Nitrous Acid Degradation of Glycosaminoglycans

Conrad, H. Edward

doi:10.1002/0471142727.mb1722as32

Cited by 15 publications

(10 citation statements)

References 5 publications

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“…Treatment of the isolated impurity with hydrazine to remove the acetyl group of the glucosamine, followed by treatment with nitrous acid, was effective in digesting the impurity, suggesting a chondroitin‐like GAG. The nitrous acid digest, when analyzed by ion‐pairing high‐performance liquid chromatography (HPLC)–negative ion MS, yielded a series of components corresponding to fully sulfated deacetylated disaccharides, tetrasaccharides, and hexasaccharides with incomplete deacetylation 11 . The nitrous acid digest of synthetically prepared FSCS, when analyzed under the same conditions, produced very similar liquid chromatography/mass spectrometry (LC/MS) data in terms of the number of components, elution pattern, and measured molecular weights of the major components 6 .…”

Section: Resultsmentioning

confidence: 99%

Structure Elucidation and Biological Activity of the Oversulfated Chondroitin Sulfate Contaminant in Baxter Heparin

McKee

Bairstow

Szabo

et al. 2010

The Journal of Clinical Pharma

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From late December 2007 to February 2008, the number of adverse responses to heparin infusions rose noticeably above baseline levels in North America, ultimately resulting in a widespread recall of all heparin vial products made by Baxter Healthcare. Using various analytical techniques and the de novo synthesis of a fully sulfated chondroitin sulfate (FSCS) derivative, the authors have confirmed the identity of the contaminant as an oversulfated chondroitin sulfate (OSCS) and have also defined the heterogeneity and concentration of this contaminant in various lots of heparin. Using both contaminated heparin products and the synthetically produced derivative, the authors have shown that the OSCS produces a dose-dependent hypotension in both pigs and rats and that the response in rats can be abrogated with bradyzide, a rodent-selective B(2) bradykinin receptor antagonist. The no observed effect level (NOEL) for this contaminant appears to be approximately 1 mg/kg, corresponding to a contamination level in finished lots of heparin of approximately 3%. Using human plasma, the OSCS derivative was shown to activate kallikrein. These data provide insight into the etiology of the adverse events, particularly refractory hypotension, observed in patients who were exposed to heparin contaminated with OSCS.

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Section: Resultsmentioning

confidence: 99%

Structure Elucidation and Biological Activity of the Oversulfated Chondroitin Sulfate Contaminant in Baxter Heparin

McKee

Bairstow

Szabo

et al. 2010

The Journal of Clinical Pharma

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“…Nitrous acid (HONO) degradation. HONO degradation was performed by the method of Conrad et al (78). 100 l of either the pH 1.5 or pH 4.0 HONO reagent was added to tubes containing ‫ف‬ 2,000 cpm of the [ 3 H] tetradecasaccharide and each pooled fraction isolated from the L-and P-selectin affinity columns.…”

Section: Methodsmentioning

confidence: 99%

“…To pursue this, aliquots of bound and unbound material from the P-and L-selectin columns were subjected to cleavage either with specific heparin lyases (I, II, and III), or by HONO treatment at pH 1.5 or 4.0. Each of these treatments can cleave heparin chains, depending upon specific structural features or modifications (72,(78)(79)(80)(81). The treated samples were analyzed by FPLC as described in Methods to evaluate the extent of cleavage (detailed data not shown).…”

Section: Analysis Of the Structural Features On [ 3 H] Tetradecasacchmentioning

confidence: 99%

Differential interactions of heparin and heparan sulfate glycosaminoglycans with the selectins. Implications for the use of unfractionated and low molecular weight heparins as therapeutic agents.

