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Allantoin as a biomarker of severity of heart disease and its relationship with myeloperoxidase
inhibited by GYY4137, AP67, AP72 (100μM; ANOVA, p<0.01 all treatments). In each assay, the potency of SHRD at preventing oxidative stress induced cytotoxicity was markedly increased by selective targeting of mitochondria (AP39, AP123; 0.05-0.1μM, ANOVA, p<0.01). SRHD also modulated Akt-ERK1/2 expression, reduced oxidative stress-inducd caspase 3, 7 and 9 activation and activity. These data suggest SHRD can inhibit/reverse oxidative stress-mediated cellular injury and highlight strategies which increase vascular H2S bioavailability, and in particular target mitochondria, represent a new therapeutic opportunity to limit MED and preserve vascular function. Keywords: mitochondria, endothelium, GYY4137
endothelial
dysfunction,
uncertainty was estimated to ± 7 µM (k = 1) based on the variance of a series of measurements. Using CP with known concentrations a linear relationship between the EPR intensity and the CP solute was obtained. •
Conclusion: It was possible to detect spinproduct (CM ) reflecting the amount of intracellular O2•-, production in myocardial tissue down to levels of approx. 10 μM. 1. Dikalov, S., K.K. Griendling, Harrison, Hypertension, 2007.
and
D.G.
Keywords: electron paramagnetic resonance, cyclic hydroxylamine spin probe, ischemia/reperfusion doi:10.1016/j.freeradbiomed.2012.08.282
doi:10.1016/j.freeradbiomed.2012.08.281 [0438] [0378] A sensitive method for quantitative measurement of the superoxide anion in frozen biopsies K. Berg1, M. Ericsson1 ,2, M. Lindgren3, H. Gustafsson*4 1 Norwegian University of Science & Technology, Norway, 2Umeå University, Sweden, 3NTNU, Norway, 4 Linköping University, Sweden Introduction: Reactive oxygen species (ROS), such as the superoxide anion (O2•-), is a molecule with a short life span, but considered very important in several biological processes like ischemic heart disease. In the present study we show how O2•-, can be detected indirectly by use of the cyclic hydroxylamine spinprobe CMH and EPR [1] of frozen myocardial tissue from rats. Methods: Approximately 45 mg frozen tissue was homogenized in oxygen free Krebs solution. Spin probe (200 µM CMH) was added and immediately incubated at 37°C for 60 min. 100 µL of the solution was transferred to a 1 mL de-capped syringe and snap frozen in liquid nitrogen. Samples for calibration were prepared from standard CP (1% DTPA; 0.9 % sterile NaCL buffer). A Bruker E500 Elexsys X-Band EPR spectrometer with a SHQE resonator and a ER 4111 VT unit, was used for EPR measurements. A sample (in its syringe) was taken from a storage Dewar and thawed into a 100 µL "tablet" and transferred into a frozen 8 mm tube and directly placed in a custom designed Dewar in the EPR resonator at 150 K. Recorded spectra were imported to MATLAB and baseline corrected. Defining the EPR signal intensity from the peak-to-peak amplitude, the quantity of ROS in µM for all samples was calculated using a calibration curve (linear from 0 to 500 µM). The
Allantoin as a biomarker of severity of heart disease and its relationship with myeloperoxidase R. Turner*1, T. Mocatta1, D. McClean2, C. Marshall2, A. Kettle1 1 University of Otago, New Zealand, 2Christchurch Hospital, New Zealand Introduction: Myeloperoxidase is elevated in patients with cardiovascular disease. Allantoin is the major oxidation product of urate in humans and has been shown to be produced when urate is exposed to myeloperoxidase in vitro. We investigated whether allantoin levels were increased in patients with cardiovascular disease. Its relationship to myeloperoxidase activity and to established markers of heart disease was determined. Methods: Myeloperoxidase activity/protein and allantoin levels in plasma from femoral arteries of healthy controls (n=31) and patients with ST elevated myocardial infarcts (n=31), stable angina (n=12) and 4 months post myocardial infarctions (n=25) were measured using an ELISA and LCMS/MS respectively. Differences between groups and for correlations between biomarkers were examined. We also measured the troponin, creatinine kinase and magnitude of ST elevation in ST-elevated myocardial infarcts (STEMI) and determined their relationship with femoral plasma allantoin concentrations. Results: Allantoin was significantly elevated (p<0.001) in patients with ST-elevated myocardial infarcts (median 6.2 µM; IQR 5.1-7.8 µM) as compared to healthy controls (2.4; 1.4-3.7), patients who had angina (4.0; 2.35.4) and patients 4 months post-myocardial infarction (3.1; 2.0-4.2). It correlated with markers of severity of
S135
