RESUMO
Sulodexide, a heparinoid medicine, is wildly used in clinic for prophylaxis and treatment of thromboembolic diseases and diabetic nephropathy. Despite its widespread use, the structure of Sulodexide remains poorly understood. It consists of various polysaccharides characterized by differing sugar compositions, linkages, and sulfonation patterns, yet they share common features such as strong hydrophilicity, high native charges, and considerable polydispersity, posing significant challenges for conventional chromatographic and online mass spectrometry (MS) characterization. In this work, a novel analytical method combining multiple-heart cut 2D-LC and in-source acid-induced dissociation (inAID) MS was developed. Three polysaccharides in Sulodexide were separated by high efficient strong-anion-exchange chromatography, followed by desalting with the second dimensional size-exclusion chromatography before MS. A novel MS strategy employing inAID technique was utilized for online analysis, leading to the initial identification of Sulodexide polysaccharide components. The results were validated through disaccharide composition analysis of those three polysaccharide components after offline preparation. This advanced strategy, merging various techniques, enable a comprehensive structural elucidation of such complex drugs and provides a viable tool for potential routine analysis of complex biomolecules.
RESUMO
Heparin-induced thrombocytopenia (HIT) is an immune complication of heparin therapy. Antibodies binding to complexes of platelet factor 4 (PF4) and heparin is the trigger of HIT. A method using size exclusion chromatography with multi-angle laser light scattering detector (SEC-MALS) was developed in this work. The soluble ultra-large complex (ULC) was separated from the small complex (SC) and their molecular weights (MWs) were firstly measured. The complexes of PF4 and three heparins with different MW, including unfractionated heparin (UFH), dalteparin (Daltep) and enoxaparin (Eno) were characterized using this method. The contents and the sizes of ULC increased gradually when heparins were added to PF4 to certain amounts. While, they reduced after more heparins were added. It is the first time to measure the MWs of the biggest ULC of PF4-heparins as millions of Dalton. at the proper ratios of PF4 to heparin (PHR). Meanwhile, those mixtures at those certain PHRs induced the higher expression of CD83 and CD14 markers on dendritic cells (DCs) suggesting that they had stronger immunogenicity and is critical for HIT.
Assuntos
Heparina , Trombocitopenia , Humanos , Heparina/farmacologia , Fator Plaquetário 4/química , Fator Plaquetário 4/metabolismo , Anticoagulantes/química , Trombocitopenia/induzido quimicamente , Fatores Imunológicos , Cromatografia em GelRESUMO
Decellularized lung scaffolds and hydrogels are increasingly being utilized in ex vivo lung bioengineering. However, the lung is a regionally heterogenous organ with proximal and distal airway and vascular compartments of different structures and functions that may be altered as part of disease pathogenesis. We previously described decellularized normal whole human lung extracellular matrix (ECM) glycosaminoglycan (GAG) composition and functional ability to bind matrix-associated growth factors. We now determine differential GAG composition and function in airway, vascular, and alveolar-enriched regions of decellularized lungs obtained from normal, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF) patients. Significant differences were observed in heparan sulfate (HS), chondroitin sulfate (CS), and hyaluronic acid (HA) content and CS/HS compositions between both different lung regions and between normal and diseased lungs. Surface plasmon resonance demonstrated that HS and CS from decellularized normal and COPD lungs similarly bound fibroblast growth factor 2, but that binding was decreased in decellularized IPF lungs. Binding of transforming growth factor ß to CS was similar in all three groups but binding to HS was decreased in IPF compared to normal and COPD lungs. In addition, cytokines dissociate faster from the IPF GAGs than their counterparts. The differences in cytokine binding features of IPF GAGs may result from different disaccharide compositions. The purified HS from IPF lung is less sulfated than that from other lungs, and the CS from IPF contains more 6-O-sulfated disaccharide. These observations provide further information for understanding functional roles of ECM GAGs in lung function and disease. STATEMENT OF SIGNIFICANCE: Lung transplantation remains limited due to donor organ availability and need for life-long immunosuppressive medication. One solution, the ex vivo bioengineering of lungs via de- and recellularization has not yet led to a fully functional organ. Notably, the role of glycosaminoglycans (GAGs) remaining in decellularized lung scaffolds is poorly understood despite their important effects on cell behaviors. We have previously investigated residual GAG content of native and decellularized lungs and their respective functionality, and role during scaffold recellularization. We now present a detailed characterization of GAG and GAG chain content and function in different anatomical regions of normal diseased human lungs. These are novel and important observations that further expand knowledge about functional GAG roles in lung biology and disease.
