- Email: [email protected]
Paraoxonase (PON1) & coronary heart disease (CHD)
Monday June 26, 2000: Poster Abstracts P:W29 Oxidation and Atherogenesis 1
MoP17:W29 J[ Platelet activation by oxygen free radicals in hypercholesterolemic patients V. Sanguigni, D. Caccese, R. Magnaterra, E. Ferrara, S. Mazzolini, E De Vito, E Violi1, R. Lauro. tDepartment of Internal Medicine University of
Rome "Tot Vergata"; Chair of Internal Medicine University of Rome "La Sapienza", Italy Objective: Previous studies provide evidence that platelets, in analogy to other circulating blood cells, are able to produce oxygen free radicals (OFR), like super oxide anion (02-). The present study was performed to evaluate a possible production of OFR from platelets of 25 hypercholesterolemic patients, with no history of coronary artery disease, hypertension, diabetes and without any hypolipidaemic terapy. Normal subjects acted as controls. Methods: 0 2 - was determined by chemilnminescience using a Bit-Orbit 1251. Studies on platelet aggregation were performed following the Born method. The stimulating effect of OFR on platelet aggregation was studied with proper "scavengers" able to inhibit OFR production leading to a decrease in platelet aggregation.
Results: Tot-Cholesterol (mg/dl) DL-Cholesterol (mg/dl) 0 2 _ production nmoles/3 × 10 cells/rain
Patients
Controls
266.15 184.55 2.11
189.54 113.08 1.05
Condusious: Data of the present study provide evidence of significantly (P < 0.02) higher platelets production of OFR (02-) in hypercholesterolemic patients, able to affect platelet activation by stimulating the arachidonic acid metabolism. Such as OFR production can play a signlficative role in the platelet hyperaggregability observed in hypercholesterolemia and may represent an important marker of atherosclerosis progression. MoP18:W29 I Paraoxonuse 0PON1) &
coronary
heart disease (CUD)
B. Mackness, P.N. Durrington, W. Turkie, G.K. Davies, M.L Mackness.
University Department of Medicine, Manchester Royal Infirmary, Oxford Road, Manchester, UK Objectives: To determine the relative importance of the PON1 genetic polymorphisms compared to PON1 quality (activity and concentration) in CHD Methods: PON1 activity with paraoxon as suhstrate, concentration by ELISA and the PON1-55 and -192 polymorphisms by PCR and restriction digestion plus serum lipid and lipoprotein concentrations were determined in 427 subjects with angiographically proven CHD and 282 healthy controls. Results: Subjects with CHD has higher serum triglycerides and lower total cholesterol and apt A1 then controls (all P < 0.05). However, there were no differences in HDL-C or apo-B between the populations. Serum PON 1 activity was in the CHD was 50% of that found in controls (122.8 (3.3-802.8) vs 214.6 (26.3-620.8) UL, P,0.001) and the PON1 concentration was also significantly lower (71.6 (11.4--489.3) vs 89.1 (16.8-527.4) #g/ml, P < 0.001) in the CHD population. Both were reduced independently of PON1 genotype. However, there were no differences in the PON1-55 and -192 gene distributions between the 2 populations and no effect of the polymorphisms on serum lipids or lipoproteins. Conclusions: These results indicate that given the importance of PONI in hydrolysing lipid-peroxides, derangement of the quality of the enzyme is more important than genotype.
