1D and 2D-HSQC NMR: Two Methods to Distinguish and Characterize Heparin From Different Animal and Tissue Sources.

The US Food and Drug Administration has encouraged the reintroduction of bovine heparin drug product to the US market to mitigate the risks of heparin shortages and potential adulteration or contamination of the primary source which is porcine heparin. Here, a 1D-NMR method was applied to compare heparin sodium of bovine intestinal origin with that of bovine lung, porcine, or ovine intestinal origin. The results showed that a simple 1D test using NMR signal intensity ratios among diagnostic signals of the proton spectra uniquely identified the origin of heparin and concomitantly could be used to assure the correct sample labeling. However, a limitation of the use of only mono-dimensional spectra is that these spectra may not provide sufficiently detailed information on the composition of heparin batches to adequately determine the quality of this complex product. As an alternative, a higher resolution quantitative 2D-HSQC method was used to calculate the percentage of mono- and disaccharides, distinguish the origin of heparin and, simultaneously, assess the heparin composition. The 2D-HSQC method is proposed to provide sufficient information to evaluate the quality of industrial production process used to make the drug substance. Together, the 1D and 2D data produced by these measurements can be used to assure the identity and purity of this widely used drug.

Structural and Functional Components of the Skate Sensory Organ Ampullae of Lorenzini.

The skate, a cartilaginous fish related to sharks and rays, possesses a unique electrosensitive sensory organ known as the ampullae of Lorenzini (AoL). This organ is responsible for the detection of weak electric field changes caused by the muscle contractions of their prey. While keratan sulfate (KS) is believed to be a component of a jelly that fills this sensory organ and has been credited with its high proton conductivity, modern analytical methods have not been applied to its characterization. Surprisingly, total glycosaminoglycan (GAG) analysis demonstrates that the KS from skate jelly is extraordinarily pure, containing no other GAGs. This KS had a molecular weight of 20 to 30 kDa, consisting primarily of N-linked KS comprised mostly of a monosulfated disaccharide repeating unit, →3) Gal (1→4) GlcNAc6S (1→. Proteomic analysis of AoL jelly suggests that transferrin, keratin, and mucin serve as KS core proteins. Actin and tropomyosin are responsible for assembling the macrostructure of the jelly, and parvalbumin α-like protein and calreticulin regulate calcium and potassium channels involved in the transduction of the electrical signal, once conducted down the AoL by the jelly, serving as the molecular basis for electroreception.

Isolation of a lectin binding rhamnogalacturonan-I containing pectic polysaccharide from pumpkin.

A rhamnogalacturonan-I (RG-I) containing pectic polysaccharide (PPc) was isolated from pumpkin following a low-temperature alkali treatment and a combination of gradual alcohol precipitation and ion-exchange. Monosaccharide compositional analysis of PPc revealed the presence of rhamnose, galacturonic acid, galactose, and arabinose in a molar ratio of 7.4: 25: 28: 2.6. Structural and linkage analysis by 1D NMR (1H NMR and 13C NMR), and 2D NMR (COSY, TOCSY, HSQC, and elevated temperature HMBC) suggested that PPc was a RG-I-like pectic polysaccharide, branched at the C-4 of some of the (about 29% of) rhamnosyl units, with relatively long ß-1,4-d-galactan side chains to which were attached, through the C-3 of ß-d-Gal, terminal non reducing α-Araf units. The results of surface plasmon resonance (SPR) show that PPc binds to two types of lectin, Ricinus communis agglutinin 120 (RCA120) and Galectin-3 (Gal-3). These binding studies show quick association and slow dissociation with a moderate binding affinity between PPc and Gal-3 of 1.26µM. The interaction between PPc and Gal-3 suggest the potential use of pumpkin pectic polysaccharide as a Gal-3 inhibitor in functional food or drug development applications.

Expanding glycosaminoglycan chemical space: towards the creation of sulfated analogs, novel polymers and chimeric constructs.