Assuntos
Glicosaminoglicanos , Doença Pulmonar Obstrutiva Crônica , Humanos , Glicosaminoglicanos/metabolismo , Pulmão/patologia , Sulfatos de Condroitina , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Matriz Extracelular/metabolismo , Dissacarídeos/análise , Dissacarídeos/metabolismoRESUMO
Heparin ï¼Hpï¼ is the most widely used anticoagulant drug in the clinicsï¼ with an annual global output of over 10 billion dollars. Hpï¼ a member of the glycosaminoglycans ï¼GAGsï¼ï¼ is prepared from porcine intestinal mucosa via extractionï¼ separationï¼ and purification. Hp is a linear polysaccharide with repeating disaccharide units. Low-molecular-weight heparins ï¼LMWHsï¼ are depolymerized from Hp via chemical or enzymatic degradation. Compared with Hpï¼ LMWHs exhibit less bleeding side effectï¼ milder immunogenicityï¼ and higher bioavailability when injected subcutaneously. In generalï¼ Hpsï¼ including LMWHsï¼ are high complex drugs with large molecular weights ï¼MWsï¼ï¼ inhomogeneous MW distributionsï¼ and structural heterogeneityï¼ including different degrees and locations of sulfonationï¼ and unique residues generated from different production processes. Thusï¼ developing efficient analytical methods to elucidate the structures of Hps and characterize or quantitate their properties is extremely challenging. Unfortunatelyï¼ this problem limits their quality controlï¼ production optimizationï¼ clinical safety monitoringï¼ and new applications. Research has constantly sought to elucidate the complicated structures of Hp drugs. Among the structural analysis and quality control methods of Hp currently availableï¼ chromatographic methods are the most widely studied and used. Howeverï¼ no literature thoroughly summarizes the specific applications of chromatographic methods in the structural analysisï¼ manufacturing processï¼ and quality control of Hp drugs. This paper systematically organizes and describes recent research progresses of the chromatographic methods used to analyze Hp drugsï¼ including the identification and composition of monosaccharidesï¼ disaccharidesï¼ oligosaccharidesï¼ and polysaccharides. The applicationsï¼ innovationsï¼ and limitations of these chromatographic methods are also summarized in this review. The insights obtained in this study will help production and quality control personnelï¼ as well as drug researchersï¼ obtain a deeper understanding of the complex structures of Hp drugs. This paper also provides a comprehensive reference for the structural analysis and quality control of Hpsï¼ proposes ideas for the development of new quality control methodsï¼ and lays a strong foundation for the in-depth structural elucidation of Hp drugs.
Assuntos
Heparina de Baixo Peso Molecular , Heparina , Animais , Suínos , Anticoagulantes , Cromatografia , Heparina Liase/metabolismo , DissacarídeosRESUMO
In this work, the first version of "Glycomapping" software is developed for the analysis of the most common low-molecular-weight heparin (LMWH), enoxaparin. Using ultrahigh-performance liquid chromatography-mass spectrometry, size exclusion chromatography is applied, and a virtual database of glycans in enoxaparin is established for the initial searching. With "Glycomapping", a complex chromatogram can be fitted, significantly improving resolution and confirming an accurate distribution range for each size of glycan within enoxaparin. In addition, randomly matched MS data can be corrected, with the constraint of the corresponding chromatographic retention time range, to remove most false positive data. The analytical stability of "Glycomapping" software was confirmed. Enoxaparin, prepared by different manufacturers and from different animal sources, was analyzed using "Glycomapping." Compared to raw data, data processed with "Glycomapping" are more robust and accurate. Another two LMWHs, nadroparin and dalteparin could also be analyzed with this software. This work lays a solid foundation for the automated analysis of heterogeneous mixtures of natural glycans, such as LMWHs and other complex oligosaccharides and polysaccharides.
