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Effect of a new synthetic free radical scavenger, 2-octadecyl ascorbic acid, on the mortality in mouse endotoxemia
Life Sciences, Vol. 47, pp. 1933-1939 Printed in the U.S.A.
Pergamon Press
EFFECT OF A NEW SYNTHETIC FREE RADICAL SCAVENGER, 2-OCTADECYL ASCORBIC ACID, ON THE MORTALITY IN MOUSE ENDOTOXEMIA Atsushi Nonaka, Tadao Manabe, Takayoshi Tobe First Department of Surgery, Faculty of Medicine, Kyoto University Kyoto, Japan (Received in final form September 18, 1990)
Summary Oxygen-derived free radicals have been implicated as mediators of cellular injury in several model systems. In order to clarify the role of oxygen radicals in endotoxemia, we measured the serial lipid peroxide changes resulting from systemic radical reactions using a newly developed color-metric method. To determine the effect of a free radical scavenger on mortality in endotoxemia, a new synthetic scavenger, 2-Octadecylascorbic acid ((X-36111, which overcome the detrimental properties (circulation half-life and cell penetration) of native SOD, was used in the model of mouse endotoxemia induced by the i. p. administration of E-colr endotoxin (10 mg/kg). Serial LPO (Lipid Peroxide) changes revealed significant elevations from the basal level of 4.52f0.79 mnol/ml to 10.5 f2.04 nmobml at 2h (P
1934
Free Radical in Mouse Endotoxemia
Vol. 47, No. 21, 1990
endotoxemia, main sources of oxyradicals are thought to be granulocytes, intracapillary, and the xanthine-xanthine oxidase system, electron transport system of mitochondria, the arachidonic acid cascade system on the cell membrane and others intracellularly. In endotoxemia, the levels of tissue malondialdehyde (MDA) have been reported to increase in the lung (6) and liver (7), which would be caused by the reaction of oxyradicals intracellularly and/or on the biomembrane. Therefore, inhibition of radical reactions in the cell would be needed. In recent studies, many investigators have experimentally administered oxyradical scavengers (SOD, CAT, etc) and measured the plasma and tissue levels of thiobarbituric acid reactive substances; i.e. malondialdehyde which corresponds with tissue oxidant injury. However, there are some problems, namely the circulation half- life of SOD and CAT is only a few minutes and their cell penetration by them is almost impossible. Furthermore, MDA is not a major component of the peroxidation products and thiobarbituric acid reacts with various kinds of aldehydes (8). In this study, we attempted to measure the circulating LPO levels by using a more sensitive and specific, newly developed coloration test and tested the effects of a new synthetic free radical scavenger (CV-3611), which has a longer circulation half life, cell penetration, and affinity of the biomembrane, on the survival rate in endotoxemic mice. Materials and Methods Female BALB/c mice, weighing 18-24gr, obtained from the Shizuoka Experimental Animal Center in Japan, were used in all of the experiments. The 2octadecylascorbic acid (CV-3611) was obtained from Takeda Chemical Industries, LTD, Osaka, Japan. A Determiner LPO kit was purchased from Kyowa Hakko LTD, Tokyo, Japan. The E-colt endotoxin (B-0111 serotype) was purchased from Aldrich biochemical corporation. Following an overnight fast, 10 mg/kg of E-coil endotoxin in saline or saline alone as a control was injected intraperitoneally, and five mice in each group were killed at 0, 2, 4, 8, 12 and 24 hr by cardiac blood sampling for plasma LPO assay: Heparinized blood obtained by cardiac puncture was centrifuged at 3,000 rpm for 15 min. Lipid peroxide in plasma was assayed by using a Determiner LPO kit which is a newly developed, sensitive and easy method. This method takes advantage of the peroxidase activity of hemoglobin which has a high substrate specificity (8) (9) (10). Coloration was carried out by incubating 0.1 ml of a sample twice at 30°C; with 1 ml of the first reagent (ascorbic acid peroxide) as a decomposer of the proton doner for 2 min, and then with 2 ml of the second reagent (chromogen and hemoglobin) for more than 5 min. Standard coloration was obtained using cumen hydroperoxide. The LPO (nmol/ml) calculation formula is as follows: = Es-Eb/Estd-Eb X 50 (Estd= absorbance at 675nm of the standard sample, Es and Eb = aborbance of the sample and blank sample, respectively). The levels of plasma LPO were not changed in the control group.
Vol.
47, No. 21,
1990
Free Radical
in Mouse Endotoxemia
1935
Another set of experimental animals were divided into four groups. Survival rate was observed as follows: control group (n = 20): saline i.p. plus 1% arabia gum s. c., treatment group (n=20); E-colt endotoxin (10 mg/kg) i.p. plus CV-3611(10 mg/kg) s.c administration just before E-co11 administration. E-cell endotoxin only i.p. administration (n=30). The survival rate from 0 to 2 days were observed for each group. The fourth group (n=30) was to measure the circulating CV-3611 level, Blood sampling was performed at 0, 1,2,4,6,10and 24 hrs after a 10 mg/kg CV-3611 S.C. injection. Blood samples (0.5 ml) were deproteinized with ethanol (4.5 ml) and centrifuged at 3,OOOrpm for 10min. The supernatant (4 ml) was concentrated in a vacuum, and the concentrate was diluted with methanol (0.15ml) and water (0.05ml). The suspension was stirred for 30sec, followed by centrifugation at 3,OOOrpm for 10min. The aliquot (0.1 ml) was analyzed by the reversed-phase HPLC (Hitachi 655A-11) which consisted of a Cl8 lnertsil ODS5p column (4.5X150 mm) with a mobile phase of CH3 CN/methanol /water /acetic acid, (1000:250:150:1). The flow rate was 1.5 ml/min and CV-3611 was detected by UV spectroscopy (Hitachi 655A) at 240nm. The amounts of CV-3611 were calculated by the use of external standards. Statistical Analysis The results in this study represent the mean f SEM. Differences between groups were calculated using an unpaired Wilcoxon test. A P value of less than 0.05 is statistically significant. The survival rate was analyzed using the x2-method with Yates correction (11). Results and Discussion In recent studies, oxygen-derived free radicals have been reported to play an important role in the shock state of endotoxemia (12) (13). However, there are controversies on the role of local infiltrated neutrophil and on the radical reactions in the cell, since direct measurement of in vivo dynamism of free radicals is almost impossible. To clarify the role of oxyradicals, there must be more reliable methods to measure the outcome of radical reactions m the tissue, ie, lipid peroxide, antioxidant level, etc, and/ or to administer the exogenous scavengers (SOD, CAT etc) and antioxidants. In this study, we observed the chronological changes of plasma LPO levels following administration of E-toll endotoxin. The levels of plasma LPO which may be caused by systemic radical reactions increased significantly from the basal level of 4.52 + 0.79 nmol/ml to 10.5 f 2.04 nmol/ml at 2h (P
Vol. 47, No. 21, 1990
Free Radical in Mouse Endotoxemia
1936
LPO (nmol/ml) 509
40-
30-
20-
10-
0I
2 I
4 I
8 I
I
12
I
24 hr
Fig. 1. Serial changes of lipid peroxide (LPO) level after i. p. administration of Ecob endotoxin (10 mg/kg). The result is represented by mean f SEM (n = 5). P values less than 0.05 are considered significant by an unpaired Wilcoxon test. *P < 0.05, compared to respective base line. E-coli endotoxin i.p. administration. s- - - -0 saline i.p. administration. kinds of aldehyde. Therefore, in this report, a newly developed, more sensitive and specific coloration test was used in which hydroperoxides, endoperoxides and peroxy radicals were reduced to their corresponding alcohol through catalysis by hemoglobin. Our results suggest that systemic radical reactions are provoked in the state of endotoxic shock. It has been reported that exogenous scavengers (SOD, CAT) have a short circulation half-life and an inability to penetrate cell membrane (14). In this study, we used a new synthetic free radical scavenger (CV-3611) which has a longer circulation half-life (T1/2=ca, 3.5h) as shown in Table 1. The level of CV-3611 in plasma reached a peak level of 0.54f 0.09 pg/ml at lh and then gradually decreased to an almost undetectable level at 24h after the s. c. administration of 10 mg/kg of
Vol. 47, No. 21, 1990
Free Radical in Mouse Endotoxemia
1937
Table 1.
