Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events


Recently, certain lots of heparin have been associated with an acute, rapid onset of serious side effects indicative of an allergic-type reaction. To identify potential causes for this sudden rise in side effects, we examined lots of heparin that correlated with adverse events using orthogonal high-resolution analytical techniques. Through detailed structural analysis, the contaminant was found to contain a disaccharide repeat unit of glucuronic acid linked β1→3 to a β-N-acetylgalactosamine. The disaccharide unit has an unusual sulfation pattern and is sulfated at the 2-O and 3-O positions of the glucuronic acid as well as at the 4-O and 6-O positions of the galactosamine. Given the nature of this contaminant, traditional screening tests cannot differentiate between affected and unaffected lots. Our analysis suggests effective screening methods that can be used to determine whether or not heparin lots contain the contaminant reported here.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


Prices may be subject to local taxes which are calculated during checkout

Figure 1: NMR analysis of contaminated heparin.
Figure 2: Chemical structures of major repeat units.
Figure 3: Detailed NMR structural analysis of the contaminant.
Figure 4: Structural assignment of the contaminant by NMR using isolated and synthetic materials.
Figure 5: Liquid chromatography–mass spectrometry (LC-MS) analysis of the contaminant.


  1. Capila, I. & Linhardt, R.J. Heparin-protein interactions. Angew. Chem. Int. Edn. Engl. 41, 391–412 (2002).

    Article  Google Scholar 

  2. Lepor, N.E. Anticoagulation for acute coronary syndromes: from heparin to direct thrombin inhibitors. Rev. Cardiovasc. Med. 8 (suppl. 3), S9–S17 (2007).

    PubMed  Google Scholar 

  3. Fischer, K.G. Essentials of anticoagulation in hemodialysis. Hemodial. Int. 11, 178–189 (2007).

    Article  Google Scholar 

  4. Contaminant detected in heparin material of specified origin in the USA and in Germany; serious adverse events reported; recall measures initiated. World Health Organization Alert No. 118 (7 March 2008). < drugalerts/Alert_118_Heparin.pdf>

  5. Notice of Recall from Rotexmedica to Bfarm (German Regulatory Authorities). Rotexmedica/Bfarm Notice (7 March 2008). <>

  6. Acute allergic-type reactions among patients undergoing hemodialysis—multiple states, 2007–2008. Morbid. Mortal. Wkly Rept. 57 (1 February 2008).

  7. 2008 Heparin Recall Information. Baxter Investigation Updates (5, 14, 19 March 2008). < biopharmaceuticals/heparin.html>

  8. Baxter provides update on investigation. Baxter Investigation Update (14 March 2008). < downloads/heparin_03-14-08.pdf>

  9. Communication. Information on heparin sodium injection. US Food and Drug Administration. <>

  10. Holme, K.R. & Perlin, A.S. Nuclear magnetic resonance spectra of heparin in admixture with dermatan sulfate and other glycosaminoglycans. 2-D spectra of the chondroitin sulfates. Carbohydr. Res. 186, 301–312 (1989).

    Article  CAS  Google Scholar 

  11. Guerrini, M., Bisio, A. & Torri, G. Combined quantitative 1H and 13C nuclear magnetic resonance spectroscopy for characterization of heparin preparations. Semin. Thromb. Hemost. 27, 473–482 (2001).

    Article  CAS  Google Scholar 

  12. Myette, J.R. et al. Molecular cloning of the heparin/heparan sulfate delta 4,5 unsaturated glycuronidase from Flavobacterium heparinum, its recombinant expression in Escherichia coli, and biochemical determination of its unique substrate specificity. Biochemistry 41, 7424–7434 (2002).

    Article  CAS  Google Scholar 

  13. Myette, J.R. et al. The heparin/heparan sulfate 2-O-sulfatase from Flavobacterium heparinum. Molecular cloning, recombinant expression, and biochemical characterization. J. Biol. Chem. 278, 12157–12166 (2003).

    Article  CAS  Google Scholar 

  14. Kuberan, B. et al. Analysis of heparan sulfate oligosaccharides with ion pair-reverse phase capillary high performance liquid chromatography-microelectrospray ionization time-of-flight mass spectrometry. J. Am. Chem. Soc. 124, 8707–8718 (2002).

    Article  CAS  Google Scholar 

  15. Maruyama, T., Toida, T., Imanari, T., Yu, G. & Linhardt, R.J. Conformational changes and anticoagulant activity of chondroitin sulfate following its O-sulfonation. Carbohydr. Res. 306, 35–43 (1998).

    Article  CAS  Google Scholar 

  16. Solvolytic desulfation of glycosaminoglycuronan sulfates with dimethyl sulfoxide containing water or methanol. Nagasawa, K., Inoue, Y. & Kamata, T. Carbohydr. Res. 58, 47–55 (1977).

