Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-12-04
2002-10-29
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
Reexamination Certificate
active
06472194
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a process for the preparation of thromboplastin from animal tissue, using an extraction with an aqueous salt solution, followed by separation of tissue material in order to obtain a thromboplastin containing salt solution.
BACKGROUND OF THE INVENTION
An extraction process is known from European patent application EP-A-0 083 773. According to the known process, an acetone powder from the animal tissue, i.e. mammal tissue, particularly brain tissue, is initially prepared. The acetone powder is then extracted with an aqueous salt solution comprising 1-20 mM calcium ions and possibly a surface active substance.
Based on recent scientific research, it appears that using bovine brain tissue and bovine nerve tissue can lead to the transfer of the causative agents of BSE (Bovine Spongiform Encephalitis); (Spongiform Encephalopathy Advisory Committee, September 1994. Transmissible Spongiform Encephalopathies. A Summary of Present Knowledge and Research.” HMSO, London, U.K.). Furthermore, it appears that, in order to prepare a satisfactory solution of thromboplastin, first preparing an acetone powder of the relevant tissue, and then carrying out the extraction with a solution of calcium salts and possibly a surface active substance is unnecessary.
SUMMARY OF THE INVENTION
The process according to the invention is characterized in that muscular tissue, obtained from mammals or from fish, is directly extracted with an aqueous salt solution.
The process according to the present invention is characterised in that muscular tissue, obtained from mammals or from fish, is directly extracted with an aqueous salt solution.
Smariga, P. E. and Maynard, J. R. (1982) Blood, 60, 140-147 relates to the effects of platelets on the tissue factor and the fibrinolytic inhibition of cultured human fibroblasts and vascular cells. From Derwent Abstract AN 92-388343-XP002089073 relating to SU 88 616 530 A, a process for the preparation of thromboplastin from brain tissue-containing pieces of tissue from 24 hours of chickens is known. Biological Abstracts (1997) 1997, no. 107757 relates to the presence of thromboplastin activity in human gastric tissues.
By using the present process, the BSE problem can be avoided. Moreover, a more simple and less laborious preparation method is obtained, since, according to the invention, the step of preparing an aceton powder is unnecessary. The muscular tissue is simply reduced to smaller pieces and is then extracted. A salt solution without calcium ions or a surface active substance is sufficient.
The muscular tissue which is used is preferably derived from mammals, especially from bovine species, as said muscular tissue can be obtained in large amounts. The process can also be applied advantageously to muscular tissue from fish.
It was experimentally demonstrated that the used muscular tissue is preferably not derived from a freshly slaughtered animal, but that it has an “age” of 4-7 days. Extracts from such an “aged” muscular tissue have a higher thromboplastin activity. A higher thromboplastin activity is also obtained with vacuum packed muscular tissue with an age of 2 weeks.
Concerning the composition of the aqueous salt solution to be used for extracting, it is preferred that it is buffered to a pH value of 6.0-7.5. Using a phosphate salt is particularly preferable, as said salt also has a good buffering action.
During the preparation according to the invention a temperature of 0-4° C. is maintained and principle. However, a higher yield of thromboplastin activity is obtained if the extraction is carried out with salt solution at a temperature of 40-50° C.
The separation of the tissue material following the extraction is preferably carried out by centrifugation. Other separation methods lead to losses and/or are more time-consuming. Thus, filtration leads to losses by adsorption of, for instance, phospholipids, if a paper filter is used, and to higher costs if synthetic filters with a special composition and a particular molecular weight threshold is used in pressurized filtration devices.
The thromboplastin obtained by using the process according to the present invention can for instance be used for nutritional purposes. For such an application, the extraction has to be carried out with a solution of salts which are authorised for nutritional products, such as cooking salt, phosphates and acetates. Concerning an application in meat products, the thromboplastin is of course preferably derived from the same animal species as the species of the meat itself.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is further illustrated by examples.
REFERENCES:
patent: 2398077 (1946-04-01), Smith et al.
patent: 4416812 (1983-11-01), Becker et al.
patent: 1671305 (1991-08-01), None
Astrup, T., “Assay and Content of Tissue Thromboplastin in Different Organs” (1965) Thrombosis Diathesis Haemorrhagica, 14, 401-416.*
Østerud et al., “Thromboplastin Content in the Vessel Walls of Different Arteries and Organs of Rabbits” (1986) Thromb. Res., 42(3), 323-329.*
Howell et al., “Comparative Studies on Thromboplastin in Various Tissues and of Factors that Modify its Procoagulant Activity” (1981) Biochem. Soc. Trans., 9(1), 70-71.*
Quick, On the quantitative estimation of prothrombin (1945) American Journal of Clinical Pathology, vol. 15, pp. 560-566.*
Magnusson, Preparation and carbohydrate analysis of bovine prothrombin (1965) Arkiv for Kemi, vol. 23, No. 28, pp. 285-298.*
Chargaff et al., Ultracentrifugation isolation from lung tissue of a macromolecular protein component with thromboplastic properties (1942) J. Biol. Chem., vol. 145, pp. 593-603.*
Fujikata et al. Blood coagulation and clotting tests in carp (1985) Nippon Suisan Gakkaishi, vol. 51, No. 6, pp. 933-939 (abstract only).*
Kuznik et al. Activities of coagulation factor and fibrinolysin in muscles before and after exertion (1975) Voprosy Meditsinskoi Khimii, vol. 21, No. 3, pp. 242-245. (abstract only).*
Eremini, E. L. Hemo coagulating and fibrinolytic properties of muscle tissue in relation to its work regime (1978) Fiziol Zh (Kiev), vol. 24, No. 4, pp. 568-570. (abstract only).*
Kuznetsov, V. I. Distribution of 5′-nucleotidase and thromboplastin activity in human tissues (1983) Kazan. Med. Zh., vol. 64, No. 1, pp. 32-35. (abstract only).*
Smargia Et Al: File Medline, abstract No. 82207075, 1982, XP002089071; “Platelet effect on tissue factor and fibrinolytic inhibition of cultured human fibroblasts and vascular cells”, Blood, vol. 60, No. 1, Jul. 1982, pp. 140-147.
Derwent Publications, AB92-388343; XP002089073, “Production of thromboplastin comprises homogeneizing brain and muscle tissues of day-old chicken with distilled water filtration followed by drying”, & SU 88 616 530 A (Animals Noncontagious Diseases Research Institute) Aug. 23, 1991.
Golyshenkov: “Blood coagulation, anticoagulant and figrinolytic characteristics of human stomach tissues”, Fiziologiya Cheloveka, vol. 22, No. 4, 1996, pp. 114-117, & Biological Abstracts, vol. 1997, abstract No. 107757, XP002089072.
Roodink Hendrikus B J
van Zeeburg Gerjan T
Harimex B.V.
Pillsbury WInthrop LLP
Weber Jon P.
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