Combined Quantitative ¹H and ¹³C Nuclear Magnetic Resonance Spectroscopy for Characterization of Heparin Preparations

ABSTRACT

The sulfation patterns of pig and bovine mucosal commercial heparin preparations can be characterized and distinguished from each other easily by analysis of their monodimensional proton and carbon nuclear magnetic resonance (¹H and ¹³C-NMR) spectra. NMR spectroscopy can detect and quantify signals associated with major sequences as well as with minor residues such as the typical ones associated with the antithrombin (AT) binding sequence and the ``linkage region.'' Contaminants arising from industrial preparation processes are also detectable.

Heparin - NMR spectra - sulfation patterns - antithrombin binding sequence

REFERENCES

• 1 Casu B. Structure and biological activities of heparin.  Adv Carbohydr Chem Biochem . 1985;  43 51-134
• 2 Lane D A, Björk I, Lindahl U. Heparin and Related Polysaccharides.  New York: Plenum Press; 1992
• 3 Thunberg L, Backstrom G, Lindahl U. Further characterization of the antithrombin-binding sequence in heparin.  Carbohydr Res . 1982;  100 393-410
• 4 Robinson H C, Horner A A, Höök M, Ogren S, Lindahl U. A proteoglycan form of heparin and its degradation to single-chain molecules.  J Biol Chem . 1978;  253 6687-6693
• 5 Coyne E. Heparin-past present and future. In: Lundblad RL, Brown WV, Mann KG, Roberts HR, eds. Chemistry and Biology of Heparin Amsterdam, The Netherlands: Elsevier-North Holland 1981: 9-17
• 6 Watt D K, Yorke S C, Slim G C. Comparison of ovine, bovine and porcine mucosal heparins by disaccharide analyses and ¹³C NMR.  Carbohydr Polym . 1997;  33 5-11
• 7 Hope J. Mice and beef and brain diseases.  Nature . 1995;  378 761-764
• 8 Mulloy B, Gray E, Barrowcliffe T W. Characterization of unfractionated heparin: comparison of materials from the last 50 years.  Thromb Haemost . 2000;  84 1052-1056
• 9 Conrad H E. Heparin-Binding Proteins.  San Diego, CA: Academic Press 1998: 61-112
• 10 Mulloy B, Johnson E A. Assignment of the ¹H-NMR spectra of heparin and heparan sulfate.  Carbohydr Res . 1987;  170 151-165
• 11 Casu B, Oreste P, Torri G. The structure of heparin oligosaccharide fragments with high anti-(factor Xa) activity containing the minimal antithrombin III-binding sequence.  Chemical and 13C-NMR studies. Biochem J . 1981;  197 599-609
• 12 Pervin A, Gallo C, Jandik K A, Han X J, Linhardt R J. Preparation and structural characterisation of large heparin-derived oligosaccharides.  Glycobiology . 1995;  5 83-95
• 13 Chai W, Hounsell E F, Bauer C J, Lawson A M. Characterisation by LSI-MS and ¹H-NMR spectroscopy of tetra-, hexa-, and octa-saccharide of porcine intestinal heparin.  Carbohydr Res . 1995;  269 139-156
• 14 Horne A, Gettins P. ¹H-NMR spectral assignments for two series of heparin-derived oligosaccharides.  Carbohydr Res . 1991;  225 43-57
• 15 Yamada S, Yamane Y, Tsuda H, Yoshida K, Sugahara K. A major common trisulfated hexasaccharide core sequence, hexuronic acid (2-sulfate)-glucosamine(N-sulfate)-iduronic acid-N-acetylglucosamine-glucuronic acid-glucosamine (N-sulfate), isolated from the low sulfated irregular region of porcine intestinal heparin.  J Biol Chem . 1998;  273 1863-1871
• 16 Desai U R, Wang H M, Kelly T R, Linhardt R J. Structure elucidation of a novel acidic tetrasaccharide and hexasaccharide derived from chemically modified heparin.  Carbohydr Res . 1995;  241 249-259
• 17 Yates E A, Santini F, Guerrini M. ¹H and ¹³C NMR spectral assignment of the major sequences of twelve systematically modified heparin derivatives.  Carbohydr Res . 1996;  294 15-27
• 18 Yates E A, Santini F, De Cristofano B. Effect of substitution pattern on ¹H, ¹³C NMR chemical shifts and ¹J_CH coupling constants in heparin derivatives.  Carbohydr Res . 2000;  329 239-247
• 19 Casu B, Johnson E A, Mantovani M. Correlation between structure, fat-clearing and anti-coagulant properties of heparins and heparan sulphates.  Arzneimittelforchung/Drug Res . 1983;  33 135-142
• 20 Casu B, Guerrini M, Naggi A. Differentiation of beef and pig mucosal heparins by NMR spectroscopy.  Thromb Haemost . 1995;  74 1205-1206
• 21 Casu B, Guerrini M, Naggi A. Characterization of sulfation patterns of beef and pig mucosal heparins by nuclear magnetic resonance spectroscopy.  Arzneimittelforchung/ Drug Res . 1996;  46 472-477
• 22 Neville A G, Mori F, Holme K R, Perlin A S. Monitoring the purity of pharmaceutical heparin preparations by high-field ¹H-nuclear magnetic resonance spectroscopy.  J Pharm Sci . 1989;  78 101-104
• 23 Iacomini M, Casu B, Guerrini M. ``Linkage Region'' sequences of heparin and heparan sulfates: detection and quantification by nuclear magnetic resonance spectroscopy.  Anal Biochem . 1999;  274 50-58
• 24 Casu B, Torri G. Structural characterisation of low molecular weight heparins.  Semin Thromb Hemost . 1999;  25 17-25
• 25 Casu B, Gennaro U. A conductimetric method for the determination of sulphate and carboxyl groups in heparin and other mucopolysaccharides.  Carbohydr Res . 1975;  39 168-176
• 26 Casu B, Naggi A, Torri G. Modulation of sulfation patterns of glycosaminoglycans. In: Fraser-Reid B, Tatsuta K, Thiem J, eds. Glycoscience: Chemistry and Chemical Biology, vol. III New York: Springer-Verlag; 2000: 1895-1904
• 27 Ruiz-Calero V, Saurina J, Galceran M T, Hernandez-Cassou S, Puignou L. Potentiality of proton nuclear magnetic resonance and multivariate calibration methods for the determination of dermatan sulfate contamination in heparin samples.  Analyst . 2000;  125 933-938
• 28 Jaseja M, Rej R N, Sauriol F, Perlin A S. Novel regio- and stereoselective modifications of heparin in alkaline solution. Nuclear magnetic resonance spectroscopic evidence.  Can J Chem . 1989;  67 1449-1456
• 29 Hricovini M, Guerrini M, Torri G, Piani S, Ungarelli F. Conformational analysis of heparin epoxide in aqueous solution. An NMR relaxation study.  Carbohydr Res . 1995;  277 11-23