Assuntos
Enoxaparina , Heparina de Baixo Peso Molecular , Animais , Anticoagulantes , Cromatografia Líquida , Dalteparina , Enoxaparina/química , Heparina/química , Heparina de Baixo Peso Molecular/análise , Nadroparina/química , SoftwareRESUMO
Heparin is a linear sulfated polysaccharide with a complex structure. It is important to figure out the sequences at the terminals of the sugar chains, as it will help us understand the heparin structure deeper and control its quality properly. The tetrasaccharide linkage region (LR) could be a tag to help us find out heparin terminals after digestion by different combinations of heparinases. In this work, orthogonal chromatographic approaches including SAX, SEC-MS and 2D-LC-MS were applied to qualitatively and quantitatively analyze the heparinase released LR-terminals. The disaccharides next to LR are those ones with low or non-sulfation, UA-GlcNAc and UA-GlcNAc6S, and then they are extended with the highly sulfated disaccharides, IdoA2S-GlcNS and IdoA2S-GlcNS6S. It is suggested that the sulfo transferases did not work at the sugar residues next to LR terminal, especially the 2-O-sulfo and N-sulfo transferases, which could be affected by steric hindrance from LR, when heparin is biosynthesized. This conclusion will be theoretical fundamental to help us understand heparin's structure deeper. The methods provided in this work could be potential ways to control heparin's quality and monitor the production processes of heparin properly.
Assuntos
Dissacarídeos , Heparina , Dissacarídeos/química , Heparina/química , Heparina Liase , Oligossacarídeos/química , TransferasesRESUMO
The TEMPO/NaBr/NaClO system was used to modify konjac glucomannan and prepare ß-1,4-linked D-mannuronic/glucuronic acid oligosaccharides. The impact of oxidant amount on the degree of oxidation, molecular weight, terminal structure and the ratio of sugar units were systematically investigated. Mannoses were transformed to mannuronic acids quantitatively, while some glucose were transformed to glucuronic acids, some degraded. The unspecific oxidation causes strong degradation of konjac glucomannan sugar chains. Multiple terminal structures were observed in the oxidized KGM products. According to those observations, the oxidation pattern of konjac glucomannan with the TEMPO/NaBr/NaClO system was speculated. It is suggested that the axial hydroxyl group at position 2 in mannose might form a hydrogen bond with the oxygen on the sugar ring. It would against the unspecific oxidation. While, both specific and unspecific oxidations were observed at glucose units with an equatorial hydroxyl group at position 2, and the degradation occurs mostly on glucose during the oxidation.
Assuntos
Mananas , Manose , Carboidratos , Óxidos N-Cíclicos , Glucose/metabolismo , Mananas/química , Manose/metabolismoRESUMO
Low molecular weight heparins (LMWHs), depolymerized from unfractionated heparin (UFH), are widely used as anticoagulant drugs in clinic. The variable degradation methods result the different types of LMWHs, such as enoxaparin prepared by alkaline degradation following benzylation and nadroparin degraded by nitrous acid and subsequent reduction. They have different anticoagulant activities, molecular weight and special oligosaccharide sequences. Oligosaccharide analysis of the heparinase-catalyzed digestion products of heparin and LMWHs is an important way to explore the fine structural composition. In this work, a MHC-2D-LC-MS system using SAX followed by SEC and tandem to MS was applied to analyze the heparinase-products of LMWHs. 15 components of enoxaparin and 20 components of nadroparin were separated and unambiguously characterized with mass spectrum, including eight common disaccharides, and the special structural domains resistant to enzyme digestion which have the 3-O sulfated residue and/or characteristic terminal residues and the linkage region tetrasaccharides.
