Heparins are widely used anticoagulant drugs. The current monitoring practice for heparin in plasma, such as the chromogenic anti-factor Xa assay, relies on heparin-triggered activation of antithrombin, an inhibitor of coagulation proteases. Such assays are not applicable to the detection of non-anticoagulant heparins, an emerging class of drug candidates for therapeutic applications unrelated to anticlotting activity. This study describes the application of a commercially available fluorescent probe assay (Heparin Red) for the direct and sensitive detection of the "chemical" heparin in plasma, independent of any anticoagulant activity. The quantification range is about 0-5 μg/mL for both unfractionated heparin (corresponding to 0-1 IU/mL) and the low molecular weight heparin enoxaparin. The Heparin Red assay is of particular value for the quantification of non-anticoagulant heparins, as exemplified by the low molecular weight heparin derivative tafoxiparin and a N-desulfated-N-reacetylated heparin. Heparin octa- and decasaccharides are also detected. Graphical abstract Heparin quantification in plasma by mixing the sample with the Heparin Red reagent and fluorescence readout.
Microbial nitrate (NO 3 − ) removal via denitrification (DNF) at high sulfide (H 2 S) concentrations was compared in sediment from a coastal freshwater pond in a developed area that receives salt-water influx during storm events, and a saline pond proximal to an undeveloped estuary. Sediments were incubated with added SO 4 2− (1,000 µg per gram dry weight basis (gdw)) to determine whether acid volatile sulfides (AVS) were formed. DNF in the sediments was measured with NO 3 -N (300 µg gdw −1 ) alone, and with NO 3 -N and H 2 S (1,000 µg S 2− gdw −1 ). SO 4 2− addition to the freshwater sediments resulted in AVS formation (970± 307 µg S gdw −1 ) similar to the wetland with no added SO 4 2− (986±156 µg S gdw −1 ). DNF rates measured with no added H 2 S were greater in the freshwater than the wetland site (10.6±0.6 vs. 6.4±0.1 µg N 2 O-N gdw −1 h −1 , respectively). High H 2 S concentrations retained NH 4 -N in the undeveloped wetland and retained NO 3 -N in the developed freshwater site, suggesting that potential salt-water influx may reduce the ability of the freshwater sediments to remove NO 3 -N.
Glycosaminoglycans are complex biomolecules of great biological and medical importance. The quantification of glycosaminoglycans, in particular in complex matrices, is challenging due to their inherent structural heterogeneity. Heparin Red, a polycationic, fluorescent perylene diimide derivative, has recently emerged as a commercial probe for the convenient detection of heparins by a mix-and-read fluorescence assay. The probe also detects glycosaminoglycans with a lower negative charge density than heparin, although with lower sensitivity. We describe here the synthesis and characterization of a structurally related molecular probe with a higher positive charge of +10 (vs. +8 of Heparin Red). The superior performance of this probe is exemplified by the quantification of low dermatan sulfate concentrations in an aqueous matrix (quantification limit 1 ng/mL) and the detection of dermatan sulfate in blood plasma in a clinically relevant concentration range. The potential applications of this probe include monitoring the blood levels of dermatan sulfate after administration as an antithrombotic drug in the absence of heparin and other glycosaminoglycans.
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