- Email: [email protected]
Measurement of paraoxonase (PON) concentrations in coronary heart desease (CHD) subjects by sandwich Elisa
Monday June 26, 2000: Poster Abstracts P:W29 Oxidation and Atherogenesis benzo-furan (BO-653), probucol and butylated hydroquinone (BHQ) were added as a DMSO solution. After 6 hr treatment, the cells were scraped and the mRNA was recovered. The expression of 6,416 genes was analyzed using a set of oligo nucleotide array (DNA chip Affymetrix). Results: Among 6,416 genes, 21 genes including genes encording mitochondrial proteins and proteins related to oxidative stress response were induced more than 3 folds by BO-653, probucol and BHQ. A gene of cytochrome P-450 1A1 isozyme which is one of drug-metabolizing phase 1 enzymes was expressed only by BHQ treatment. For other genes, BO-653 showed a similar induction pattern to BHQ rather than probucol.
MoP8:W29
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I S e r u m bilirubin a n d albumin levels are markers of the extent of atherosclerosls in dyslipidaemic patients
J
J.A. Papadakis 1, E.S. Ganotakis I , G. Vrentzos 1, D. Emmanouel I , D.P. Mikhailidis2 . I Dept. of Internal Medicine, University Hospital of
Heraklion, Greece; 2Dept. of Molecular Pathology & Clinical Biochemistry, Royal Free and University College Medical School (UCL), London, UK There is evidence that bilirubin (Bil) acts as an antioxidant and that its circulating levels are reduced in patients with extensive coronary disease. Serum albumin (Alb) levels may also act as an antioxidant, possibly in collaboration with Bil. We compared, in a retrospective study, serum Bil concentrations in 443 patients with primary dyslipidaemia; 61 (13.8%) patients had ischaemic heart disease (1HD), 111 (25.1%) had peripheral vascular disease (PVD) and the remaining 271 (61.1%) patients had no clinically evident cardiovascular disease (CVD). Patients with PVD had significantly (p < 0.01) lower serum Bil levels when compared with those with IHD or without CVD. Serum Bil levels correlated significantly (rs = 0.12; p = 0.01) with Alb concentrations, when all patients were considered. Smokers had significantly lower Bil concentrations than non-smokers in both the PVD and without CVD subgroups (p = 0.01 and p = 0.02, respectively). Men with PVD had significantly lower the [Bill × [Alb] product (a proposed combined index of Bil and Alb) when compared with those without CVD, in both non-smoker and smoker subgroups (p = 0.009 and p = 0.04, respectively). Bil, Alb and the [Bill × [Alb] product correlated with lipids and fibrinogen. All the results varied depending on the patient subgroup (e.g. divided by gender or smoking status) evaluated. Conclusion: This study supports the concept that low serum Bil and AIb levels are associated with more extensive CVD.
MoP9:W29 ] Measurement of paraoxonase (PON) concentrations in coronary heart desease (CHD) subjects by sandwich Elba T. Kuiiraoka I , M. Ishihara 1, T. Fujioka 2, E. Saitu 2, S. Saito 2, T. Egashira I , H. Hattori I . l Research Department, R&D Center, BML Inc., Kawagoe;
2Second Department of Internal Medicine, Nihon University, Tokyo, Japan Serum paraoxonase (PON) is associated with high density lipoproteins, and has been shown to prevent the peroxidation of low density lipoprotein phospbolipids. We have developed a sensitive sandwich ELISA, using specific monoclonal antibodies against PON to measure serum PON concentration. We have measured serum PON in healthy Japanese subjects (n = 87) and CHD patients (n = 33) diagnosed by angiography. Serum PON concentrations were 59.3 -4- 12.5 /~g/mL (mean =E SD) and 52.0 =E 13.6 #g/mL in healthy subjects and CHD patients, respectively. Serum PON concentrations in healthy subjects at 192 polymorphism were 69.5 4- 10.3 (QQ), 63.0 -4- 11.0 (QR), 52.8 =t= 10.8 (RR)/~g/mL, and in CHD patients were 56.8 -4- 18.2 (QQ), 56.7 =1= 15.5 (QR), 46.9 -4- 9.44 (R~)/~g/mL. Serum PON concentrations in CHD patients were lower than those in healthy subjects, especially in RR (P < 0.001). Both paraoxonase specific activity and arylesterase specific activity in CHD patients were also lower, especially in RR (P < 0.001). These results suggested that PON in CHD patients must be inactivated due to being exposed to high oxidative stress. The measurement of PON concentration can be useful tool for the detection of oxidative stress and the progress of atherosclerosis. I
MoP10:W29 J Rapid isolation of VLDL subfractions: Assessment of composition and susceptibility to oxidation J. McEneny, E.R. Trimble, I.S. Young. Department of Clinical Biochemistry,
The Queen's University of Belfast, Belfast, UK Objective: To establish a procedure for the subfractionation of VLDL by rapid ultracentrifugation.
