A colorimetric assay for chemical heparin in plasma

Klein, Michael D.; Drongowski, Robert A.; Linhardt, Robert J.; Langer, Robert

doi:10.1016/0003-2697(82)90219-6

Cited by 53 publications

(25 citation statements)

References 15 publications

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“…The stability of the sulfate groups on the sugar backbone was monitored using (7-aminophenothiazin-3-ylidene)-dimethylazanium chloride (Azure A), which binds the sulfated groups in a polysaccharide chain [58]. Briefly, 200 µL of a 10 mg/L solution of aqueous Azure A were added to 20 µL of diluted sulfated polysaccharide samples.…”

Section: Methodsmentioning

confidence: 99%

Assessment of Heparanase-Mediated Angiogenesis Using Microvascular Endothelial Cells: Identification of λ-Carrageenan Derivative as a Potent Anti Angiogenic Agent

et al. 2017

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Heparanase is overexpressed by tumor cells and degrades the extracellular matrix proteoglycans through cleavage of heparan sulfates (HS), allowing pro-angiogenic factor release and thus playing a key role in tumor angiogenesis and metastasis. Here we propose new HS analogs as potent heparanase inhibitors: Heparin as a positive control, Dextran Sulfate, λ-Carrageenan, and modified forms of them obtained by depolymerization associated to glycol splitting (RD-GS). After heparanase activity assessment, 11 kDa RD-GS-λ-Carrageenan emerged as the most effective heparanase inhibitor with an IC50 of 7.32 ng/mL compared to 10.7 ng/mL for the 16 kDa unfractionated heparin. The fractionated polysaccharides were then tested in a heparanase-rich medium-based in vitro model, mimicking tumor microenvironment, to determine their effect on microvascular endothelial cells (HSkMEC) angiogenesis. As a preliminary study, we identified that under hypoxic and nutrient poor conditions, MCF-7 cancer cells released much more mature heparanase in their supernatant than in normal conditions. Then a MatrigelTM assay using HSkMEC cultured under hypoxic conditions in the presence (or not) of this heparanase-rich supernatant was realized. Adding heparanase-rich media strongly enhanced angiogenic network formation with a production of twice more pseudo-vessels than with the control. When sulfated polysaccharides were tested in this angiogenesis assay, RD-GS-λ-Carrageenan was identified as a promising anti-angiogenic agent.

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Section: Methodsmentioning

confidence: 99%

Assessment of Heparanase-Mediated Angiogenesis Using Microvascular Endothelial Cells: Identification of λ-Carrageenan Derivative as a Potent Anti Angiogenic Agent

et al. 2017

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“…The entrapment efficiency of the formulations was determined indirectly by measuring the amount of unentrapped LMWH in the external aqueous solution recovered after centrifugation and washing of the microparticles using an azure A colorimetric method, which is based on the metachromatic reaction between heparin and azure A dye [32]. Briefly, an aliquot of EAP samples was mixed with a fixed amount of azure A dye solution (17.8 μg/ml) in a 96-well microtiter plate and absorbance was measured at 620 nm (Synergy MX Microplate Reader, Biotech, Winnoski, VT).…”

Section: Entrapment Efficiencymentioning

confidence: 99%

“…An aliquot of samples (200 μl) was withdrawn following centrifugation over a period of 48 h at various time intervals and the amount of drug released from the microparticles was measured using an azure A colorimetric method as described earlier [32].…”

Section: In Vitro Lmwh Release Studymentioning

confidence: 99%

PEG–PLGA based large porous particles for pulmonary delivery of a highly soluble drug, low molecular weight heparin

Patel

Gupta

Ahsan

2012

Journal of Controlled Release

130

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“…Azure A dye is known to undergo a shift of its adsorption spectrum from blue to purple in the presence of heparin (6). Indeed, this unique metachtomatic characteristic of azure A dye has been used to measure the chemical quantities of heparin in plasma samples (13). Since the binding between azure A and heparin is reversible and being relatively weak compared to that between protamine and heparin, when protamine is added to the heparin-azure A dye complex, it will displace azure A dye from the complex and subsequently restore the dye's original adsorption spectrum.…”

Section: Discussionmentioning

confidence: 99%

A method for the quantitation of protamine in plasma

Yang

Teng

et al. 1994

Thrombosis Research

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A unique and simple colorimetric method for the quantitation of plasma protamine levels has been developed. The method is established on the competitive binding displacement mechanism between protamine and heparin-azure A dye complex, and the metachromatic color change of azure A dye in the presence of heparin. Because the method is based on the clinical specificity of protamine as the heparin antagonist, it is specific for protamine quantitation. Plasma protamine levels determined by this method are within 94% of accuracy when compared with their aqueous counterparts determined by the conventional Lowry protein assay. Since the method measures the protamine excess after heparin neutralization, it potentially could be employed during clinical heparin reversal with protamine to monitor protamine excess. In addition, the method may provide a useful means to identify the mechanism of the so-called "heparin rebound".

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A colorimetric assay for chemical heparin in plasma

Cited by 53 publications

References 15 publications

Assessment of Heparanase-Mediated Angiogenesis Using Microvascular Endothelial Cells: Identification of λ-Carrageenan Derivative as a Potent Anti Angiogenic Agent

Assessment of Heparanase-Mediated Angiogenesis Using Microvascular Endothelial Cells: Identification of λ-Carrageenan Derivative as a Potent Anti Angiogenic Agent

PEG–PLGA based large porous particles for pulmonary delivery of a highly soluble drug, low molecular weight heparin

A method for the quantitation of protamine in plasma

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