Assuntos
Heparina de Baixo Peso Molecular , Heparina , Anticoagulantes/química , Enoxaparina/química , Heparina/análise , Heparina Liase/metabolismo , Heparina de Baixo Peso Molecular/análiseRESUMO
Carrageenan (CGN) belongs to the sulfated polysaccharides family that is commonly used in the food industry. For oligosaccharide analysis, a liquid chromatography quadrupole time-of-flight/mass spectrometry strategy was developed using a hypothetical database. There are 2100 structures in the developed hypothetical κ-CGN database. To eliminate false-positive results, three approaches were used, including size exclusion chromatography with mass spectrometry, which differentiates the loss of sulfated groups caused by the hydrolysis process or the ionization process. Profiling of acidic hydrolysis products of κ-CGN was found that after 12 h of HCl cultivation, the κ-CGN was hydrolyzed to oligosaccharides lower than the degree of polymerization 10, breaking the α-1,3-glycoside linkage and producing even-numbered oligosaccharides. Another finding was that the pH at which acidic hydrolysis is terminated affects the generation of even and odd oligosaccharides. Peeling reaction occurs at the reduction end 4-linked-3,6-anhydrous-d-galactose when adjusted to alkaline conditions, thus generating odd oligosaccharides.
Assuntos
Oligossacarídeos , Espectrometria de Massas em Tandem , Carragenina/química , Cromatografia em Gel , Hidrólise , Oligossacarídeos/química , Sulfatos/química , Espectrometria de Massas em Tandem/métodosRESUMO
Heparin is a linear sulfated polysaccharide. It is composed of a repeating disaccharide unit with different sulfo patterns. The compositional analysis after heparin was decomposed to disaccharides and enzyme resistant domains is an important way to delve into its structure. Strong anion exchange (SAX) chromatography is commonly used for the compositional analysis due to its high resolution, stability and capability of quantitation. However, nonvolatile salt in mobile phase is not compatible with MS, then the structural domains cannot be identified without standards. Here, a new two-dimensional liquid chromatography system, multiple heart cut (MHC), was developed and linked to mass spectrometry (MS) directly to provide a comprehensive analysis of enzyme digested heparin. SAX was applied as the first dimensional chromatography, in which 17 peaks were observed and integrated in the digested heparin. Size-exclusion chromatography (SEC) was used as the second dimensional chromatography to desalt efficiently. Structural information of each component was then obtained with MS, including eight common disaccharides, eight enzyme resistant tetrasaccharides and a heparin-core protein linkage domain. The comparison of enzyme digested heparins obtained from different vendors using this system suggested their similar major structure and activity, but slightly different production processes.