STEMI disease such as creatinine kinase MB, peak troponin I levels and magnitude of ST elevation (P<0.05).The allantoin concentration was correlated with myeloperoxidase activity (CC= 0.504; p=0.0047) and MPO protein (CC = 0.452; p=0.01). In patients with STEMI, allantoin was also found to be inversely correlated with neutrophil intracellular myeloperoxidase mean fluorescence intensity (CC = -0.450; p=0.001), a measure of neutrophil degranulation. Discussion: We propose that higher plasma allantoin levels indicate increased oxidative stress in patients with STEMI and allantoin is good biomarker of severity of heart disease. The correlation of myeloperoxidase with plasma allantoin suggests that it is partially responsible for oxidation of urate to allantoin in vivo. Keywords: Allantoin, Myeloperoxidase, STEMI, Heart Disease
prevented the palmitate dependent increase in CD11b (p<0.05) and CD36 (p<0.001). Ceramide converting inhibitors 1S,2R-D-erythro-2-N-myristoylamino)-1phenyl-1-propanol (d-erythro MAPP 20 μM) and 1phenyl-2-palmitoylamino-3-morpholino-1propanol (PPMP 0.05 μM) were used to determine if ceramides or downstream sphingolipids are required for palmitate induced phenotype changes in monocytes. In the presence of MAPP and PPMP, palmitate induced increase in CD11b are sustained suggesting sphingolipids are key players in the observed phenotypic changes. The present study demonstrates in monocytes that palmitate but not oleate increases cell surface marker expression, accompanied by increased mitochondrial ROS and decreased metabolic viability. MnTBap could ameliorate changes in mitochondrial ROS and metabolic viability, but surface antigen expression decreased only by fumonisin B1 suggesting changes require de novo ceramide synthesis
doi:10.1016/j.freeradbiomed.2012.08.283
Keywords: Monocytes, Fatty acids, Ceramides, reactive oxygen species
[0501]
doi:10.1016/j.freeradbiomed.2012.08.284
Palmitate induces phenotype changes in monocytes via de novo ceramide synthesis
[0525]
C. Pararasa*, D. Gao, C.R. Dunston, C.J. Bailey, H.R. Griffiths Aston University, UK
Heme Oxygenase 1 Induction In The Peri-Infarct Region After Cerebral Ischemia-Reperfusion Injury In Rats Is Associated With Reduced Blood-Brain Barrier Breakdown
Ageing is associated with physiological changes such as altered adipose tissue distribution and redox status, elevated metabolic disorder risk and increased free fatty acids. Saturated fatty acids can induce insulin resistance in endothelial cells and monocytes, in part explaining the increase cardiovascular disease risk with elevated fatty acids. The aim of this study is to investigate the effects of the two major physiological fatty acids palmitate and oleate (saturated and monounsaturated fatty acids respectively) on monocyte phenotype. THP-1 monocyte cell surface marker expression, mitochondrial reactive oxygen species, cell viability and caspase-3 were evaluated following 24h treatment with palmitate, oleate or bovine serum albumin. A concentration dependent increase in CD11b (p<0.01), CD36 (p<0.001) and mitochondrial reactive oxygen species (p<0.05) following palmitate but not oleate treatment was determined by flow cytometry. Decreased metabolic viability (p<0.01) was observed with palmitate (300μM), whilst no significant change in caspase-3 was observed. The superoxide dismutase mimetic MnTBAP (200μM) ameliorated the reduced metabolic viability and increased mitochondrial ROS due to palmitate, whilst increased CD11b and CD36 were unaffected. De novo ceramide synthesis inhibitor fumonisin B1 (50μM)
A. Alfieri*1, S. Srivastava1, R.C.M. Siow1, M.R. Duchen2, P.A. Fraser1, G.E. Mann1 et al 1 King's College London, UK, 2University College London, UK
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Disruption of the blood-brain barrier (BBB) followed by macromolecular leak and cell infiltration plays a critical role in stroke and the development of neurological dysfunction. Heme oxygenase-1 (HO-1) serves as an inducible protective factor in neurodegenerative disorders by means of its endogenous antioxidant, antiinflammatory and anti-apoptotic actions. In the present study we have investigated HO-1 expression in the brain after cerebral ischemia-reperfusion injury. Male Sprague-Dawley rats (250-300g) were subjected to right intraluminal middle cerebral artery occlusion (MCAo) for 70 min under isoflurane anaesthesia and followed by 24 or 72 h of reperfusion. Brain coronal sections were fluorescently stained for HO-1 along with cell markers and endogenous IgG as marker of BBB breakdown. HO1 expression at 24h was induced in microvessels and astrocytes of the peri-infarct region, while activated microglia in the infarct core mainly expressed HO-1 72h after ischemia-reperfusion. Macromolecular IgG leakage
endothelial
dysfunction,
uncertainty was estimated to ± 7 µM (k = 1) based on the variance of a series of measurements. Using CP with known concentrations a linear relationship between the EPR intensity and the CP solute was obtained. •
Conclusion: It was possible to detect spinproduct (CM ) reflecting the amount of intracellular O2•-, production in myocardial tissue down to levels of approx. 10 μM. 1. Dikalov, S., K.K. Griendling, Harrison, Hypertension, 2007.