CV-3611
(fig/ml)
Sample No.
N=l
N=2
N=3
N=4
N=5
Time lhr
0.48
0.35
0.51
0.80
0.54
0.54f 0.10
2hr
0.28
0.37
0.91
0.51
0.53
0.52f 0.20
4hr
0.12
0.14
0.23
0.09
0.16
0.15f 0.04
6hr
0.03
0.04
0.02
0.05
0.04
0.04f 0.004
1Ohr
0.05
ND
ND
ND
0.02
24hr
ND
ND
ND
ND
ND
Mean*SEM
Changes of plasma CV-3611 concentration after S.C.administration of CV3611(10 mg/kg). The circulation half life of CV-3611 is approximately 3.5 hours and almost disappears at 12h after S.C.administration. ND = Non Detectable.
CV-3611. The molecular weight of CV-3611 is very small (428.6) and the penetration of the biomembrane was confirmed in the experiment using 14C-labelled CV-3611. The percentages of 14C-labelled CV-3611 in the subcellular fraction of liver cells 6h after administration were; 100% in the homogenate, 18.7% in the nucleus; 13.1% in the mitochondria, 22.1% in the microsome and 58.4% in the cytosol (unpublished data). Therefore, it can be concluded that CV-3611 is able to scavenge free radicals in the extra and/or intracellular space. 2-octadecylascorbic acid (CV-3611) is a novel free radical scavenger which scavenges several free radicals including 02-, OH, LOO*, LO , L*, etc. and has a high affinity for the biomembrane by introducing lipophilic groups on the hydroxyls of the 2-, or 3-carbon in ascorbic acid (15). The scavenging effect of CV-3611 has been demonstrated in both the model of PMN 02- producing system and the inhibition of lipid peroxidation of linoleic acid caused by 02‘, OH and LOO (16). Kuzuya et al. (17) demonstrated that CV-3611 exhibited a dose dependent inhibition of free radical generation, assessed by chemiluminescence assay and SOD inhibitable reduction of ferricytochrome C, in A23187 stimulated neutrophils m v&o and noticed that CV3611 rn VIVOadministration was also effective on myocardial reperfusion injury in the canine. We have shown in this study that when administered just before an E. colr endotoxin i.p. injection, CV-3611 remarkably elevated the two day survival rate in endotoxin shock mice compared to the no treatment group as shown in Fig. 2. These
Free Radical in Mouse Endotoxemia
1938
Vol. 47, No. 21, 1990
Survival Rate (%>
60 W
: saline i.p.+arabia
M
: E-coli i.p.+CV-3611
W
: E-coli i.p.
gum S.C. S.C.
40
Fig. 2. Effect of CV-3611 on survival rate in endotoxemic mouse. Cl - 0 : control group (saline i.p. plus arabia gum s.c.) n = 20 0 -0: treatment group (E-colr endotoxin 10 mg/kg i. p. plus CV3611 lOmg/kgs.c.)n=20 0 - 0 : no treatment group (E-coli endotoxin 10 mg/kg i. p. ) n = 20 The difference between treatment and no treatment group was analysed by x2-method with yate’s correction. P values less than 0.05 are statistically significant. *P
findings radicals further therapy
give indirect evidence to the relationship between oxygen derived free and the diseased state of endotoxin shock. They also provide a basis for work to investigate main organs in endotoxin shock and for more effective of endotoxic state and multiple organ failure.
Vol.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
47,
No.