Assuntos
Heparina Liase/metabolismo , Heparina/análise , Espectrometria de Massas/métodos , Cromatografia em Gel , Cromatografia por Troca Iônica , Cromatografia Líquida , Dissacarídeos/química , Heparina/química , Oligossacarídeos/químicaRESUMO
The use of electrospun scaffolds for neural tissue engineering applications allows a closer mimicry of the native tissue extracellular matrix (ECM), important for the transplantation of cells in vivo. Moreover, the role of the electrospun fiber mat topography on neural stem cell (NSC) differentiation remains to be completely understood. In this work REN-VM cells (NSC model) were differentiated on polycaprolactone (PCL) nanofibers, obtained by wet/wet electrospinning, and on flat glass lamellas. The obtained differentiation profile of NSCs was evaluated using immunofluorescence and qPCR analysis. Glycosaminoglycan (GAG) analysis was successfully emplyed to evaluate changes in the GAG profile of differentiating cells through the use of the highly sensitive liquid chromatography-tandem mass/mass spectrometry (LC-MS/MS) method. Our results show that both culture platforms allow the differentiation of REN-VM cells into neural cells (neurons and astrocytes) similarly. Moreover, LC-MS/MS analysis shows changes in the production of GAGs present both in cell cultures and conditioned media samples. In the media, hyaluronic acid (HA) was detected and correlated with cellular activity and the production of a more plastic extracellular matrix. The cell samples evidence changes in chondroitin sulfate (CS4S, CS6S, CS4S6S) and heparan sulfate (HS6S, HS0S), similar to those previously described in vivo studies and possibly associated with the creation of complex structures, such as perineural networks. The GAG profile of differentiating REN-VM cells on electrospun scaffolds was analyzed for the first time. Our results highlight the advantage of using platforms obtain more reliable and robust neural tissue-engineered transplants.
Assuntos
Diferenciação Celular , Sulfatos de Condroitina/biossíntese , Heparitina Sulfato/biossíntese , Células-Tronco Neurais/metabolismo , Alicerces Teciduais/química , Linhagem Celular Transformada , Humanos , Células-Tronco Neurais/citologiaRESUMO
A water-soluble ß-glucan from fruiting body of Hericium erinaceus was obtained after water extraction, purification and fractionation. Analyses of monosaccharide composition, molecular weight and linkage mode demonstrated that it is a ß-glucan with 1â3 and 1â6 linkage modes and a molecular weight of 13.3 kDa. An endo-1,6-ß-d-glucanase was used to digest the ß-glucan and the digested products over time were analyzed with a HPAEC-PAD-MS platform. The linkage mode of each glycosidic bond in the digested oligosaccharides were confirmed with MS/MS. At the end of digestion, enzyme resistant oligosaccharides were observed as several 1â6 linked glucoses with one or two 1â3 linked glucose residues. All these domains are more like constructional pieces from a branch-on-branch glucan, in which multiple 1â6 linked glucan chains are hooked through one or two 1â3 linked glucose residues. Averagely, there is a 1â3 linkage per six 1â6 linked glucoses in this branch-on-branch ß-glucan.
Assuntos
Carpóforos/química , Hericium/química , Monossacarídeos/análise , Oligossacarídeos/química , Espectrometria de Massas em Tandem/métodos , beta-Glucanas/química , Configuração de Carboidratos , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia por Troca Iônica/métodos , Peso MolecularRESUMO
Two bottom-up strategies, disaccharide and oligosaccharide analyses, were applied to elucidate the structure of a fucosylated chondroitin sulfate (FCS). The FCS was hydrolyzed with mild acid. The remained part was digested with CS lyase for disaccharide analysis. The products from each step were analyzed and the results revealed that mild sulfuric acid mainly released sulfated fucose branches, but also affected some residues and sulfo-groups on the backbone. Over 140 oligosaccharide fragments were generated by catalytic oxidation and identified by HPSEC-MS, including sulfated fucose oligosaccharides exclusively from branches, sulfated backbone fragments, and junctional fragments. Based on the results provided by these two methods, the proposed backbone of the FCS is mainly composed of GlcAâGalNAc4S6S and GlcAâGalNAc6S, and the branch is mainly located at GalNAc. The longest branch observed is nonasaccharide, and most of the fucose on the branches are mono and/or di-sulfated. NMR results supported the conclusion.
