Blair A. Fraser

Contaminated Heparin Associated with Adverse Clinical Events and Activation of the Contact System

BACKGROUND There is an urgent need to determine whether oversulfated chondroitin sulfate (OSCS), a compound contaminating heparin supplies worldwide, is the cause of the severe anaphylactoid reactions that have occurred after intravenous heparin administration in the United States and Germany. METHODS Heparin procured from the Food and Drug Administration, consisting of suspect lots of heparin associated with the clinical events as well as control lots of heparin, were screened in a blinded fashion both for the presence of OSCS and for any biologic activity that could potentially link the contaminant to the observed clinical adverse events. In vitro assays for the activation of the contact system and the complement cascade were performed. In addition, the ability of OSCS to recapitulate key clinical manifestations in vivo was tested in swine. RESULTS The OSCS found in contaminated lots of unfractionated heparin, as well as a synthetically generated OSCS reference standard, directly activated the kinin-kallikrein pathway in human plasma, which can lead to the generation of bradykinin, a potent vasoactive mediator. In addition, OSCS induced generation of C3a and C5a, potent anaphylatoxins derived from complement proteins. Activation of these two pathways was unexpectedly linked and dependent on fluid-phase activation of factor XII. Screening of plasma samples from various species indicated that swine and humans are sensitive to the effects of OSCS in a similar manner. OSCS-containing heparin and synthetically derived OSCS induced hypotension associated with kallikrein activation when administered by intravenous infusion in swine. CONCLUSIONS Our results provide a scientific rationale for a potential biologic link between the presence of OSCS in suspect lots of heparin and the observed clinical adverse events. An assay to assess the amidolytic activity of kallikrein can supplement analytic tests to protect the heparin supply chain by screening for OSCS and other highly sulfated polysaccharide contaminants of heparin that can activate the contact system.

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.

Isolation and primary structure of a peptide from the corpora cardiaca of Heliothis zea with adipokinetic activity

An adipokinetic hormone was isolated from the corpora cardiaca of the corn ear worm moth, Heliothis zea, and purified by reversed phase high performance liquid chromatography. The primary structure, pGlu-Leu-Thr-Phe-Thr-Ser-Ser-Trp-Gly-NH2, was determined by automated gas-phase Edman degradation of the peptide deblocked with pyroglutamic aminopeptidase, and by fast atom bombardment mass spectrometry. The hormone was synthesized and the natural and synthetic material had identical chromatographic, spectroscopic, and biological properties. The peptide was found to have lipid mobilizing activity in H. zea adults.

The structure of the repeating oligosaccharide unit of the pneumococcal capsular polysaccharide type 18C

The structure of the repeating oligosaccharide of the pneumococcal capsular polysaccharide type 18C has been investigated. The repeating oligosaccharide, isolated from an aqueous hydrofluoric acid hydrolyzate of the polysaccharide, was shown, by fast atom bombardment-mass spectrometry, to have a molecular weight of 928, and to contain an O-acetyl group and a glycerol residue. Information about the sequence in the per-O-methylated oligosaccharide was derived from electron-impact mass spectrometry. Supporting data were obtained from methylation analysis, periodate and chromium trioxide oxidations, and enzymic and acid hydrolyses of the oligosaccharide. These studies indicated that the polysaccharide consists of the following pentasaccharide repeating unit. (formula see text)

Synthesis and fast-atom-bombardment-mass spectrometry of N-acetylmuramoyl-l-alanyl-d-isoglutamine (MDP)

N-Acetylmuramoyl-L-alanyl-D-isoglutamine (MDP) was synthesized by a series of condensations of appropriate reagents, followed by hydrogenolysis. Each intermediate step resulted in a stable, crystalline product. D-Isoglutamine 4-benzyl ester was condensed with N-(tert-butoxycarbonyl)-L-alanine N-hydroxysuccinimide ester, to give N-(tert-butoxycarbonyl)-L-alanyl-D-isoglutamine benzyl ester. Condensation of L-alanyl-D-isoglutamine benzyl ester with N-acetyl-1-O-benzyl-4,6-O-benzylidenemuramic acid, followed by hydrogenolysis, gave MDP. The synthetic scheme was shown to be capable of producing gram quantities of highly pure MDP, as well as a few of its analogs. The synthetic MDP was characterized by analytical and biological methods, and it was found that the use of fast-atom-bombardment-mass spectrometry may greatly simplify the characterization process.

Fast Atom Bombardment Mass Spectrometry: Application to Peptide Structural Analysis

Study of structure-function relationships for biological molecules is a central theme of contemporary biomedical research. Once isolated and purified, these molecules require structural characterization before they can be used effectively to study their function in the living organism. One important subset, polypeptides, are often only available in picomole quantities. To structurally analyze these small quantities has been one goal of protein chemistry. Several methods have been developed to determine the amino acid composition, amino, acid sequence, and covalent chemical structure of polypeptides. Amino acid analysis requires only picomoles of polypeptide 1. The amino acid sequence for a polypeptide can be determined from picomole amounts by automated sequential Edman degradation followed by high performance liquid chromatographic identification of the resulting phenylthiohydantoin amino acids 2. The molecular weight and other covalent structures can be inferred from the mass spectrum of picomoles of the polypeptide. Integration of these technologies is useful in providing structural information about these small quantities.