Glycosaminoglycans (GAGs) have therapeutic potential in areas ranging from angiogenesis, inflammation, hemostasis and cancer. GAG bioactivity is conferred by intrinsic structural features, such as disaccharide composition, glycosidic linkages and sulfation pattern. Unfortunately, the in vitro enzymatic synthesis of defined GAGs is quite restricted by a limited understanding of current GAG synthases and modifying enzymes. Our work provides insights into GAG-active enzymes through the creation of sulfated oligosaccharides, a new polysaccharide and chimeric polymers. We show that a C6-sulfonated uridine diphospho (UDP)-glucose (Glc) derivative, sulfoquinovose, can be used as an uronic acid donor, but not as a hexosamine donor, to cap hyaluronan (HA) chains by the HA synthase from the microbe Pasteurella multocida. However, the two heparosan (HEP) synthases from the same species, PmHS1 and PmHS2, could not employ the UDP-sulfoquinovose under similar conditions. Serendipitously, we found that PmHS2 co-polymerized Glc with glucuronic acid (GlcA), creating a novel HEP-like polymer we named hepbiuronic acid [-4-GlcAß1-4-Glcα1-]n. In addition, we created chimeric block polymers composed of both HA and HEP segments; in these reactions GlcA-, but not N-acetylglucosamine-(GlcNAc), terminated GAG acceptors were recognized by their noncognate synthase for further extension, likely due to the common ß-linkage connecting GlcA to GlcNAc in both of these GAGs. Overall, these GAG constructs provide new tools for studying biology and offer potential for future sugar-based therapeutics.

Parent heparin and daughter LMW heparin correlation analysis using LC-MS and NMR.

Heparin is a structurally complex, polysaccharide anticoagulant derived from livestock, primarily porcine intestinal tissues. Low molecular weight (LMW) heparins are derived through the controlled partial depolymerization of heparin. Increased manufacturing and regulatory concerns have provided the motivation for the development of more sophisticated analytical methods for determining both their structure and pedigree. A strategy, for the comprehensive comparison of parent heparins and their LMW heparin daughters, is described that relies on the analysis of monosaccharide composition, disaccharide composition, and oligosaccharide composition. Liquid chromatography-mass spectrometry is rapid, robust, and amenable to automated processing and interpretation of both top-down and bottom-up analyses. Nuclear magnetic resonance spectroscopy provides complementary top-down information on the chirality of the uronic acid residues and glucosamine substitution. Principal component analysis (PCA) was applied to the normalized abundance of oligosaccharides, calculated in the bottom-up analysis, to show parent and daughter correlation in oligosaccharide composition. Using these approaches, six pairs of parent heparins and their daughter generic enoxaparins from two different manufacturers were comprehensively analyzed. Enoxaparin is the most widely used LMW heparin and is prepared through controlled chemical ß-eliminative cleavage of porcine intestinal heparin. Lovenox®, the innovator version of enoxaparin marketed in the US, was analyzed as a reference for the daughter LMW heparins. The results, show similarities between LMW heparins from two different manufacturers with Lovenox®, excellent lot-to-lot consistency of products from each manufacturer, and detects a correlation between each parent heparin and daughter LMW heparin.

Comparison of Low-Molecular-Weight Heparins Prepared From Bovine Heparins With Enoxaparin.

Heparin and its low-molecular-weight heparin (LMWH) derivatives are widely used clinical anticoagulants. These drugs are critical for the practice of medicine in applications including kidney dialysis, cardiopulmonary bypass, and in the management of venous thromboembolism. Currently, these drugs are derived from livestock, primarily porcine intestine. The worldwide dependence on a single animal species has made the supply chain for this critical drug quite fragile, leading to the search for other sources of these drugs, including bovine tissues such as bovine intestine or lung. A number of laboratories are currently examining the similarities and differences between heparins prepared from porcine and bovine tissues. The current study is designed to compare LMWH prepared from bovine heparins through chemical ß-elimination, a process currently used to prepare the LMWH, enoxaparin, from porcine heparin. Using top-down, bottom-up, compositional analysis and bioassays, LMWHs, derived from bovine lung and intestine, are shown to closely resemble enoxaparin.

Structural Analysis of Heparin-Derived 3-O-Sulfated Tetrasaccharides: Antithrombin Binding Site Variants.