Assuntos
Sulfatos de Condroitina/análise , Pepinos-do-Mar/química , Animais , Sulfatos de Condroitina/metabolismo , Hidrólise , Pepinos-do-Mar/metabolismoRESUMO
Despite progress in use of decellularized lung scaffolds in ex vivo lung bioengineering schemes, including use of gels and other materials derived from the scaffolds, the detailed composition and functional role of extracellular matrix (ECM) proteoglycans (PGs) and their glycosaminoglycan (GAG) chains remaining in decellularized lungs, is poorly understood. Using a commonly utilized detergent-based decellularization approach in human autopsy lungs resulted in disproportionate losses of GAGs with depletion of chondroitin sulfate/dermatan sulfate (CS/DS) > heparan sulfate (HS) > hyaluronic acid (HA). Specific changes in disaccharide composition of remaining GAGs were observed with disproportionate loss of NS and NS2S for HS groups and of 4S for CS/DS groups. No significant influence of smoking history, sex, time to autopsy, or age was observed in native vs. decellularized lungs. Notably, surface plasmon resonance demonstrated that GAGs remaining in decellularized lungs were unable to bind key matrix-associated growth factors FGF2, HGF, and TGFß1. Growth of lung epithelial, pulmonary vascular, and stromal cells cultured on the surface of or embedded within gels derived from decellularized human lungs was differentially and combinatorially enhanced by replenishing specific GAGs and FGF2, HGF, and TGFß1. In summary, lung decellularization results in loss and/or dysfunction of specific GAGs or side chains significantly affecting matrix-associated growth factor binding and lung cell metabolism. GAG and matrix-associated growth factor replenishment thus needs to be incorporated into schemes for investigations utilizing gels and other materials produced from decellularized human lungs. STATEMENT OF SIGNIFICANCE: Despite progress in use of decellularized lung scaffolds in ex vivo lung bioengineering schemes, including use of gels and other materials derived from the scaffolds, the detailed composition and functional role of extracellular matrix (ECM) proteoglycans (PGs) and their glycosaminoglycan (GAG) chains remaining in decellularized lungs, is poorly understood. In the current studies, we demonstrate that glycosaminoglycans (GAGs) are significantly depleted during decellularization and those that remain are dysfunctional and unable to bind matrix-associated growth factors critical for cell growth and differentiation. Systematically repleting GAGs and matrix-associated growth factors to gels derived from decellularized human lung significantly and differentially affects cell growth. These studies highlight the importance of considering GAGs in decellularized lungs and their derivatives.
Assuntos
Células Epiteliais/efeitos dos fármacos , Matriz Extracelular/química , Glicosaminoglicanos/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Brônquios/citologia , Técnicas de Cultura de Células , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Feminino , Fator 2 de Crescimento de Fibroblastos/farmacologia , Glicosaminoglicanos/análise , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Engenharia Tecidual/métodos , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Sepsis patients are at increased risk for hospital-acquired pulmonary infections, potentially due to postseptic immunosuppression known as the compensatory anti-inflammatory response syndrome (CARS). CARS has been attributed to leukocyte dysfunction, with an unclear role for endothelial cells. The pulmonary circulation is lined by an endothelial glycocalyx, a heparan sulfate-rich layer essential to pulmonary homeostasis. Heparan sulfate degradation occurs early in sepsis, leading to lung injury. Endothelial synthesis of new heparan sulfates subsequently allows for glycocalyx reconstitution and endothelial recovery. We hypothesized that remodeling of the reconstituted endothelial glycocalyx, mediated by alterations in the endothelial machinery responsible for heparan sulfate synthesis, contributes to CARS. Seventy-two hours after experimental sepsis, coincident with glycocalyx reconstitution, mice demonstrated impaired neutrophil and protein influx in response to intratracheal lipopolysaccharide (LPS). The postseptic reconstituted glycocalyx was structurally remodeled, with enrichment of heparan sulfate disaccharides sulfated at the 6-O position of glucosamine. Increased 6-O-sulfation coincided with loss of endothelial sulfatase-1 (Sulf-1), an enzyme that specifically removes 6-O-sulfates from heparan sulfate. Intravenous administration of Sulf-1 to postseptic mice restored the pulmonary response to LPS, suggesting that loss of Sulf-1 was necessary for postseptic suppression of pulmonary inflammation. Endothelial-specific knockout mice demonstrated that loss of Sulf-1 was not sufficient to induce immunosuppression in non-septic mice. Knockdown of Sulf-1 in human pulmonary microvascular endothelial cells resulted in downregulation of the adhesion molecule ICAM-1. Taken together, our study indicates that loss of endothelial Sulf-1 is necessary for postseptic suppression of pulmonary inflammation, representing a novel endothelial contributor to CARS.