MoP19:W29 [ The influence of different doses n-3 fatty acids (n-3 FA) with and without supplementation of vitamin E (vit E) on the levels of reactive oxygen species (ROS) in circulating human leukocytes H. Olsen, I. Seljeflot, T. Lyberg, H. Arnesen. UUevaal University Hospital,
Oslo, Norway Objective: To investigate the degree of peroxidation assessed as levels of different intracellular ROS in circulating leukocytes, after treatment with low and high doses of n-3 FA with and without supplementation of vit E. Methods: The effects were studied in non-diabetic men (40-73 yrs), with serum total cholesterol concentration 6-9 mmol/L and triglycerides > 1.5 mmol/L and without any medication. They were randomised to n-3 FA 1.0 g/d (group 1), n-3 FA 6.0 g/d (group 2) or n-3 FA 6.0 g/d combined with vit E 400 mg/d (group 3). Fasting citrated whole blood was collected
57
before and after 6 weeks intervention. Applying flow cytometric techniques, the basal levels of ROS as well as the levels obtained in the absence and presence of phorbol 12-myristate 13-acetate (PMA, 100 ng/mL) for 90 rain were measured in circulating monocytes (MO) and granulocytes (GR). The mean fluorescense intensity (MFI) in 104 leukocytes of the fluorochromes dihydroethidium (DHE, 5 #M), dihydrorhodamine 123 (DHR, 5 /~M) and dichlorofluorescein-diacetate (DCF, 5/.tM), reflecting mainly the levels of superoxide anion (O~), peroxynitrite (ONTO-) and hydrogen peroxide (H202), respectively, was recorded. When evaluating the PMA-stimulated samples, a stimulation index was calculated in which the autofluorescence of the cells and the procedure-related influences, were taken into consideration. Results: After 6 weeks intervention there were no significant changes in the intracellular leukocyte levels of ROS in group 1. In group 2 a significant increase was observed in the basal levels of ONTO- in GR (p = 0.011), whereas no changes were found after in vitro PMA-stimulation. In group 3 no changes were observed in the basal condition, but a significant decrease could be demonstrated in the PMA-stimulated level of 0 2 in MO (p < 0.001). With the letter variable the difference in changes between the groups was also statistically significant (p = 0.017). Conclusion: Although small changes were noted regarding intracellular ROS levels in circulating leukocytes, no important influence on the degree of peroxidation could be demonstrated subsequent to 6 weeks supplementation with low and high doses of n-3 FA with or without vit E. MoP20:W29 J Increased leukocyte levels of reactive oxygen species (ROS) in populations at risk for atherosclerotic disease H. Olsen, I. Seljeflot, H. Kahler, J. Larsen, H. Arnesen, T. Lyberg. Ullevaal
University Hospital, Oslo, Norway Objective: To investigate the intracellular levels of ROS in circulating leukocytes in populations at risk for atherosclerosis as compared to healthy individuals, with the purpose to estimate the potential usefulness of leukocyte ROS as markers for the disease. Methods: The reference group (ref-group) consisted of 6 individuals (aged 35-65 yrs, 50% women) with normal serum total cholesterol levels, using no medication. The risk populations consisted of 27 non-diabetic men (aged 40-73 yrs), with un-treated hypercholestemlemia and without any medication, and 12 individuals (aged 39-56 yrs, 42% women) with well-controlled insulin-dependent diabetes mellitus. Citrated whole blood was collected at fasting condition. Applying flow cytometric techniques, the basal levels and the phorbol 12-myristate 13-acetate (PMA, 100 ng/mL) stimulated production of ROS, were measured in circulating monocytes (MO) and granulocytes (GR). The mean fluorescence intensity (MFI) in 104 leukocytes of the fluorochromes dihydroethidium (DHE, 5 #M), dihydrorhodamin 123 (DHR, 5 /zM) and dichlorofluorescein-diacetate (DCE 5 /zM), reflecting mainly the levels of i.e. superoxide anion (O2-), peroxynitrite (ONTO-) and hydrogen peroxide (H202), respectively, was recorded. Results: As no significant differences in leukocyte ROS were observed between the two risk populations, these data are grouped together (n = 39). Except for H202 levels in MO, the basal levels of all types of ROS in both MO and GR were significantly higher in the risk population as compared to the ref-group (p < 0.01). In PMA-stimulated whole blood there were significantly increased levels o f t 2 in MO (p < 0.0001) and of H202 in GR (p < 0.05) in the risk population compared to the ref-group. Conclusion: Increased levels of different ROS could be demonstrated in circulating leukocytes from a population at risk for atherosclerotic disease compared to healthy controls, which may indicate a higher degree for oxidative reactions. 1
MOP21 :W29 / Hemolysis increases atherogenesis despite induction of hemoxygenase in cholesterol-fed rabbits A. Femadez I , F. L6pez, E. Hurt-Camejo2 , E. Romano ] , G. Camejo2,
~lnstituto Venezolano de lnvestigaciones Cientificas (IVIC), Caracas, Venezuela; 2Wallenberg Laboratory, Gi~teborg, Sweden Objectives: To explore if intravascular hemolysis that can induce heme-mediated oxidation of apoB lipoproteins in the arterial intima may contribute to atherogenlcity. Methods and Results: We used a model of increased intravascular hemolysis (IVH) caused by intravenous phenylhydrazine administration in rabbits with and without diet-induced moderate hypercholesterolemia (HC). Evaluation of the antioxidant status of plasma, free radical generation, indicated that at the end of the treatment period this was compromised by the IVH. After
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000
MoP17:W29 J[ Platelet activation by oxygen free radicals in hypercholesterolemic patients V. Sanguigni, D. Caccese, R. Magnaterra, E. Ferrara, S. Mazzolini, E De Vito, E Violi1, R. Lauro. tDepartment of Internal Medicine University of
Rome "Tot Vergata"; Chair of Internal Medicine University of Rome "La Sapienza", Italy Objective: Previous studies provide evidence that platelets, in analogy to other circulating blood cells, are able to produce oxygen free radicals (OFR), like super oxide anion (02-). The present study was performed to evaluate a possible production of OFR from platelets of 25 hypercholesterolemic patients, with no history of coronary artery disease, hypertension, diabetes and without any hypolipidaemic terapy. Normal subjects acted as controls. Methods: 0 2 - was determined by chemilnminescience using a Bit-Orbit 1251. Studies on platelet aggregation were performed following the Born method. The stimulating effect of OFR on platelet aggregation was studied with proper "scavengers" able to inhibit OFR production leading to a decrease in platelet aggregation.