and
D.G.
Keywords: electron paramagnetic resonance, cyclic hydroxylamine spin probe, ischemia/reperfusion doi:10.1016/j.freeradbiomed.2012.08.282
doi:10.1016/j.freeradbiomed.2012.08.281 [0438] [0378] A sensitive method for quantitative measurement of the superoxide anion in frozen biopsies K. Berg1, M. Ericsson1 ,2, M. Lindgren3, H. Gustafsson*4 1 Norwegian University of Science & Technology, Norway, 2Umeå University, Sweden, 3NTNU, Norway, 4 Linköping University, Sweden Introduction: Reactive oxygen species (ROS), such as the superoxide anion (O2•-), is a molecule with a short life span, but considered very important in several biological processes like ischemic heart disease. In the present study we show how O2•-, can be detected indirectly by use of the cyclic hydroxylamine spinprobe CMH and EPR [1] of frozen myocardial tissue from rats. Methods: Approximately 45 mg frozen tissue was homogenized in oxygen free Krebs solution. Spin probe (200 µM CMH) was added and immediately incubated at 37°C for 60 min. 100 µL of the solution was transferred to a 1 mL de-capped syringe and snap frozen in liquid nitrogen. Samples for calibration were prepared from standard CP (1% DTPA; 0.9 % sterile NaCL buffer). A Bruker E500 Elexsys X-Band EPR spectrometer with a SHQE resonator and a ER 4111 VT unit, was used for EPR measurements. A sample (in its syringe) was taken from a storage Dewar and thawed into a 100 µL "tablet" and transferred into a frozen 8 mm tube and directly placed in a custom designed Dewar in the EPR resonator at 150 K. Recorded spectra were imported to MATLAB and baseline corrected. Defining the EPR signal intensity from the peak-to-peak amplitude, the quantity of ROS in µM for all samples was calculated using a calibration curve (linear from 0 to 500 µM). The
Allantoin as a biomarker of severity of heart disease and its relationship with myeloperoxidase R. Turner*1, T. Mocatta1, D. McClean2, C. Marshall2, A. Kettle1 1 University of Otago, New Zealand, 2Christchurch Hospital, New Zealand Introduction: Myeloperoxidase is elevated in patients with cardiovascular disease. Allantoin is the major oxidation product of urate in humans and has been shown to be produced when urate is exposed to myeloperoxidase in vitro. We investigated whether allantoin levels were increased in patients with cardiovascular disease. Its relationship to myeloperoxidase activity and to established markers of heart disease was determined. Methods: Myeloperoxidase activity/protein and allantoin levels in plasma from femoral arteries of healthy controls (n=31) and patients with ST elevated myocardial infarcts (n=31), stable angina (n=12) and 4 months post myocardial infarctions (n=25) were measured using an ELISA and LCMS/MS respectively. Differences between groups and for correlations between biomarkers were examined. We also measured the troponin, creatinine kinase and magnitude of ST elevation in ST-elevated myocardial infarcts (STEMI) and determined their relationship with femoral plasma allantoin concentrations. Results: Allantoin was significantly elevated (p<0.001) in patients with ST-elevated myocardial infarcts (median 6.2 µM; IQR 5.1-7.8 µM) as compared to healthy controls (2.4; 1.4-3.7), patients who had angina (4.0; 2.35.4) and patients 4 months post-myocardial infarction (3.1; 2.0-4.2). It correlated with markers of severity of
S135
STEMI disease such as creatinine kinase MB, peak troponin I levels and magnitude of ST elevation (P<0.05).The allantoin concentration was correlated with myeloperoxidase activity (CC= 0.504; p=0.0047) and MPO protein (CC = 0.452; p=0.01). In patients with STEMI, allantoin was also found to be inversely correlated with neutrophil intracellular myeloperoxidase mean fluorescence intensity (CC = -0.450; p=0.001), a measure of neutrophil degranulation. Discussion: We propose that higher plasma allantoin levels indicate increased oxidative stress in patients with STEMI and allantoin is good biomarker of severity of heart disease. The correlation of myeloperoxidase with plasma allantoin suggests that it is partially responsible for oxidation of urate to allantoin in vivo. Keywords: Allantoin, Myeloperoxidase, STEMI, Heart Disease