55
Methods: Four subfractions of VLDL (A, B, C & D; where A denotes the largest and most buoyant) were isolated by sequential ultracentrifugation requiring a total preparation time of less than 3.5 hours, compared to 18 hours reported by other procedures. Each subfraction was assessed for; lipid composition, preformed hydroperoxides (HPOs; a marker of in viva peroxidation) and conjugated diene production (a marker of in vitro peroxidation). Results: As the subfractions decreased in size and increased in density (A-+D) the percent triglyceride decreased 86-+62% while cholesterol increased 14---,38%. With increasing density of the subfraction the distribution of fatty acid changed; SFA decreased 39.3--~37.1%, MUFA remained unchanged 38.4-+37.6% and PUFA increased 23.2--+27.0%. HPOs were not different between the subfractions 7.4--> 6.4 nmol/mg protein. Lag time results demonstrated that the subfractions became more susceptible to oxidation { 162-+96" min (p < 0.05)} as they decreased in size and increased in density. Conclusions: Four VLDL subfractions (A->D) were isolated by sequential density gradient ultracentrifugation requiring 1/5th of the time of previously reported procedures. For the first time we have shown how, like LDL, VLDL subfractions become more susceptible to oxidation with decreasing size and increasing density. This method may be applied to patient groups for the detection of abnormalities within their VLDL subfractions that may not be detectable when examining whole VLDL.
I MoP11 :W29 I Evidence of ATP and glucose depleted areas within the atherosclerotic plaque in viva M. Levin, M. Evaldsson, O. Wiklund, G. Bondjers, T. Bjtmheden. The
Wallenberg Laboratory for Cardiovascular Research, GSteborg, Sweden According to the Anoxemia theory ofatherosclerosis, an imbalance between the demand for and supply of oxygen and nutrients in the arterial wall is a key factor in the development and progression of atherosclerotic lesions. However, the energymetabolic situation of the arterial wall in viva is largely unknown. At our lab the prescence of hypoxic areas at depth in the atherosclerotic plaque in viva has been demonstrated in lesions > 4 - 5 0 0 / z m thick. Objective: The aim of the present study was to determine local concentrations of ATP (adenosine triphosphate), glucose and lactate within the atherosclerotic arterial wall in viva at high spatial resolution. Methods: The bioluminescence method metabolic imaging was used to study local concentrations of ATP, glucose and lactate. To get a reflection of the in viva situation, aortas from rabbits (n = 9) with experimental atherosclerosis were snap-frozen in situ in the anestethized animal and consecutive cryosections were used for the different analyses. Results: In plaques exceeding a certain thickness, ATP (>4-500/~m) and glucose depleted (>5-600/~m) areas were demonstrated in the central parts of the plaque. Lactate concentrations were homogenous in the plaque. Conclusions: ATP and glucose depleted areas were demonstrated at depth in the athemsclerotic plaque. We believe that this is a result of an insufficient diffusion of glucose and oxygen due to the thickness of the lesion, maybe in combination with an increased local metabolic demand within the plaque. These results lend support to the Anoxemia theory ofatherosclerosis.