Results: Tot-Cholesterol (mg/dl) DL-Cholesterol (mg/dl) 0 2 _ production nmoles/3 × 10 cells/rain
Patients
Controls
266.15 184.55 2.11
189.54 113.08 1.05
Condusious: Data of the present study provide evidence of significantly (P < 0.02) higher platelets production of OFR (02-) in hypercholesterolemic patients, able to affect platelet activation by stimulating the arachidonic acid metabolism. Such as OFR production can play a signlficative role in the platelet hyperaggregability observed in hypercholesterolemia and may represent an important marker of atherosclerosis progression. MoP18:W29 I Paraoxonuse 0PON1) &
coronary
heart disease (CUD)
B. Mackness, P.N. Durrington, W. Turkie, G.K. Davies, M.L Mackness.
University Department of Medicine, Manchester Royal Infirmary, Oxford Road, Manchester, UK Objectives: To determine the relative importance of the PON1 genetic polymorphisms compared to PON1 quality (activity and concentration) in CHD Methods: PON1 activity with paraoxon as suhstrate, concentration by ELISA and the PON1-55 and -192 polymorphisms by PCR and restriction digestion plus serum lipid and lipoprotein concentrations were determined in 427 subjects with angiographically proven CHD and 282 healthy controls. Results: Subjects with CHD has higher serum triglycerides and lower total cholesterol and apt A1 then controls (all P < 0.05). However, there were no differences in HDL-C or apo-B between the populations. Serum PON 1 activity was in the CHD was 50% of that found in controls (122.8 (3.3-802.8) vs 214.6 (26.3-620.8) UL, P,0.001) and the PON1 concentration was also significantly lower (71.6 (11.4--489.3) vs 89.1 (16.8-527.4) #g/ml, P < 0.001) in the CHD population. Both were reduced independently of PON1 genotype. However, there were no differences in the PON1-55 and -192 gene distributions between the 2 populations and no effect of the polymorphisms on serum lipids or lipoproteins. Conclusions: These results indicate that given the importance of PONI in hydrolysing lipid-peroxides, derangement of the quality of the enzyme is more important than genotype.