Assuntos
Células Endoteliais/enzimologia , Pulmão/imunologia , Pneumonia/prevenção & controle , Sepse/complicações , Sulfotransferases/deficiência , Animais , Feminino , Glicocálix/metabolismo , Lipopolissacarídeos/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia/etiologia , Pneumonia/metabolismo , Sepse/induzido quimicamente , Sepse/patologiaRESUMO
Enoxaparin, one of the most important low-molecular-weight heparins (LMWHs), is widely used as a clinical anticoagulant. Different production processes and animal sources of its precursor (unfractionated heparin) can result in the structural diversity of enoxaparin. In this study, 38 lots of enoxaparin prepared at different times, from different providers and animal sources, were systematically analyzed. SEC and SAX were used to analyze the oligosaccharide dispersity and structural compositions (disaccharide domains) of enoxaparins by size and charge, respectively. The results provide clues as to whether the structural variations in enoxaparin, observed in oligosaccharide mapping and/or disaccharide analysis, are attributable to differences in the animal sources of its heparin precursor or enoxaparin production processes based on times or brands. The representative enoxaparins were fingerprinted with online multiple heart-cut two-dimensional liquid chromatography-mass spectrometry (MHC-2DLC-MS). The profiles in MHC-2DLC-MS showed the detailed structural information of enoxaparins. In addition, the binding capacities to antithrombin III (AT) of these 38 lots of enoxaparins were detected using surface plasmon resonance (SPR) with the competitive inhibition mode. The results showed that the glycan size distribution of an enoxaparin is more related to its production process. The disaccharide composition, sequence and the variety of glycans of an enoxaparin are more related to its AT binding-based anticoagulant activity.
RESUMO
Heparin is a polypharmacological agent with anticoagulant activity. Periodate oxidation of the nonsulfated glucuronic acid residue results in non-anticoagulant heparin derivative (NACH) of reduced molecular weight. Similar treatment of a low molecular weight heparin, dalteparin, also removes its anticoagulant activity, affording a second heparin derivative (D-NACH). A full structural characterization of these two derivatives reveals their structural differences. SPR studies display their ability to bind to several important heparin-binding proteins, suggesting potential new therapeutic applications.
Assuntos
Heparina de Baixo Peso Molecular/química , Preparações Farmacêuticas/química , Animais , Anticoagulantes/química , Ligação Competitiva , Cromatografia Líquida de Alta Pressão , Dalteparina/química , Heparina de Baixo Peso Molecular/análise , Espectrometria de Massas , Oxirredução , Ácido Periódico/química , Ressonância de Plasmônio de Superfície , SuínosRESUMO
Glycosaminoglycans (GAGs) are biologically and pharmacologically important linear, anionic polysaccharides containing various repeating disaccharides sequences. The analysis of these polysaccharides generally relies on their chemical or enzymatic breakdown to disaccharide units that are separated, by chromatography or electrophoresis, and detected, by UV, fluorescence, or mass spectrometry (MS). Isoelectric focusing (IEF) is an important analytical technique with high resolving power for the separation of analytes exhibiting differences in isoelectric points. One format of IEF, the capillary isoelectric focusing (cIEF), is an attractive approach in that it can be coupled with mass spectrometry (cIEF-MS) to provide online focusing and detection of complex mixtures. In the past three decades, numerous studies have applied cIEF-MS methods to the analysis of protein and peptide mixtures by positive-ion mode mass spectrometry. However, polysaccharide chemists largely rely on negative-ion mode mass spectrometry for the analysis of highly sulfated GAGs. The current study reports a negative-ion mode cIEF-MS method using an electrokinetically pumped sheath liquid nanospray capillary electrophoresis-mass spectrometry (CE-MS) coupling technology. The feasibility of this negative-ion cIEF-MS method and its potential applications are demonstrated using chondroitin sulfate and heparan sulfate oligosaccharides mixtures.