I MOP12:W29
J Modification at" high density lipoproteins: Effects on OxLDL cytotoxicity and on cholesterol effiux
A. Pirillo 1, P. Uboldi I , S. Marcovina 3 , A.L. Catapano 1,2. llnstitute of
Pharmacological Sciences, Milan; 2Centro per It Studio, la Prevenzione e la Terapia delle Vasculopatie Aterosclerotiche, Ospedale Bassini, Milan, Italy; 3Department of Medicine, University of Washington, Seattle, USA Elevated plasma levels of high density lipoproteins (HDL) are believed to be antiatherogenic. Among the mechanisms by which HDL might exert their effects, attention has been paid to the role of HDL in reverse cholesterol transport. Furthermore, HDL may be antiatherogenic by preventing the cytotoxicity of oxidized low density lipoproteins (OxLDL). HDL also undergo modifications that may affect their biological activities. Lipoxygenases (LOX) belong to a family of enzymes which may play a role in atherogenesis because of their ability to oxidize lipoproteins. In this study we investigated the effect of lipoxygenase-mediated modification of HDL (LOX-HDL) on their ability to prevent the cytotoxicity of OxLDL and on cholesterol removal from ceils and aimed at relating these effects to changes in apo AI structure. OxLDL are cytotoxic to endothelial cells; this effect could be reverted by the presence of increasing concentrations of HDL (70.83% -t- 2.3 of control in the presence of OxLDL versus 141.33% :i: 2.43 in the presence of OxLDL + HDL 400 /zg protein/ml). When LOX-HDL were added to cells incubated with OxLDL, the improvement of cell viability was lower than in presence of native HDL
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000
MoP8:W29
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I S e r u m bilirubin a n d albumin levels are markers of the extent of atherosclerosls in dyslipidaemic patients
J
J.A. Papadakis 1, E.S. Ganotakis I , G. Vrentzos 1, D. Emmanouel I , D.P. Mikhailidis2 . I Dept. of Internal Medicine, University Hospital of
Heraklion, Greece; 2Dept. of Molecular Pathology & Clinical Biochemistry, Royal Free and University College Medical School (UCL), London, UK There is evidence that bilirubin (Bil) acts as an antioxidant and that its circulating levels are reduced in patients with extensive coronary disease. Serum albumin (Alb) levels may also act as an antioxidant, possibly in collaboration with Bil. We compared, in a retrospective study, serum Bil concentrations in 443 patients with primary dyslipidaemia; 61 (13.8%) patients had ischaemic heart disease (1HD), 111 (25.1%) had peripheral vascular disease (PVD) and the remaining 271 (61.1%) patients had no clinically evident cardiovascular disease (CVD). Patients with PVD had significantly (p < 0.01) lower serum Bil levels when compared with those with IHD or without CVD. Serum Bil levels correlated significantly (rs = 0.12; p = 0.01) with Alb concentrations, when all patients were considered. Smokers had significantly lower Bil concentrations than non-smokers in both the PVD and without CVD subgroups (p = 0.01 and p = 0.02, respectively). Men with PVD had significantly lower the [Bill × [Alb] product (a proposed combined index of Bil and Alb) when compared with those without CVD, in both non-smoker and smoker subgroups (p = 0.009 and p = 0.04, respectively). Bil, Alb and the [Bill × [Alb] product correlated with lipids and fibrinogen. All the results varied depending on the patient subgroup (e.g. divided by gender or smoking status) evaluated. Conclusion: This study supports the concept that low serum Bil and AIb levels are associated with more extensive CVD.
MoP9:W29 ] Measurement of paraoxonase (PON) concentrations in coronary heart desease (CHD) subjects by sandwich Elba T. Kuiiraoka I , M. Ishihara 1, T. Fujioka 2, E. Saitu 2, S. Saito 2, T. Egashira I , H. Hattori I . l Research Department, R&D Center, BML Inc., Kawagoe;
2Second Department of Internal Medicine, Nihon University, Tokyo, Japan Serum paraoxonase (PON) is associated with high density lipoproteins, and has been shown to prevent the peroxidation of low density lipoprotein phospbolipids. We have developed a sensitive sandwich ELISA, using specific monoclonal antibodies against PON to measure serum PON concentration. We have measured serum PON in healthy Japanese subjects (n = 87) and CHD patients (n = 33) diagnosed by angiography. Serum PON concentrations were 59.3 -4- 12.5 /~g/mL (mean =E SD) and 52.0 =E 13.6 #g/mL in healthy subjects and CHD patients, respectively. Serum PON concentrations in healthy subjects at 192 polymorphism were 69.5 4- 10.3 (QQ), 63.0 -4- 11.0 (QR), 52.8 =t= 10.8 (RR)/~g/mL, and in CHD patients were 56.8 -4- 18.2 (QQ), 56.7 =1= 15.5 (QR), 46.9 -4- 9.44 (R~)/~g/mL. Serum PON concentrations in CHD patients were lower than those in healthy subjects, especially in RR (P < 0.001). Both paraoxonase specific activity and arylesterase specific activity in CHD patients were also lower, especially in RR (P < 0.001). These results suggested that PON in CHD patients must be inactivated due to being exposed to high oxidative stress. The measurement of PON concentration can be useful tool for the detection of oxidative stress and the progress of atherosclerosis. I
MoP10:W29 J Rapid isolation of VLDL subfractions: Assessment of composition and susceptibility to oxidation J. McEneny, E.R. Trimble, I.S. Young. Department of Clinical Biochemistry,
The Queen's University of Belfast, Belfast, UK Objective: To establish a procedure for the subfractionation of VLDL by rapid ultracentrifugation.