prevented the palmitate dependent increase in CD11b (p<0.05) and CD36 (p<0.001). Ceramide converting inhibitors 1S,2R-D-erythro-2-N-myristoylamino)-1phenyl-1-propanol (d-erythro MAPP 20 μM) and 1phenyl-2-palmitoylamino-3-morpholino-1propanol (PPMP 0.05 μM) were used to determine if ceramides or downstream sphingolipids are required for palmitate induced phenotype changes in monocytes. In the presence of MAPP and PPMP, palmitate induced increase in CD11b are sustained suggesting sphingolipids are key players in the observed phenotypic changes. The present study demonstrates in monocytes that palmitate but not oleate increases cell surface marker expression, accompanied by increased mitochondrial ROS and decreased metabolic viability. MnTBap could ameliorate changes in mitochondrial ROS and metabolic viability, but surface antigen expression decreased only by fumonisin B1 suggesting changes require de novo ceramide synthesis
doi:10.1016/j.freeradbiomed.2012.08.283
Keywords: Monocytes, Fatty acids, Ceramides, reactive oxygen species
[0501]
doi:10.1016/j.freeradbiomed.2012.08.284
Palmitate induces phenotype changes in monocytes via de novo ceramide synthesis
[0525]
C. Pararasa*, D. Gao, C.R. Dunston, C.J. Bailey, H.R. Griffiths Aston University, UK
Heme Oxygenase 1 Induction In The Peri-Infarct Region After Cerebral Ischemia-Reperfusion Injury In Rats Is Associated With Reduced Blood-Brain Barrier Breakdown
Ageing is associated with physiological changes such as altered adipose tissue distribution and redox status, elevated metabolic disorder risk and increased free fatty acids. Saturated fatty acids can induce insulin resistance in endothelial cells and monocytes, in part explaining the increase cardiovascular disease risk with elevated fatty acids. The aim of this study is to investigate the effects of the two major physiological fatty acids palmitate and oleate (saturated and monounsaturated fatty acids respectively) on monocyte phenotype. THP-1 monocyte cell surface marker expression, mitochondrial reactive oxygen species, cell viability and caspase-3 were evaluated following 24h treatment with palmitate, oleate or bovine serum albumin. A concentration dependent increase in CD11b (p<0.01), CD36 (p<0.001) and mitochondrial reactive oxygen species (p<0.05) following palmitate but not oleate treatment was determined by flow cytometry. Decreased metabolic viability (p<0.01) was observed with palmitate (300μM), whilst no significant change in caspase-3 was observed. The superoxide dismutase mimetic MnTBAP (200μM) ameliorated the reduced metabolic viability and increased mitochondrial ROS due to palmitate, whilst increased CD11b and CD36 were unaffected. De novo ceramide synthesis inhibitor fumonisin B1 (50μM)
A. Alfieri*1, S. Srivastava1, R.C.M. Siow1, M.R. Duchen2, P.A. Fraser1, G.E. Mann1 et al 1 King's College London, UK, 2University College London, UK
S136
Disruption of the blood-brain barrier (BBB) followed by macromolecular leak and cell infiltration plays a critical role in stroke and the development of neurological dysfunction. Heme oxygenase-1 (HO-1) serves as an inducible protective factor in neurodegenerative disorders by means of its endogenous antioxidant, antiinflammatory and anti-apoptotic actions. In the present study we have investigated HO-1 expression in the brain after cerebral ischemia-reperfusion injury. Male Sprague-Dawley rats (250-300g) were subjected to right intraluminal middle cerebral artery occlusion (MCAo) for 70 min under isoflurane anaesthesia and followed by 24 or 72 h of reperfusion. Brain coronal sections were fluorescently stained for HO-1 along with cell markers and endogenous IgG as marker of BBB breakdown. HO1 expression at 24h was induced in microvessels and astrocytes of the peri-infarct region, while activated microglia in the infarct core mainly expressed HO-1 72h after ischemia-reperfusion. Macromolecular IgG leakage