21,
1990
Free Radical
in Mouse Endotoxemia
1939
A. M. MICHELON, Superoxide and Superoxide Dismutase. J. M. McCORD, and I. FRIDOVICH (Eds): London Academic Press (1976). J. M. McCORD, N. Engl. J. Med. 313,159-163 (1985). L. A. SMEDLY, M. G. TONNESEN, R. A. SANDHAUS. J. Clin. Invest. 77, 1233-1243 (1986). P. M. HENSON, G. L. LARSON, R. 0. WEBSTER. Ann NY Acad. Sci. 384, 287-300 (1982). J. VARANI, S. E. G. FLIGIEL, G. 0. TILL. Lab. Invest. 53,656-663 (1985). R. H. DEMLING, J. LaLONDE, P. LI-JUAN. J. Appl. Physiol. 240, H348H353 (1981). F. KUNIMOTO, T. MORITA, R. OGAWA. Circ Shock 21, 15-22 (1987). Agric. Biol. Chem. 49, K. KANAZAWA, S. MINAMOTO, AND H. ASHIDA, 2799-2801(1985). S. MINAMOTO, K. KANAZAWA, H. ASHIDA. Agric. Bio. Chem. 49, 27472751(1985). N. ONISHI, H. OHKAWA, A. MIIKE. Biochem. lnt. 10, 205-211(1985). Feinberg SE, The analysis of cross-classified data, Cambridge, The MIT press, (1977). C. W. BRONER, J. L. SHENEP, G. L. STIDHAM. Lab. Invest. 16, 848-851 (1988). N. C. OLSON, M. K. GRIZZLE, and D. L. ANDERSON. J. Appl. Physiol. 63, 1526-1532 (1987). A. M. MICHELSON, and K. PUGET, Acta-physiol Stand, 492, 67-80 (1980). K. WONG, Agents and Actions, L. G. CLELAND, and M. J. POZNANSKY, 10,231-239 (1980). K. KATO, S. TERAO, N. SHIMAMOTO, and M. HIRATA, J. Med. Chem., 31 793-798, (1988). S. TERAO, The fourth biennial general meeting of society for free radical research, 36 (1988). K. KAZUYA, S. HOSHIDA, M. NISHIDA, Y. KIM Cardiovascular Research, 23, 323-330 (1989).
Pergamon Press
EFFECT OF A NEW SYNTHETIC FREE RADICAL SCAVENGER, 2-OCTADECYL ASCORBIC ACID, ON THE MORTALITY IN MOUSE ENDOTOXEMIA Atsushi Nonaka, Tadao Manabe, Takayoshi Tobe First Department of Surgery, Faculty of Medicine, Kyoto University Kyoto, Japan (Received in final form September 18, 1990)
Summary Oxygen-derived free radicals have been implicated as mediators of cellular injury in several model systems. In order to clarify the role of oxygen radicals in endotoxemia, we measured the serial lipid peroxide changes resulting from systemic radical reactions using a newly developed color-metric method. To determine the effect of a free radical scavenger on mortality in endotoxemia, a new synthetic scavenger, 2-Octadecylascorbic acid ((X-36111, which overcome the detrimental properties (circulation half-life and cell penetration) of native SOD, was used in the model of mouse endotoxemia induced by the i. p. administration of E-colr endotoxin (10 mg/kg). Serial LPO (Lipid Peroxide) changes revealed significant elevations from the basal level of 4.52f0.79 mnol/ml to 10.5 f2.04 nmobml at 2h (P
1934
Free Radical in Mouse Endotoxemia
Vol. 47, No. 21, 1990
endotoxemia, main sources of oxyradicals are thought to be granulocytes, intracapillary, and the xanthine-xanthine oxidase system, electron transport system of mitochondria, the arachidonic acid cascade system on the cell membrane and others intracellularly. In endotoxemia, the levels of tissue malondialdehyde (MDA) have been reported to increase in the lung (6) and liver (7), which would be caused by the reaction of oxyradicals intracellularly and/or on the biomembrane. Therefore, inhibition of radical reactions in the cell would be needed. In recent studies, many investigators have experimentally administered oxyradical scavengers (SOD, CAT, etc) and measured the plasma and tissue levels of thiobarbituric acid reactive substances; i.e. malondialdehyde which corresponds with tissue oxidant injury. However, there are some problems, namely the circulation half- life of SOD and CAT is only a few minutes and their cell penetration by them is almost impossible. Furthermore, MDA is not a major component of the peroxidation products and thiobarbituric acid reacts with various kinds of aldehydes (8). In this study, we attempted to measure the circulating LPO levels by using a more sensitive and specific, newly developed coloration test and tested the effects of a new synthetic free radical