55
Methods: Four subfractions of VLDL (A, B, C & D; where A denotes the largest and most buoyant) were isolated by sequential ultracentrifugation requiring a total preparation time of less than 3.5 hours, compared to 18 hours reported by other procedures. Each subfraction was assessed for; lipid composition, preformed hydroperoxides (HPOs; a marker of in viva peroxidation) and conjugated diene production (a marker of in vitro peroxidation). Results: As the subfractions decreased in size and increased in density (A-+D) the percent triglyceride decreased 86-+62% while cholesterol increased 14---,38%. With increasing density of the subfraction the distribution of fatty acid changed; SFA decreased 39.3--~37.1%, MUFA remained unchanged 38.4-+37.6% and PUFA increased 23.2--+27.0%. HPOs were not different between the subfractions 7.4--> 6.4 nmol/mg protein. Lag time results demonstrated that the subfractions became more susceptible to oxidation { 162-+96" min (p < 0.05)} as they decreased in size and increased in density. Conclusions: Four VLDL subfractions (A->D) were isolated by sequential density gradient ultracentrifugation requiring 1/5th of the time of previously reported procedures. For the first time we have shown how, like LDL, VLDL subfractions become more susceptible to oxidation with decreasing size and increasing density. This method may be applied to patient groups for the detection of abnormalities within their VLDL subfractions that may not be detectable when examining whole VLDL.
I MoP11 :W29 I Evidence of ATP and glucose depleted areas within the atherosclerotic plaque in viva M. Levin, M. Evaldsson, O. Wiklund, G. Bondjers, T. Bjtmheden. The
Wallenberg Laboratory for Cardiovascular Research, GSteborg, Sweden According to the Anoxemia theory ofatherosclerosis, an imbalance between the demand for and supply of oxygen and nutrients in the arterial wall is a key factor in the development and progression of atherosclerotic lesions. However, the energymetabolic situation of the arterial wall in viva is largely unknown. At our lab the prescence of hypoxic areas at depth in the atherosclerotic plaque in viva has been demonstrated in lesions > 4 - 5 0 0 / z m thick. Objective: The aim of the present study was to determine local concentrations of ATP (adenosine triphosphate), glucose and lactate within the atherosclerotic arterial wall in viva at high spatial resolution. Methods: The bioluminescence method metabolic imaging was used to study local concentrations of ATP, glucose and lactate. To get a reflection of the in viva situation, aortas from rabbits (n = 9) with experimental atherosclerosis were snap-frozen in situ in the anestethized animal and consecutive cryosections were used for the different analyses. Results: In plaques exceeding a certain thickness, ATP (>4-500/~m) and glucose depleted (>5-600/~m) areas were demonstrated in the central parts of the plaque. Lactate concentrations were homogenous in the plaque. Conclusions: ATP and glucose depleted areas were demonstrated at depth in the athemsclerotic plaque. We believe that this is a result of an insufficient diffusion of glucose and oxygen due to the thickness of the lesion, maybe in combination with an increased local metabolic demand within the plaque. These results lend support to the Anoxemia theory ofatherosclerosis.
I MOP12:W29
J Modification at" high density lipoproteins: Effects on OxLDL cytotoxicity and on cholesterol effiux
A. Pirillo 1, P. Uboldi I , S. Marcovina 3 , A.L. Catapano 1,2. llnstitute of
Pharmacological Sciences, Milan; 2Centro per It Studio, la Prevenzione e la Terapia delle Vasculopatie Aterosclerotiche, Ospedale Bassini, Milan, Italy; 3Department of Medicine, University of Washington, Seattle, USA Elevated plasma levels of high density lipoproteins (HDL) are believed to be antiatherogenic. Among the mechanisms by which HDL might exert their effects, attention has been paid to the role of HDL in reverse cholesterol transport. Furthermore, HDL may be antiatherogenic by preventing the cytotoxicity of oxidized low density lipoproteins (OxLDL). HDL also undergo modifications that may affect their biological activities. Lipoxygenases (LOX) belong to a family of enzymes which may play a role in atherogenesis because of their ability to oxidize lipoproteins. In this study we investigated the effect of lipoxygenase-mediated modification of HDL (LOX-HDL) on their ability to prevent the cytotoxicity of OxLDL and on cholesterol removal from ceils and aimed at relating these effects to changes in apo AI structure. OxLDL are cytotoxic to endothelial cells; this effect could be reverted by the presence of increasing concentrations of HDL (70.83% -t- 2.3 of control in the presence of OxLDL versus 141.33% :i: 2.43 in the presence of OxLDL + HDL 400 /zg protein/ml). When LOX-HDL were added to cells incubated with OxLDL, the improvement of cell viability was lower than in presence of native HDL
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000