    Article  CAS  Google Scholar 

  17. Linhardt, R.J. Analysis of glycosaminoglycans with polysaccharide lyases. in Current Protocols in Molecular Biology (eds. Ausubel, F.A. et al.) 17.13B.1–17B.13.16 (Wiley, New York, 1994).

    Google Scholar 

  18. Kinoshita, A. et al. Novel tetrasaccharides isolated from squid cartilage chondroitin sulfate E contain unusual sulfated disaccharide units GlcA(3-O-sulfate)β1-3GalNAc(6-O-sulfate) or GlcA(3-O-sulfate)β1-3GalNAc. J. Biol. Chem. 272, 19656–19665 (1997).

    Article  CAS  Google Scholar 

  19. Hadding, U. et al. Ability of the T cell-replacing polyanion dextran sulfate to trigger the alternate pathway of complement activation. Eur. J. Immunol. 3, 527–529 (1973).

    Article  CAS  Google Scholar 

  20. Kang, O.H. et al. Suppressive effect of non-anaphylactogenic anti-IgE antibody on the development of dextran sulfate sodium-induced colitis. Int. J. Mol. Med. 18, 893–899 (2006).

    CAS  PubMed  Google Scholar 

  21. Siebeck, M. et al. Dextran sulfate activates contact system and mediates arterial hypotension via B2 kinin receptors. J. Appl. Physiol. 77, 2675–2680 (1994).

    Article  CAS  Google Scholar 

  22. Hojima, Y., Cochrane, C.G., Wiggins, H.C., Austen, K.F. & Steven, R.L. In vitro activation of the contact (Hageman factor) system of plasma by heparin and chondroitin sulfate E. Blood 63, 1453–1459 (1984).

    CAS  PubMed  Google Scholar 

  23. Naggi, A. et al. Sulfamino-galactosaminoglycans, a new class of semi-synthetic polysaccharides. in Biomedical and Biotechnological Advances in Industrial Polysaccharides (eds. Crescenzi, V., Dea, I.C.M. & Paoletti, S.) 101–108 (Gordon & Breach Science, New York, 1989).

    Google Scholar 

  24. Siegmenth, W. & Radi, I. Comparison of glycosaminoglycan polysulfate (Arteparon) and physiological saline solution in arthrosis of the large joints. Results of a multicenter double-blind study. Z. Rheumatol. 42, 223–228 (1983).

    Google Scholar 

  25. Greinacher, A., Michels, I., Schäfer, M., Kiefel, V. & Mueller-Eckhardt, C. Heparin-associated thrombocytopenia in a patient treated with polysulphated chondroitin sulphate: evidence for immunological crossreactivity between heparin and polysulphated glycosaminoglycan. Br. J. Haematol. 81, 252–254 (1992).

    Article  CAS  Google Scholar 

  26. Weimann, G. et al. Glucosamine sulfate does not crossreact with the antibodies of patients with heparin-induced thrombocytopenia. Eur. J. Haematol. 66, 195–199 (2001).

    Article  CAS  Google Scholar 

  27. Cicero, O., Barbato, G. & Bazzo, R. Sensitivity enhancement of a two-dimensional experiment for the measurement of heteronuclear long-range coupling constants, by a new scheme of coherence selection by gradients. J. Magn. Reson. 148, 209–213 (2001).

    Article  CAS  Google Scholar 

  28. Cifonelli, J.C. Reaction of heparitin sulfate with nitrous acid. Carbohydr. Res. 8, 233–242 (1968).

    Article  CAS  Google Scholar 

  29. Wishart, D.S. et al. 1H, 13C and 15N chemical shift referencing in biomolecular NMR. J. Biomol. NMR 6, 135–140 (1995).

    Article  CAS  Google Scholar 

Download references


The authors would like to thank Sucharita Roy for work on the chemical synthesis of oversulfated chondroitin sulfate standards, Scott Bailey for assistance in the analysis of selected samples by one- and two-dimensional NMR and Laura Citterio for work in various separation and analytical steps. We also thank James Anderson, Josh Sorafine and Andre Jones for design of composition experiments and HPLC analysis of the heparinase-digested samples. Finally, we thank Ada Ziolkowski for help with manuscript preparation. This work was supported in part by US National Institute of General Medical Sciences grants GM57073 (R.S.) and GM38060 (R.J.L).

Author information

Authors and Affiliations


Corresponding author

Correspondence to Ram Sasisekharan.

Ethics declarations

Competing interests

R.L. and R.S. have been directors of Momenta Pharmaceuticals since 2001 and own stock in the company, and both of them have served as scientific advisors to Scientific Protein Labs (SPL) since the end of February 2008. R.J.L. has served as a scientific advisor to Baxter International since mid-February 2008.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 (PDF 1470 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Guerrini, M., Beccati, D., Shriver, Z. et al. Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events. Nat Biotechnol 26, 669–675 (2008).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing