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山本正樹2010年学会発表

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  • 1. Bilirubin Oxidation reflects the influence of Nitric Oxide after Myocardial Ischemia Reperfusion Masaki Yamamoto, Hideaki Nishimori, Seiichirou Wariishi, Hironori Maeda, Takayuki Sato † , Tokio Yamaguchi † † , Shiro Sasaguri Kochi University, Dept. of Surgery 2, Dept. of Physiology † Tokyo Medical and Dental University †† American college of surgeon the 96 th clinical congress. Washington DC, October 3- 7, 2010
  • 2. Therapeutic intervention or coronary artery bypass grafting can recover coronary blood flow. Though myocardial damage after blood flow reperfusion, namely myocardial ischemia reperfusion (MIR) injury remains to be resolved. Background Furthermore, the reaction between nitric oxide (NO) and reactive oxygen spesies (ROS) produce peroxynitrite and results in tissue injury. We reported that the influence of oxidative stress induced by myocardial ischemia reperfusion (MIR) was expressed numerically with oxidized Bilirubin, namely Biopyrrin . (Yamamoto M. Circulation J . 2008, 72(9), 1520- 1527) (Yamamoto M. Am. J Transplantation 2007; 7:, 2008, 72(9), 1-10
  • 3. 1. Xanthine oxidase system ATP ADP AMP Adenosine Adenosine Hypoxanthine Xanthine Xanthine Oxidase O 2 ( reperfusion ) H 2 O 2 ,・ OH 2. NADPH oxidase system O 2 O 2 - H 2 O 2 ClO - NADPH NADP + + H + NADPH Oxidase Myeloperoxidase C l - H 2 O 3. Nitric oxide system NO  +  O 2 - ONOO - ; Deficient ATP + re-oxygenation ; Leukocyte infiltration Ischemic cardiomyocyte Endothelial cell Leukocyte Myocardial Ischemia Reperfusion injury and Oxidative Stress O 2 - ・ + 2H +
  • 4. Reactive Oxygen Species Heme oxygenase-1 (HO-1) Heme Biliverdin Bilirubin Biopyrrin scavenger Excretion into urine Myocardial Ischemia Reperfusion Bilirubin synthesis is regulated by heme oxygenase-1 (HO-1) which is rapidly induced by oxidative stress and inflammatory reactions. Bilirubin Oxidation
  • 5. What is Biopyrrin ? Biotripyrrin-a : R 1 = Me , R 2 = -CH = CH 2 Biotripyrrin-b : R 1 = -CH = CH 2 , R 2 = Me Oxidized bilirubin forms at least seven hydrophilic metabolites, called biopyrrins , whch can be detected using the anti-bilirubin monoclonal antibody 24G7. Due to their hydrophiic properties, the biopyrrins are immediately excreted into the urine and can indicate the intensity of oxidation. (Yamaguchi T et.al., J Biochem , 1994; 116: 298) N H CH O R 2 R 1 1 2 3 4 5 15 N H CH COOH Me 6 7 8 9 10 16 8a 8b N H COOH Me 11 12 13 14 17 O 12a 12b
  • 6. Furthermore, this study investigate that NO generation was associated with the biopyrrins elevation after MIR. Purpose The present study examined levels of tissue and urinary biopyrrins to determine the influence of oxidative stress on the reperfused heart and other organs after MIR.
