纳豆激酶溶血栓作用及作用机理的研究
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摘要
心脑血管疾病是目前危害人类及动物健康的严重疾病，寻找及研制价廉、无毒、高效的溶栓药物成为当务之急。日本传统食品纳豆，在民间一直被用来预防和治疗心脑血管疾病。自从日本学者须见洋行首次发现纳豆中含有溶栓激酶以来，纳豆这一食品引起了世界的广泛关注，其中重要功能因子—纳豆激酶，以其易于提取，无任何毒副作用，溶栓效果好，作用时间长等特点得到了深入研究。纳豆激酶有望开发成为新一代溶栓药物。本研究通过对纳豆激酶纤维蛋白溶解活性的研究；纳豆激酶的体内血栓溶解实验；纳豆激酶溶栓作用机理的研究；纳豆激酶的安全性评价等一系列实验，对纳豆激酶的溶栓作用及作用机理做了全面系统的研究。对纤维蛋白溶解活性的研究结果显示：高、中、低剂量的纳豆激酶均能显著地缩短血浆优球蛋白的溶解时间、显著地降低血浆纤维蛋白原的含量、显著地增加纤维蛋白裂解产物的含量，表明纳豆激酶具有较强的纤维蛋白溶解活性，且同等剂量的纳豆激酶和尿激酶相比，前者的纤溶活性显著高于后者；对体内血栓溶解的实验结果显示：纳豆激酶可明显地延长血栓形成时间、显著减少股动脉血栓的湿重及长度，并明显提高血栓的再通率、显著减少肺内血栓的形成，表明纳豆激酶具有较强的体内溶栓作用，且同等剂量的纳豆激酶和尿激酶相比，前者的溶栓作用大于后者；对纳豆激酶溶栓作用机理的研究显示：纳豆激酶可显著提高t-PA的含量及活性，而对PAI-1的含量无明显影响，表明纳豆激酶是通过激活t-PA来发挥溶栓作用的；对纳豆激酶的安全性评价实验结果表明：纳豆激酶无毒无副作用。
Heart and brain vessel disease is an important disease which do harm to human health. At present, urgent affair is to look for a kind of cheaply, innocuously, effciently thrombolytic medicine. Natto which is a typical soybean food eaten in Japan was used to preventing and treating heart and brain vessel disease previously. Since the nattokinase(NK) was discovered by Sumi in Japan, the Natto have become an important food in the world. So the functional factor - nattokinase which has a strong fibrinolytic activity was researched deeply. This paper included several experiments below: first, studies on fibrinolytic activity of NK; second, thrombolytic experiment of NK in vivo; third, thrombolytic mechanism of NK; forth, safety evaluation of NK. Results: first experiment showed that high, middle, low dosage NK could decrease significantly dissolve time of euglobulin in plasma, and reduce significantly the content of fibrinogen in plama and increase the content of fibrin decompose product. The first experiment ind
icated that NK has a strong fibrinolytic activity, and it has more fibrinolytic activity than Urokinase(UK) at the same dosage. The second experiment showed that NK could prolong thrombus forming time in blood vessel remarkedly, and could lessen the length and weight of thrombus and promote the ratio of recirculation of the femoral artery significantly, decrease the weight of thrombus in lung. This experiment indicated that NK has a strong thrombolytic action in vivo, and its potency was superior than UK at the same dosage. The third experiment showed that NK could decrease significantly the content and activity of t-PA, but NK hadnt obvious effects on contents of PAI-1. This experiment indicated that thrombolytic activity of NK was obtained by activating t-PA The last experiment showed that NK has innocuity, and no side-effect.