scavenger (CV-3611), which has a longer circulation half life, cell penetration, and affinity of the biomembrane, on the survival rate in endotoxemic mice. Materials and Methods Female BALB/c mice, weighing 18-24gr, obtained from the Shizuoka Experimental Animal Center in Japan, were used in all of the experiments. The 2octadecylascorbic acid (CV-3611) was obtained from Takeda Chemical Industries, LTD, Osaka, Japan. A Determiner LPO kit was purchased from Kyowa Hakko LTD, Tokyo, Japan. The E-colt endotoxin (B-0111 serotype) was purchased from Aldrich biochemical corporation. Following an overnight fast, 10 mg/kg of E-coil endotoxin in saline or saline alone as a control was injected intraperitoneally, and five mice in each group were killed at 0, 2, 4, 8, 12 and 24 hr by cardiac blood sampling for plasma LPO assay: Heparinized blood obtained by cardiac puncture was centrifuged at 3,000 rpm for 15 min. Lipid peroxide in plasma was assayed by using a Determiner LPO kit which is a newly developed, sensitive and easy method. This method takes advantage of the peroxidase activity of hemoglobin which has a high substrate specificity (8) (9) (10). Coloration was carried out by incubating 0.1 ml of a sample twice at 30°C; with 1 ml of the first reagent (ascorbic acid peroxide) as a decomposer of the proton doner for 2 min, and then with 2 ml of the second reagent (chromogen and hemoglobin) for more than 5 min. Standard coloration was obtained using cumen hydroperoxide. The LPO (nmol/ml) calculation formula is as follows: = Es-Eb/Estd-Eb X 50 (Estd= absorbance at 675nm of the standard sample, Es and Eb = aborbance of the sample and blank sample, respectively). The levels of plasma LPO were not changed in the control group.
Vol.
47, No. 21,
1990
Free Radical
in Mouse Endotoxemia
1935
Another set of experimental animals were divided into four groups. Survival rate was observed as follows: control group (n = 20): saline i.p. plus 1% arabia gum s. c., treatment group (n=20); E-colt endotoxin (10 mg/kg) i.p. plus CV-3611(10 mg/kg) s.c administration just before E-co11 administration. E-cell endotoxin only i.p. administration (n=30). The survival rate from 0 to 2 days were observed for each group. The fourth group (n=30) was to measure the circulating CV-3611 level, Blood sampling was performed at 0, 1,2,4,6,10and 24 hrs after a 10 mg/kg CV-3611 S.C. injection. Blood samples (0.5 ml) were deproteinized with ethanol (4.5 ml) and centrifuged at 3,OOOrpm for 10min. The supernatant (4 ml) was concentrated in a vacuum, and the concentrate was diluted with methanol (0.15ml) and water (0.05ml). The suspension was stirred for 30sec, followed by centrifugation at 3,OOOrpm for 10min. The aliquot (0.1 ml) was analyzed by the reversed-phase HPLC (Hitachi 655A-11) which consisted of a Cl8 lnertsil ODS5p column (4.5X150 mm) with a mobile phase of CH3 CN/methanol /water /acetic acid, (1000:250:150:1). The flow rate was 1.5 ml/min and CV-3611 was detected by UV spectroscopy (Hitachi 655A) at 240nm. The amounts of CV-3611 were calculated by the use of external standards. Statistical Analysis The results in this study represent the mean f SEM. Differences between groups were calculated using an unpaired Wilcoxon test. A P value of less than 0.05 is statistically significant. The survival rate was analyzed using the x2-method with Yates correction (11). Results and Discussion In recent studies, oxygen-derived free radicals have been reported to play an important role in the shock state of endotoxemia (12) (13). However, there are controversies on the role of local infiltrated neutrophil and on the radical reactions in the cell, since direct measurement of in vivo dynamism of free radicals is almost impossible. To clarify the role of oxyradicals, there must be more reliable methods to measure the outcome of radical reactions m the tissue, ie, lipid peroxide, antioxidant level, etc, and/ or to administer the exogenous scavengers (SOD, CAT etc) and antioxidants. In this study, we observed the chronological changes of plasma LPO levels following administration of E-toll endotoxin. The levels of plasma LPO which may be caused by systemic radical reactions increased significantly from the basal level of 4.52 + 0.79 nmol/ml to 10.5 f 2.04 nmol/ml at 2h (P