  • 7. Male Wistar rats (BW250 ~ 300g) 2. Sham group 1. MI/R group Models 3. L-NMMA group ; undergo the same surgical procedures except for LCA ligation ; occlude the left coronary artery (LCA)for 30 min followed by reperfusion. ; r eceived L-NMMA i.v.at a dose of 30 mg/kg immediately before LCA reperfusion N G -monomethyl-L-arginine (NOS inhibitor) ※※ LCA Left atrium 8-0 silk Left ventricle ※ ※ Myocardial Ischemia Reperfusion Methods ※※
  • 8. 8 24 16 36 48 h Measurement of urine samples 1. MIR Individual urine samples collected every 2 h up to 48 h after MIR Urinary biopyrrins were measured using an ELISA kit containing the anti-bilirubin monoclonal antibody. The concentration of urinary biopyrrins was collected to the urinary creatinine concentration. Experimental Protocol
  • 9. 8 24 16 36 48 h Assessment of myocardial infarct size, lung edema and hemodynamic changes (immunohistochemical study, ELISA assay, Western blotting ) 3. 2. ( myocardial TTC staining, lung wet-dry weight ratio, pressure study) MIR Pathological assessment and measurement of biopyrrin & Heme Oxygenase-1(HO-1) The heart, lungs, brain, liver, kidneys, spleen and femoral muscles of the rats were separately harvested at 8 or 24 h after MIR
  • 10. Urinary Biopyrrins        (mmol/g·creatinine) 9 8 7 6 5 4 3 2 1 0 10 20 30 40 50 0 Reperfusion time   (h) Urinary biopyrrins after MIR ( n = 6 ) Sham group MIR group L-NMMA group
  • 11. Brain Heart Lung Liver Spleen Kidney Muscle 8 h Brain Heart Lung Liver Spleen Kidney Muscle 24 h (MIR group) (MIR group) Local bilirubin oxidation
  • 12. Bilirubin Oxidation in Myocardium HO-1 8 h 24G7 24 h MIR 24 h Sham 20  m B
  • 13. HO-1 8 h 24G7 24 h MIR 24 h Sham 20  m Bilirubin Oxidation in Lung
  • 14. Levels of biopyrrin and HO-1 in heart and lung tissue Biopyrrins (mmol/ g·protein) 2.5 2.0 1.5 1.0 0.5 0 6 5 4 3 2 0 1 8h 24h 8h 24h heart lung HO-1 /  -actin ratio * # * * # # 8h 24h 8h 24h heart lung * # * * # P<0.05 vs. sham P<0.05 vs. MIR * # P<0.05 vs. sham P<0.05 vs. MIR ( n = 5 ) ( n = 5 ) * Sham MIR L-NMMA Sham MIR L-NMMA
  • 15. NO 2 - /NO 3 - (mmol/ g·protein) 5 3 0 4 2 1 # # * 8h 24h 8h 24h heart lung ( n = 5 ) * # P<0.05 vs. sham P<0.05 vs. MIR 6 * * * # Nitric oxide in heart and lung Sham MIR L-NMMA
  • 16. 0 10 20 30 40 50 60 Infarct area / area at risk (%) 70 # 8h 24h # Myocardial infarct area and lung edema Area at risk / left ventricle (%) 8h 24h * # P<0.05 vs. sham P<0.05 vs. MIR ( n = 5 ) 8h 24h 0 10 20 30 40 50 60 70 Lung wet– dry weight ratio 4.5 4.9 4.7 5.1 5.3 5.5 * * # # # Sham MIR L-NMMA
  • 17. Measurement of cardiac function after MIR
  • 18. Bilirubin oxidation measured as urinary biopyrrins showed biphasical elevation. Biopyrrin synthesis of heart and lungs was the origin of urinary biopyrrins. Though, its origin cannot be explained solely by synthesis in the lungs and heart. 1. Discussion Several investigator suggested the influence of that serum bilirubin oxidation or hemolysis during the inflammatory process after MIR. Morita Y. Int J Cardiol 2001; 80 . Yamaguchi T. Eur J Biochem 1997; 245 . ( )
  • 19. Hemodynamic findings indicating high LVEDP and the lung WD ratio suggested that pulmonary edema influences oxidative stress in the lungs after MIR. Immunohistochemistry revealed biopyrrin expression in relatively large mononuclear cells located in thickened alveolar walls. The NO radical production causes severe myocardial and pulmonary injuries in sub-acute phase of MIR. Bilirubin scavenges NO radicals directly and quenches excess oxidants, therefore, biopyrrins reflects NO radical as well as ROS in tissues. 2. 3. Pulmonary oxidative stress is evoked by the NADPH pathway in cells of the alveolar wall after lung congestion, lung edema could cause bilirubin oxidation by superoxide generated in mononuclear cells.
  • 20. The dynamics of urinary biopyrrins might reflect earlier biopyrrin generation in the lungs and delayed formation both in the lungs and heart where NO is involved. The urinary biopyrrins showed the generation of oxidative stress in sub-acute phase of MIR. 2. 1. Conclusion

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