656
ORGAN, TISSUE, AND CELL DAMAGE
[70]
aldehyde (a photolytic breakdown contaminant of commercial folic acid preparations), methylxanthines, urea, and guanidinium ion. 1,~9 In addition, cyanide, arsenite, formaldehyde, and methanol also interfere with the assay by reacting at or vicinal to the molybdenum active site.~,2° Considerations. Although it is possible that HPLC assay of XO could be rendered less sensitive by the presence of uricase in certain tissues, xanthine is a relatively avid competitive inhibitor of uricase (urate oxidase). 21 However, in tissues with substantial uricase activity, it may be necessary to add a uricase inhibitor, such as oxonic acid. 22 Another consideration is that reversible (but not irreversible) XO will be largely converted to XD by DTE, so this assay is relatively insensitive to reversible XO. The considerable and increasing interest in the possibility that XOderived 02 metabolites may contribute to a variety of processes in normal and disease states warrants careful measurement of this enzyme in biological tissues. The techniques described herein offer one approach to this challenge. 19 I. Fridovich, J. Biol. Chem. 239, 3519 (1964). 2o V. M a s s e y and D. E d m o n d s o n , J. Biol. Chem. 245, 6595 (1970). 21 j. F. V a n Pilsum, J. Biol. Chem. 204, 613 (1953). 22 j. M. B a g n a s c o , H. P. Friedl, K. S. Guice, K. T. Oldham, and G. O. Till, FASEB J. 3, A628 (1989).
[70] O x y R a d i c a l s in D i s s e m i n a t e d I n t r a v a s c u l a r C o a g u l a t i o n By TOSHIKAZU YOSHIKAWA
Disseminated intravascular coagulation (DIC) is a dynamic, potentially disastrous complication arising from a variety of disease processes. It is a consequence of tissue damage-induced activation of the coagulation cascade. Excess thrombin thus generated overwhelms the normal antithrombin activities of the body and results in consumption of certain clotting elements. Fibrin deposition within the circulation causes ischemic damage, while consumption of clotting factors and activation of the fibrinolytic system lead to a multisite bleeding diathesis. Increasing experimental and clinical evidence now demonstrates that the generation of oxygen radicals can mediate, and is largely responsible I E. J. W. Bowie a n d C. A. O w e n , Biol. Haematol. 49, 217 (1983).
METHODS IN ENZYMOLOGY,VOL. 186
Copyright © 1990by AcademicPress, Inc. All rights of reproduction in any form reserved.
[70]
oxY RADICALS IN DISSEMINATED INTRAVASCULAR COAGULATION
657
for, several forms of tissue damage and the resulting organ dysfunction. A generation of oxygen free radicals seems to be implicated in the pathogenesis of DIC caused by endotoxin, z-5
Endotoxin-Induced Disseminated Intravascular Coagulation Endotoxin (Escherichia coli 055:B5 lipopolysaccharide B; Difco Lab., Detroit, MI) is dissolved in pyrogen-free physiological saline before every experiment. Experimental DIC is induced by a sustained infusion of 100 mg/kg of endotoxin, diluted in 11.4 ml of saline, into the femoral vein for 4 hr using a syringe. For i.v. infusions the femoral vein of the animal is cannulated in light Nembutal (5 mg/100 g, i.p.) anesthesia. The severity of DIC is determined with various parameters, such as fibrinogen and fibrin degradation products (FDP), fibrinogen, prothrombin time (PT), partial thromboplastin time (PTT), platelet counts, and percent glomerular fibrin deposits (%GFD). One hundred glomeruli are counted and those having fibrin thrombi are expressed as a percentage. Four hours after the infusion of endotoxin (100 mg/kg), fibrinogen levels and platelet counts are significantly decreased, PT and PTT are prolonged, and FDP and %GFD are increased) ,3 Serum thiobarbituric acid (TBA)-reactive substances are significantly increased 3 and 4 hr after the infusion of endotoxin. 3 TBA-Reactive Substances. TBA reactants are determined in several tissues of rats infused with 100 mg/kg of endotoxin for 4 hr. The levels of TBA-reactive substances in serum, abdominal aortic wall, and ileum mucosa are significantly increased in rats infused for 4 hr with 100 mg/kg of endotoxin 3 (Table I). Effects of Superoxide Dismutase and Catalase. Superoxide dismutase (SOD) or catalase significantly inhibits the increase in FDP levels and %GFD, the prolongation of PT and PTT, and the reduction of fibrinogen levels and platelet counts 2 (Table II). These findings suggest that active oxygen species, such as superoxide or hydrogen peroxide, can affect the endotoxin-induced DIC. Effects of Vitamin E. The changes in coagulation parameters are significantly greater in vitamin E-deficient rats when compared to those in 2 T. Yoshikawa, M. Murakami, N. Yoshida, and M. Kondo, Thromb. Haemostasis 50, 869 (1983). 3 T. Yoshikawa, M. Murakami, Y. Furukawa, H. Kato, S. Takemura, and M. Kondo, Thromb. Haemostasis 49, 214 (1983). 4 T. Yoshikawa, M. Murakami, and M. Kondo, Toxicol. Appl. Pharmacol. 74, 173 (1984). 5 T. Yoshikawa, Y. Furukawa, M. Murakami, K. Watanabe, and M. Kondo, Thromb. Haemostasis 48, 235 (1982).
