Highly sensitive real-time PCR method to identify species origin in heparinoids.

Heparinoids are the starting material for sulodexide production, a drug used as intravenous anti-coagulant, as an alternative to heparin. The origin determination in the starting material for sulodexide, heparin, and derivatives is crucial for safety (including the impact related to bovine spongiform encephalopathy) and efficacy of the final products. Therefore, European countries have decided to approve the production of heparin only from porcine intestinal mucosa. PCR (polymerase chain reaction) methods are available to evaluate the origin species of crude heparin, during heparin production process, while they lack for the same analysis in heparinoids during sulodexide manufacturing processes. Notably, two main critical issues occur during the origin determination by using PCR for heparinoid analysis: first, heparin has been known to inhibit DNA polymerase activity and, second, the DNA amounts are very low in these samples. To overcome these critical issues, our proposed method is based on two fundamental steps, the DNA concentration by glycogen treatment and DNA purification, which occur before and after DNA extraction, respectively. Finally, by applying real-time PCR, we amplify three specific DNA sequences of ruminant species (bovine, ovine, and caprine), to assess possible contamination, and one from swine, to confirm the origin species. To date, such a method is the only one that determines origin species by PCR for heparinoids that guarantee quality, safety, and traceability of heparin-derived pharmaceutical products. In conclusion, our proposed method is an alternative to nuclear magnetic resonance and ELISA methods, because real-time PCR offers significant advantages in sensitivity, specificity, and robustness. Graphical Abstract.

An uncommon cause of postoperative bleeding.

The postoperative phase can be associated with an increased risk of thrombosis and abnormal bleeding. We report the case of a patient admitted to our intensive care unit for postoperative monitoring who developed two episodes of hemorrhagic shock. A thromboelastogram performed with heparinase I digestion demonstrated the action of endogenous heparin-like substances whose presence was subsequently confirmed at the histological examination of the tumor. Heparinase-modified thromboelastography has proven to be an effective and rapid point-of-care coagulation test and in our case allowed early detection of circulating heparinoids.

Comparative study of Factor Xa fluorogenic substrates and their influence on the quantification of LMWHs.

Low molecular weight heparins (LMWHs) are recognised as the preferred anticoagulants in the prevention and treatment of venous thromboembolism. Anti-Factor Xa (anti-FXa) levels are used to monitor the anticoagulant effect of LMWHs and such assays are routinely employed in hospital diagnostic laboratories. In this study, a fluorogenic anti-FXa assay was developed using a commercially available fluorogenic substrate with an attached 6-amino-1-naphthalene-sulfonamide (ANSN) fluorophore and was used for the determination of two LMWHs, enoxaparin and tinzaparin and the heparinoid, danaparoid. The assay was based on the complexation of heparinised plasma with 100 nM exogenous FXa and 25 µM of the fluorogenic substrate Mes-D-LGR-ANSN (C(2)H(5))(2) (SN-7). The assay was tested with pooled plasma samples spiked with anticoagulant concentrations in the range 0-1.6 U mL(-1). The statistically sensitive assay range was 0-0.4 U mL(-1) for enoxaparin and tinzaparin and 0-0.2 U mL(-1) for danaparoid, with assay variation typically below 10.5%. This assay was then compared with a previously published fluorogenic anti-FXa assay developed with the peptide substrate, methylsulfonyl-D: -cyclohexylalanyl-glycyl-arginine-7-amino-4-methylcoumarin acetate (Pefafluor FXa). Both assays were compared in terms of fluorescence intensity, lag times and sensitivity to anticoagulants.

Improved hydrophilic interaction chromatography LC/MS of heparinoids using a chip with postcolumn makeup flow.

Heparan sulfate (HS) and heparin are linear, heterogeneous carbohydrates of the glycosaminoglycan (GAG) family that are modified by N-acetylation, N-sulfation, O-sulfation, and uronic acid epimerization. HS interacts with growth factors in the extracellular matrix, thereby modulating signaling pathways that govern cell growth, development, differentiation, proliferation, and adhesion. High-performance liquid chromatography (HPLC)-chip-based hydrophilic interaction liquid chromatography/mass spectrometry has emerged as a method for analyzing the domain structure of GAGs. However, analysis of highly sulfated GAG structures decasaccharide or larger in size has been limited by spray instability in the negative-ion mode. This report demonstrates that addition of postcolumn makeup flow to the amide-HPLC-chip configuration permits robust and reproducible analysis of extended GAG domains (up to degree of polymerization 18) from HS and heparin. This platform provides quantitative information regarding the oligosaccharide profile, degree of sulfation, and nonreducing chain termini. It is expected that this technology will enable quantitative, comparative glycomics profiling of extended GAG oligosaccharide domains of functional interest.