Koenig¹,

Norgard-Sumnicht²,

Linhardt³

et al. 1998

J. Clin. Invest.

377

288

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The selectins are calcium-dependent C-type lectins that bind certain sialylated, fucosylated, sulfated glycoprotein ligands. L-selectin also recognizes endothelial proteoglycans in a calcium-dependent manner, via heparan sulfate (HS) glycosaminoglycan chains enriched in unsubstituted glucosamine units. We now show that these HS chains can also bind P-selectin, but not E-selectin. However, while L-selectin binding requires micromolar levels of free calcium, P-selectin recognition is largely divalent cation-independent. Despite this, HS chains bound to P-selectin are eluted by ethylenediamine tetraacetic acid (EDTA), but only at high concentrations. Porcine intestinal mucosal (mast cell-derived) heparin (PIM-heparin) shows similar properties, with no binding to E-selectin, calcium-dependent binding of a subfraction to L-selectin and to P-selectin, and calcium-independent binding of a larger fraction to P-selectin, the latter being disrupted by high EDTA concentrations. Analysis of defined heparin fragment pools shows a size dependence for interaction, with tetradecasaccharides showing easily detectable binding to L- and P-selectin affinity columns. L-selectin binding fragments include more heavily sulfated and epimerized regions and, as with the endothelial HS chains, they are enriched in free amino groups. The P-selectin binding component includes this fraction as well as some less highly modified regions. Thus, endothelium-derived HS chains and mast cell-derived heparins could play a role in modulating the biology of selectins in vivo. Notably, P- and L-selectin binding to sialyl-Lewisx and to HL-60 cells (which are known to carry the native ligand PSGL-1) is inhibited by unfractionated pharmaceutical heparin preparations at concentrations 12-50-fold lower than those recommended for effective anticoagulation in vivo. In contrast, two low molecular weight heparins currently considered as clinical replacements for unfractionated heparin are much poorer inhibitors. Thus, patients undergoing heparin therapy for other reasons may be experiencing clinically significant inhibition of L- and P-selectin function, and the current switchover to low-molecular weight heparins may come at some loss of this effect. Low-dose unfractionated heparin should be investigated as a treatment option for acute and chronic diseases in which P- and L-selectin play pathological roles.

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“…As a consequence, controlled enzymatic or chemical depolymerization is often required to lower their molecular weight and simplify these polydisperse mixtures before analysis 32,33 . Although enzymatic depolymerization offers advantages based on enzyme specificity, chemical depolymerization using reagents with high chemoselectivity is also quite valuable 32,33 . Electrospray ionization (ESI)-MS can be interfaced with HPLC and thus offers the possibility to analyze mixtures of GAG-derived oligosaccharides.…”

Section: Introductionmentioning

confidence: 99%

High-performance liquid chromatography-mass spectrometry for mapping and sequencing glycosaminoglycan-derived oligosaccharides

Volpi

Linhardt

2010

Nat Protoc

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Glycosaminoglycans (GAGs) have proven to be very difficult to analyze and characterize because of their high negative charge density, polydispersity and sequence heterogeneity. As the specificity of the interactions between GAGs and proteins results from the structure of these polysaccharides, an understanding of GAG structure is essential for developing a structure–activity relationship. Electrospray ionization (ESI) mass spectrometry (MS) is particularly promising for the analysis of oligosaccharides chemically or enzymatically generated by GAGs because of its relatively soft ionization capacity. Furthermore, on-line high-performance liquid chromatography (HPLC)-MS greatly enhances the characterization of complex mixtures of GAG-derived oligosaccharides, providing important structural information and affording their disaccharide composition. A detailed protocol for producing oligosaccharides from various GAGs, using controlled, specific enzymatic or chemical depolymerization, is presented, together with their HPLC separation, using volatile reversed-phase ion-pairing reagents and on-line ESI-MS structural identification. This analysis provides an oligosaccharide map together with sequence information from a reading frame beginning at the nonreducing end of the GAG chains. The preparation of oligosaccharides can be carried out in 10 h, with subsequent HPLC analysis in 1–2 h and HPLC-MS analysis taking another 2 h.

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Nitrous Acid Degradation of Glycosaminoglycans

Cited by 15 publications

References 5 publications

Structure Elucidation and Biological Activity of the Oversulfated Chondroitin Sulfate Contaminant in Baxter Heparin

Structure Elucidation and Biological Activity of the Oversulfated Chondroitin Sulfate Contaminant in Baxter Heparin

Differential interactions of heparin and heparan sulfate glycosaminoglycans with the selectins. Implications for the use of unfractionated and low molecular weight heparins as therapeutic agents.

High-performance liquid chromatography-mass spectrometry for mapping and sequencing glycosaminoglycan-derived oligosaccharides

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