658
ORGAN, TISSUE, AND CELL DAMAGE
[70]
TABLE I TBA-REACTIVE SUBSTANCES IN SERUM AND TISSUES OF RATS INDUCED BY SUSTAINED INFUSION OF ENDOTOXIN a
Source Serum e Aortic w a l l g
Liverg Kidneyg Gastrointestinal mucosae Stomach Jejunum Ileum
Colon
Control b (n (n (n (n
= = = =
p value d
DIC ~
4.0 28.5 14.7 17.8
+-- 0.9 - 7.9 - 2.1 - 1.9
15)f 12) 8) 8)
6.3 11.5 14.4 7.1
- 0.7 (n = 8) ± 2.2 (n = 12) ± 3.1 (n = 10) ±2.1(n=8)
5.8 58.8 14.6 17.6
1.0 (n = - 13.2 (n = ± 1.2 (n = - 0.8 (n =
20) 12) 8) 8)
6.6 ± 1.2 (n = 8) 12.2 ± 2.1 (n = 12) 19.9 +- 4.3 (n = 10) 6.9-+ 1.2(n=8)
ORGAN, TISSUE, AND CELL DAMAGE
[70]
aldehyde (a photolytic breakdown contaminant of commercial folic acid preparations), methylxanthines, urea, and guanidinium ion. 1,~9 In addition, cyanide, arsenite, formaldehyde, and methanol also interfere with the assay by reacting at or vicinal to the molybdenum active site.~,2° Considerations. Although it is possible that HPLC assay of XO could be rendered less sensitive by the presence of uricase in certain tissues, xanthine is a relatively avid competitive inhibitor of uricase (urate oxidase). 21 However, in tissues with substantial uricase activity, it may be necessary to add a uricase inhibitor, such as oxonic acid. 22 Another consideration is that reversible (but not irreversible) XO will be largely converted to XD by DTE, so this assay is relatively insensitive to reversible XO. The considerable and increasing interest in the possibility that XOderived 02 metabolites may contribute to a variety of processes in normal and disease states warrants careful measurement of this enzyme in biological tissues. The techniques described herein offer one approach to this challenge. 19 I. Fridovich, J. Biol. Chem. 239, 3519 (1964). 2o V. M a s s e y and D. E d m o n d s o n , J. Biol. Chem. 245, 6595 (1970). 21 j. F. V a n Pilsum, J. Biol. Chem. 204, 613 (1953). 22 j. M. B a g n a s c o , H. P. Friedl, K. S. Guice, K. T. Oldham, and G. O. Till, FASEB J. 3, A628 (1989).
[70] O x y R a d i c a l s in D i s s e m i n a t e d I n t r a v a s c u l a r C o a g u l a t i o n By TOSHIKAZU YOSHIKAWA
Disseminated intravascular coagulation (DIC) is a dynamic, potentially disastrous complication arising from a variety of disease processes. It is a consequence of tissue damage-induced activation of the coagulation cascade. Excess thrombin thus generated overwhelms the normal antithrombin activities of the body and results in consumption of certain clotting elements. Fibrin deposition within the circulation causes ischemic damage, while consumption of clotting factors and activation of the fibrinolytic system lead to a multisite bleeding diathesis. Increasing experimental and clinical evidence now demonstrates that the generation of oxygen radicals can mediate, and is largely responsible I E. J. W. Bowie a n d C. A. O w e n , Biol. Haematol. 49, 217 (1983).