Vol. 47, No. 21, 1990
Free Radical in Mouse Endotoxemia
1936
LPO (nmol/ml) 509
40-
30-
20-
10-
0I
2 I
4 I
8 I
I
12
I
24 hr
Fig. 1. Serial changes of lipid peroxide (LPO) level after i. p. administration of Ecob endotoxin (10 mg/kg). The result is represented by mean f SEM (n = 5). P values less than 0.05 are considered significant by an unpaired Wilcoxon test. *P < 0.05, compared to respective base line. E-coli endotoxin i.p. administration. s- - - -0 saline i.p. administration. kinds of aldehyde. Therefore, in this report, a newly developed, more sensitive and specific coloration test was used in which hydroperoxides, endoperoxides and peroxy radicals were reduced to their corresponding alcohol through catalysis by hemoglobin. Our results suggest that systemic radical reactions are provoked in the state of endotoxic shock. It has been reported that exogenous scavengers (SOD, CAT) have a short circulation half-life and an inability to penetrate cell membrane (14). In this study, we used a new synthetic free radical scavenger (CV-3611) which has a longer circulation half-life (T1/2=ca, 3.5h) as shown in Table 1. The level of CV-3611 in plasma reached a peak level of 0.54f 0.09 pg/ml at lh and then gradually decreased to an almost undetectable level at 24h after the s. c. administration of 10 mg/kg of
Vol. 47, No. 21, 1990
Free Radical in Mouse Endotoxemia
1937
Table 1.
CV-3611
(fig/ml)
Sample No.
N=l
N=2
N=3
N=4
N=5
Time lhr
0.48
0.35
0.51
0.80
0.54
0.54f 0.10
2hr
0.28
0.37
0.91
0.51
0.53
0.52f 0.20
4hr
0.12
0.14
0.23
0.09
0.16
0.15f 0.04
6hr
0.03
0.04
0.02
0.05
0.04
0.04f 0.004
1Ohr
0.05
ND
ND
ND
0.02
24hr
ND
ND
ND
ND
ND
Mean*SEM
Changes of plasma CV-3611 concentration after S.C.administration of CV3611(10 mg/kg). The circulation half life of CV-3611 is approximately 3.5 hours and almost disappears at 12h after S.C.administration. ND = Non Detectable.
CV-3611. The molecular weight of CV-3611 is very small (428.6) and the penetration of the biomembrane was confirmed in the experiment using 14C-labelled CV-3611. The percentages of 14C-labelled CV-3611 in the subcellular fraction of liver cells 6h after administration were; 100% in the homogenate, 18.7% in the nucleus; 13.1% in the mitochondria, 22.1% in the microsome and 58.4% in the cytosol (unpublished data). Therefore, it can be concluded that CV-3611 is able to scavenge free radicals in the extra and/or intracellular space. 2-octadecylascorbic acid (CV-3611) is a novel free radical scavenger which scavenges several free radicals including 02-, OH, LOO*, LO , L*, etc. and has a high affinity for the biomembrane by introducing lipophilic groups on the hydroxyls of the 2-, or 3-carbon in ascorbic acid (15). The scavenging effect of CV-3611 has been demonstrated in both the model of PMN 02- producing system and the inhibition of lipid peroxidation of linoleic acid caused by 02‘, OH and LOO (16). Kuzuya et al. (17) demonstrated that CV-3611 exhibited a dose dependent inhibition of free radical generation, assessed by chemiluminescence assay and SOD inhibitable reduction of ferricytochrome C, in A23187 stimulated neutrophils m v&o and noticed that CV3611 rn VIVOadministration was also effective on myocardial reperfusion injury in the canine. We have shown in this study that when administered just before an E. colr endotoxin i.p. injection, CV-3611 remarkably elevated the two day survival rate in endotoxin shock mice compared to the no treatment group as shown in Fig. 2. These
Free Radical in Mouse Endotoxemia
1938
Vol. 47, No. 21, 1990
Survival Rate (%>
60 W
: saline i.p.+arabia
M
: E-coli i.p.+CV-3611
W
: E-coli i.p.
