Electrophoresis for the analysis of heparin purity and quality

Volpi, Nicola; Maccari, Francesca; Suwan, Jiraporn; Linhardt, Robert J.

doi:10.1002/elps.201100479

Cited by 39 publications

(26 citation statements)

References 33 publications

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“…Although heparin and HS are both polydisperse and heterogeneous in nature, heparin is intrinsically more heavily sulfated than HS, 101,102 which is likely responsible for its strong inhibitory effect on GDF5 activity. While the unfractionated HS preparation did not inhibit GDF5 activity, it did not significantly promote the activity of the growth factor either.…”

Section: Discussionmentioning

confidence: 99%

Growth Differentiation Factor 5-Mediated Enhancement of Chondrocyte Phenotype Is Inhibited by Heparin: Implications for the Use of Heparin in the Clinic and in Tissue Engineering Applications

Ayerst

Smith

Nurcombe

et al. 2017

Tissue Engineering Part A

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The highly sulfated glycosaminoglycan (GAG) heparin is widely used in the clinic as an anticoagulant, and researchers are now using it to enhance stem cell expansion/differentiation protocols, as well as to improve the delivery of growth factors for tissue engineering (TE) strategies. Growth differentiation factor 5 (GDF5) belongs to the bone morphogenetic protein family of proteins and is vital for skeletal formation; however, its interaction with heparin and heparan sulfate (HS) has not been studied. We identify GDF5 as a novel heparin/HS binding protein and show that HS proteoglycans are vital in localizing GDF5 to the cell surface. Clinically relevant doses of heparin (≥10 nM), but not equivalent concentrations of HS, were found to inhibit GDF5's biological activity in both human mesenchymal stem/stromal cell-derived chondrocyte pellet cultures and the skeletal cell line ATDC5. We also found that heparin inhibited both GDF5 binding to cell surface HS and GDF5-induced induction of Smad 1/5/8 signaling. Furthermore, GDF5 significantly increased aggrecan gene expression in chondrocyte pellet cultures, without affecting collagen type X expression, making it a promising target for the TE of articular cartilage. Importantly, this study may explain the variable (and disappointing) results seen with heparin-loaded biomaterials for skeletal TE and the adverse skeletal effects reported in the clinic following long-term heparin treatment. Our results caution the use of heparin in the clinic and in TE applications, and prompt the transition to using more specific GAGs (e.g., HS derivatives), with better-defined structures and fewer off-target effects.

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Section: Discussionmentioning

confidence: 99%

Growth Differentiation Factor 5-Mediated Enhancement of Chondrocyte Phenotype Is Inhibited by Heparin: Implications for the Use of Heparin in the Clinic and in Tissue Engineering Applications

Ayerst

Smith

Nurcombe

et al. 2017

Tissue Engineering Part A

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“…Bottom-up analysis of LMWHs include disaccharide compositional analysis 16–22 and oligosaccharide mapping. 23,24 In these methods LMWH is further broken down to its constituent disaccharides or oligosaccharides by more complete chemical or enzymatic treatment and these fragments are characterized and quantified using LC, 16,18 capillary electrophoresis (CE), 21,25 polyacrylamide gel electrophoresis, 24,26 or hyphenated techniques, 27 such as LC-MS 27–30 or CE-laser induced fluorescence (LIF). 21 While these methods allow the sensitive detection of minor components, such as 1,6-anhydro amino sugar residues, they result in the loss of much of the information required to understand the primary structure or sequence of the individual polysaccharide chains comprising a LMWH.…”

Section: Introductionmentioning

confidence: 99%

Top-Down Approach for the Direct Characterization of Low Molecular Weight Heparins Using LC-FT-MS

Zhang²,

et al. 2012

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Low molecular heparins (LMWHs) are structurally complex, heterogeneous, polydisperse, and highly negatively charged mixtures of polysaccharides. The direct characterization of LMWH is a major challenge for currently available analytical technologies. Electrospray ionization (ESI) liquid chromatography-mass spectrometry (LC-MS) is a powerful tool for the characterization complex biological samples in the fields of proteomics, metabolomics and glycomics. LC-MS has been applied to the analysis of heparin oligosaccharides, separated by size exclusion, reversed phase ion-pairing chromatography and by chip-based amide hydrophilic interaction chromatography (HILIC). However, there have been limited applications of ESI-LC-MS for the direct characterization of intact LMWHs (top-down analysis) due to their structural complexity, low ionization efficiency and sulfate loss. Here we present a simple and reliable HILIC-Fourier transform (FT)-ESI-MS platform to characterize and compare two currently marketed LMWH products using the top-down approach requiring no special sample preparation steps. This HILIC system relies on cross-linked diol rather than amide chemistry, affording highly resolved chromatographic separations using a relatively high percentage of acetonitrile in the mobile phase, resulting in stable and high efficiency ionization. Bioinformatics software (GlycerSoft 1.0) was used to automatically assign structures within 5-ppm mass accuracy.

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“…Many researchers have focused on developing the detection method for heparin based on analytical methods, such as anion exchange chromatography, capillary electrophoresis, potentiometric methods, colorimetric method,, electrochemistry, ion mobility mass spectrometry and Surface‐Enhanced Raman Scattering (SERS) . These methods exhibit high sensitivity towards heparin, however, is complicated and time‐consuming which limit their practical applications…”

Section: Introductionmentioning

confidence: 99%

Sensitive Detection of Coralyne and Heparin Using a Singly Labeled Fluorescent Oligonucleotide Probe

Zhu

Chen

2019

ChemistrySelect

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Herein, we designed and developed a simple, rapid and sensitive method for detection of coralyne and heparin using a singly labeled fluorescent probe, based on photoinduced electron transfer (PIET). A specific oligonucleotide strand was labeled with fluorophore (6‐FAM) at its 5’ end and four guanines at its 3’ end. In the presence of coralyne, homo‐adenine DNA duplex is formed via A2‐coralyne‐A2 coordination, thus leading to the fluorescence quenching due to PIET between guanine and fluorophore. Upon the addition of heparin, the formation of heparin‐coralyne complex can prevent coralyne from forming A2‐coralyne‐A2 coordination, separating fluorophore from guanines; as a result, the fluorescence intensity can be recovered. The detection limits of the method with respect to coralyne and heparin were 3.1 nM and 0.04 nM (S/N=3), respectively. Moreover, the proposed method was successfully applied to detect heparin in human serum sample, further demonstrating that it can have promising practical applications.

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Electrophoresis for the analysis of heparin purity and quality

Cited by 39 publications

References 33 publications

Growth Differentiation Factor 5-Mediated Enhancement of Chondrocyte Phenotype Is Inhibited by Heparin: Implications for the Use of Heparin in the Clinic and in Tissue Engineering Applications

Growth Differentiation Factor 5-Mediated Enhancement of Chondrocyte Phenotype Is Inhibited by Heparin: Implications for the Use of Heparin in the Clinic and in Tissue Engineering Applications

Top-Down Approach for the Direct Characterization of Low Molecular Weight Heparins Using LC-FT-MS

Sensitive Detection of Coralyne and Heparin Using a Singly Labeled Fluorescent Oligonucleotide Probe

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