METHODS IN ENZYMOLOGY,VOL. 186
Copyright © 1990by AcademicPress, Inc. All rights of reproduction in any form reserved.
[70]
oxY RADICALS IN DISSEMINATED INTRAVASCULAR COAGULATION
657
for, several forms of tissue damage and the resulting organ dysfunction. A generation of oxygen free radicals seems to be implicated in the pathogenesis of DIC caused by endotoxin, z-5
Endotoxin-Induced Disseminated Intravascular Coagulation Endotoxin (Escherichia coli 055:B5 lipopolysaccharide B; Difco Lab., Detroit, MI) is dissolved in pyrogen-free physiological saline before every experiment. Experimental DIC is induced by a sustained infusion of 100 mg/kg of endotoxin, diluted in 11.4 ml of saline, into the femoral vein for 4 hr using a syringe. For i.v. infusions the femoral vein of the animal is cannulated in light Nembutal (5 mg/100 g, i.p.) anesthesia. The severity of DIC is determined with various parameters, such as fibrinogen and fibrin degradation products (FDP), fibrinogen, prothrombin time (PT), partial thromboplastin time (PTT), platelet counts, and percent glomerular fibrin deposits (%GFD). One hundred glomeruli are counted and those having fibrin thrombi are expressed as a percentage. Four hours after the infusion of endotoxin (100 mg/kg), fibrinogen levels and platelet counts are significantly decreased, PT and PTT are prolonged, and FDP and %GFD are increased) ,3 Serum thiobarbituric acid (TBA)-reactive substances are significantly increased 3 and 4 hr after the infusion of endotoxin. 3 TBA-Reactive Substances. TBA reactants are determined in several tissues of rats infused with 100 mg/kg of endotoxin for 4 hr. The levels of TBA-reactive substances in serum, abdominal aortic wall, and ileum mucosa are significantly increased in rats infused for 4 hr with 100 mg/kg of endotoxin 3 (Table I). Effects of Superoxide Dismutase and Catalase. Superoxide dismutase (SOD) or catalase significantly inhibits the increase in FDP levels and %GFD, the prolongation of PT and PTT, and the reduction of fibrinogen levels and platelet counts 2 (Table II). These findings suggest that active oxygen species, such as superoxide or hydrogen peroxide, can affect the endotoxin-induced DIC. Effects of Vitamin E. The changes in coagulation parameters are significantly greater in vitamin E-deficient rats when compared to those in 2 T. Yoshikawa, M. Murakami, N. Yoshida, and M. Kondo, Thromb. Haemostasis 50, 869 (1983). 3 T. Yoshikawa, M. Murakami, Y. Furukawa, H. Kato, S. Takemura, and M. Kondo, Thromb. Haemostasis 49, 214 (1983). 4 T. Yoshikawa, M. Murakami, and M. Kondo, Toxicol. Appl. Pharmacol. 74, 173 (1984). 5 T. Yoshikawa, Y. Furukawa, M. Murakami, K. Watanabe, and M. Kondo, Thromb. Haemostasis 48, 235 (1982).
658
ORGAN, TISSUE, AND CELL DAMAGE
[70]
TABLE I TBA-REACTIVE SUBSTANCES IN SERUM AND TISSUES OF RATS INDUCED BY SUSTAINED INFUSION OF ENDOTOXIN a
Source Serum e Aortic w a l l g
Liverg Kidneyg Gastrointestinal mucosae Stomach Jejunum Ileum
Colon
Control b (n (n (n (n
= = = =
p value d
DIC ~
4.0 28.5 14.7 17.8
+-- 0.9 - 7.9 - 2.1 - 1.9
15)f 12) 8) 8)
6.3 11.5 14.4 7.1
- 0.7 (n = 8) ± 2.2 (n = 12) ± 3.1 (n = 10) ±2.1(n=8)
5.8 58.8 14.6 17.6
1.0 (n = - 13.2 (n = ± 1.2 (n = - 0.8 (n =
20) 12) 8) 8)
6.6 ± 1.2 (n = 8) 12.2 ± 2.1 (n = 12) 19.9 +- 4.3 (n = 10) 6.9-+ 1.2(n=8)