gum S.C. S.C.
40
Fig. 2. Effect of CV-3611 on survival rate in endotoxemic mouse. Cl - 0 : control group (saline i.p. plus arabia gum s.c.) n = 20 0 -0: treatment group (E-colr endotoxin 10 mg/kg i. p. plus CV3611 lOmg/kgs.c.)n=20 0 - 0 : no treatment group (E-coli endotoxin 10 mg/kg i. p. ) n = 20 The difference between treatment and no treatment group was analysed by x2-method with yate’s correction. P values less than 0.05 are statistically significant. *P
findings radicals further therapy
give indirect evidence to the relationship between oxygen derived free and the diseased state of endotoxin shock. They also provide a basis for work to investigate main organs in endotoxin shock and for more effective of endotoxic state and multiple organ failure.
Vol.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
47,
No.
21,
1990
Free Radical
in Mouse Endotoxemia
1939
A. M. MICHELON, Superoxide and Superoxide Dismutase. J. M. McCORD, and I. FRIDOVICH (Eds): London Academic Press (1976). J. M. McCORD, N. Engl. J. Med. 313,159-163 (1985). L. A. SMEDLY, M. G. TONNESEN, R. A. SANDHAUS. J. Clin. Invest. 77, 1233-1243 (1986). P. M. HENSON, G. L. LARSON, R. 0. WEBSTER. Ann NY Acad. Sci. 384, 287-300 (1982). J. VARANI, S. E. G. FLIGIEL, G. 0. TILL. Lab. Invest. 53,656-663 (1985). R. H. DEMLING, J. LaLONDE, P. LI-JUAN. J. Appl. Physiol. 240, H348H353 (1981). F. KUNIMOTO, T. MORITA, R. OGAWA. Circ Shock 21, 15-22 (1987). Agric. Biol. Chem. 49, K. KANAZAWA, S. MINAMOTO, AND H. ASHIDA, 2799-2801(1985). S. MINAMOTO, K. KANAZAWA, H. ASHIDA. Agric. Bio. Chem. 49, 27472751(1985). N. ONISHI, H. OHKAWA, A. MIIKE. Biochem. lnt. 10, 205-211(1985). Feinberg SE, The analysis of cross-classified data, Cambridge, The MIT press, (1977). C. W. BRONER, J. L. SHENEP, G. L. STIDHAM. Lab. Invest. 16, 848-851 (1988). N. C. OLSON, M. K. GRIZZLE, and D. L. ANDERSON. J. Appl. Physiol. 63, 1526-1532 (1987). A. M. MICHELSON, and K. PUGET, Acta-physiol Stand, 492, 67-80 (1980). K. WONG, Agents and Actions, L. G. CLELAND, and M. J. POZNANSKY, 10,231-239 (1980). K. KATO, S. TERAO, N. SHIMAMOTO, and M. HIRATA, J. Med. Chem., 31 793-798, (1988). S. TERAO, The fourth biennial general meeting of society for free radical research, 36 (1988). K. KAZUYA, S. HOSHIDA, M. NISHIDA, Y. KIM Cardiovascular Research, 23, 323-330 (1989).