Heparin is a polysaccharide that is widely used as an anticoagulant drug. The mechanism for heparin's anticoagulant activity is primarily through its interaction with a serine protease inhibitor, antithrombin III (AT), that enhances its ability to inactivate blood coagulation serine proteases, including thrombin (factor IIa) and factor Xa. The AT-binding site in the heparin is one of the most well-studied carbohydrate-protein binding sites and its structure is the basis for the synthesis of the heparin pentasaccharide drug, fondaparinux. Despite our understanding of the structural requirements for the heparin pentasaccharide AT-binding site, there is a lack of data on the natural variability of these binding sites in heparins extracted from animal tissues. The present work provides a detailed study on the structural variants of the tetrasaccharide fragments of this binding site afforded following treatment of a heparin with heparin lyase II. The 5 most commonly observed tetrasaccharide fragments of the AT-binding site are fully characterized, and a method for their quantification in heparin and low-molecular-weight heparin products is described.

Top-down and bottom-up analysis of commercial enoxaparins.

A strategy for the comprehensive analysis of low molecular weight (LMW) heparins is described that relies on using an integrated top-down and bottom-up approach. Liquid chromatography-mass spectrometry, an essential component of this approach, is rapid, robust, and amenable to automated processing and interpretation. Nuclear magnetic resonance spectroscopy provides complementary top-down information on the chirality of the uronic acid residues comprising a low molecular weight heparin. Using our integrated approach four different low molecular weight heparins prepared from porcine heparin through chemical ß-eliminative cleavage were comprehensively analyzed. Lovenox™ and Clexane™, the innovator versions of enoxaparin marketed in the US and Europe, respectively, and two generic enoxaparins, from Sandoz and Teva, were analyzed. The results which were supported by analysis of variation (ANOVA), while showing remarkable similarities between different versions of the product and good lot-to-lot consistency of each product, also detects subtle differences that may result from differences in their manufacturing processes or differences in the source (or parent) porcine heparin from which each product is prepared.

Quantitative analysis of the major linkage region tetrasaccharides in heparin.

Heparin is a polysaccharide based anticoagulant drug composed of a complex mixture of glycosaminoglycan chains and peptidoglycosaminoglycan chains. In an effort to better characterize this important polysaccharide based drug, we examined the peptide components of the minor peptidoglycosaminoglycan chains comprising heparin. Three different the glycan-peptide linkage regions tetrasaccharide fragments were isolated from pharmaceutical heparin using heparin lyase II and characterized the structure of these tetrasaccharides using nuclear magnetic resonance spectroscopy and mass spectrometry. A sensitive and quantitative assay was developed for these linkage regions using multiple reaction-monitoring tandem mass spectrometry. These three different linkage regions were found in heparins coming from porcine intestine and bovine lung. Two of these were also present in the low molecular weight heparin, enoxaparin.

Heparin and anticoagulation.

Heparin, a sulfated polysaccharide, has been used as a clinical anticoagulant for over 90 years. Newer anticoagulants, introduced for certain specialized applications, have not significantly displaced heparin and newer heparin-based anticoagulants in most medical procedures. This chapter, while reviewing anticoagulation and these newer anticoagulants, focuses on heparin-based anticoagulants, including unfractionated heparin, low molecular weight heparins and ultra-low molecular weight heparins. Heparin's structures and its biological and therapeutic roles are discussed. Particular emphasis is placed on heparin's therapeutic application and its adverse effects. The future prospects are excellent for new heparins and new heparin-based therapeutics with improved properties.

Analysis of Heparins Derived From Bovine Tissues and Comparison to Porcine Intestinal Heparins.

Heparin is a widely used clinical anticoagulant. It is also a linear glycosaminoglycan with an average mass between 10 and 20 kDa and is primarily made up of trisulfated disaccharides comprised of 1,4-linked iduronic acid and glucosamine residues containing some glucuronic acid residues. Heparin is biosynthesized in the Golgi of mast cells commonly found in the liver, intestines, and lungs. Pharmaceutical heparin currently used in the United States is primarily extracted from porcine intestines. Other sources of heparin including bovine intestine and bovine lung are being examined as potential substitutes for porcine intestinal heparin. These additional sources are intended to serve to diversify the heparin supply, making this lifesaving drug more secure. The current study examines bovine heparins prepared from both intestines and lung and compares these to porcine intestinal heparin. The structural properties of these heparins are examined using nuclear magnetic resonance, gel permeation chromatography, and disaccharide analysis of heparinase-catalyzed depolymerized heparin. The in vitro functional activities of these heparins have also been determined. The goal of this study is to establish the structural and functional similarities and potential differences between bovine and porcine heparins. Porcine and bovine heparins have structural and compositional similarities and differences.