Heart and brain vessel disease is an important disease which do harm to human health. At present, urgent affair is to look for a kind of cheaply, innocuously, effciently thrombolytic medicine. Natto which is a typical soybean food eaten in Japan was used to preventing and treating heart and brain vessel disease previously. Since the nattokinase(NK) was discovered by Sumi in Japan, the Natto have become an important food in the world. So the functional factor - nattokinase which has a strong fibrinolytic activity was researched deeply. This paper included several experiments below: first, studies on fibrinolytic activity of NK; second, thrombolytic experiment of NK in vivo; third, thrombolytic mechanism of NK; forth, safety evaluation of NK. Results: first experiment showed that high, middle, low dosage NK could decrease significantly dissolve time of euglobulin in plasma, and reduce significantly the content of fibrinogen in plama and increase the content of fibrin decompose product. The first experiment ind
icated that NK has a strong fibrinolytic activity, and it has more fibrinolytic activity than Urokinase(UK) at the same dosage. The second experiment showed that NK could prolong thrombus forming time in blood vessel remarkedly, and could lessen the length and weight of thrombus and promote the ratio of recirculation of the femoral artery significantly, decrease the weight of thrombus in lung. This experiment indicated that NK has a strong thrombolytic action in vivo, and its potency was superior than UK at the same dosage. The third experiment showed that NK could decrease significantly the content and activity of t-PA, but NK hadnt obvious effects on contents of PAI-1. This experiment indicated that thrombolytic activity of NK was obtained by activating t-PA The last experiment showed that NK has innocuity, and no side-effect.
引文
1 木内斡，铃木英也 高活性纳豆开发的现状[J] 食品科学，1991，33(6)：85
2 Sumi H. A noval fibrinolytic enzyme in the vegetable natto: a typical and popular soybean food in the Japanese diet.Experience, 1987, 43(20): 1110～1111
3 康明官。中外著名发酵食品生产工艺手册。北京：化学工业出版社，1997
4 王正刚，丁贵平，蔡正森。纳豆激酶的研究进展。中国生化药物杂志，1998，19(6)：401～403
5 Sumi H. Experient, 1988, 43(18): 111～115
6 付利等。生物工程进展，1995，15(5)：46～49
7 徐涛等。中国生化药物杂志，1995，16(4)：196
8 李宁。微生物学报，1996，16(2)：43～47
9 张淑梅，张云湖，赵晓祥等。纳豆激酶基因的克隆与表达。中国生物化学与分子生物学报，1999，(12) 15(6)：912～915
10 Nakamura T et al.Biosci. Biotech. Biochem, 1992, 56(11): 1869
11 须见洋行。化学与生物，1991，29(2)：119～123
12 Mitsugu F.et al,Biochemical and Biophisical Research Communication, 1993, 197(3): 1340～1347
13 凌均建，罗立新，杨汝德。纳豆激酶的分子生物学研究进展。广东药学院学报，1992，12(15) 4：300～303
14 徐涛，宋文延。一种新型纤溶活性物质——纳豆激酶。生物药物杂志，1995，16(4)：196
15 Ichishima E. et al. Biochemica and Biophysica Acta, 1986, 869: 178
16 Hirotoshi K,et al. Agric Biol Chem, 1989, 53(10): 2695～2702, 62(7): 2482
17 Yuki Y, et al. Biosci Biotech Biochem, 1994, 58(2): 366
18 Kim W, et al. Appl Environ Microbiol, 1996
19 李荣萍等。生物技术。1996，6(3)：21～23
20 Mitsugu F, et al. Biol Pharm Bul, 1995, 8(6): 1387～1391
21 Fujita M, et al. Biol Pharm Bull, 1995, 18(9): 1194～1196
22 Sumi H, et al. Acta Haematol, 1990, 84: 139
23 Fujita M, et al. Biol Pharm Bull, 1995, 18(10): 1387
24 YuKi Y, Nakagama T, Fujita M. A Sandwich enzymelinked immunosorbent assay for natto, Biotech Biochem, 1994, 58(2): 366～370
25 Murametsu K, et al. Nippon Shokuhin Kagaka Kogaku Kaishi, 1995, 42(8): 575～582
26 Sumi H, et al. Nippon Shokuhin Kagaka Kogaku Kaishi, 1996, 43(10): 1124～1127
27 徐叔云，卞如濂，陈修。药理实验方法学。第2版，北京：人民卫生出版社，1994，1114
28 毛腾敏等。血瘀病理模型探索(二)——衰老血瘀模型观测。北京医科大学学报，1993，23
(2): 100
29 Stassen JM, Lijnen HR, Kieckens L et al. Small animal thrombosis models for the evaluation of thrombolytic agents. Circulation. 1991, 83(supple Ⅳ): Ⅳ-65.
