Detection and Extraction of Heparin from Camel Lungs

Author(s): Ahmad Almeman*, Kasem Abdulmajed, Eltayeb E. Eid .

Journal Name: Current Pharmaceutical Biotechnology

Volume 20 , Issue 6 , 2019

Become EABM
Become Reviewer

Graphical Abstract:


Abstract:

Background: Heparin is an essential drug used as an anticoagulant. Access to raw material suitable for heparin extraction is critical for creating a viable business opportunity. In Saudi Arabia, large amounts of raw material with potential for heparin extraction are wasted.

Objective: To extract heparin and low-molecular-weight heparin (LMWH) from the camel lung, and measure its potency and activity.

Methods: Heparin preparation included three steps: extraction, electrophoretic identification, and activity measurement. Fresh lung tissue (100 g) was minced and homogenized in a blender. Crude heparin extracts were prepared using Charles’s or Volpi’s method with slight modifications. Heparin was purified by electrophoresis using high-purity agarose gels in barium acetate buffer. The heparin activity of purified samples was assayed spectrophotometrically using commercial heparin kits.

Results: Charles’s and Volpi’s extraction methods were simple and easy to establish. The yield was 90 mg crude heparin per 100 g of camel lung tissue following Volpi’s extraction protocol, whereas Charles’s method did not yield any heparin. The separation of heparin and LMWH by gel electrophoresis resulted in sharp and clear product bands using material prepared according to Volpi’s method. The heparin preparation had an anti-factor Xa activity of 37 IU/mg, indicating weak potency.

Conclusion: Preparation of active heparin from camel lung tissue is a technology applicable in manufacturing. Further method development is needed to increase heparin purity and potency.

Keywords: Heparin, LMWH, camel, dromedary, lung, electrophoresis, factor X.