MoP19:W29 [ The influence of different doses n-3 fatty acids (n-3 FA) with and without supplementation of vitamin E (vit E) on the levels of reactive oxygen species (ROS) in circulating human leukocytes H. Olsen, I. Seljeflot, T. Lyberg, H. Arnesen. UUevaal University Hospital,
Oslo, Norway Objective: To investigate the degree of peroxidation assessed as levels of different intracellular ROS in circulating leukocytes, after treatment with low and high doses of n-3 FA with and without supplementation of vit E. Methods: The effects were studied in non-diabetic men (40-73 yrs), with serum total cholesterol concentration 6-9 mmol/L and triglycerides > 1.5 mmol/L and without any medication. They were randomised to n-3 FA 1.0 g/d (group 1), n-3 FA 6.0 g/d (group 2) or n-3 FA 6.0 g/d combined with vit E 400 mg/d (group 3). Fasting citrated whole blood was collected
57
before and after 6 weeks intervention. Applying flow cytometric techniques, the basal levels of ROS as well as the levels obtained in the absence and presence of phorbol 12-myristate 13-acetate (PMA, 100 ng/mL) for 90 rain were measured in circulating monocytes (MO) and granulocytes (GR). The mean fluorescense intensity (MFI) in 104 leukocytes of the fluorochromes dihydroethidium (DHE, 5 #M), dihydrorhodamine 123 (DHR, 5 /~M) and dichlorofluorescein-diacetate (DCF, 5/.tM), reflecting mainly the levels of superoxide anion (O~), peroxynitrite (ONTO-) and hydrogen peroxide (H202), respectively, was recorded. When evaluating the PMA-stimulated samples, a stimulation index was calculated in which the autofluorescence of the cells and the procedure-related influences, were taken into consideration. Results: After 6 weeks intervention there were no significant changes in the intracellular leukocyte levels of ROS in group 1. In group 2 a significant increase was observed in the basal levels of ONTO- in GR (p = 0.011), whereas no changes were found after in vitro PMA-stimulation. In group 3 no changes were observed in the basal condition, but a significant decrease could be demonstrated in the PMA-stimulated level of 0 2 in MO (p < 0.001). With the letter variable the difference in changes between the groups was also statistically significant (p = 0.017). Conclusion: Although small changes were noted regarding intracellular ROS levels in circulating leukocytes, no important influence on the degree of peroxidation could be demonstrated subsequent to 6 weeks supplementation with low and high doses of n-3 FA with or without vit E. MoP20:W29 J Increased leukocyte levels of reactive oxygen species (ROS) in populations at risk for atherosclerotic disease H. Olsen, I. Seljeflot, H. Kahler, J. Larsen, H. Arnesen, T. Lyberg. Ullevaal
University Hospital, Oslo, Norway Objective: To investigate the intracellular levels of ROS in circulating leukocytes in populations at risk for atherosclerosis as compared to healthy individuals, with the purpose to estimate the potential usefulness of leukocyte ROS as markers for the disease. Methods: The reference group (ref-group) consisted of 6 individuals (aged 35-65 yrs, 50% women) with normal serum total cholesterol levels, using no medication. The risk populations consisted of 27 non-diabetic men (aged 40-73 yrs), with un-treated hypercholestemlemia and without any medication, and 12 individuals (aged 39-56 yrs, 42% women) with well-controlled insulin-dependent diabetes mellitus. Citrated whole blood was collected at fasting condition. Applying flow cytometric techniques, the basal levels and the phorbol 12-myristate 13-acetate (PMA, 100 ng/mL) stimulated production of ROS, were measured in circulating monocytes (MO) and granulocytes (GR). The mean fluorescence intensity (MFI) in 104 leukocytes of the fluorochromes dihydroethidium (DHE, 5 #M), dihydrorhodamin 123 (DHR, 5 /zM) and dichlorofluorescein-diacetate (DCE 5 /zM), reflecting mainly the levels of i.e. superoxide anion (O2-), peroxynitrite (ONTO-) and hydrogen peroxide (H202), respectively, was recorded. Results: As no significant differences in leukocyte ROS were observed between the two risk populations, these data are grouped together (n = 39). Except for H202 levels in MO, the basal levels of all types of ROS in both MO and GR were significantly higher in the risk population as compared to the ref-group (p < 0.01). In PMA-stimulated whole blood there were significantly increased levels o f t 2 in MO (p < 0.0001) and of H202 in GR (p < 0.05) in the risk population compared to the ref-group. Conclusion: Increased levels of different ROS could be demonstrated in circulating leukocytes from a population at risk for atherosclerotic disease compared to healthy controls, which may indicate a higher degree for oxidative reactions. 1
MOP21 :W29 / Hemolysis increases atherogenesis despite induction of hemoxygenase in cholesterol-fed rabbits A. Femadez I , F. L6pez, E. Hurt-Camejo2 , E. Romano ] , G. Camejo2,
~lnstituto Venezolano de lnvestigaciones Cientificas (IVIC), Caracas, Venezuela; 2Wallenberg Laboratory, Gi~teborg, Sweden Objectives: To explore if intravascular hemolysis that can induce heme-mediated oxidation of apoB lipoproteins in the arterial intima may contribute to atherogenlcity. Methods and Results: We used a model of increased intravascular hemolysis (IVH) caused by intravenous phenylhydrazine administration in rabbits with and without diet-induced moderate hypercholesterolemia (HC). Evaluation of the antioxidant status of plasma, free radical generation, indicated that at the end of the treatment period this was compromised by the IVH. After
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000