30 lozel J.P,, Holvoet P, Tschopp T. Experimental pulmonary embolus in the rat: a new in vivo model to test thrombolytic drugs. J Cardiovase Pharmacol, 1988, 12(5): 520
31 Oseph LR, et al: Prevention of occlusive coronary artery thrombosis by prostacyclin imfusion in the dog, Circulation, 1981, 64(5): 906
32 Collen D, Lijnen HR. Staphylokinase, a fibrin specific plasminogen activator with the rapeutic potential Blood, 1994, 84(3): 680-686
33 陈飞虎等。重组葡激酶对犬股动脉血栓的再通作用。中国药理学通报，1999，oct；15(5) 447-449
34 Nowak A and Gurewich V:Thrombolysis of streptokinase in rabbits. Dose response, fibrin specificity and laboratory evaluation of fibrinolytic effect. Thromb Diath Haemorrh 31: 265, 1974, 35
35 现代药理实验方法 张均田主编。北京医科大学。中国协和医科大学联合出版社。1818-1826
36 Iltzius P and Hofmann V: Conformational identity of human fibrinogen and monomeric fibrin. Thromb Res 19: 793, 1980
37 Ouyang C and Teng CM: In vivo effects of the purified thrombin-like and anticoagulant principles of agkistrodon acutus (hundred pace snake) venom.Toxicon 16: 583, 1978
38 Uyang C and Huang TF: Alpha and beta-fibrinogenases from Trimeresurus gramineus snake venom. Biochim Biophys Acta 571: 270, 1979
39 宫村英宏，竹中阳子等纳豆血栓溶解素生成条件及性质[J]。Nippon shokuhin Kagaku Kogku Kaishi, 1998, 45(20): 100-107
40 Lindgren A, Lindoff C, Norrving B, et al. plasminogen activator and plasminogen activator inhibitor-1 in stroke patients. Stroke patients. Stroke, 1996, 27(6): 1066-1071
41 Ayashi T.Tissue-type plasminogen activator(t-PA) and Plasminogen activator inhibitor(PAI). Rinsho Byori, 1994, 42(4): 346-351
42 Otto AJ, Carter AM, Stickland M, et al. Plasminogen activator inhibitor-1 (PAI-1) 4G/5G promoter polymorphisn and levels in subjects with cerebrovascular disease. Thromb Haemost, 1997, 7(4); 730-734
43 Wakamatsu S, Banno T, Tohnai M, et al. Change of the plasma levels of Tissue plaminogen activator and plasminogen activator inhibitor-1 in cerebrai infarction induced by the venous occlusion. Nippon Ronen Igakkai Zasshi, 1995, 32(8-9): 566-570
44 Dietzmann k, von Bossanyi P, Krause D, et al.Expression of the plasminogen activator system and the inhibitors PAI-1 and PAI-2 in posttraumatic lesions of the CNS and the brain injuries following dramatic circulatory arrests: an immunohistochemical study.Pathol Res Pract, 2000, 199(1): 15-21
45 OhtaS,Sakuragawa N.Tissue-phsminogen activator(t-PA).Nippon Rinsho,1999, 57Suppl(-HD-):671-675
46 Zunker P, Schick A, Padr T, et al. Tissue plasminogen activator and plasminogen activator inhibitor in patients with acute ischemic stroke:relation to stroke etiology. Neurol Res, 1999, 21(8): 727-732
47 Ueda T, Hatakeyama T, Sakaki S, et al. Changes in coagulation and fibrinolytic system after locai intra-arterial thrombolysis for acute ischemic stroke. Neurol Res, 1999, 21(8): 727-732.
48 Ernando B, Carlos CB, Aurora DL, et al. Prothrombotic state in young people with iodiopathic stroke a prospective study. Stroke, 1994, 25: 287-289
49 Ridker PM, Hennnekens CH, Stampfer MJ, et al. Prospective study of endogenous tissue plasminogen activator and risk of stroke. Lancet, 1994, 343: 940-943.
50 Collen D. On the regulation and control of fibrinolysis[J]. Thromb Hae most, 1980, 43: 77.
51 Rijken DC. Relationship between structure and function t-PA [J]. Klin Wochenschr, 1988, 265: 109.
52 Lupu F, Bergonelli GE, Heim DA, et al. Localication and production of plasminogen activator inhibitor-1 in human healthy and atherosclerotic arteries. Arterioscler Thromb, 1993, 13: 1090-1100.