[1]
WHO Model List of Essential Medicines (19th List). World Health Organization. April 2015. Archived (PDF) from the original on 13 December 2016. Retrieved 8 December 2016
[2]
Hylek, E.M.; Regan, S.; Henault, L.E.; Gardner, M.; Chan, A.T.; Singer, D.E.; Barry, M.J. Challenges to the effective use of unfractionated heparin in the hospitalized management of acute thrombosis. Arch. Intern. Med., 2003, 163(5), 621-627.
[3]
Streiff, M.B.; Agnelli, G.; Connors, J.M.; Crowther, M.; Eichinger, S.; Lopes, R.; McBane, R.D.; Moll, S.; Ansell, J. Guidance for the treatment of deep vein thrombosis and pulmonary embolism. J. Thromb, 2016, 41, 32-67.
[4]
Li, J.J.; Corey, E.J. Drug discovery: Practices, Processes, and Perspectives; Wiley & Sons: New York, 2013.
[5]
United States Food and Drug Administration (2010) Generic enoxaparin questions and answers. http://www.fda.gov/Drugs/ DrugSafety/PostmarketDrugSafetyInformationforPatientsand Providers/ucm220037.htm Accessed 15 Jan 2019.
[6]
Fareed, J.; Hoppensteadt, D.; Jeske, W.; Clarizio, R.; Walenga, J.M. Low molecular weight heparins: A developmental perspective. Expert Opin. Investig. Drugs, 1997, 6(6), 705-733.
[8]
Yu, Y.; Chen, Y.; Mikael, P.; Zhang, F.; Stalcup, A.M.; German, R.; Gould, F.; Ohlemacher, J.; Zhang, H.; Linhardt, R.J. Surprising absence of heparin in the intestinal mucosa of baby pigs. Glycobiology, 2017, 27(1), 57-63.
[9]
Fu, L.; Suflita, M.; Linhardt, R.J. Bioengineered heparins and heparan sulfates. Adv. Drug Deliv. Rev., 2016, 97, 237-249.
[10]
Lindhal, U. Structure and biosynthesis of heparin like polysaccharides. Fed. Proc., 1977, 36, 19.
[11]
Volpi, N. Extraction, purification and evaluation of structures and physiochemical properties of glycosaminoglycans. Boll. Chim. Farm., 1993, 132(5), 153-160.
[12]
Sasiekharan, R.; Venkataraman, G. Heparin and heparin sulfate: Biosynthesis, structure and function. Curr. Opin. Chem. Biol., 2000, 4(6), 626-631.
[13]
Sarwar, M.I.; Hussain, M.S.; Leghari, A.R. Heparin can be isolated and purified from bovine intestine by different techniques. Int. J. Pharm. Sci. Invention, 2013, 2319-6718.
[14]
Sarwar, M.I.; Hussain, M.S.; Manzoor, M.A.; Ahmad, M.; Hakeem, A. Isolation and purification of heparin from bovine pancreas by different methods. JSZMC, 2015, 6(4), 873-877.
[15]
NIHS, 2008. Heparin Sodium Heparinum natricum, www.nihs.go.jp/dbcb/Heparin/EP-heparin_Na.pdf
[16]
Volpi, N. Disaccharides analysis and molecular mass determination to microgram level of single sulfated glycosaminoglycan species in mixtures following agarose gel electrophoresis. Anal. Biochem., 1999, 273(2), 229-239.
[17]
Volpi, N. Purification of heparin, dermatan sulfate and chondroitin sulfate from mixtures by sequential precipitation with various organic solvents. J. Chromatogr. B Biomed. Appl., 1996, 685(1), 27-34.
[18]
Lamari, F.N.; Militsopoulou, M.; Mitropoulou, T.N.; Hjerpe, A.; Karamanos, N.K. Analysis of glycosaminoglycan-derived disaccharides in biologic samples by capillary electrophoresis and protocol for sequencing glycosaminoglycans. Biomed. Chromatogr., 2002, 16(2), 95-102.
[19]
WHO Pharmaceuticals Newsletter – No. 2 2008. Heparin Sodium.
[21]
Volpi, N.; Buzzega, D. Agarose-gel electrophoresis for the quality assurance and purity of heparin formulations. J. Pharm. Biomed. Anal., 2012, 67-68, 144-147.
[22]
Scot, D.A.; Charles, A.F. Studies on heparin: III. The purification of heparin. J. Biol. Chem., 1933, 102, 437-448.
[23]
Volpi, N.; Maccari, F.; Suwan, J.; Linhardt, R.J. Electrophoresis for the analysis of heparin purity and quality. Electrophoresis, 2012, 33(11), 1531-1537.
[24]
Warda, M.; Gouda, E.M.; Toida, T.; Chi, L.; Linhardt, R.J. Isolation and characterization of raw heparin from dromedary intestine: evaluation of a new source of pharmaceutical heparin. Comp. Biochem. Physiol. C Toxicol. Pharmacol., 2003, 136(4), 357-365.
[25]
Warda, M.; Mao, W.; Toida, T.; Linhardt, R.J. Turkey intestine as a commercial source of heparin? Comparative structural studies of intestinal avian and mammalian glycosaminoglycans. Comp. Biochem. Physiol. B Biochem. Mol. Biol., 2003, 134, 189-197.
[26]
Grant, A.C.; Linhardt, R.J.; Fitzgerald, G.; Park, J.J.; Langer, R. Metachromatic activity of heparin and heparin fragments. Anal. Biochem., 1984, 17, 25-32.
[27]
Kooy, F.K.; Ma, M.; Beeftink, H.H.; Eggink, G.; Tramper, J.; Boeriu, C.G. Quantification and characterization of enzymatically produced hyaluronan with fluorophore-assisted carbohydrate electrophoresis. Anal. Biochem., 2009, 384(2), 329-336.
[28]
Bhilocha, S.; Amin, R.; Pandya, M.; Yuan, H.; Tank, M.; LoBello, J.; Shytuhina, A.; Wang, W.; Wisniewski, H.G.; de la Motte, C.; Cowmana, M.K. Agarose and polyacrylamide gel electrophoresis methods for molecular mass analysis of 5-500 kDa hyaluronan. Anal. Biochem., 2011, 417(1), 41-49.
[29]
Volpi, N.; Maccari, F. Detection of submicrogram quantities of glycosaminoglycans on agarose gels by sequential staining with toluidine blue and Stains-All. Electrophoresis, 2002, 23(24), 4060-4066.


Rights & PermissionsPrintExport Cite as

Article Details

VOLUME: 20
ISSUE: 6
Year: 2019
Page: [476 - 482]
Pages: 7
DOI: 10.2174/1389201020666190401145544
Price: $58

Article Metrics

PDF: 17
HTML: 2