생약학회지 (Korean Journal of Pharmacognosy)

  • 제40권2호
  • /
  • Pages.109-117
  • /
  • 2009
  • /
  • 0253-3073(pISSN)
  • /
  • 2288-9299(eISSN)
한국생약학회 (The Korean Society of Pharmacognosy)
권호 표지

글리코사미노글칸 분해 효소와 핵자기공명을 이용한 오염된 헤파린 시료의 분석

Analysis of Contaminated Heparins by the Combination of Glycosaminoglycan Degrading Enzymes and Nuclear Magnetic Resonance Spectroscopy

  • 임아랑 (서울대학교 약학대학/천연물과학연구소) ;
  • 박유미 (서울대학교 약학대학/천연물과학연구소) ;
  • 홍정희 (식품의약품안전청) ;
  • 이수정 (식품의약품안전청) ;
  • 이선희 (식품의약품안전청) ;
  • 김영식 (서울대학교 약학대학/천연물과학연구소)
  • Im, A-Rang (Natural Products Research Institute, College of Pharmacy, Seoul National University) ;
  • Park, You-Mie (Natural Products Research Institute, College of Pharmacy, Seoul National University) ;
  • Hong, Chong-Hui (Korea Food Drug Administration) ;
  • Lee, Su-Jung (Korea Food Drug Administration) ;
  • Lee, Sun-Hee (Korea Food Drug Administration) ;
  • Kim, Yeong-Shik (Natural Products Research Institute, College of Pharmacy, Seoul National University)
  • 발행 : 2009.06.30
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

We examined the purity of six heparin samples by using heparinase, chondroitinase, $^{1}H-NMR$, and polyacrylamide gel electrophoresis. To obtain high molecular weight contaminants from heparin samples, heparinase I - digested samples were subjected to the exhaustive microcon filtration. The filtration process removed heparin-derived di- and oligosaccharides effectively. By combining chondroitinase ABC treatment and strong anion exchange - high performance liquid chromatography, the result showed all six samples contained chondroitin sulfate as a contaminant ranging from 1.3 to 14.9%. Among them, sample S3 showed the highest content of 14.9%, which was further analyzed by chondroitinase AC treatment to confirm chondroitin sulfate B (dermatan sulfate). $^{1}H-NMR$ chemical shifts of N-acetyl groups clearly suggested the existence of chondroitin sulfate B (sample S3) and oversulfated chondroitin sulfate (samples S2 and S4) as contaminants. In addition, polyacrylamide gel electrophoresis was useful for qualitative detection on the sample's purity. These results suggest that the tools of heparinase I and chondroitinase ABC in combination with NMR spectroscopy would give very useful information for investigation of heparin contaminants such as oversulfated chondroitin sulfate and dermatan sulfate in heparin samples.