53 Altes A, Abellan MF, Nateo J, et al. Hemastatic disturbances in acute ischemic strokes: a study of 86 patients. Acta Haematol, 1995, 94: 10-15
54 Mrgaglione M, Di Minno G, Grandone E, et al. Abnormally high circulation levels of tissue plasminogen activator and plasminogen activator inhibitor-1 in patients with a history of ischemic stroke. Arterioscler Thromb, 1994, 14(11): 1741-1745
55 Zhang YD, Shi JP, Li ZH, et al. Analysis of risk factors for recurrent cerebral infarction. J Neurol, 1998, 245(6-7): 373
56 Nagahiro S, Uno M, Sato K, et al. Pathophysiology and treatment of cerebral ischemia. J Med Iinvest, 1998, 45: 57-70
57 Buchan AM, Barber PA, Newcommin N, et al. Effectiveness of t-PA in acute ischemic stroke: outcome relates to appropriateness. Neurology, 2000, 54(3): 679-684
58 Gebel JM, Sila CA, Sloan MA, et al. Thrombolysis-related intracranial hemorrhage: a radiographic analysis of 244 cases from the GUSTO-trial with clinical correlation. Global Utilization of Streptokinase and Tissue Plasminogen Activator for Occluded Coronary Arteries. Stroke, 1998, 29(30): 563-569
59 Joho A. Tissue-type plasmingen activator [J]. N Eng J Med, 1988, 319: 925
60 Miles, Binding and activation of plasmimogen on the platelet surface [J]. J Biolchem, 1986, 261: 11656
61 Sprenger ED. Plaminogen and activator inhibitors [J]. Blood, 1987, 69: 381
62 Mimuro J. Plasminogen activator inhibitor 1(PAI-1). Nippon Rinsho, 1999, 57(-HD-): 678-681
63 Collen D. Mechanisms of inhibition of tissue-type plasminogen actovator in blood [J]. Thromb Hae most, 1983, 50; 678
64 王结义。人血浆t-PA及其抑制物活性的测定[J]。中华医学检验杂志，1989，12：120
65 Lindahl TL. The mechanism of the reaction between human plsminogen activator inhibitor 1 and tissue
plasminogen activator[J].J Biochem, 1990, 265: 109
66 Bass R, Gunzel.p, Henschler D, et al. LD_(50) versus acute toxicity. Atch, Tox: 601, 1982, 51: 183-186
67 Muller H and Kley HP. Retrospective stady on the reliality of an "approximate LD_(50)" determined with a small number of animals. Arch Toxicol, 1982, 51: 189-196
68 Schutz E and Fuchs H. A new approach to minimizing the number of animal used in acute toxicity testing and optimizing the information of test results. Arch Toxicology, 1982, 51: 197-220
69 Philips TD and Hayes AW. Techniques in membrane Toxicology,Hayes AW ed. Principles and methodz of Toxicology. Second Edition. New York: Raven Presz, 1989, 761-776
2 Sumi H. A noval fibrinolytic enzyme in the vegetable natto: a typical and popular soybean food in the Japanese diet.Experience, 1987, 43(20): 1110～1111
3 康明官。中外著名发酵食品生产工艺手册。北京：化学工业出版社，1997
4 王正刚，丁贵平，蔡正森。纳豆激酶的研究进展。中国生化药物杂志，1998，19(6)：401～403
5 Sumi H. Experient, 1988, 43(18): 111～115
6 付利等。生物工程进展，1995，15(5)：46～49
7 徐涛等。中国生化药物杂志，1995，16(4)：196
8 李宁。微生物学报，1996，16(2)：43～47
9 张淑梅，张云湖，赵晓祥等。纳豆激酶基因的克隆与表达。中国生物化学与分子生物学报，1999，(12) 15(6)：912～915
10 Nakamura T et al.Biosci. Biotech. Biochem, 1992, 56(11): 1869
11 须见洋行。化学与生物，1991，29(2)：119～123
12 Mitsugu F.et al,Biochemical and Biophisical Research Communication, 1993, 197(3): 1340～1347
13 凌均建，罗立新，杨汝德。纳豆激酶的分子生物学研究进展。广东药学院学报，1992，12(15) 4：300～303
14 徐涛，宋文延。一种新型纤溶活性物质——纳豆激酶。生物药物杂志，1995，16(4)：196
15 Ichishima E. et al. Biochemica and Biophysica Acta, 1986, 869: 178
16 Hirotoshi K,et al. Agric Biol Chem, 1989, 53(10): 2695～2702, 62(7): 2482
17 Yuki Y, et al. Biosci Biotech Biochem, 1994, 58(2): 366
18 Kim W, et al. Appl Environ Microbiol, 1996
19 李荣萍等。生物技术。1996，6(3)：21～23
20 Mitsugu F, et al. Biol Pharm Bul, 1995, 8(6): 1387～1391