Assuntos
Dissacarídeos/análise , Focalização Isoelétrica/métodos , Espectrometria de Massas/métodos , Proteínas de Bactérias/química , Sequência de Carboidratos , Condroitina ABC Liase/química , Sulfatos de Condroitina/análise , Sulfatos de Condroitina/química , Dissacarídeos/química , Escherichia coli/enzimologia , Heparina Liase/química , Heparitina Sulfato/análise , Heparitina Sulfato/química , Ponto Isoelétrico , Pedobacter/enzimologia , Proteus vulgaris/enzimologiaRESUMO
There is a critical need to generate functional hepatocytes to aid in liver repair and regeneration upon availability of a renewable, and potentially personalized, source of human hepatocytes (hHEP). Currently, the vast majority of primary hHEP are obtained from human tissue through cadavers. Recent advances in stem cell differentiation have opened up the possibility to obtain fully functional hepatocytes from embryonic or induced pluripotent stem cells, or adult stem cells. With respect to the latter, human bone marrow mesenchymal stromal cells (hBMSCs) can serve as a source of autogenetic and allogenic multipotent stem cells for liver repair and regeneration. A major aspect of hBMSC differentiation is the extracellular matrix (ECM) composition and, in particular, the role of glycosaminoglycans (GAGs) in the differentiation process. In this study, we examine the influence of four distinct culture conditions/protocols (T1-T4) on GAG composition and hepatic markers. α-Fetoprotein and hepatocyte nuclear factor-4α were expressed continually over 21 days of differentiation, as indicated by real time quantitative PCR analysis, while albumin (ALB) expression did not begin until day 21. Hepatocyte growth factor (HGF) appears to be more effective than activin A in promoting hepatic-like cells through the mesenchymal-epithelial transition, perhaps due to the former binding to the HGF receptor to form a unique complex that diversifies the biological functions of HGF. Of the four protocols tested, uniform hepatocyte-like morphological changes, ALB secretion, and glycogen storage were found to be highest with protocol T2, which involves both early- and late-stage combinations of growth factors. The total GAG profile of the hBMSC ECM is rich in heparan sulfate (HS) and hyaluronan, both of which fluctuate during differentiation. The GAG profile of primary hHEP showed an HS-rich ECM, and thus, it may be possible to guide hBMSC differentiation to more mature hepatocytes by controlling the GAG profile expressed by differentiating cells.
Assuntos
Diferenciação Celular , Glicosaminoglicanos/metabolismo , Hepatócitos/citologia , Células-Tronco Mesenquimais/metabolismo , Ativinas/farmacologia , Osso e Ossos/citologia , Células Cultivadas , Técnicas de Reprogramação Celular/métodos , Fator de Crescimento de Hepatócito/farmacologia , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Hepatócitos/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , alfa-Fetoproteínas/genética , alfa-Fetoproteínas/metabolismoRESUMO
Heparin is a polysaccharide anticoagulant drug isolated from animal tissues. There have been concerns on the safety and security of the heparin supply chain since 2007-8 when a contamination crisis led to its disruption. The current study applies a suite of modern analytical techniques to porcine, bovine and ovine intestinal mucosal heparins. These techniques include structural analysis by nuclear magnetic resonance spectrometry, disaccharide compositional analysis, bottom-up analysis of tetrasaccharides corresponding to heparin's antithrombin III binding site. Chemometric analysis was then applied to understand how these structural differences to predict the animal/tissue source of heparin and to help detect blending of heparins from various sources.