may contribute to the proatherogenic effect of oxidative modified low density lipoprotein. (Supported in part by the Research Committee of the University of Ioannina and the Biomed 2 Program PL 963191 ) THE INFLUENCE OF LOW-DENSITY L I P O P R O T E I N MODIFIED W I T H PEROXYNITRITE ON T H E ACTIVATION OF TISSUE FACTOR IN H U M A N THP-I CELLS

K.R. Bruckdorfer, J.A. Adam, C. Ettelaie, N. James, K.M. Naseem. Department of Biochemistry and Molecular Biolog): Royal & University College Medical School Rowland Hill Street, London NW3 2PE UK The aim is to establish whether LDL nitrate with peroxynitfite induces Tissue Factor (TF) activity in monocyte/macrophages. TF activity of cells was measured by one stage prothrombin time assay, lipid oxidation with the Xylenol Orange method and nitration of protein

71st EAS.Congress and Satellite Symposia