21 Fujita M, et al. Biol Pharm Bull, 1995, 18(9): 1194～1196
22 Sumi H, et al. Acta Haematol, 1990, 84: 139
23 Fujita M, et al. Biol Pharm Bull, 1995, 18(10): 1387
24 YuKi Y, Nakagama T, Fujita M. A Sandwich enzymelinked immunosorbent assay for natto, Biotech Biochem, 1994, 58(2): 366～370
25 Murametsu K, et al. Nippon Shokuhin Kagaka Kogaku Kaishi, 1995, 42(8): 575～582
26 Sumi H, et al. Nippon Shokuhin Kagaka Kogaku Kaishi, 1996, 43(10): 1124～1127
27 徐叔云，卞如濂，陈修。药理实验方法学。第2版，北京：人民卫生出版社，1994，1114
28 毛腾敏等。血瘀病理模型探索(二)——衰老血瘀模型观测。北京医科大学学报，1993，23
(2): 100
29 Stassen JM, Lijnen HR, Kieckens L et al. Small animal thrombosis models for the evaluation of thrombolytic agents. Circulation. 1991, 83(supple Ⅳ): Ⅳ-65.
30 lozel J.P,, Holvoet P, Tschopp T. Experimental pulmonary embolus in the rat: a new in vivo model to test thrombolytic drugs. J Cardiovase Pharmacol, 1988, 12(5): 520
31 Oseph LR, et al: Prevention of occlusive coronary artery thrombosis by prostacyclin imfusion in the dog, Circulation, 1981, 64(5): 906
32 Collen D, Lijnen HR. Staphylokinase, a fibrin specific plasminogen activator with the rapeutic potential Blood, 1994, 84(3): 680-686
33 陈飞虎等。重组葡激酶对犬股动脉血栓的再通作用。中国药理学通报，1999，oct；15(5) 447-449
34 Nowak A and Gurewich V:Thrombolysis of streptokinase in rabbits. Dose response, fibrin specificity and laboratory evaluation of fibrinolytic effect. Thromb Diath Haemorrh 31: 265, 1974, 35
35 现代药理实验方法 张均田主编。北京医科大学。中国协和医科大学联合出版社。1818-1826
36 Iltzius P and Hofmann V: Conformational identity of human fibrinogen and monomeric fibrin. Thromb Res 19: 793, 1980
37 Ouyang C and Teng CM: In vivo effects of the purified thrombin-like and anticoagulant principles of agkistrodon acutus (hundred pace snake) venom.Toxicon 16: 583, 1978
38 Uyang C and Huang TF: Alpha and beta-fibrinogenases from Trimeresurus gramineus snake venom. Biochim Biophys Acta 571: 270, 1979
39 宫村英宏，竹中阳子等纳豆血栓溶解素生成条件及性质[J]。Nippon shokuhin Kagaku Kogku Kaishi, 1998, 45(20): 100-107
40 Lindgren A, Lindoff C, Norrving B, et al. plasminogen activator and plasminogen activator inhibitor-1 in stroke patients. Stroke patients. Stroke, 1996, 27(6): 1066-1071
41 Ayashi T.Tissue-type plasminogen activator(t-PA) and Plasminogen activator inhibitor(PAI). Rinsho Byori, 1994, 42(4): 346-351
42 Otto AJ, Carter AM, Stickland M, et al. Plasminogen activator inhibitor-1 (PAI-1) 4G/5G promoter polymorphisn and levels in subjects with cerebrovascular disease. Thromb Haemost, 1997, 7(4); 730-734
43 Wakamatsu S, Banno T, Tohnai M, et al. Change of the plasma levels of Tissue plaminogen activator and plasminogen activator inhibitor-1 in cerebrai infarction induced by the venous occlusion. Nippon Ronen Igakkai Zasshi, 1995, 32(8-9): 566-570
44 Dietzmann k, von Bossanyi P, Krause D, et al.Expression of the plasminogen activator system and the inhibitors PAI-1 and PAI-2 in posttraumatic lesions of the CNS and the brain injuries following dramatic circulatory arrests: an immunohistochemical study.Pathol Res Pract, 2000, 199(1): 15-21
45 OhtaS,Sakuragawa N.Tissue-phsminogen activator(t-PA).Nippon Rinsho,1999, 57Suppl(-HD-):671-675
46 Zunker P, Schick A, Padr T, et al. Tissue plasminogen activator and plasminogen activator inhibitor in patients with acute ischemic stroke:relation to stroke etiology. Neurol Res, 1999, 21(8): 727-732
47 Ueda T, Hatakeyama T, Sakaki S, et al. Changes in coagulation and fibrinolytic system after locai intra-arterial thrombolysis for acute ischemic stroke. Neurol Res, 1999, 21(8): 727-732.