Heparins: process-related physico-chemical and compositional characteristics, fingerprints and impurities.

During the past 25 years, heparin extraction and purification processes have changed. The results of these changes are reflected by the continuous increase in potency of the International Standard for heparin. This increase is due not only to a higher purity, but also to a number of changes in the physico-chemical characteristics of heparin. For long time, all these changes have been disregarded as non-critical by regulatory authorities. Heparin marketing authorisation was reviewed only two years ago and Pharmacopoeia monographs were reviewed just for the addition of new tests, mainly aimed at tackling the oversulfated chondroitin sulfate (OSCS) crisis. Currently, heparin monographs are again under revision. Such changes, different for each manufacturer, have caused a further increase in the heterogeneity of individual batches of heparin. This review aims at showing that chemical, physical and biological characteristics of heparin (such as disaccharide composition, amount of low sulfated and high sulfated sequences, molecular weight profiles [MW], activities, structural artifacts, fingerprints and glycosaminoglycans impurities) are all process-dependent and may significantly vary when different processes are used to minimise the content of dermatan sulfate. The wide heterogeneity of the physico-chemical characteristics of currently marketed heparin and the lack of suitable and shareable reference standards for the identification/quantification of process-related impurities caused, and are still causing, heated debates among scientific institutions, companies and authorities.

Analysis of pharmaceutical heparins and potential contaminants using (1)H-NMR and PAGE.

In 2008, heparin (active pharmaceutical ingredient, API) lots were associated with anaphylactoid-type reactions. Oversulfated chondroitin sulfate (OSCS), a semi-synthetic glycosaminoglycan (GAG), was identified as a contaminant and dermatan sulfate (DS) as an impurity. While DS has no known toxicity, OSCS was toxic leading to patient deaths. Heparins, prepared before these adverse reactions, needed to be screened for impurities and contaminants. Heparins were analyzed using high-field (1)H-NMR spectroscopy. Heparinoids were mixed with a pure heparin and analyzed by (1)H-NMR to assess the utility of (1)H-NMR for screening heparin adulterants. Sensitivity of heparinoids to deaminative cleavage, a method widely used to depolymerize heparin, was evaluated with polyacrylamide gel electrophoresis to detect impurities and contaminants, giving limits of detection (LOD) ranging from 0.1% to 5%. Most pharmaceutical heparins prepared between 1941 and 2008 showed no impurities or contaminants. Some contained DS, CS, and sodium acetate impurities. Heparin prepared in 2008 contained OSCS contaminant. Heparin adulterated with heparinoids showed additional peaks in their high-field (1)H-NMR spectra, clearly supporting NMR for monitoring of heparin API with an LOD of 0.5-10%. Most of these heparinoids were stable to nitrous acid treatment suggesting its utility for evaluating impurities and contaminants in heparin API.

Determination of low molecular weight heparin in clinical laboratory.

The applicability was investigated of automated spectrophotometric heparin assays and three clotting assays for determination of two low molecular weight (LMW) heparin fractions: Org 10172 and DxN10 and two infractionated commercially available heparins. The relative activity of the two commercially available heparins was similar in the anti-Xa assay, in the anti-IIa assay and in 3 clotting assays. The LMW heparins showed markedly different relative activity in all 5 assays. The activities of those heparin preparations relative to the standard heparin were compared in the 5 assays, but standardization against a standard heparin preparation appeared impossible. Methods of heparin determination can be used to monitor treatment with a heparin preparation only if the same preparation is used as a reference substance.

Anticoagulant activity of a synthetic heparinoid in relation to molecular weight and N-sulfate content.

Addition of chlorosulfonyl isocyanate to C==C bonds in cis-1,4-polyisoprene and reaction of the adduct with NaOH resulted in the formation of a water-soluble polyelectrolyte with N-sulfate and carboxylate groups. The polyelectrolyte showed anticoagulant activity and it was found, just as with heparin, that the activity was related to molecular weight and N-sulfate content.

Glycosaminoglycans from Ateroid and bovine duodenal mucosa and pancreas.

Glycosaminoglycans (GG) were isolated from commercial Ateroid and compared with those from bovine duodenal mucosa and pancreas. The major GG in Ateroid is heparin. Heparan sulfate (HS) and dermatan sulfate were also found. HS, chondroitin sulfates, and heparin were isolated from duodenal mucosa after papain digestion, but a residue, non-digestible, was mostly heparin. Pancreas contains very little GG, and the GG composition is similar to that of mucosa. The heparin isolated from Ateroid and mucosa have similar lipoprotein lipase-releasing activity, but the former has considerably less anticoagulant activity. Interestingly, papain digestion of mucosa and pancreas did not release all heparin from the tissue, suggesting that the protein to which heparin is linked is not readily accessible to the enzyme.