키워드

참고문헌

  1. Guerrini, M., Beccati, D., Shriver, Z., Naggi, A., Viswanathan, K., Bisio, A., Capila, I., Lansing, J. C., Guglieri, S., Fraser, B., Al-Hakim, A., Gunay, N. S., Zhang, Z., Robinson, L., Buhse, L., Nasr, M., Woodcock, J., Langer, R., Venkataraman, G., Linhardt, R. J., Casu, B., Torri, G. and Sasisekharan, R. (2008) Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events. Nat. Biotechnol. 26: 669-675 https://doi.org/10.1038/nbt1407
  2. Li, B., Suwan, J., Martin, J. G., Zhang, F., Zhang, Z., Hoppensteadt, D., Clark, M., Fareed, J. and Lindhardt, R. J. (2009) Oversulfated chondroitin sulfate interaction with heparin-binding proteins: New insights into adverse reactions from contaminated heparins. Biochem. Pharamcol. 78: 292-300 https://doi.org/10.1016/j.bcp.2009.04.012
  3. Maruyama, T., Toida, T., Imanari, T., Yu, G. and Linhardt, R. J. (1998) Conformational changes and anticoagulant activity of chondroitin sulfate following its O-sulfonation. Carbohydr. Res. 306: 35-43 https://doi.org/10.1016/S0008-6215(97)10060-X
  4. Kishimoto, T. K., Viswanathan, K., Ganguly, T., Elankumaran, S., Smith, S., Pelzer, K., Lansing, J. C., Sriranganathan, N., Zhao, G., Galcheva-Gargova, Z., Al-Hakim, A., Bailey, G. S., Fraser, B., Roy, S., Rogers-Cotrone, T., Buhse, L., Whary, M., Fox, J., Nasr, M., Dal Pan, G. J., Shriver, Z., Langer, R. S., Venkataraman, G., Austen, K. F., Woodcock, J. and Sasisekharan, R. (2008) Contaminated heparin associated with adverse clinical events and activation of the contact system. N. Engl. J. Med. 308: 2457-2467 https://doi.org/10.1056/NEJMoa0803200.
  5. Blossom, D. B., Kallen, A. J., Patel, P. R., Elward, A., Robinson, L., Gao, G., Langer, R., Perkins, K. M., Jaeger, J. L., Kurkjian, K. M., Jones, M., Schillie, S. F., Shehab, N., Ketterer, D., Venkataraman, G., Kishimoto, T. K., Shriver, Z., McMahon, A. W., Austen, K. F., Kozlowski, S., Srinivasan, A., Turabelidze, G., Gould, C. V., Arduino, M. J. and Sasisekharan, R. (2008) Outbreak of adverse reactions associated with contaminated heparin. ibid 359: 2674-2684
  6. Liu, H., Zhang, Z. and Linhardt, R. J. (2009) Lessons learned from the contamination of heparin. Nat. Prod. Rep. 26: 313-321 https://doi.org/10.1039/b819896a
  7. Han, Y. H., Garron, H. L., Kim, H. Y., Kim, W., Zhang, Z., Ryu, K. S., Shaya, D., Xiao, Z., Cheong, C., Kim, Y. S., Linhardt, R. J., Jeon, Y. H. and Cygler, M. J. Biol. Chem. submitted (under revision)
  8. Rice, K. G., Rottink M. K. and Linhardt, R. J. (1987) Fractionation of heparin-derived oligosaccharides by gradient polyacrylamide-gel electrophoresis. Biochem. J. 244: 515-522
  9. Zhang, Z., Weiwer, M., Li, Boyangzi., Kemp, M. M., Daman, T. H. and Linhardt, R. J. (2008) Oversulfated Chondroitin sulfate: Impact of a heparin impurity, associated with adverse clinical events, on low-molecular-weight heparin preparation. J. Med. Chem 51: 5498-5501 https://doi.org/10.1021/jm800785t
  10. Beyer, T., Diehl, B., Randel, G., Humpfer, E., Schafer, H., Spraul, M., Schollmayer, C. and Holzgrabe, U. (2008) Quality assessment of unfractionated heparin using $^1H$ nuclear magnetic resonance spectroscopy. J. Pharm. Biomed. Anal. 48: 13-19 https://doi.org/10.1016/j.jpba.2008.06.014
  11. Tollefsen, D. M., Majerus, D. W. and Blank, M. K. (1982) Heparin cofactor II, Purification and properties of a heparindependent inhibitor of thrombin in human plasma. J. Biol. Chem. 257: 2162-2169
  12. Tollefsen, D. M., Pestka, C. A. and Monafo, W. J. (1983) Activation of heparin cofactor II by dermatan sulfate. ibid, 258: 6713-6716
  13. Kyogashinma, M., Onaya, J., Miyauchi, S., Arai, M., Shibata, Y., Suda, A., Sakai, T., Takada, Y. and Takada, A. (1999) Antithrombotic activity of avian crown dermatan sulfate. Thromb. Res. 96: 459-465 https://doi.org/10.1016/S0049-3848(99)00138-3
  14. Fernandez, F., Van Ryn, J., Ofosu, F. A., Hirsh, J. and Buchanan, M. R. (1986) The harmorrhagic and antithrombotic effects of dermatan sulphate. Brit. J. Haemotol. 64: 309-317 https://doi.org/10.1111/j.1365-2141.1986.tb04124.x