48 Ernando B, Carlos CB, Aurora DL, et al. Prothrombotic state in young people with iodiopathic stroke a prospective study. Stroke, 1994, 25: 287-289
49 Ridker PM, Hennnekens CH, Stampfer MJ, et al. Prospective study of endogenous tissue plasminogen activator and risk of stroke. Lancet, 1994, 343: 940-943.
50 Collen D. On the regulation and control of fibrinolysis[J]. Thromb Hae most, 1980, 43: 77.
51 Rijken DC. Relationship between structure and function t-PA [J]. Klin Wochenschr, 1988, 265: 109.
52 Lupu F, Bergonelli GE, Heim DA, et al. Localication and production of plasminogen activator inhibitor-1 in human healthy and atherosclerotic arteries. Arterioscler Thromb, 1993, 13: 1090-1100.
53 Altes A, Abellan MF, Nateo J, et al. Hemastatic disturbances in acute ischemic strokes: a study of 86 patients. Acta Haematol, 1995, 94: 10-15
54 Mrgaglione M, Di Minno G, Grandone E, et al. Abnormally high circulation levels of tissue plasminogen activator and plasminogen activator inhibitor-1 in patients with a history of ischemic stroke. Arterioscler Thromb, 1994, 14(11): 1741-1745
55 Zhang YD, Shi JP, Li ZH, et al. Analysis of risk factors for recurrent cerebral infarction. J Neurol, 1998, 245(6-7): 373
56 Nagahiro S, Uno M, Sato K, et al. Pathophysiology and treatment of cerebral ischemia. J Med Iinvest, 1998, 45: 57-70
57 Buchan AM, Barber PA, Newcommin N, et al. Effectiveness of t-PA in acute ischemic stroke: outcome relates to appropriateness. Neurology, 2000, 54(3): 679-684
58 Gebel JM, Sila CA, Sloan MA, et al. Thrombolysis-related intracranial hemorrhage: a radiographic analysis of 244 cases from the GUSTO-trial with clinical correlation. Global Utilization of Streptokinase and Tissue Plasminogen Activator for Occluded Coronary Arteries. Stroke, 1998, 29(30): 563-569
59 Joho A. Tissue-type plasmingen activator [J]. N Eng J Med, 1988, 319: 925
60 Miles, Binding and activation of plasmimogen on the platelet surface [J]. J Biolchem, 1986, 261: 11656
61 Sprenger ED. Plaminogen and activator inhibitors [J]. Blood, 1987, 69: 381
62 Mimuro J. Plasminogen activator inhibitor 1(PAI-1). Nippon Rinsho, 1999, 57(-HD-): 678-681
63 Collen D. Mechanisms of inhibition of tissue-type plasminogen actovator in blood [J]. Thromb Hae most, 1983, 50; 678
64 王结义。人血浆t-PA及其抑制物活性的测定[J]。中华医学检验杂志，1989，12：120
65 Lindahl TL. The mechanism of the reaction between human plsminogen activator inhibitor 1 and tissue
plasminogen activator[J].J Biochem, 1990, 265: 109
66 Bass R, Gunzel.p, Henschler D, et al. LD_(50) versus acute toxicity. Atch, Tox: 601, 1982, 51: 183-186
67 Muller H and Kley HP. Retrospective stady on the reliality of an "approximate LD_(50)" determined with a small number of animals. Arch Toxicol, 1982, 51: 189-196
68 Schutz E and Fuchs H. A new approach to minimizing the number of animal used in acute toxicity testing and optimizing the information of test results. Arch Toxicology, 1982, 51: 197-220
69 Philips TD and Hayes AW. Techniques in membrane Toxicology,Hayes AW ed. Principles and methodz of Toxicology. Second Edition. New York: Raven Presz, 1989, 761-776