Profiling glycol-split heparins by high-performance liquid chromatography/mass spectrometry analysis of their heparinase-generated oligosaccharides

Abstract

Glycol-split (gs) heparins, obtained by periodate oxidation / borohydride reduction of heparin currently used as anticoagulant and antithrombotic drug, are arousing increasing interest in anti-cancer and anti-inflammation therapies. These new medical uses are favored by the loss of anticoagulant activity associated with glycol-splitting-induced inactivation of the antithrombin III (AT) binding site. The structure of gs-heparins has not been studied yet in detail. In this work, an ion-pair reversed-phase chromatography (IPRP-HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) widely used for unmodified heparin has been adapted to the analysis of oligosaccharides generated by digestion with heparinases of gs-heparins usually prepared from porcine mucosal heparin. The method has been also found very effective in analyzing glycol-split derivatives obtained from heparins of different animal and tissue origin. Besides the major 2-O-sulfated disaccharides, heparinase digests of gs-heparins mainly contain tetra- and hexasaccharides incorporating one or two gs residues, with distribution patterns typical for individual gs-heparins. A heptasulfated, mono-N-acetylated hexasaccharide with two gs residues has been shown to be a marker of the gs-modified AT binding site within heparin chains.

Analysis of 3-O-sulfo group-containing heparin tetrasaccharides in heparin by liquid chromatography–mass spectrometry

“…Due to the sensitivity of detection, only the main peak (ΔUA-GlcNAc6S-GlcA-GlcNS3S6S) was detected and the other two compounds were out of the limit of detection [29]. RPIP-HPLC separation coupled to MS detection enhanced the detection sensitivity by an order of magnitude and offered a second method of analyte identification [30]. In our laboratory, RPIP-UPLC-MS has been applied successfully for analysis of chondroitin sulfate (CS)/dermatan sulfate (DS) disaccharides [31] and heparin/heparan sulfate (HS) disaccharides [32].…”

“…The 3 major 3- O -sulfo tetrasaccharides in the heparin and LMWH samples were quantified for the first time [30]. The tetrasaccharide composition of the heparin and LMWH samples are presented in Table 2.…”

Anti-heparanase activity of ultra-low-molecular-weight heparin produced by physicochemical depolymerization

“…Glycol-split heparins were prepared by exhaustive periodate oxidation followed by 165 borohydride reduction of heparin (Alekseeva et al, 2013). Reaction mixture (3 mL) 166 containing 50 mg of heparin and 0.1 M NaIO 4 was incubated in the dark at 4°C under stirring 167 at 500 rpm.…”

Susceptibility of enoxaparin reducing end amino sugars to periodate oxidation

“…Notably, we have previously observed the presence of ΔU 2S -A NS6S -I 2S -R am in trace amount in the heparinase-digests of gs-heparins 5 and in relatively high quantity in the digest of gs-enoxaparin. 12 In the first case its formation was hypothesized to be the result of oxidation of non- N -substituted amino sugars that could be present in heparins, while in the case of gs-enoxaparin its generation was mainly associated with oxidation of terminal N -sulfated mannosamine.…”

“…5 The new medical uses of gs-heparins are favored by the loss of anticoagulant activity associated with glycol-splitting of the D-glucuronic acid residue within the ATBR sequence, which is essential for strong binding to, and activation of, antithrombin (AT). 6,7 Other biological activities of heparins, which are not critically dependent on the intact AT-binding sequence, usually are not significantly impaired by glycol-splitting.…”